Principles of cell signaling - UT Southwestern Medical Center at Dallas
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39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 589 TICAM-1 P TRIF/ TICAM-1 P TRAM y Tyr Tyr JAK1 Tyr P y y Tyr1007 J y Tyr1007 P P P P 3y y IL-10R Tyr IL-10R TRAM Tyr706 Tyr706 P P P chain Tyr Tyr P SHP-1 Tyr542 Tyr518 Tyr544 Tyr974Tyr921 SHP-2 Tyr580 Tyk2 Tyr P SHP-1 Tyr542 SHP-2 Tyr580 SHP-1 Tyr Tyr JAK1 MD-2 TLR4 y P P TRIF/ TICAM-1 Tyr759 Tyr915 IFN R1 Tyr915 Tyr759 IL-6R Tyr Tyr JAK1 Tyr542 Tyr807 Tyr721 Tyr706 Fc RIa chain Tyr SOCS1 /JAB Tyr Tyr518 Tyr JAK1 Tyr JAK3 Tyr Gab2 P Tyr771 Tyr783 Tyr519 Tyr771 PLC Tyr1254 Tyr783 Ser IRAK1 Thr TBK-1 Lys IL-10R Tyr JAK1 Tyk2 Tyr Tyr Tyr759 P Tyr Tyr915 IL-6R IL-10R IL-10R Tyr542 SOCS1 /JAB IFN R2 Tyr Tyk2 Tyr gp130 P Tyr518 common Tyr chain Tyr440 IL-6R SHP-2 Tyr580 P Gab2 JAK2Tyr1007 IFN R1 Tyr767 Tyr905 Tyr814 P PP2A P Tyr JAK1 Tyr JAK1 IKK Syk PLC Tyr1254 P IRAK-M Syk chain common chainTyr IL-4R JAK3Tyr P JAK2Tyr1007 Tyk2 Tyr Tyr580SHP-2 TyrJAK1 Tyr559 Tyr697 P P Tyr JAK1 IKK Tyr IFN R2 Tyr440 IL-6R Tyr767 Tyr905 Tyr814 Tyr767 Tyr905 Tyr814 P Tyr519 M-CSFR SHIP common chainTyr IL-4R TBK-1 Principles of cell signaling Tyk2 Tyr 14 Syk Tyr519 Tyr542SHP-2 Tyr580 P P JAK3 Tyr P Tyr Gab2 RasGAP RasGAP P STAT5 Tyr SOCS1 /JAB P Tyr759 P Ser385 Ser Thr IRF-3 P P Ser386 P IRAK1 Tyr759 Tyr915 Ser TAB1 P Tyr701 STAT1 Ser727 P P P Tyr Thr184 Ser192 TAK1Thr187 IRAK1 Ser Thr P Ub IRAK1 Lys Tyr JAK1 Tyr542 P Thr Tyr SHP-2 P JNK Tyr580 Tyr Uev1A STAT3 Tyr P Tyr Ser338 IRS P Tyr Raf Ser62 Ras Thr Tyr341 Ser338 GDP Ser312(307:R) STAT3 Ser4 Tyr341 GTP Ser SOS Ser312(307:R) P Tyr P Ser473 Akt/PKB Thr Grb2 IRS PI3K P Thr38 TAB1 Ser GTP Fyn Tyr p38MAPK Thr184 Ser192 TAK1Thr187 Lys63TRAF6 Src Pi Ras IL-4R Tyr TAB2 Ub GDP P Thr TAB2 P Lys ys63TRAF6 Tyr542 Tyr580 SHP-2 P SOCS3 Ser Thr P Gab2 P Tyr Tyr JAK2 Tyr1007 SOCS1 /JAB SOCS3 STAT5 IFN R2 IFN R1 Tyr440 P Tyr JAK1 Tyk2 Tyr Tyr JAK1 Lys Tyr915 IL-6R IL-6R Tyr767 Tyr905 Tyr767 Tyr905 Tyr814 P Tyr814 PP P P Ser385 IRF-3 Ser386 P P P P Ser4 Raf Ser62 Fyn Ubc13 Grb2 Ser Thr38 Ser473 Akt/PKB Grb2 PI3K P P Ser Thr IRAK1 Lys Ub P P TRAF2 TRAF1 A20 Ub Ser Thr P IRAK1 Lys Lys63TRAF6 TAB2 Ub P Lys63TRAF6 Ub Thr184 Ser192 TAK1Thr187 PP P Thr P P Lys PSer385 Ser385 IRF-3 Ser386 P Ser386 P Ub Lys63TRAF6 P P Tyr IFN STAT6 P IL-4 IL-1ra Thr184 Ser192 TAK1Thr187 Ser32 Ser42 P P TAB1 Ser Thr TAB1 Ser Grb2 Thr P Ser70 P Ser42 P P Tyr STAT3 P Tyr SEK2/MKK7 P Ser73 Ser63 Ser73 c-Jun P proteasome Tyr STAT6 P Ser369 Thr577 RSK Ser227 Ser386 P P P Thr Tyr P PI3K Tyr JNK JNK MEKK ERK2 P Tyr STAT6 P Tyr P Thr R Ser386 P ERK1 Thr183 Tyr185 P c-Jun P Tyr701 Tyr701 STAT1 Ser727 Ser727 P P Ser374 c-Fos Ser113 Ser70 P P Ser63 SEK2/MKK7 ASK Ser369 Ser21 Ser32 c-FosSer113 P The University of Texas Southwestern Medical Center at Dallas SOS PP2A P Ser21 P Ser374 Thr P P P Ser SOS Thr IRS Ser312(307:R) PP2B 0 Melanie H. Cobb and Elliott M. Ross Ser Thr Tyr Tyr701 STAT1 Ser727 P TAB2 P Thr184 Ser192 TAK1Thr187 TAB1 IRAK1 SOS P SOCS3 Ser Thr P TAB2 P P Ser P Ub P SEK1/MKK4 SOCS3 IL-1ra IKK Ser176 Ser181 SEK1/MKK4 Thr IL-4R P Lys21 Lys22 Elk-1 Ser389 P P c-jun PP2B P I B Ser36 p50 IFN p50 P Tyr STAT3 P Tyr NF- B p65+p50 Ser529 PIAS3 p60 LXR MKP NF- B p65+p50 Ser529 NF- B p65+p50 Ser529 STAT6 Ser276 P Lys21 Lys22 I B Ser276 Ser276 P P P P Ser529 P P P Ub Tyr542 SHP-2 Tyr580 Lys22 I B Tyr185 ERK2 Tyr185 Ser32 P Ser36 Ub P P Lys21 Lys22 Ser529 I B Ser36 P SCF TrCP P I B NF- B p65+p50 Ser529 PKA P Lys22 Ser32 Lys22 I B UbcH5 Ser32 Ser70 P CREB RXR P LXR LXR SREBP1c / bHLH IRF-1 IRF-9 P c-Jun STAT2 IRF-2 Ser36 P Ser133 c-FosSer113 p53 Ser727 Tyr701 STAT1 P Tyr P Lys21 Ser276 Ser36 P P P P P Ser63 Ser73 Ser63 Ser73 PIAS1 P Lys21 P Ser21 P Ser374 PTyr701 Tyr701 STAT1 Ser727 Ser727 P P PKA Ser32 P PIAS1 Ser276 PKA IKK CAPK P Ser42 NF- B p65+p50 Ser529 Ser32 P Ub PTyr701 Tyr701 STAT1 Ser727 P Ser727 P P Tyr STAT5 P Tyr PKA Ub NF- B p65+p50 Ser276 P Thr183 ERK1 Thr183 P Lys21 PAFR Tyr185 ERK2 P IKK Tyr701 Tyr701 STAT1 Ser727 Ser727 P P Thr183 ERK1 CK II MKP P Tyr STAT3 P Tyr P Ser385 Ser385 IRF-3 Ser386 Ser386 P P Ser36 IKK P Ser176 Ser181 P CAPK NF- B p65+p50 Ser529 Ser276 Ser276 P P Ser529 P P PIAS3 Ser32 PKA NF- B p65+p50 Ser276 Ser529 Tyr185 ERK2 Tyr185 P Ser389Elk-1 Ser133 CREB PKA P Tyr P Thr183 ERK1 Thr183 Ser383 P Tyr STAT6 P Tyr SOCS1/JAB Ser276 This image represents about 10% of the map of the known signaling interactions and reactions in the mouse macrophage. Preparing such a map in a computable format is the first step in analyzing a large signaling network. This map was prepared by the group led by Hiroaki Kitano at the Systems Biology Institute, Tokyo, using their CellDesigner program. Map courtesy of Kanae Oda, Yukiko Matsuoka, and Hiroaki Kitano (The Systems Biology Institute). Ser383 Thr577 RSK Ser227 Ser386 P P Ser73 c-Jun Ser32 I B P Ser369 IL-4 P Ser63 IKK P c-fos Tyr STAT6 Tyr A20 Tyr JNK IKK PKA NF- B p65+p50 Ser529 NF- B p65+p50 Ser276 Ser276 TNF AP-1 c-Fos+c-Jun Ser529 TNF 9 r 27-hydroxyChol acetyl CoA carboxylase IL-1 LXR fatty acid synthetase P Ser484 IFN- IRF-7 Ser485 IL-10 P acyl CoA synthetase IRF-7 IL-6 IFNGM-CSF IRF-9 IRF-2 NOSII/iNOS IRF-1 CPT1 nucleus IFN- CHAPTER OUTLINE P IFN- Ser484 P GM-CSF P IRF-7 SREBP1c / bHLH Ser485 PTyr701 Tyr701 STAT1 Ser727 Ser727 P P TyrJAK1 P Tyr STAT5 P Tyr GM-CSFR IRF-2 acetyl CoA carboxylase Ser484 IRF-1 IRF-7 Ser485 Tyr P GM-CSFR P Ser727 STAT1 Tyr701 P Tyr ? STAT2 JAK2 Tyr1007 P Tyr 14.1 14.2 14.3 14.4 14.5 14.6 14.7 14.8 14.9 14.10 14.11 14.12 14.13 14.14 14.15 14.16 14.17 14.18 14.19 14.20 Introduction Cellular signaling is primarily chemical Receptors sense diverse stimuli but initiate a limited repertoire of cellular signals Receptors are catalysts and amplifiers Ligand binding changes receptor conformation Signals are sorted and integrated in signaling pathways and networks Cellular signaling pathways can be thought of as biochemical logic circuits Scaffolds increase signaling efficiency and enhance spatial organization of signaling Independent, modular domains specify protein-protein interactions Cellular signaling is remarkably adaptive Signaling proteins are frequently expressed as multiple species Activating and deactivating reactions are separate and independently controlled Cellular signaling uses both allostery and covalent modification Second messengers provide readily diffusible pathways for information transfer Ca2+ signaling serves diverse purposes in all eukaryotic cells Lipids and lipid-derived compounds are signaling molecules PI 3-kinase regulates both cell shape and the activation of essential growth and metabolic functions Signaling through ion channel receptors is very fast Nuclear receptors regulate transcription G protein signaling modules are widely used and highly adaptable IRF-9 STAT5 P Tyr 14.21 acetyl CoA TG TG STAT3 Site-2 protease malonyl CoA Heterotrimeric G proteins regulate a wide variety of effectors Heterotrimeric G proteins are controlled by a regulatory GTPase cycle Small, monomeric GTP-binding proteins are multiuse switches Protein phosphorylation/dephosphorylation is a major regulatory mechanism in the cell Two-component protein phosphorylation systems are signaling relays Pharmacological inhibitors of protein kinases may be used to understand and treat disease Phosphoprotein phosphatases reverse the actions of kinases and are independently regulated Covalent modification by ubiquitin and ubiquitin-like proteins is another way of regulating protein function The Wnt pathway regulates cell fate during development and other processes in the adult Diverse signaling mechanisms are regulated by protein tyrosine kinases Src family protein kinases cooperate with receptor protein tyrosine kinases MAPKs are central to many signaling pathways Cyclin-dependent protein kinases control the cell cycle Diverse receptors recruit protein tyrosine kinases to the plasma membrane What’s next? Summary References acyl-CoA malate STAT5 Tyr STAT3 citrate liase malate dehydrogenase glycerol 3P carnitine Site Golgi fatty acid CoASH CoASH oxaloacetate SCAP fatty acid synthetase lipid droplet IRF-9 Tyr R SREBP1c / bHLH GM-CSFR HOCl MPO acyl CoA synthetase NADPH oxidase acylcarnitine citrate NADPH SOD NADP+ CPT I K Thr38 Cl- CACT Ser473 Akt/PKB P NOSII/iNOS Tyr701 STAT1 Ser727 calpain P 14.22 malic enzyme PI3K Thr38 pyruvate carrier P Tyr SHP-2 Tyr580 14.26 14.27 14.28 14.29 14.30 14.31 14.32 14.33 14.34 14.35 14.36 pyruvate dehydrogenase Akt/PKB Tyr701 STAT1 Ser727 P P H2O2 ATP pyruvate dehydrogenase ATP synthetase acylcarnitine ADP STAT2 NAD+ pyruvate Tyr542 P 14.25 .O 2 O2 carnitine P Ser473 P 14.24 e- CPT II CoASH P P -2 14.23 PDH kinase PDH kinase F hypoxanthine Fe3+ NADH+H+ pyruvate acetyl CoA pyruvate carboxylase acyl-CoA H+ H+ O2 e- e- xanthine oxidase 589 LOOH xanthine 39057_ch14_cellbio.qxd 8/28/06 5:11 PM 14.1 Page 590 Introduction All cells, from prokaryotes through plants and animals, sense and react to stimuli in their environments with stereotyped responses that allow them to survive, adapt, and function in ways appropriate to the needs of the organism. These responses are not simply direct physical or metabolic consequences of changes in the local environment. Rather, cells express arrays of sensing proteins, or receptors, that recognize specific extracellular stimuli. In response to these stimuli, receptors regulate the activities of diverse intracellular regulatory proteins that in turn initiate appropriate responses by the cell. The process of sensing external stimuli and conveying the inherent information to intracellular targets is referred to as cellular signal transduction. Cells respond to all sorts of stimuli. Microbes respond to nutrients, toxins, heat, light, and chemical signals secreted by other microbes. Cells in multicellular organisms express receptors specific for hormones, neurotransmitters, autocrine and paracrine agents (hormonelike compounds from the secreting cell or cells Overview of major receptor types in a cell Transmembrane scaffold (GPCR) Ion G protein Receptor coupled protein channel receptor kinase Twocomponent complex Heterotrimeric G protein E1 E1 E2 E2 Guanylyl cyclase Sensor ( Histidine kinase ( Response regulator Transcription factor NUCLEUS FIGURE 14.1 Receptors form a rather small number of families that share common mechanisms of action and overall similar structures. 590 CHAPTER 14 Principles of cell signaling nearby), odors, molecules that regulate growth or differentiation, and proteins on the outside of adjacent cells. A mammalian cell typically expresses about fifty distinct receptors that sense different inputs, and, overall, mammals express several thousand receptors. Despite the diversity of cellular lifestyles and the enormous number of substances sensed by different cells, the general classes of proteins and mechanisms involved in signal transduction are conserved throughout living cells, as shown in FIGURE 14.1. • G protein-coupled receptors, composed of seven membrane-spanning helices, promote activation of heterotrimeric GTP-binding proteins called G proteins, which associate with the inner face of the plasma membrane and convey signals to multiple intracellular proteins. • Receptor protein kinases are often dimers of single membrane-spanning proteins that phosphorylate their intracellular substrates and, thus, change the shape and function of the target proteins. These protein kinases frequently contain protein interaction domains that organize complexes of signaling proteins on the inner surface of the plasma membrane. • Phosphoprotein phosphatases reverse the effect of protein kinases by removing the phosphoryl groups added by protein kinases. • Other single membrane-spanning enzymes, such as guanylyl cyclase, have an overall architecture similar to the receptor protein kinases but different enzymatic activities. Guanylyl cyclase catalyzes the conversion of GTP to 3′:5′cyclic GMP, which is used to propagate the signal. • Ion channel receptors, although diverse in detailed structure, are usually oligomers of subunits that each contain several membrane-spanning segments. The subunits change their conformations and relative orientations to permit ion flux through a central pore. • Two-component systems may either be membrane spanning or cytosolic. The number of their subunits is also variable, but each two-component system contains a histidine kinase domain or subunit that is regulated by a signaling molecule and a response regulator that 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 591 contains a phosphorylatable aspartate (Asp) residue. • Some receptors are transmembrane scaffolds that change either the conformation or oligomerization of their intracellular scaffold domains in response to extracellular signaling molecules, or ligands, and, thus, recruit interacting regulatory proteins to a common site on the membrane. • Nuclear receptors are transcription factors, often heterodimers, that may reside in the cytoplasm until activated by agonists or may be permanently located in the nucleus. The biochemical processes of signal transduction are strikingly similar among cells. Bacteria, fungi, plants, and animals use similar proteins and multiprotein modules to detect and process signals. For example, evolutionarily conserved heterotrimeric G proteins and G protein-coupled receptors are found in plants, fungi, and animals. Similarly, 3′:5′ cyclic AMP (cAMP) is an intracellular signaling molecule in bacteria, fungi, and animals; and Ca2+ serves a similar role in all eukaryotes. Protein kinases and phosphoprotein phosphatases are used to regulate enzymes in all cells. Although the basic biochemical components and processes of signal transduction are conserved and reused, they are often used in wildly divergent patterns and for many different physiological purposes. For example, cAMP is synthesized by distantly related enzymes in bacteria, fungi, and animals, and acts on different proteins in each organism; it is a pheromone in some slime molds. Cells often use the same series of signaling proteins to regulate a given process, such as transcription, ion transport, locomotion, and metabolism. Such signaling pathways are assembled into signaling networks to allow the cell to coordinate its responses to multiple inputs with its ongoing functions. It is now possible to discern conserved reaction sequences in and between pathways in signaling networks that are analogous to devices within the circuits of analog computers: amplifiers, logic gates, feedback and feed-forward controls, and memory. This chapter discusses the principles and strategies of cellular signaling first and then discusses the conserved biochemical components and reactions of signaling pathways and how these principles are applied. 14.2 Cellular signaling is primarily chemical Key concepts • Cells can detect both chemical and physical signals. • Physical signals are generally converted to chemical signals at the level of the receptor. Most signals sensed by cells are chemical, and, when physical signals are sensed, they are generally detected as chemical changes at the level of the receptor. For example, the visual photoreceptor rhodopsin is composed of the protein opsin, which binds to a second component, the colored vitamin A derivative cis-retinal (the chromophore). When cis-retinal absorbs a photon, it photoisomerizes to trans-retinal, which is an activating ligand of the opsin protein. (For more on rhodopsin signaling see 14.20 G protein signaling modules are widely used and highly adaptable). Similarly, plants sense red and blue light using the photosensory proteins phytochrome and cryptochrome, which detect photons that are absorbed by their tetrapyrrole or flavin chromophores. Cryptochrome homologs are also expressed in animals, where they probably mediate adjustment of the diurnal cycle. A few receptors do respond directly to physical inputs. Pressure-sensing channels, which exist in one form or another in all organisms, mediate responses to pressure or shear by changing their ionic conductance. In mammals, hearing is mediated indirectly by a mechanically operated channel in the hair cell of the inner ear. The extracellular domain of a protein called cadherin is pulled in response to acoustic vibration, generating the force that opens the channel. Cells sense mechanical strain through a number of cell surface proteins, including integrins. Integrins provide signals to cells based on their attachment to other cells and to molecular complexes in the external milieu. One major group of physically responsive receptors is made up of channels that sense electric fields. Another interesting group are heat/pain-sensing ion channels; several of these heat-sensitive ion channels also respond to chemical compounds, such as capsaicin, the “hot” lipid irritant in hot peppers. Whether a signal is physical or chemical, the receptor initiates the reactions that change the behavior of the cell. We will discuss how these effects are generated in the rest of the chapter. 14.2 Cellular signaling is primarily chemical 591 39057_ch14_cellbio.qxd 8/28/06 5:11 PM 14.3 Page 592 Receptors sense diverse stimuli but initiate a limited repertoire of cellular signals Key concepts • Receptors contain a ligand-binding domain and an effector domain. • Receptor modularity allows a wide variety of signals to use a limited number of regulatory mechanisms. • Cells may express different receptors for the same ligand. • The same ligand may have different effects on the cell depending on the effector domain of its receptor. Receptors mediate responses to amazingly diverse extracellular messenger molecules; hence, the cell must express a large number of receptor varieties, each able to bind its extracellular ligand. In addition, each receptor must be able to initiate a cellular response. Receptors, thus, contain two functional domains: a ligandbinding domain and an effector domain, which may or may not correspond to definable structural domains within the protein. The separation of ligand-binding and effector functions allows receptors for diverse ligands to produce a limited number of evolutionarily conserved intracellular signals through the action of a few effector domains. In fact, there are Receptors have a ligand-binding domain and an effector domain CHIMERIC RECEPTOR Ligand A LBD1 ED1 Output 1 Ligand A LBD1 ED2 Output 2 Ligand B LBD1 ED1 Output 1 LBD2 ED1 Output 1 Ligand C LBD3 ED2 Output 2 FIGURE 14.2 Receptors can be thought of as composed of two functional domains, a ligand-binding domain (LBD) and an effector domain (ED). The twodomain property implies that two receptors that respond to different ligands (middle) could initiate the same function by activating similar effector domains, or that a cell could express two receptor isoforms (left) that respond to the same ligand with distinct cellular effects mediated by different effector domains. It also implies that one can create an artificial chimeric receptor with novel properties. 592 CHAPTER 14 Principles of cell signaling only a limited number of receptor families, which are related by their conserved structures and signaling functions (see Figure 14.1). There are several useful correlates to the two-domain nature of receptors. For example, a cell can control its responsiveness to an extracellular signal by regulating the synthesis or degradation of a receptor or by regulating the receptor’s activity (see 14.10 Cellular signaling is remarkably adaptive). In addition, the nature of a response is generally determined by the receptor and its effector domain rather than any physicochemical property of the ligand. FIGURE 14.2 illustrates the concept that a ligand may bind to more than one kind of receptor and elicit more than one type of response, or several different ligands may all act identically by binding to functionally similar receptors. For example, the neurotransmitter acetylcholine binds to two classes of receptors. Members of one class are ion channels; members of the other regulate G proteins. Similarly, steroid hormones bind both to nuclear receptors, which bind chromatin and regulate transcription, and to other receptors in the plasma membrane. Conversely, when multiple ligands bind to receptors of the same biochemical class, they generate similar intracellular responses. For example, it is not uncommon for a cell to express several distinct receptors that stimulate production of the intracellular signaling molecule cAMP. The effect of the receptor on the cell will also be determined significantly by the biology of the cell and its state at any given time. Ligand binding and effector domains may evolve independently in response to varied selective pressures. For example, mammalian and invertebrate rhodopsins transduce their signal through different effector G proteins (Gt and Gq, respectively). Another example is calmodulin, a small calcium-binding regulatory protein in animals, which in plants appears as a distinct domain in larger proteins. The receptor’s two-domain nature allows the cell to regulate the binding of ligand and the effect of ligand independently. Covalent modification or allosteric regulation can alter ligand-binding affinity, the ability of the ligand-bound receptor to generate its signal or both. We will discuss these concepts further in 14.13 Cellular signaling uses both allostery and covalent modification. Receptors can be classified either according to the ligands they bind or the way in which they signal. Signal output, which is character- 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 593 istic of the effector domain, usually correlates best with overall structure and sequence conservation. (Receptor families grouped by their functions are the organizational basis of the second half of this chapter.) However, classifying receptors pharmacologically, according to their specificity for ligands, is particularly useful for understanding the organization of endocrine and neuronal systems and for categorizing the multiple physiological responses to drugs. Expression of a receptor that is not normally expressed in a cell is often sufficient to confer responsiveness to that receptor’s ligand. This responsiveness often occurs because the cell expresses the other components necessary for propagating the intracellular signal from the receptor. The precise nature of the response will reflect the biology of the cell. Experimentally, responsiveness to a compound can be induced by introducing the cDNA that encodes the receptor. For example, mammalian receptors may be expressed in yeast, such that the yeast respond visibly to receptor ligands, thus providing a way to screen for new chemicals (drugs) that activate the receptor. Finally, it is possible to create chimeric receptors by fusing the ligand-binding domain from one receptor with the effector domain from a different receptor (Figure 14.2). Such chimeras can mediate novel responses to the ligand. With genetic modification of the ligandbinding domain, receptors can be reengineered to respond to novel ligands. Thus, scientists can manipulate cell functions with nonbiological compounds. 14.4 Receptors are catalysts and amplifiers Key concepts • Receptors act by increasing the rates of key regulatory reactions. • Receptors act as molecular amplifiers. Receptors act to accelerate intracellular functions and are, thus, functionally analogous to enzymes or other catalysts. Some receptors, including the protein kinases, protein phosphatases, and guanylate cyclases, are themselves enzymes and thus classical biochemical catalysts. More generally, however, receptors use the relatively small energy of ligand binding to accelerate reactions that are driven by alternative energy sources. For example, receptors that are ion channels catalyze the movement of ions across membranes, a process driven by the electrochemical potential developed by distinct ion pumps. G protein-coupled receptors and other guanine nucleotide exchange factors catalyze the exchange of GDP for GTP on the G protein, an energetically favored process dictated by the cell’s nucleotide energy balance. Transcription factors accelerate the formation of the transcriptional initiation complex, but transcription itself is energetically driven by multiple steps of ATP and dNTP hydrolysis. As catalysts, receptors enhance the rates of reactions. Most signaling involves kinetic rather than thermodynamic regulation; that is, signaling events change reaction rates rather than their equilibria (see the next section). Thus, signaling is similar to metabolic regulation, in which specific reactions are chosen according to their rates, with thermodynamic driving forces playing only a supportive role. In all signaling reactions, receptors use their catalytic activities to function as molecular amplifiers. Directly or indirectly, a receptor generates a chemical signal that is huge, both energetically and with respect to the number of molecules recruited by a single receptor. Molecular amplification is a hallmark of receptors and many other steps in cellular signaling pathways. 14.5 Ligand binding changes receptor conformation Key concepts • Receptors can exist in active or inactive conformations. • Ligand binding drives the receptor toward the active conformation. A central mechanistic question in receptor function is how the binding of a signaling molecule to the ligand-binding domain increases the activity of the effector domain. The key to this question is that receptors can exist in multiple molecular conformations, some active for signaling and others inactive. Ligands shift the conformational equilibrium among these conformations. The structural changes that occur during the receptor’s inactive-active isomerization and how ligand binding drives these changes are exciting areas of biophysical research. However, the basic concept can be described simply in terms of coupling the conformational isomerizations of the ligandbinding and effector domains. 14.5 Ligand binding changes receptor conformation 593 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 594 How do ligands activate (or not activate) a receptor? Most of the basic regulatory activities of receptors can be described by a simple scheme that considers the receptors as having two interconvertible conformations, inactive (R) and active (R*). R and R* are in equilibrium, which is described by the equilibrium constant J. J R R* Because unliganded receptors are usually minimally active, J<<1 and an unliganded receptor spends most of its time in the R state. When a signaling molecule (L) binds, it drives the receptor toward the active conformation, R*, in which the effector domain is functional. The ligand-bound receptor thus spends most of its time in the active R* state. J R + L R*+ L K K* J* R L R* L The mechanism whereby a ligand can activate receptor is a simple consequence of its relative affinities for the receptor’s active and inactive conformations. A ligand can bind to the receptor in either of its conformations, described here by association constants K for the R state and K* for the R* state. Any ligand that binds with higher affinity for the R* conformation than for R will be an activator. If K* is greater than K, the ligand is an agonist. According to the Second Law of Thermodynamics, a system of Receptor ligands can vary in their activities and potencies Fractional activity of receptor Fractional activity of receptor 1.0 0.8 0.6 0.012 High affinity agonist Lower affinity agonist 0.4 Partial agonist 0.2 0 Log [L] 0.010 0.008 0.006 Inverse agonist 0.004 0.002 0 Log [L] FIGURE 14.3 The simple two-state model shown here can describe a wide variety of behaviors displayed by receptors and their various regulatory ligands. The left panel shows fractional activity of a receptor exposed to two agonists with different affinities and one partial agonist. The right panel shows the effect of an inverse agonist. If the low fractional activity of unliganded receptor is detected as significant biological activity, then its inhibition by the inverse agonist would be easily detectable. 594 CHAPTER 14 Principles of cell signaling coupled equilibria displays path independence: the net free energy difference between two states is independent of which intermediary reactions take place. For the receptor, any path from R to R*L therefore has the same free energy change, and the products of the equlibrium constants along each path are equal. For the example above, path independence means that: J•K* = K•J* Therefore, J* / J = K* / K. Thus, if binding to the R* configuration is preferred (i.e., K*/K>>1), then ligand binding will shift the conformation to the R* state to an equivalent extent (i.e., J*/J>>1). The relative activation by a saturating concentration of ligand, J*/J, will exactly equal the ligand’s relative selectivity for the active receptor conformation, K*/K. This argument is generally valid for the regulation of a protein’s activity by any regulatory ligand. This model explains many properties of receptors and their ligands both simply and quantitatively. • First, J must be greater than zero for the equilibrium to exist. Thus, even unliganded receptor has some activity. Overexpressed receptors frequently display their intrinsic low activity. • Because physiological receptors are nearly inactive in the absence of ligand, J must be much less than 1 and is probably less than 0.01; most receptors are less than 1% active without agonist. • Ligands can vary in their selectivities between R and R*. Their abilities to activate will also vary. Some ligands, referred to as agonists, can drive formation of appreciable R*. Others, known as partial agonists, will promote submaximal activation. Chemical manipulation of a ligand’s structure will often alter its activity as an agonist. These relationships are depicted graphically in FIGURE 14.3. • A ligand that binds equally well to both the R and R* states will not cause activation. However, such a ligand may still occupy the binding site and thereby competitively inhibit binding of an activating ligand. Such competitive inhibitors, referred to as antagonists, are frequently used as drugs to block unwanted activation of a receptor in various disease states. • A ligand that binds preferentially to R 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 595 relative to R* will further shift the conformational equilibrium to the inactive state and cause net inhibition. Such ligands are called inverse agonists. Because J is already low, effects of inverse agonists may only be noticeable if a receptor is overexpressed or if the receptor is mutated to increase its intrinsic activity (i.e., the mutation increases J). • The extent to which an agonist stimulates a receptor is unrelated to its affinity. Both agonists and antagonists may bind with either high or low affinity. Affinity does determine the receptor’s sensitivity—that is, how low a concentration of ligand can the receptor detect. Affinities of receptors for natural regulatory ligands vary enormously, with physiologic Kd values ranging from <10-12 M for some hormones to about 10-3 M for some bacterial chemoattractants. Another aspect of sensitivity is how abruptly or gradually the receptor is activated as the concentration of agonist increases. The above model predicts that a receptor is activated significantly at agonist concentrations between 0.1 and 10 times its Kd. A variety of cellular mechanisms can convert such a conventional response range of about 100-fold to either a more gradual response or a very steep, switchlike response. • This model only describes equilibria. It makes no predictions about the rates of ligand binding or release, or of the conformational isomerization that leads to activation. This model shows how three important aspects of receptor action are independently determined. As mentioned above, affinity for ligand, which determines the concentration range over which the ligand functions, is independent of the ligand’s net effectiveness at driving receptor activation. The rate of response is also largely independent of these other two properties. Each aspect of receptor function can thus be independently regulated in response to other incoming signals or by the metabolic or developmental state of the cell. Such control of signal input is central to whole-cell coordination of signal transduction. Examples and mechanisms will recur throughout this chapter. 14.6 Signals are sorted and integrated in signaling pathways and networks Key concepts • Signaling pathways usually have multiple steps and can diverge and/or converge. • Divergence allows multiple responses to a single signal. • Convergence allows signal integration and coordination. Receptors rarely act directly on the intracellular processes that they ultimately regulate. Rather, receptors typically initiate a sequence of regulatory events that involve intermediary proteins and small molecules. The use of multistep signaling pathways allows cells to amplify signals, adjust signaling kinetics, insert control points, integrate multiple signals, and route signals to distinct effectors. Branched pathways give cells the ability to integrate multiple incoming signals and to direct information to the correct control points. As FIGURE 14.4 illustrates, branching can be either convergent, with multiple signals regulating common end points, or divergent, with a single pathway branching to control more than one process. In multicellular organisms, divergent branching allows a single hormone receptor to initiate distinct cell-appropriate patterns of responses in different cells and tissues. Divergent signaling also allows a receptor to regulate qualitatively different cellular responses with quantitatively distinct intensities, each dependent on signal amplification in the intermediary pathway. Convergent branching—when several receptors activate the same pathway to elicit the same regulatory responses—is also common. Convergent branching allows multiple incoming signals, both stimulatory and inhibitory, to be integrated and coordinately regulated at a common site downstream of the receptors. Receptors for several different hormones frequently initiate similar or overlapping patterns of signaling in a single target cell. Overlapping converging and diverging signaling pathways create signaling networks within cells that coordinate responses to multiple inputs (Figure 14.4). Typically, such pathways are complex in the number and diversity of their components and in the topology of their circuit 14.6 Signals are sorted and integrated in signaling pathways and networks 595 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 596 Convergent and divergent signaling pathways RECEPTORS TRANSDUCERS EFFECTORS Linear, parallel Convergent Divergent Multiply branched FIGURE 14.4 Signaling pathways use convergent and divergent branching to coordinate information flow. The diagrams at top show how even a simple, threelevel signaling network can sort information. Convergence or divergence can take place at multiple points along a signaling pathway. As an example of complexity, the lower portion of the figure shows a small segment (~10%) of the G protein-mediated signaling network in a mouse macrophage cell line. It omits several interpathway regulatory mechanisms and completely ignores inputs from non-G protein-coupled receptors. Pathway map courtesy of Lily Jiang, University of Texas Southwestern Medical Center. maps. Signaling networks are also spatially complex. They may include components in various subcellular locations, with initial receptors and associated proteins in the plasma membrane, but with downstream proteins in the cytoplasm or intracellular organelles. Such complexity is necessary to allow the cells to integrate and sort incoming signals and to regulate multiple intracellular functions simultaneously. The complexity and adaptability of signaling networks, like the one shown in the lower half of Figure 14.4, make their dynamics at the whole-cell level difficult or impossible to grasp intuitively. Signaling networks resemble large 596 CHAPTER 14 Principles of cell signaling analog computers, and investigators are increasingly depending on computational tools to understand cellular information flow and its regulation. First, many signaling interactions that include only two or three proteins exert functions analogous to traditional computational logic circuits (see the next section). The theory and experience with such circuits in electronics facilitate understanding biological signaling functions as well. The enormous complexity of cellular signaling networks can be simplified by considering them to be composed of interacting signaling modules, i.e., groups of proteins that process signals in well-understood ways. A cellular signaling module is analogous to an integrated circuit in an electronic instrument that performs a known function, but whose exact components could be changed for similar use in another device. The concept of modular construction facilitates both qualitative and quantitative understanding of signaling networks. We will refer to many standard signaling modules later in the chapter. Examples include monomeric and heterotrimeric G protein modules, MAPK cascades, tyrosine (Tyr) kinase receptors and their binding proteins, and Ca2+ release/uptake modules. In each case, despite the numerous phylogenetic, developmental, and physiologic variations, understanding the basic function of that class of module conveys understanding of all its incarnations. Last, the evolutionary importance of modules is significant; once the architecture of a module is established it can be reused. For larger-scale networks, multiplexed, high-throughput measurements on living cells have been combined with powerful kinetic modeling strategies to allow an increasingly accurate quantitative depiction of information flow within signaling modules or entire networks. Such models, with sound and experimentally based parameter sets, can describe signaling processes in systems too complex for intuitive or ad hoc analysis. They are also vital as tests of understanding because they can predict experimental results in ways that can be used to test the validity of the model. Well-grounded models can then be used (cautiously) to suggest the mechanisms of systems for which data sets remain unattainable. At even greater levels of complexity, the theories and tools of computer science are increasingly giving useful systemslevel analyses of signal flow in cells. Using computational tools to analyze large arrays of quantitative data allows us to understand cellular information flow and its regulation. 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 597 Developing quantitative models of signaling networks is a frontier in signaling biology. These models both help describe network function and pinpoint experiments to clarify mechanism. 14.7 Cellular signaling pathways can be thought of as biochemical logic circuits Simple logic circuits Logical (Boolean) Quantitative (Analog) Additive A OR B Response A Response B Response A + B Response A + fixed [B] A B log (agonist concentration) More than additive A AND B Response A + B Key concepts • Signaling networks are composed of groups of biochemical reactions that function as mathematical logic functions to integrate information. • Combinations of such logic functions combine as signaling networks to process information at more complex levels. A B A + B A B Response log (agonist concentration) Less than additi ve A NOT B Response As introduced in the preceding section, processes that signaling pathways use to integrate and direct information to cellular targets are strikingly analogous to the mathematical logic functions that are used to design the individual circuits of electronic computers. Indeed, there are biological equivalents of essentially all of the functional components that computer scientists and engineers consider in the design of computers and electronic control devices. To understand signaling pathways, it is, therefore, useful to consider groups of reactions within a pathway as constituting logic circuits of the sort used in electronic computing, as illustrated in FIGURE 14.5. The simplest example is when two stimulatory pathways converge. If sufficient input from either is adequate to elicit the response, the convergence would constitute an “OR” function. If neither input is sufficient by itself but the combination of the two elicits the response, then the converging pathways would create “AND” functions. AND circuits are also referred to as coincidence detectors—a response is elicited only when two stimulating pathways are activated simultaneously. AND functions can result from the combination of two similar but quantitatively inadequate inputs. Alternatively, two mechanistically different inputs might both be required to elicit a response. An example of the latter would be a target protein that is allosterically activated only when phosphorylated, or that is activated by phosphorylation but is only functional when recruited to a specific subcellular location. The opposite of an AND circuit is a NOT function, where one pathway blocks the stim- A A Response B A + B A + B B log (agonist concentration) FIGURE 14.5 Signaling networks use simple logic functions to process information. Boolean OR, AND, and NOT functions (left) correspond to the quantitative interactions between converging signals that are shown on the right. ulatory effect of another. Simple logic gates are observed at many locations in cellular signaling pathways. We can also think about convergent signaling in quantitative rather than Boolean terms by considering the additivity of inputs to a distinct process (see Figure 14.5, right). The OR function referred to above can be considered to be the additive positive inputs of two pathways. Such additivity could represent the ability of several receptors to stimulate a pool of a particular G protein or the ability of two protein kinases to phosphorylate a single substrate. Additivity may be positive, as in the examples above, or negative, such as when two inhibitory inputs combine. Inhibition and stimulation may also combine additively to yield an algebraically balanced output. Alternatively, multiple inputs can combine with either more or less than an additive effect. The NOT function, discussed above, is analogous to describing a blockade of stimulation. The AND function describes synergism, where one input potentiates another but alone has little effect. Even simple signaling networks can display complex patterns of information processing. One 14.7 Cellular signaling pathways can be thought of as biochemical logic circuits 597 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 598 good example is the creation of “memory”: making the effect of a transient signal more or less permanent. Signaling pathways have multiple ways of setting memories, and of forgetting. One mechanism, common in protein kinase pathways, is the positive feedback loop, illustrated in the top panel of FIGURE 14.6. In a positive feedback loop, the input stimulates a transducer (T), which in turn stimulates the effector protein (E) to create the output. If the effector can also acSignal processing circuits Positive feedback loop : irreversible ON switch Output Input Output E T + Input strength Positive feed-forward loop : responds to prolonged input input Output Input T Output E + Time Conformational lock - Dual control switch OH P Output Kinase E G E G P OH E G OH P E E E G Phosphatase G K G K P G P Time FIGURE 14.6 Relatively complex signal processing can be executed by simple multi-protein modules. The figure depicts three types of signaling modules (left) and their behavior in response to agonist (right). (top) In a positive feed-back module, a transducer protein (T) stimulates an effector (E) to produce a cellular output, but the effector also stimulates the activity of the transducer. The result can be an all-or-none switch, where input up to a threshold has little effect, but then becomes committed when feedback from the effector is sufficient to maintain transducer activity even in the absence of continued input from the receptor. (center) In a positive feed-forward module, the effector requires input both from the transducer and from upstream in the pathway. When stimulation is brief (short horizontal bar under trace at right), significant amounts of active transducer do not accumulate and output is minimal. When stimulation is prolonged (longer bar), signal output is substantial. (bottom) In some dual-control switching modules, the binding of one regulator (G) can both activate the effector and expose another regulatory site, shown here as a Ser substrate site (-OH) for a protein kinase. The effector can only be phosphorylated or dephosphorylated when G is bound. Therefore, as shown at the right, addition of G alone will activate but activation of the kinase (K) alone will not. If kinase is active while G is bound, phosphorylation is resistant to phosphatase activity unless G is again present to reexpose the phosphoserine residue (shown on the graph at the right as a bold P). 598 CHAPTER 14 Principles of cell signaling tivate the transducer, sufficient initial signal can be fed back to the transducer that it can maintain the effector's full signal output even when input is removed. Such systems typically display a threshold behavior, as shown on the right. A positive feed-forward loop can generate memory of another type (Figure 14.6, middle panel), indicating the duration of input. In such circuits, the effector requires simultaneous input from both the receptor and from the intermediary transducer. If the pathway from receptor through transducer is relatively slow, or if it requires the accumulation of a substantial amount of transducer, only a prolonged input will trigger a response, as shown in the time-base output diagram at the right. A third way to establish memory is to allow one input to control the reversibility of a second regulatory event (Figure 14.6, bottom panel). WASP, a protein that initiates the polymerization of actin to drive cellular motion and shape change, is activated both by phosphorylation and by the binding of Cdc42, a small GTP-binding protein (G). However, the phosphorylation site on WASP is only exposed when WASP is bound to Cdc42. Phosphorylation thus requires both activated Cdc42 and activated protein kinase. If Cdc42 dissociates, the phosphorylated state of WASP persists until another signaling molecule, whose identity remains uncertain, binds again to expose the site to a protein phosphatase. As shown in the time-base graph, exposure to Cdc42 will activate, but exposure to kinase alone will not. If Cdc42 is present, then the kinase can activate WASP. Phospho-WASP is relatively insensitive to protein phosphatase (P) alone, but can be dephosphorylated if Cdc42 or another G protein binds to expose the site to phosphatase. 14.8 Scaffolds increase signaling efficiency and enhance spatial organization of signaling Key concepts • Scaffolds organize groups of signaling proteins and may create pathway specificity by sequestering components that have multiple partners. • Scaffolds increase the local concentration of signaling proteins. • Scaffolds localize signaling pathways to sites of action. 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 599 Scaffolds concentrate and insulate signaling proteins The INAD signaling complex Pheromone TRP GPCR Rhodopsin Cdc42p Cdc42p Ste20p Ste20p PKC PDZ - G protein PKC PDZ PDZ INAD PDZ PDZ CaM PDZ PDZ Z INAD PDZ PDZ Ste11p PDZ Ste7p Ste5p Ste Ste CaM CYTOSOL Fus FIGURE 14.7 The scaffold InaD organizes proteins that transmit visual signals in the fly photoreceptor cell. InaD is localized to the photoreceptor membrane and coordinates light sensing and visual transduction. In invertebrate eyes, the visual signaling pathway goes from rhodopsin through Gq to a phospholipase C-, and Ca2+ release triggered by PLC action initiates depolarization. This system is specialized for speed, and requires that the relevant proteins are nearby. InaD contains five PDZ domains, each of which binds to the C terminus of a signal transducing protein. The TRP channel, which mediates Ca2+ entry, PLC-, and a protein kinase C isoform that is involved in rapid desensitization all bind constitutively to InaD. Rhodopsin and a myosin (NinaC) also bind, and Gq binds indirectly. 7p 3p Scaffold organizes MAPK cascade Pheromone Mating response High osmolarity Cdc42p Cdc42p Ste20p Ste20p Ste11p Ste11p Ste5p Pbs2p Ste7p Hog1p Fus3p Mating response The proteins in a signaling pathway are frequently colocalized within cells such that their mutual interactions are favored and their interactions with other proteins are minimized. Many signaling pathways are organized on scaffolds. Scaffolds bind several components of a signaling pathway in multiprotein complexes to enhance signaling efficiency. Scaffolds promote interactions of proteins that have a low affinity for each other, accelerate activation (and often inactivation) of the associated components, and localize the signaling proteins to appropriate sites of action. Colocalization may be tonic or regulated, and stimulus-dependent scaffolding often determines signaling outputs. The binding sites on a scaffolding protein are often localized in distinct modular proteinbinding domains, giving the impression that the protein is designed simply to hold the components of the pathway together. Many scaffolding proteins do lack intrinsic enzymatic activity, but some signaling enzymes also act as scaffolds. Binding to a scaffold facilitates signaling by increasing the local concentrations of the components, so that diffusion or transport of molecules to their sites of action is not necessary. In the photoreceptor cells of Drosophila, scaffolding of signaling components is critical for rapid signal transmission. These cells contain the InaD Fus3p 11p Scaffold determines specificity of Ste11p signaling Osmoadaptation FIGURE 14.8 The scaffold Ste5p organizes the components of the MAPK cascade that mediates the pheromone-induced mating response in Saccharomyces cerevisiae. In the top left panel, Ste5p brings the components of the MAPK cascade to the membrane in response to pheromone. In the top right panel, binding to the heterotrimeric G protein brings loaded Ste5p in proximity to the protein kinase Ste20p bound to the activated small GTP binding protein Cdc42p. Their colocalization facilitates the sequential activation of the cascade components, resulting in activation of the MAPK Fus3p and the mating response. The MAP3K Ste11p can regulate not only the MAPK Fus3p in the mating pathway, but also the MAPK Hog1p in the high osmolarity pathway, as shown in the bottom two panels. The scaffold to which Ste11p binds, either Ste5p or Pbs2 (both a scaffold and a MAP2K), determines which MAPK and downstream events are activated as the output. scaffolding protein, which has five modular binding domains, known as PDZ domains. Each of its PDZ domains binds to a C-terminal motif of a target protein, thereby facilitating interactions among the associated proteins. FIGURE 14.7 shows a model for how InaD organizes the signaling proteins. The mutational loss of InaD produces a nearly blind fly, and deletion of a single PDZ domain can yield a fly with a distinct visual defect characteristic of the protein that binds to the missing domain. A second example is Ste5p, a scaffold for the pheromone-induced mating response pathway in S. cerevisiae. FIGURE 14.8 illustrates how Ste5p binds and organizes components of a mitogen- 14.8 Scaffolds increase signaling efficiency and enhance spatial organization of signaling 599 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 600 activated protein kinase (MAPK) cascade, including a MAP3K (Ste11p), a MAP2K (Ste7p) and a MAPK (Fus3p). (The MAPK cascade will be discussed in 14.32 MAPKs are central to many signaling pathways). The function of Ste5p is partially retained even if the positions of its binding sites for the kinases are shuffled in the linear sequence of the protein, indicating that a major role is to bring the enzymes into proximity, rather than to precisely orient them. Ste5p also binds to the subunits of the heterotrimeric G protein that mediates the actions of mating pheromones, linking the membrane signal to the intracellular transducers. Yeast that lack Ste5p cannot mate, demonstrating that Ste5p is required for this biological function (but not all functions) carried out by the pathway. In addition to facilitating signaling in their own pathways, scaffolds can enhance signaling specificity by limiting interactions with other signaling proteins. Scaffolds thus insulate components of a signaling pathway both from activation by inappropriate signals and from producing incorrect outputs. For example, the mating and osmosensing pathways in yeast share several components, including the MAP3K Ste11p, but each pathway maintains specificity because it employs different scaffolds that restrict signal transmission. In contrast, the presence of excess scaffold can inhibit signaling because the individual signaling components will more frequently bind to distinct scaffold proteins rather than forming a functional complex. Such dilution among scaffolds causes separation rather than concentration of the components, preventing their productive interaction. 14.9 Independent, modular domains specify proteinprotein interactions Key concepts • Protein interactions may be mediated by small, conserved domains. • Modular interaction domains are essential for signal transmission. • Adaptors consist exclusively of binding domains or motifs. Modular protein interaction domains or motifs occur in many signaling proteins and confer the ability to bind structural motifs in other molecules, including proteins, lipids, and nucleic 600 CHAPTER 14 Principles of cell signaling acids. Some of these domains are listed in FIGURE 14.9. In contrast to scaffolds, which bind specific proteins with considerable selectivity, modular interaction domains generally recognize not a single molecule but a group of targets that share related structural features. Modular interaction domains important for signal transduction were first discovered in the protein tyrosine kinase proto-oncogene Src, which contains a protein tyrosine kinase domain and two domains named Src homology (SH) 2 and 3 domains. The modular SH2 and SH3 domains were originally identified by comparison of Src to two other tyrosine kinases, Fps and Abl. One or both of these domains appear in numerous proteins and both are critically involved in protein-protein interactions. SH3 domains, which consist of approximately 50 residues, bind to specific short proline-rich sequences. Many cytoskeletal proteins and proteins found in focal adhesion complexes contain SH3 domains and proline rich sequences, suggesting that this targeting motif may send proteins with these domains to these sites of action within cells. In contrast to phosphotyrosine-SH2 binding, the proline-rich binding sites for SH3 domains are present in resting and activated cells. However, SH3-proline interactions may be negatively regulated by phosphorylation within the proline-rich motif. SH2 domains, which consist of approximately 100 residues, bind to Tyr phosphorylated proteins, such as cytoplasmic tyrosine kinases and receptor tyrosine kinases. Thus, Tyr phosphorylation regulates the appearance of SH2 binding sites and, thereby, regulates a set of protein-protein interactions in a stimulusdependent manner. A clever strategy was used to identify the binding specificity of SH2 domains. An isolated recombinant SH2 domain was incubated with cell lysates and then recovered from the lysates using a purification tag. The proteins associated with the SH2 domain were some of the same proteins that were recognized by antiphosphotyrosine antibodies. By this and other methods, it was discovered that SH2 domains recognize sequences surrounding Tyr phosphorylation sites and require phosphorylation of the included Tyr for high affinity binding. Information on specific amino acid sequences that recognize and bind to modular binding domains is being accumulated as these individual interactions are identified. In addition, screening programs using cDNA and/or peptide libraries to assess binding capabilities 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 601 Characteristics of some common modular protein domains Domain Characteristics Cellular involvement 14-3-3 Binds protein phosphoserine or phosphothreonine Protein sequestration Bromo Binds acetylated lysine residues Chromatin-associated proteins Dimerization Caspase activation C1 Binds phorbol esters or diacylglycerol Recruitment to membranes C2 Binds phospholipids Signal transduction, vesicular trafficking Binds calcium Calcium-dependent processes F-Box Binds Skp1 in a ubiquitin-ligase complex Ubiquitination FHA Binds protein phosphothreonine or phosphoserine Various; DNA damage FYVE Binds to PI(3)P Membrane trafficking, TGF- signaling HECT Binds E2 ubiquitin-conjugating enzymes to transfer ubiquitin to the substrate or to ubiquitin chains Ubiquitination LIM Zinc-binding cysteine-rich motif that forms two tandemly repeated zinc fingers Wide variety of processes PDZ Binds to the C-terminal 4-5 residues of proteins that have a hydrophobic residue at the terminus; may bind to PIP2 Scaffolding diverse protein complexes often at the membrane PH Binds to specific phosphoinositides, esp. PI-4,5-P2, PI-3,4-P2 or PI-3,4,5-P3. Recruitment to membranes and motility Binds zinc and may be found in E3 ubiquitin ligases Ubiquitination, transcription SAM Homo- and heterooligomerization Wide variety of processes SH2 Binds to protein phosphotyrosine Tyrosine protein kinase (pY) signaling SH3 Binds to PXXP motifs Various processes TPR Degenerate sequence of ~34 amino acids with residues WL/GYAFAP; forms a scaffold Wide variety of processes WW Binds proline-rich sequences Alternative to SH3; vesicular trafficking CARD EF hand RING FIGURE 14.9 The table describes a subset of known modular protein interaction domains found in many proteins. Interactions mediated by these domains are essential to controlling cell function. Few if any of these domains exist in prokaryotes. Adapted from the Pawson Lab, Protein Interaction Domains, Mount Sinai Hospital (http://pawsonlab.mshri.on.ca/). yield such motifs. Consensus target sequences for individual domains have been identified based on the sequence specificity of their binding to arrayed sequences. These consensus sequences can then be used to predict whether the domain will bind a site in a candidate protein. Adaptor proteins, which lack enzymatic activity, link signaling molecules and target them in a manner that is responsive to extracellular signals. Adaptor proteins are generally made up of two or more modular interaction domains or the complementary recognition motifs. Unlike scaffolds, adaptors are usually 14.9 Independent, modular domains specify protein-protein interactions 601 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 602 multifunctional because their modular interaction domains and motifs are not as highly specific. Adaptors bind to two or more other signaling proteins via their protein-protein interaction domains to colocalize them or to facilitate additional interactions. Grb2 is a prototypical adaptor protein that was identified as a protein that bound to the Cterminal region of the EGF receptor. Grb2 has one SH2 and two SH3 domains. It binds constitutively to specific proline-rich segments of proteins through its SH3 domain, although this binding can be negatively regulated. One target of Grb2 is SOS, a guanine nucleotide exchange factor that activates the small GTP-binding protein Ras in response to EGF signaling. Through its SH2 domain, Grb2 binds Tyr-phosphorylated proteins, including the receptors themselves in a stimulus-dependent manner. Thus, Tyr phosphorylation of these receptors in response to ligand will enable the binding of Grb2 to the receptors, which, in turn, will recruit SOS to the membrane-localized receptor. Once at the membrane, SOS can activate its target, Ras. FIGURE 14.10 Top: Upon exposure to a stimulus, signaling pathways adjust their sensitivities to adapt to the new level of input. Thus, the response decays after initial stimulation. A second similar stimulus will elicit a smaller response unless adequate time is allowed for recovery. Bottom: Some adaptation mechanisms feed back only on the receptor that is stimulated and do not alter parallel pathways. Such mechanisms are referred to as homologous. At left, agonist a for receptor R1 can initiate either of two feedback events that desensitize R1 alone. In other cases, a stimulus will also cause parallel or related systems to desensitize. At the right, agonist a initiates desensitization of both R1 and R2. The response to agonist b, which binds to R2, is also desensitized. Such heterologous desensitization is common. Cellular signaling is remarkably adaptive Key concepts • Sensitivity of signaling pathways is regulated to allow responses to change over a wide range of signal strengths. • Feedback mechanisms execute this function in all signaling pathways. • Most pathways contain multiple adaptive feedback loops to cope with signals of various strengths and durations. A universal property of cellular signaling pathways is adaptation to the incoming signal. Cells continuously adjust their sensitivity to signals to maintain their ability to detect changes in input. Typically, when a cell is exposed to a new input, it initiates a process of desensitization that dampens the cellular response to a new plateau lower than the initial peak response, as illustrated in FIGURE 14.10. When the stimulus is removed, the desensitized state can persist, with sensitivity slowly returning to normal. Similarly, the removal of a tonic stimulus can hypersensitize signaling systems. Patterns of adaptation in signaling networks R esponse Initial response Desensitization Agonist Agonist Agonist Time Homologous desensitization a K R2 R1 X2 Y Z CHAPTER 14 Principles of cell signaling Heterologous desensitization a R esponse X1 602 14.10 R1 Time Reapply a or b R2 R1 R2 Agonist a for R1 R esponse R1 R 1 or R 2 b a X2 X1 Y Z Agonist a for R1 Time Reapply a or b 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 603 Adaptation in signaling is one of the best examples of biological homeostasis. The adaptability of cellular signaling can be quite impressive. Cells commonly regulate their sensitivity to physiological stimuli over more than a 100-fold range, and the mammalian visual response can adapt to incoming light over a 107-fold range. This remarkable ability allows a photoreceptor cell to detect a single photon, and allows a person to read in both very dim light and intense sunlight. Adaptability is observed in bacteria, plants, fungi, and animals. Many of its properties are conserved throughout biology, although the most complex adaptive mechanisms are found in animals. The general mechanism for adaptation is the negative feedback loop, which biochemically samples the signal and controls the adaptive process. Adaptation varies with both the intensity and the duration of the incoming signal. Stronger or more persistent inputs tend to drive greater adaptive change and, often, adaptation that persists for a longer time. Cells can modulate adaptation in this way because adaptation is exerted by a succession of independent mechanisms, each with its own sensitivity and kinetic parameters. G protein pathways offer excellent examples of adaptation. FIGURE 14.11 shows that the earliest step in adaptation is receptor phosphorylation, which is catalyzed by G protein-coupled receptor kinases (GRKs) that selectively recognize the receptor’s ligand-activated conformation. Phosphorylation inhibits the receptor’s ability to stimulate G protein activation and also promotes binding of arrestin, a protein that further inhibits G protein activation. Moreover, arrestin binding primes receptors for endocytosis, which removes them from the cell surface. Endocytosis can also be the first step in receptor proteolysis. Along with these direct effects, many receptor genes display feedback inhibition of transcription, such that signaling by a receptor decreases its own expression. Stimulation thus causes multiple adaptive processes that range from immediate (phosphorylation, arrestin binding) through delayed (transcriptional regulation), and include both reversible and irreversible events. This array of adaptive events has been demonstrated for many G protein-coupled receptors, and many cells may use all of them to control output from one receptor. The speed, extent, and reversibility of adaptation are selected by a cell’s developmental program. Cells can change their patterns of adaptation both qualitatively and quantitatively by altering the points in a pathway where feedback is initiated and exerted. In a linear pathway, changing Multiple adaptation processes occur after a stimulus Relative response 1 Receptor phosphorylation 2 Arrestin binding 3 Receptor endocytosis 4 Endosomal receptor degradation 5 Receptor transcription inhibited 0 1 10 100 1000 Time (seconds) Agonist added Agonist binds Agonist GPCR GRK G protein G protein active 1 Receptor Arrestin phosphorylation 2 Arrestin binding EFFECTORS 3 Receptor endocytosis CYTOPLASM Receptor recycling Early endosome 4 Receptor degradation 5 Receptor NUCLEUS Lysosome DNA transcription inhibited G P C R gene FIGURE 14.11 Multiple adaptation processes are invoked during a stimulus, and multiple nested mechanisms for adaptation are the rule. They are usually invoked sequentially according to the duration and intensity of the stimulus. For GPCRs, at least five desensitizing mechanisms are known, with others acting on the G protein and effectors. these points will alter the kinetics or extent of adaptation (Figure 14.10). In branched pathways, changing these points can determine whether adaptation is unique to one input or is exerted for many similar inputs. If receptor activation triggers its desensitization directly, or if an event downstream on an unbranched pathway triggers desensitization, then only signals that initiate with that receptor will be altered. Receptor-selective adaptation is referred to as homologous adaptation (Figure 14.10). 14.10 Cellular signaling is remarkably adaptive 603 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 604 Cells increase the richness, adaptability, and regulation of their signaling pathways by expressing multiple species of individual signaling proteins that display distinct biochemical properties. These species may be encoded by multiple genes or by multiple mRNAs derived from a single gene by alternative splicing or mRNA editing. The numerical complexity implicit in these choices is impressive. Consider the neurotransmitter serotonin: In mammals, there are thirteen serotonin receptors, each of which stimulates a distinct spectrum of G proteins of the Gi, Gs, and Gq families. (A fourteenth serotonin receptor is an ion channel.) FIGURE 14.12 shows the relationship of serotonin receptors to these G protein families. There is also tremendous diversity among the G proteins and adenylyl cyclases. There are three genes for Gαi and one each for the closely related Gαz and Gαo. Furthermore, the Gαo mRNA is multiply spliced. There are four Gq members. In addition, there are five genes for Gβ and twelve for Gγ, and most of the possible Gβγ dimers are expressed naturally. There are ten genes for adenylyl cyclases, which are direct targets of Gs and either direct or indirect targets of the other G proteins. While all nine membrane-bound adenylyl cyclase isoforms are stimulated by Gαs, they display diverse stimulatory and inhibitory responses to Gβγ, Gαi, Ca2+, calmodulin, and several protein kinases, as illustrated in FIGURE 14.13. Thus, stimulation by serotonin can lead to diverse responses depending upon the various forms of the proteins that are engaged at a particular time and location. Alternatively, feedback control can initiate downstream from multiple receptors in a convergent pathway and thus regulate both the initiating receptor and the others. Such heterologous adaptation regulates all the possible inputs to a given control point. A common example is the phosphorylation of G proteincoupled receptors by either protein kinase A or protein kinase C, which are activated by downstream signals cAMP or Ca2+ plus the lipid diacylglycerol, respectively. Like GRK, these kinases both attenuate receptor activity and promote arrestin binding. Cells also alter their responses to incoming signals for homeostatic reasons. These considerations include phase of the cell cycle, metabolic status, or other aspects of cellular activity. Again, all these adaptive processes may be displayed to a greater or lesser extent in different cells, different pathways within a cell or different situations during the cell’s lifetime. 14.11 Signaling proteins are frequently expressed as multiple species Key concepts • Distinct species (isoforms) of similar signaling proteins expand the regulatory mechanisms possible in signaling pathways. • Isoforms may differ in function, susceptibility to regulation or expression. • Cells may express one or several isoforms to fulfill their signaling needs. FIGURE 14.12 Receptors for serotonin have evolved in mammals as a family of 13 genes that regulate three of the four major classes of G proteins. While all respond to the natural ligand serotonin, the binding sites have evolved sufficient differences that drugs have been developed that specifically target one or more isoforms. The type 3 serotonin receptors, not shown here, are ligand-gated ion channels and are not obviously related to the others. Evolutionary relationship of serotonin receptor isoforms Isoforms G protein 1B 1D Gi 1E 1F 1A 5A 5B Gs 7 4 2A Gq 2C 2B 6 120 100 Gs 80 60 40 Nucleotide substitution distance 604 CHAPTER 14 Principles of cell signaling 20 0 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 605 Different isoforms of adenylyl cyclase are regulated differently Gαs Gβγ Ca2+ NO PKC PKA Regulators CaMK inhibit Gαi activate CaM FIGURE 14.13 All of the mammalian membrane-bound adenylyl cyclases are structurally homologous and catalyze the same reaction, and all are stimulated by Gs. Their responses to other inputs (protein kinases CaMK, PKA and PKC; Ca2+; calmodulin (CaM); NO•) are specific to each isoform, allowing a rich combinatoric input to cellular cAMP signaling. Sometimes isoforms of a signaling protein are subject to quite different kinds of inputs. For example, all of the members of the phospholipase C family (PLC) hydrolyze phosphatidylinositol-4,5-bisphosphate to form two second messengers, diacylglycerol and inositol-1,4,5 trisphosphate (see 14.16 Lipids and lipid-derived compounds are signaling molecules). The distinct isoforms may be regulated by diverse combinations of Gαq, Gβγ, phosphorylation, monomeric G proteins, or Ca2+. Because a cell has multiple options when expressing a form of a signaling protein, it can use expression of particular isoforms to alter how it performs otherwise identical signaling functions. Different cells express one or more isoforms to allow appropriate responses, and expression can vary according to other inputs or the cell’s metabolic status. In addition, signaling pathways are remarkably resistant to mutational or other injuries because loss of a single species or isoform of a signaling protein can often be compensated for by increased expression or activity of another species. Similarly, engineered overexpression can result in the reduced expression of endogenous proteins. The existence of multiple receptor species can, thus, substantially add to adaptability and the consequent resistance of signaling networks to damage. 14.12 Activating and deactivating reactions are separate and independently controlled Key concepts • Activating and deactivating reactions are usually executed by different regulatory proteins. • Separating activation and inactivation allows for fine-tuned regulation of amplitude and timing. In signaling networks, individual proteins are frequently activated and deactivated by distinct reactions, a feature that facilitates separate regulation. Common examples include using protein kinases and phosphoprotein phosphatases 14.12 Activating and deactivating reactions are separate and independently controlled 605 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 606 to catalyze protein phosphorylation and dephosphorylation; using adenylyl cyclase to create cAMP while using phosphodiesterases to hydrolyze it or anion transporters to pump it out of the cell; or using GTP/GDP exchange factors (GEFs) to activate G proteins and GTPaseactivating proteins (GAPs) to deactivate them. Depending on stoichiometry and detailed mechanism, these strategies can convey either additive or nonadditive inputs while maintaining fine control over the kinetics of activation and deactivation of a signaling pathway. The use of distinct reactions for activation and deactivation is analogous to the use of distinct anabolic and catabolic enzymes in reversible metabolic pathways. 14.13 Cellular signaling uses both allostery and covalent modification Key concepts • Allostery refers to the ability of a molecule to alter the conformation of a target protein when it binds noncovalently to that protein. • Modification of a protein’s chemical structure is also frequently used to regulate its activity. Cellular signaling uses almost every imaginable mechanism for regulating the activities of intracellular proteins, but most can be described as either allosteric or covalent. Individual signaling proteins typically respond to multiple allosteric and covalent inputs. Allostery refers to the ability of a molecule to alter the conformation of a target protein when it binds noncovalently to that protein. Because a protein’s activity reflects its conformation, the binding of any molecule that alters conformation can change the target protein’s activity. Any molecule can have allosteric effects: protons or Ca2+, small organic molecules, or other proteins. Allosteric regulation can be both inhibitory or stimulatory. Covalent modification of a protein’s chemical structure is also frequently used to regulate its activity. The change in the protein’s chemical structure alters its conformation and, thus, its activity. Most regulatory covalent modification is reversible. The classic and most common regulatory covalent event is phosphorylation, in which a phosphoryl group is transferred from ATP to the protein, most often to the hydroxyl group of serine (Ser), threonine(Thr), or tyrosine (Tyr). Enzymes that phosphorylate proteins 606 CHAPTER 14 Principles of cell signaling are known as protein kinases. Their actions are opposed by phosphoprotein phosphatases, which catalyze the hydrolysis of the phosphoryl group to yield free phosphate and restore the unmodified hydroxyl residue. Other forms of covalent modification are also common and will be addressed throughout the chapter. 14.14 Second messengers provide readily diffusible pathways for information transfer Key concepts • Second messengers can propagate signals between proteins that are at a distance. • cAMP and Ca2+ are widely used second messengers. Signaling pathways make use of both proteins and small molecules according to their distinctive attributes. A small molecule used as an intracellular signal, or second messenger, has a number of advantages over a protein as a signaling intermediary. Small molecules can be synthesized and destroyed quickly. Because they can be made readily, they can act at high concentrations so that their affinities for target proteins can be low. Low affinity permits rapid dissociation, such that their signals can be terminated promptly when free second messenger molecules are destroyed or sequestered. Because second messengers are small, they also can diffuse quickly within the cell, although many cells have developed mechanisms to spatially restrict such diffusion. Second messengers are, thus, superior to proteins in mediating fast responses, particularly at a distance. Second messengers are also useful when signals have to be addressed to large numbers of target proteins simultaneously. These advantages often overcome their lack of catalytic activity and their inability to bind multiple molecules simultaneously. FIGURE 14.14 lists intracellular second messengers developed through evolution. This number is surprisingly low. Several are nucleotides synthesized from major metabolic nucleotide precursors. They include cAMP, cyclic GMP, ppGppp, and cyclic ADP-ribose. Other soluble second messengers include a sugar phosphate, inositol-1,4,5-trisphosphate (IP3), a divalent metal ion Ca2+, and a free radical gas nitric oxide (NO•). Lipid second messengers include diacylglycerol and phosphatidylinositol-3,4,5-trisphosphate, 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 607 phosphatidylinositol-4,5-diphosphate, sphingosine-1-phosphate and phosphatidic acid. The first signaling compound to be described as a second messenger was cAMP. The name arose because cAMP is synthesized in animal cells as a second, intracellular signal in response to numerous extracellular hormones, the first messengers in the pathway. cAMP is used by prokaryotes, fungi, and animals to convey information to a variety of regulatory proteins. (Its occurrence in higher plants has still not been proved.) Adenylyl cyclases, the enzymes that synthesize cAMP from ATP, are regulated in various ways depending on the organism in which they occur. In animals, adenylyl cyclase is an integral protein of the plasma membrane whose multiple isoforms are stimulated by diverse agents (see Figure 14.13). In animal cells, adenylyl cyclase is generally stimulated by Gs, which was originally discovered as an adenylyl cyclase regulator. Some fungal adenylyl cyclases are also stimulated by G proteins. Bacterial cyclases are far more diverse in their regulation. cAMP is removed from cells in two ways. It may be extruded from cells by an ATP-driven anion pump but is more often hydrolyzed to 5′AMP by members of the cyclic nucleotide phosphodiesterase family, a large group of proteins that are themselves under multiple regulatory controls. The prototypical downstream regulator for cAMP in animals is the cAMP-dependent protein kinase, but a bacterial cAMP-regulated transcription factor was discovered shortly thereafter, and other effectors are now known (Figure 14.14). The cAMP system remains the prototypical eukaryotic signaling pathway in that its components exemplify almost all of the recognized varieties of signaling molecules and their interactions: hormone, receptor, G protein, adenylyl cyclase, protein kinase, phosphodiesterase, and extrusion pump. The second messenger-stimulated protein kinase PKA is a tetramer composed of two catalytic (C) subunits and two regulatory (R) subunits, as illustrated in FIGURE 14.15. The R subunit binds to the catalytic subunit in the substratebinding region, maintaining C in an inhibited state. Each R subunit binds two molecules of cAMP, four cAMP molecules per PKA holoenzyme. When these sites are filled, the R subunit dimer dissociates rapidly, leaving two free catalytic subunits with high activity. The difference in affinity of R for C in the presence and absence of cAMP is ~10,000-fold. The strongly cooper- Second messengers Second messenger Targets Protein kinase A Synthesis/ PreRelease cursor Adenylyl cyclase ATP Organic anion transporter Bacterial transcription factors 3':5'-cyclic AMP (cAMP) Removal Phosphodiesterase Cation channel Cyclic nucleotide phosphodiesterase Rap GDP/GTP exchange factor (Epac) RNA polymerase Magic spot (ppGpp, ppGppp) ObgE transcription arrest detector Inositol-1,3,52+ trisphosphate IP3-gated Ca (IP 3) channel Protein Diacylglycerol kinase C (DAG) Trp cation channel Phosphatidyl- Ion channel inositol-4,5bisphosphate (PIP 2 ) Transporters Protein kinase G 3':5'-Cyclic GMP (cGMP) GTP SpoTcatalyzed hydrolysis Phospholipase C PIP2 Phosphatase Phospholipase C PIP2 Diacylglycerol kinase Rel1A SpoT Diacylglycerol lipase PIP 5-kinase PI-4-P Phospholipase C Phosphatase Guanylyl cyclase GTP Phosphodiesterase ADP-ribose cyclase NAD Hydrolysis Diguanylate cyclase GTP Cyclic di-GMP phosphodiesterase Cation channel Cyclic nucleotide phosphodiesterase Cyclic ADP-ribose Ca2+ channel Cyclic Various two diguanosine- component monophosphate system proteins Nitric oxide (NO. ) Guanylyl cyclase NO. synthase arginine Reduction Ca2+ Numerous calmodulin Release from storage organelles Stored or plasma Ca2+ membrane channels Akt (protein Phosphatidyl- kinase B) PI 3-kinase inositol-3,4,5trisphosphate Other PH domains/proteins PIP2 Reuptake and extrusion pumps Phosphatase FIGURE 14.14 Major second messengers, some of the proteins that they regulate, their sources and their disposition. ative binding of cAMP generates a very steep activation curve with an apparent threshold below which no significant activation of PKA occurs, as illustrated in Figure 14.15. PKA activity, thus, increases dramatically over a narrow range of cAMP concentrations. PKA is also regulated 14.14 Second messengers provide readily diffusible pathways for information transfer 607 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 608 Activation of PKA by cAMP Activated PKA PKA - cAMP R - cAMP C (C) Catalytic subunits R C R (R) Regulatory subunits 4 cAMP R 2 C 2 + 4 cAMP R R2 . - cAMP - cAMP C C cAMP4 + 2C 14.15 Kinase activity as a function of [cAMP] (%) 100 90% 80 60 40 20 10% 2 x 10-9 2 x 10-8 2 x 10-7 [cAMP] FIGURE 14.15 PKA is a heterotetramer composed of two catalytic (C) and two regulatory (R) subunits. Binding of four molecules of cAMP to the regulatory subunits induces dissociation of two molecules of C, the active form of PKA, from the cAMP-bound regulatory subunit dimer. In the bottom panel, the cooperative binding of four molecules of cAMP generates a steep activation profile. Activity increases from approximately 10% to 90% as the cAMP concentration increases only 10-fold. An apparent threshold is introduced because there is little change in activity at low concentrations of cAMP. by phosphorylation of its activation loop. Phosphorylation occurs cotranslationally, and the activation loop phosphorylation is required for assembly of the R2C2 tetramer. The PKAs are mostly cytosolic and are also targeted to specific locations by binding organelle-associated scaffolds (A-kinase anchoring proteins, or AKAPs). These AKAPs facilitate phosphorylation of membrane proteins including GPCRs, transporters, and ion channels. AKAPs can also target PKA to other cellular locations including mitochondria, the cytoskeleton, and the centrosome. AKAPs often harbor binding sites for other regulatory molecules such as phosphoprotein phosphatases and additional protein kinases, which allows for coordination of multiple signaling pathways and integration of their outputs. PKA generally phosphorylates substrates with a primary consensus motif of Arg-ArgXaa-Ser-Hydrophobic, placing it in a large group of kinases that recognize basic residues preceding the phosphorylation site. PKA regulates pro608 CHAPTER 14 Principles of cell signaling teins throughout the cell ranging from ion channels to transcription factors, and its conserved substrate preference frequently permits prediction of substrates by sequence analysis. The cAMP response element binding protein CREB is phosphorylated by PKA on Ser 133 and is largely responsible for the impact of cAMP on transcription of numerous genes. Ca2+ signaling serves diverse purposes in all eukaryotic cells Key concepts • Ca2+ serves as a second messenger and regulatory molecule in essentially all cells. • Ca2+ acts directly on many target proteins and also regulates the activity of a regulatory protein calmodulin. • The cytosolic concentration of Ca2+ is controlled by organellar sequestration and release. Ca2+ is used as a second messenger in all cells, and is, thus, an even more widespread second messenger than cAMP. Many proteins bind Ca2+ with consequent allosteric changes in their enzymatic activities, subcellular localization, or interaction with other proteins or with lipids. Direct targets of Ca2+ regulation include almost all classes of signaling proteins described in this chapter, numerous metabolic enzymes, ion channels and pumps, and contractile proteins. Most noteworthy may be muscle actomyosin fibers, which are triggered to contract in response to cytosolic Ca2+ (see 8.21 Myosin-II functions in muscle contraction). Although free Ca2+ is found at concentrations near 1 mM in most extracellular fluids, intracellular Ca2+ concentrations are maintained near 100 nanomolar levels by the combined action of pumps and transporters that either extrude free Ca2+ or sequester it in the endoplasmic reticulum or mitochondria. Ca2+ signaling is initiated when Ca2+-selective channels in the endoplasmic reticulum or plasma membrane are opened to allow Ca2+ to enter the cytoplasm. The most important entrance channels include electrically gated channels in animal plasma membranes; a Ca2+ channel in the endoplasmic reticulum that is opened by another second messenger, inositol 1,4,5-trisphosphate (see below); and an electrically gated channel in the endoplasmic (sarcoplasmic) reticulum of muscle that opens in response to depolarization of nearby plasma membrane, a process known as excitation-contraction coupling (see 2.9 Plasma mem- 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 609 Calcium binding causes a conformational change in calmodulin Calcium-bound calmodulin bound to target peptide of CaMK Calcium-free calmodulin Ca 2+ (Ca 2+)4 . calmodulin calmodulin free + 4 Ca 2+ target . active FIGURE 14.16 Ribbon diagrams representing the crystal structures of calmodulin free of Ca2+ and bound to four Ca2+ ions reveal the huge conformational change that calmodulin undergoes upon Ca2+ binding. Ca2+-calmodulin causes activity changes in target proteins. The bottom panel shows the activation of a target by calmodulin as a function of the intracellular free Ca2+ concentration. The requirement for binding four Ca2+ ions to induce the conformational transition results in cooperative activation of targets. Activity increases from 10% to 90% as the Ca2+ concentration increases only 10-fold. Structures generated from Protein Data Bank files 1CFD and 1MXE. target Activation of target by calmodulin (%) 100 90% 80 60 40 20 3 x 10-8 10% 3 x 10-7 brane Ca2+ channels activate intracellular functions). In addition to the proteins that are regulated by binding Ca2+ directly, many other proteins respond to Ca2+ by binding a widespread Ca2+ sensor, the small, ~17 kDa protein calmodulin. Calmodulin requires the binding of four molecules of Ca2+ to become fully active, and binding is highly cooperative, generating a sigmoid activation profile illustrated in FIGURE 14.16. Calmodulin generally binds its targets in a Ca2+dependent manner, but Ca2+-free calmodulin may remain bound but inactive in some cases. For example, calmodulin is a constitutive subunit of phosphorylase kinase that is activated upon Ca2+ binding. Higher plants again make major modifications to this paradigm. Calmodulin is not expressed as a distinct protein but, instead, is found as a domain in Ca2+-regulated proteins. In yet another variation, the adenylyl cyclase secreted by the pathogenic bacterium Bordetella pertussis is inactive outside cells but is activated by Ca2+-free calmodulin in animal cells, where its rapid production of cAMP is highly toxic. [Ca2+] 3 x 10-6 14.16 Lipids and lipid-derived compounds are signaling molecules Key concepts • Multiple lipid-derived second messengers are produced in membranes. • Phospholipase Cs release soluble and lipid second messengers in response to diverse inputs. • Channels and transporters are modulated by different lipids in addition to inputs from other sources. • PI 3-kinase synthesizes PIP3 to modulate cell shape and motility. • PLD and PLA2 create other lipid second messengers. Signals that originate at the plasma membrane may have soluble regulatory targets in the cytoplasm or intracellular organelles, but integral plasma membrane proteins are also subject to acute controls. For these targets, lipid second messengers may be primary inputs. Lipids derived from membrane phospholipids or other 14.16 Lipids and lipid-derived compounds are signaling molecules 609 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 610 lipid species play numerous roles in cell signaling. Because their analysis has been more difficult than for soluble messengers, many probably remain to be discovered and understood. FIGURE 14.17 shows the structure of some of these lipids. Phospholipase Cs (PLCs) are the prototypical lipid signaling enzymes. PLC isoforms catalyze the hydrolysis of phospholipids between the 3-sn-hydroxyl and the phosphate group to yield a diacylglycerol and phosphate ester. In animals and fungi, PLCs specific for the substrate Structures of some lipid second messengers O Phosphatidylinositol (PI) O O O O- O P OH O O OH 6 2 1 4 5 3 HO OH OH O Phosphatidylinositol-3,4,5-trisphosphate (PIP3) O O O O- O P OH O O H-O3PO OH 6 2 1 4 OPO3H- 5 3 OPO3HO Diacylglycerol (DAG) O O O OH Inositol trisphosphate (IP3) OH OPO3H2 1 4 3 HO OH 6 5 OPO3H- OPO3HO Phosphatidic acid (PA) O O O O- O P O- O FIGURE 14.17 Structures of some lipid second messengers and the common precursor phosphatidylinositol. The acyl side chain structures shown here are the most common for mammalian PI lipids. Much of the PA in cells is derived from PC, and its acyl chains may differ from those shown. 610 CHAPTER 14 Principles of cell signaling 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 611 phosphatidylinositol-4,5-bisphosphate (PIP2) hydrolyze PIP2 to form two second messengers: 1,2-sn-diacylglycerol (DAG) and inositol-1,4,5trisphosphate (IP3). The PLC substrate PIP2 is itself an important regulatory ligand that modulates the activity of several ion channels, transporters, and enzymes. Thus, PLC alters concentration of three second messengers; its net effect depends on the net turnover of the substrate and products. DAG is probably the best known lipid second messenger; its hydrophobicity limits it to action in membranes. DAG activates some isoforms of protein kinase C (PKC), modulates the activity of several cation channels and activates at least one other protein kinase. DAG can be further hydrolyzed to release arachidonic acid, which can regulate some ion channels. Arachidonic acid is also the precursor of oxidation products, such as prostaglandins and thromboxanes, which are potent extracellular signaling agents. In addition to DAG, PKCs require interaction with Ca2+ and an acidic phospholipid, such as phosphatidylserine, to become activated. Thus, activation of PKC requires the coincidence of multiple inputs both to generate DAG and to increase intracellular Ca2+. There are more than a dozen PKCs, classified together according to highly conserved sequences in the catalytic domain. Three subgroups of PKCs, also identifiable by sequence, share different patterns of regulation. Their regulation provides examples of many ways in which other mammalian protein kinases are regulated. The first of these groups, canonical PKCs, are generally soluble or very loosely associated with membranes prior to the appearance of DAG. DAG causes their association with membranes and permits activation upon binding of other regulators. The second group of PKCs requires similar lipids but not Ca2+, and the third group requires other lipids but neither DAG nor Ca2+ for activation. The N-terminal region of PKCs contains a pseudosubstrate domain, a sequence that resembles that of a typical substrate except that the target Ser is replaced with Ala. The pseudosubstrate region binds to the active site to inhibit the kinase. Activators cause the pseudosubstrate domain to flip out of the active site. PKCs are also activated by proteolysis, as are many protein kinases with discrete autoinhibitory domains. Proteases clip a flexible hinge region, which results in loss of the regulatory domain and consequent activation of the kinase. PKC is the major receptor for phorbol esters, a class of powerful tumor promoters. Phorbol esters mimic DAG and cause a more massive and prolonged activation than physiological stimuli. This massive stimulation can induce proteolysis of PKC, resulting in downregulation, or loss of the kinase. (For a personal description on the discovery of protein kinase C see EXP : 14-0001 ) IP3, the second product of the PLC reaction, is a soluble second messenger. The most significant IP3 target is a Ca2+ channel in the endoplasmic reticulum. IP3 causes this channel to open and release stored Ca2+ into the cytoplasm, thereby rapidly elevating the cytosolic Ca2+ over 100-fold and, in turn, causing the activation of numerous targets of Ca2+ signaling. There are at least six families of PIP2-selective PLC enzymes, defined by their distinct forms of regulation, domain compositions, and overall sequence conservation. Their catalytic domains are all quite similar. The PLC-βs are stimulated primarily by Gαq and Gβγ (to individually varying extents). Several are also modulated by phosphorylation. PLC-γ isoforms are stimulated by phosphorylation on Tyr residues, frequently by receptor tyrosine kinases. The PLC-ε isoforms are regulated by small, monomeric G proteins of the Rho family. The regulation of the PLC-δs is still incompletely understood. Two other classes similar to the PLCδs, PLC-η and -ζ, have also been defined recently. (There is no PLC-α.) In addition to their distinct modes of regulation, all of the PLCs are stimulated by Ca2+, and Ca2+ often acts synergistically with other stimulatory inputs. This synergy underlies the intensification and prolongation of Ca2+ signaling observed in many cells. Phospholipases A2 and D (PLA2 and PLD) also hydrolyze glycerol phospholipids in cell membranes to form important signaling compounds. PLA2 hydrolyzes the fatty acid at the sn2 position of multiple phospholipids to produce the cognate lysophospholipid and the free fatty acid, which is generally unsaturated. The free fatty acid is often arachidonic acid, a precursor of extracellular signals. The biological roles of free lysophospholipids are not understood in detail but have been linked to effects on the structure of the membrane bilayer. PLD catalyzes a reaction much like that of PLC but instead hydrolyzes the phosphodiester on the substituent side of the phosphate group to form 3-sn-phosphatidic acid. Cellular PLDs act on multiple glycerol phospholipid substrates, but phosphatidylcholine is probably the sub- 14.16 Lipids and lipid-derived compounds are signaling molecules 611 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 612 strate most relevant to signaling functions. The functions of the phosphatidic acid product, which is also formed by phosphorylation of DAG, remain poorly understood but appear to include a role in secretion and the fusion of intracellular membranes. 14.17 PI 3-kinase regulates both cell shape and the activation of essential growth and metabolic functions Key concepts • Phosphorylation of some lipid second messengers changes their activity. • PIP3 is recognized by proteins with a pleckstrin homology domain. FIGURE 14.18 Activated PI 3kinase phosphorylates PIP2 to produce PIP3. The PH domain-containing protein kinases PDK1 and Akt bind to PIP3 at the plasma membrane. Their colocalization facilitates the phosphorylation of Akt by PDK1. A second phosphorylation within a hydrophobic motif results in Akt activation by one of several candidate protein kinases. The Akt-2 isoform is required to elicit hallmark actions of insulin. Lipid second messengers may also be modified by phosphorylation. PI 3-kinase phosphorylates PIP2 on the 3-position of the inositol ring to form PI 3,4,5-P3, another lipid second messenger. The total activity of PI 3-kinase is too low to significantly deplete total PIP2, but formation of small amounts of PIP3 in localized membrane domains is vital for altering cell shape and cellular motility. PIP3 acts by recruiting proteins that contain PIP3 binding domains, including pleckstrin homology (PH) and FYVE domains, to sites where they regulate cytoskeletal remodeling, contractile protein function, or other regulatory events. These proteins anchor and/or orient the structural or motor proteins involved in cellular movement and localize signaling proteins to sites of action at the membrane. PIP3 PIP 3 binding brings Akt and PDK1 to the membrane Akt and PDK1 bind PIP 3 through PH domains PIP 2 phosphorylated PIP 2 Akt is activated by phosphorylation PIP 3 p85 p110 PI 3-kinase Akt PDK1 PH domains Akt Other kinase PDK1 Glucose uptake Glycogen synthesis Antilipolysis Antiapoptosis 612 CHAPTER 14 Principles of cell signaling signaling can be fast and dramatic; it largely accounts for directing the mobility of motile mammalian cells. Lipid mediators are essential in the insulin signaling pathway. The binding of insulin stimulates the Tyr autophosphorylation of its receptor and the activation of effectors through insulin receptor substrate (IRS) proteins (see 14.30 Diverse signaling mechanisms are regulated by protein tyrosine kinases). PI 3-kinase is activated when its p85 subunit binds to IRS1. The PIP3 generated by PI3kinase binds the protein kinases Akt and phosphoinositide-dependent kinase-1 (PDK-1) via their PH domains. This interaction results in the localization of Akt to the membrane where it is activated by PDK1, as illustrated in FIGURE 14.18. Akt phosphorylates downstream targets, including protein kinases, GAPs, and transcription factors. Activation of Akt, specifically Akt-2, is required for the hallmark actions of insulin including regulation of glucose transporter translocation, enhanced protein synthesis, and expression of gluconeogenic and lipogenic enzymes. 14.18 Signaling through ion channel receptors is very fast Key concepts • Ion channels allow the passage of ions through a pore, resulting in rapid (microsecond) changes in membrane potential. • Channels are selective for particular ions or for cations or anions. • Channels regulate intracellular concentrations of regulatory ions, such as Ca2+. Ligand-gated ion channels are multisubunit, membrane-spanning proteins that create and regulate a water-filled pore through the membrane, as illustrated in the X-ray crystal structure of the nicotinic acetylcholine receptor in FIGURE 14.19. When stimulated by extracellular agonists, the subunits rearrange their conformations and orientations to open the pore and, thus, connect the aqueous spaces on either side of the membrane. The pore has a diameter that allows ions to diffuse freely from one side of the membrane to the other, driven by the electrical and chemical gradients that have been established by ion pumps and transporters. (For more about channel, pump and transporter mechanics see 2 Transport of ions and small molecules across membranes.) Channels maintain selectivity among ions by regulating the pore diameter precisely 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 613 and by lining the walls of the pore with appropriate hydrophilic residues. Receptor ion channels can, thus, provide a diffusion path for only cations or anions, or select among different ions. Ligand-gated ion channels provide the fastest signal transduction mechanism found in biology. Upon binding an agonist ligand, channels open within microseconds. At synapses, where neurotransmitters need to diffuse less than 0.1 micron, a signal in the postsynaptic cell can be generated in 100 microseconds. In contrast, receptor-stimulated G proteins require about 100 milliseconds to exchange GDP for GTP, and the action of receptor protein kinases is even slower. Ligand-gated ion channels are important receptors in many cells in addition to neurons and muscle, and other ion channels play equally vital roles in signaling pathways triggered by other classes of ligands. Ion channel signaling differs from that of the other receptors mentioned in this chapter in that there is no immediate protein target nor, in most cases, is there a specific second messenger involved. In most cases, channel-mediated ion flow acts to increase or decrease the cell’s membrane potential and, thus, modulates all transport processes for metabolites or ions that are electrically driven. Animal cells maintain an inside-negative membrane potential by pumping out Na+ ions and pumping in K+ ions (for more on membrane potential see 2.4 Electrochemical gradients across the cell membrane generate the membrane potential). The opening of a channel selective for Na+ will thus depolarize cells, and the opening of a channel for K+ will hyperpolarize cells. Similarly, because Cl- is primarily extracellular, opening Cl- channels will also cause hyperpolarization. These electrical effects convey information to effector proteins that are energetically coupled to the membrane potential, or to specific ion gradients, or that bind a specific ion (such as Ca2+) whose concentration changes upon channel opening. The nicotinic acetylcholine receptor is the prototypical receptor ion channel and was the first receptor that was shown to be a channel. It is a relatively unselective cation channel that causes depolarization of the target cell by allowing Na+ influx. It is best known as the excitatory receptor at the neuromuscular synapse, where it triggers contraction, but alternative isoforms are also active in neurons and many other cells. In muscles, nicotinic depolarization acts via a voltage-sensitive Ca2+ channel to allow Ca2+ release from the sarcoplasmic reticulum into the cytosol. Calcium acts Nicotinic acetylcholine receptor structure CLOSED OPEN Pore Pore CYTOSOL FIGURE 14.19 The nicotinic cholinergic receptor is a cation-selective channel that is composed of five homologous but usually nonidentical subunits that oligomerize to form a primarily -helical membrane-spanning core. The channel itself is created within this core, and its opening and closing are executed by cooperative changes in subunit arrangement. Structure generated from Protein Data Bank file 2BG9. as a second (or third) messenger to initiate contraction (see 2.13 Cardiac and skeletal muscles are activated by excitation-contraction coupling). Nicotinic receptors promote exocytosis in some secretory cells by a similar mechanism, where Ca2+ triggers the exocytic event. In neurons, where nicotinic stimulation causes an action potential (depolarization that is rapidly propagated along the neuron), the initial depolarization is sensed by voltage-sensitive Na+ channels. Their opening (along with the action of other channels) propagates the action potential along the neuron. The nervous system is rich in receptor cation channels that respond to other neurotransmitters, the most common of which is the amino acid glutamate (Glu). The three different families of glutamate receptors share the property of cation conductance, but each family has its own spectrum of drug responses. All operate as neuronal activators, with one interesting twist: The NMDA family of receptors, named for their response to a selective drug, is permeant to Ca2+ in addition to Na+. A significant component of its activity is to permit the inward flow of Ca2+, which acts as a second messenger on a wide variety of targets. Persistant stimulation of NMDA channels by glutamate released during injury, or by drugs, can cause toxic amounts of Ca2+ to enter, resulting in neuronal death. A second functional group of receptor channels is selective for anions and, by allowing inward flux of Cl-, hyperpolarizes the target cell. Anion-selective receptors include those for γaminobutyric acid (GABA) and glycine (Gly). In neurons, hyperpolarization can inhibit the initiation of an action potential and/or neurotransmitter release. 14.18 Signaling through ion channel receptors is very fast 613 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 614 Perhaps the most diverse family of ligandgated channels is that of the TRP and TRP-like family, of which about 30 have been found in mammals. Distinct forms are found in invertebrates. The TRP channels are Ca2+-selective channels that are formed by tetramers of identical subunits that surround the central channel. Each subunit is composed of a homologous bundle of six membrane-spanning helices, but the N and C termini contain a diverse collection of regulatory and protein interaction domains, including protein kinase domains (whose substrates are currently unknown). All TRP channels allow transmembrane flux of Ca2+ to permit its action as a second messenger, but different TRP isoforms serve numerous physiological functions. The prototypical TRP, found in invertebrate photoreceptors, gates Ca2+ flow from intracellular stores into the cytoplasm to initiate visual signaling. Others admit Ca2+ from outside the cell, and still others allow Ca2+ to enter the endoplasmic reticulum virtually directly from the extracellular space because they form a bridge between the plasma membrane and channels in the endoplasmic reticulum at points where the membranes abut each other. Regulation of TRP channels is perhaps even more diverse. Various TRP channels respond to heat, cold, painful stimuli, pressure, and high or low osmolarity. Many TRPs are regulated either positively or negatively by lipids, such as eicosanoids, diacylglycerol, and PIP2. For example, capsaicin, the hot compound in chilis, is an agonist for some vanilloid receptors (TRPVs). Still other TRP channels are mechanosensors that allow cilia to sense fluid flow. The most famous of these is the sensory channel of the hair cell of the inner ear. This channel opens when the apical cilia on the hair cell are bent in response to sound-driven fluid flow. 14.19 Nuclear receptors regulate transcription Key concepts • Nuclear receptors modulate transcription by binding to distinct short sequences in chromosomal DNA known as response elements. • Receptor binding to other receptors, inhibitors, or coactivators leads to complex transcriptional control circuits. • Signaling through nuclear receptors is relatively slow, consistent with their roles in adaptive responses. Nuclear receptors are unique among cellular 614 CHAPTER 14 Principles of cell signaling receptors in that their ligands pass unaided through the plasma membrane. These receptors, when complexed with their ligands, enter the nucleus and regulate gene transcription. Ligands for nuclear receptors include sex steroids (estrogen and testosterone) and other steroid hormones, vitamins A and D, retinoids and other fatty acids, oxysterols, and bile acids. Nuclear receptors are structurally conserved. They consist of a C-terminal ligand binding domain, an N-terminal interaction region that recognizes components of the transcriptional machinery and acts as a transactivation domain, a centrally located zinc finger domain that binds DNA, and, often, another transactivation domain nearer the C-terminus. In the absence of ligand, these receptors are bound to corepressor proteins that suppress their activity. Upon hormone binding, corepressors dissociate and the receptors are assembled in multiprotein complexes with coactivators that modulate receptor action and facilitate transcriptional regulation. As illustrated in FIGURE 14.20, agonists and antagonists bind to distinct receptor conformations (see 14.5 Ligand binding changes receptor conformation). Receptor agonists favor the binding of receptors to coactivators and DNA, and antagonists favor conformations that block coactivator-receptor binding. Nuclear receptors bind with high specificity to hormone response elements in the 5’ untranscribed region of regulated genes. Response elements are typically short direct or inverted repeat sequences, and a gene may contain response elements for several different receptors in addition to binding sites for other transcriptional regulatory proteins. The sex steroid estrogen can bind to two different nuclear receptors, the estrogen receptors ER and ER. Coactivator and corepressor proteins differentially regulate ER and ER in transcriptional complexes that are expressed in specific cell types. Other ligands that bind to these receptors include valuable therapeutic agents. For example, 4 hydroxy-tamoxifen is an estrogen receptor antagonist used in the therapy of estrogen-receptor-positive breast cancer to inhibit growth of residual cancer cells. However, unlike its antagonistic effects on the estrogen receptor in breast, 4 hydroxy-tamoxifen displays weak partial agonist activity in uterus. In the estrogen receptor system, partial agonists are known as selective estrogen receptor modulators (SERMs). Properties that contribute to partial agonist activity include the relative expression of the two estrogen recep- 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 615 Estrogen receptor conformation depends on which ligand is bound Agonist-bound conformation Antagonist-bound conformation N N K362 H11 H5 K362 H5 545 C H11 H6 545 H3 542 H6 538 542 H3 538 FIGURE 14.20 The estrogen receptor adopts different conformations when bound to agonists and antagonists. The ligand-binding domain of the estrogen receptor is bound to the agonist estradiol on the left and to the antagonist raloxifene on the right. Note the marked difference in position of helix 12, shown in blue in the active structure and green in the inhibited structure. Reproduced from Brzozowski, A. M., et al. 1997. Molecular basis of agonism and antagonism in the oestrogen receptor. Nature. 389: 753–758. Photo courtesy of M. Brzozowski, University of New York. tors, ER and ER, as well as the expression of repressors and coactivators that interact with each receptor type. Thus, the behavior of nuclear receptor ligands must be considered in the tissue, cellular, and signaling context. 14.20 G protein signaling modules are widely used and highly adaptable Key concepts • The basic module is a receptor, a G protein and an effector protein. • Cells express several varieties of each class of proteins. • Effectors are heterogeneous and initiate diverse cellular functions. Activation of G protein-coupled receptors (GPCRs) and their associated heterotrimeric G proteins is one of the most widespread mechanisms of communicating extracellular signals to the intracellular environment. G protein signaling modules are found in all eukaryotes. Depending on the species, mammals express 500-1000 GPCRs that respond to hormones, neurotransmitters, pheromones, metabolites, local signaling substances, and other regulatory molecules. Essentially all chemical classes are represented among the GPCR ligands. In addition, a roughly equal number of olfactory GPCRs are expressed in olfactory neurons and work in combination to screen compounds in the animal’s environment via the sense of smell. Because GPCRs are involved in many kinds of physiologic responses, they are also one of the most widely used targets for drugs. A minimal G protein signaling module consists of three proteins: a G protein-coupled receptor, the heterotrimeric G protein, and an effector protein, as illustrated in FIGURE 14.21. The receptor activates the G protein on the inner face of the plasma membrane in response to an extracellular ligand. The G protein then activates (or occasionally inhibits) an effector protein that propagates a signal within the cell. Thus, signal conduction in the simplest G protein module is linear. However, as depicted in FIGURE 14.22, a typical animal cell may express a dozen GPCRs, more than six G proteins, and a dozen effectors. Each GPCR regulates one or more G proteins, and each G protein regulates several effectors. Moreover, distinct efficiencies and rates govern each interaction. Thus, a cell’s G protein network is actually a signal-integrating computer whose 14.20 G protein signaling modules are widely used and highly adaptable 615 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 616 output is a spectrum of cellular signals that is complex in both amplitude and kinetics. Because of their conserved parts list, G protein modules are well suited to initiating a wide variety of intracellular signals in response to diverse molecular inputs and can do so over a wide range of time scales (milliseconds to minutes). GPCRs are integral plasma membrane proteins composed of a bundle of seven hydrophobic membrane-spanning helices with an extracellular N terminus and cytosolic C terminus, as depicted in FIGURE 14.23. Based on the three-dimensional structure of rhodopsin and on copious biochemical and genetic data, it is likely that all GPCRs share the same basic mechanism of conformational activation and deactivation in response to activating ligands (see 14.5 Ligand binding changes receptor conformation). Binding of agonist ligand on the extracellular face of the receptor drives realignment of the helices to alter the structure of a binding site for the heterotrimeric G protein on the cytoplasmic face, and this altered conformation of the G proteinbinding surface promotes G protein activation. Heterotrimeric G protein signaling Agonist GPCR PIP 2 DAG Trimeric G protein Receptor activated Activated G protein dissociates Hydrolysis of PIP2 to IP 3 and DAG IP 3 IP 3-gated Ca2+ channel Release of Ca2+ CYTOSOL ENDOPLASMIC RETICULUM Ca2+ FIGURE 14.21 G protein-mediated signal transduction follows a path of agonist to receptor to heterotrimeric G protein to effector to the effector's output. Both G and G subunits regulate distinct effectors. In the example shown here, Gq regulates a phospholipase C- to produce two second messengers, diacyglycerol (DAG) and inositol-trisphosphate (IP3). IP3 triggers Ca2+ release from the endoplasmic reticulum. FIGURE 14.22 A portion of the G protein-mediated signaling network in macrophages highlights some of the complexity of interactions possible in such systems. Several receptors and G protein subunits are omitted. Where a named G protein is shown, its signaling output is probably mediated by its G subunit. Activation of any G protein also activates its G subunit, although Gmediated signaling is usually most prominent from Gi trimers. In addition, several G proteins modulate the activities of others through poorly understood pathways. Only a small sampling of effectors is shown, and the only adaptive mechanism shown is GRK-catalyzed phosphorylation of receptors. Data from Paul Sternweis, Alliance for Cellular Signaling. Partial G protein signaling network in mouse macrophages Agonist C5a ISO PGE S1P UDP UTP PAF LPA GPCR C5aR β 2 AR E2R EDG P2YR P2YR PAFR EDG G Protein Gi Gs G 12 Gq Ad Cyc ?? ATP Effector PI 3Kinase cAMP PIP 3 Ca 2+ cAMP PDE AMP Ca 2+ pump Inactivation mechanisms GRK 616 G 12 CHAPTER 14 Principles of cell signaling PIP 2 PLC- β DAG + IP 3 IP 3 R Phosphatase IP 2 + P i 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 617 Structure of rhodopsin Heterotrimeric G protein structure C YTOPLASM MEMBRAN E Retinal FIGURE 14.23 The figure shows the crystal structure of the GPCR rhodopsin. Each membrane-spanning helix is a different color; most structures on the cytoplasmic face are not shown. The retinal chromophore is shown within the helix bundle. GPCR sequence similarity separates the mammalian GPCRs into at least four structural families that are so diverse that there may be little sequence similarity among the classes. Within a family, similarity is greatest in the membrane-spanning helices, less in the interhelical loops, and least in the N- and C-terminal domains and in the cytoplasmic loop that connects spans five and six. Regardless, the generalizations about functional domains in receptors seem to hold true within different families. GPCRs frequently form dimers, occasionally heterodimers, and dimerization can be crucial for function. Structure generated from Protein Data Bank file 1F88. The heterotrimeric G proteins to which GPCRs are coupled are composed of a nucleotide-binding Gα subunit and a Gβγ subunit dimer, as illustrated in FIGURE 14.24. The structure of the trimer and each subunit is known for several states of activation and in complex with several interacting proteins. A Gαβγ heterotrimer is named according to its α subunit, which largely defines the G protein’s selectivity among receptors. Each subunit also regulates a distinct group of effector proteins. Gα subunits are globular, two-domain proteins of 38-44 kDa. The GTP-binding domain belongs to the GTP-binding protein superfamily that includes the small, monomeric G proteins (such as Ras, Rho, Arf, Rab; see 14.23 Small, monomeric GTP-binding proteins are multiuse switches) as well as the GTP-binding translational initiation and elongation factors. A second domain modulates GTP binding and hydrolysis. Gα subunits are only slightly hydrophobic, but they are predominantly membrane-associated FIGURE 14.24 The structure of the nonactivated Gi heterotrimer, the G protein that is responsible for inhibition of adenylyl cyclase and for most G-mediated signaling, is shown with each subunit colored as shown. GDP is shown bound to the Gi subunit. Structure generated from Protein Data Bank file 1GP2. G protein targets EFFECTOR PROTEIN G protein Stimulated Gs G olf Adenylyl cyclase G i (3) Go Gz K + channel, PI 3-kinase G gus Other cation channel G t (2) Cyclic GMP phosphodiesterase G q (4) Phospholipase-Cβ G 12 G 13 Rho GEF Inhibited Adenylyl cyclase FIGURE 14.25 G protein-regulated effectors do not share structural similarities. They may be ion channels or membrane spanning enzymes in the plasma membrane, peripheral proteins on the inner face of the membrane, or fundamentally soluble proteins that can bind to G subunits. The chart shows the major groups of G proteins, sorted according to sequence similarity, and some of the effectors that they are known to regulate. because of constitutive N-terminal fatty acylation and because they bind to the membraneattached Gβγ subunits. Mammals have 16 Gα genes that are grouped in subfamilies according to similar sequence and function (e.g., s, i, q, and 12). These subfamilies are listed in FIGURE 14.25. Gβ and Gγ subunits associate irreversibly soon after translation to form stable Gβγ dimers, which then associate reversibly with a Gα. Gβ subunits are 35 kDa proteins composed of seven 14.20 G protein signaling modules are widely used and highly adaptable 617 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 618 -strand repeats that form a cylindrical structure known as a propeller. There are five Gβ genes in mammals. Four encode strikingly similar proteins that naturally dimerize with the twelve Gγ subunits (Figure 14.24). The fifth, Gβ5, is less closely related to the others and interacts primarily with a Gγ-like domain in other proteins rather than with Gγ subunits themselves. Gγ subunits are smaller (~7 kDa) and far more diverse in sequence than are the Gβ’s. The last three amino acid residues of Gγ subunits are proteolyzed to leave a conserved C-terminal cysteine that is irreversibly S-prenylated and carboxymethylated, helping to anchor Gβγ to the membrane. Gβ and Gγ subunits can associate in most possible combinations. Because almost all cells express multiple Gβ and Gγ subunits, it has been difficult to assign specific roles to individual Gβγ combinations. The best recognized interactions of Gβγ subunits occur at sites on Gβ, although distinct functions of Gγ have also been supported. 14.21 Heterotrimeric G proteins regulate a wide variety of effectors Key concepts • G proteins convey signals by regulating the activities of multiple intracellular signaling proteins known as effectors. • Effectors are structurally and functionally diverse. • A common G-protein binding domain has not been identified among effector proteins. • Effector proteins integrate signals from multiple G protein pathways. G protein-regulated effectors include enzymes that create or destroy intracellular second messengers (adenylyl cyclase, cyclic GMP phosphodiesterase, phospholipase C-β, phosphatidylinositol-3-kinase), protein kinases, ion channels (K+, Ca2+) and possibly membrane transport proteins (see Figure 14.25). Effectors may be integral membrane proteins or intrinsically soluble proteins that bind G proteins at the membrane surface. No conserved G protein-binding domain or sequence motif has been identified among effector proteins, and most effectors are related to proteins that have similar functions but that are not regulated by G proteins. Sensitivity to G protein regulation, thus, evolved independently in multiple families of regulatory proteins. 618 CHAPTER 14 Principles of cell signaling Because they can respond to a variety of Gα and Gβγ subunits, effector proteins can integrate signals from multiple G protein pathways. The different Gα or Gβγ subunits may have opposite or synergistic effects on a given effector. For example, some of the membranebound adenylyl cyclases in mammals are stimulated by Gαs and inhibited by Gαi (see Figure 14.13). Many effectors are further regulated by other allosteric ligands (e.g., lipids, calmodulin) and by phosphorylation, contributing even more to integration of information. Effectors are usually represented as multiple isoforms, and each isoform may be regulated differently, adding to the complexity of G protein networks. For example, some isoforms of adenylyl cyclase are stimulated by Gβγ, whereas others are inhibited. All phospholipase C-βs are stimulated both by Gαq family members and by Gβγ, but the potency and maximal effect of these two inputs vary dramatically among the four PLC-β isoforms. 14.22 Heterotrimeric G proteins are controlled by a regulatory GTPase cycle Key concepts • Heterotrimeric G proteins are activated when the Gα subunit binds GTP. • GTP hydrolysis to GDP inactivates the G protein. • GTP hydrolysis is slow, but is accelerated by proteins called GAPs. • Receptors promote activation by allowing GDP dissociation and GTP association; spontaneous exchange is very slow. • RGS proteins and phospholipase C-βs are GAPs for G proteins. The key event in heterotrimeric G protein signaling is the binding of GTP to the Gα subunit. GTP binding activates the Gα subunit, which allows both it and the Gβγ subunit to bind and regulate effectors. The Gα subunit remains active as long as GTP is bound, but Gα also has GTPase activity and hydrolyzes bound GTP to GDP. Gα-GDP is inactive. G proteins thus traverse a GTPase cycle of GTP binding/activation and hydrolysis/deactivation, as depicted in FIGURE 14.26. Therefore, the control of G protein signaling is intrinsically kinetic. The relative signal strength, or amplitude, is proportional to the fraction of G protein that is in the active, GTP-bound form. This fraction equals the balance of the rates of GTP binding and GTP hydrolysis, the activating 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 619 and deactivating arms of the GTPase cycle. Both limbs are highly regulated over a range of rates greater than 1000-fold. Receptors promote G protein activation by opening the nucleotide-binding site on the G protein, thus accelerating both GDP dissociation and GTP association. This process is referred to as GDP/GTP exchange catalysis. Exchange proceeds in the direction of activation because the affinity of G proteins for GTP is much higher than that for GDP and because the cytosolic concentration of GTP is about 20-fold higher than that of GDP. Spontaneous GDP/GTP exchange is very slow for most G proteins (many minutes), which maintains basal signal output at a low level. In contrast, receptor-catalyzed exchange can take place in a few tens of milliseconds, which allows rapid responses in cells such as visual photoreceptors, other neurons, or muscle. Because receptors are not directly required for a G protein’s signaling activity, a receptor can dissociate after GDP/GTP exchange and catalyze the activation of additional G protein molecules. In this way, a single receptor may maintain the activation of multiple G proteins, providing molecular amplification of the incoming signal. Other receptors may remain bound to their G protein targets, which means that they do not act as amplifiers. However, more tightly bound receptors can initiate signaling more quickly and promote G protein reactivation when hydrolysis of bound GTP is rapid. In the absence of stimulus, Gα subunits hydrolyze bound GTP slowly. The average activation lifetime of the G α-GTP complex is about 10-150 seconds, depending on the G protein. This rate is far slower than rates of deactivation often observed in cells when an agonist is removed. For example, visual signaling terminates in about 10 ms after stimulation by a photon, and many other G protein systems are almost as fast. GTP hydrolysis is accelerated by GTPase-activating proteins (GAPs), which directly bind Gα subunits. In some cases acceleration exceeds 2000-fold. Such speed is necessary in systems like vision or neurotransmission, which must respond to quickly changing stimuli. Because G protein signaling is a balance of activation and deactivation, GAPs deplete the pool of GTP-activated G protein and can thereby also act to inhibit G protein signaling. GAPs can thus inhibit signaling, quench output upon signal termination, or both. What behavior predominates depends on the GAP’s intrinsic activity and its regulation. The regulatory GTPase cycle Receptor + agonist Receptor - agonist G protein GTP GDP Effector protein G protein-GTP G protein-GDP *ACTIVE* G protein-GTPEffector protein *ACTIVE* Pi GAP FIGURE 14.26 G proteins are activated when GTP binds to the G subunit, such that both G-GTP and G can bind and regulate the activities of appropriate effector proteins. G subunits also have intrinsic GTPase activities, and the primary deactivating reaction is hydrolysis of bound GTP to GDP (rather than GTP dissociation). Thus, the steady-state signal output from a receptor-G protein module is the fraction of the G protein in the GTP-bound state, which reflects the balance of the activation and deactivation rates. Both GTP binding and GTP hydrolysis are intrinsically slow and highly regulated. GDP binds tightly to G, such that GDP dissociation is rate-limiting for binding of a new molecule of GTP and consequent reactivation. Both GDP release and GTP binding are catalyzed by GPCRs. Hydrolysis of bound GTP is accelerated by GTPase-activating proteins (GAPs). Receptors and GAPs coordinately control both the steady-state level of signal output and the rates of activation and deactivation of the module. There are two families of GAPs for heterotrimeric G proteins. The RGS proteins (regulators of G protein signaling) are a family of about 30 proteins, most or all of which have GAP activity and regulate G protein signaling rates and amplitudes. The role of RGS proteins in terminating the G protein signal can be seen in FIGURE 14.27. Some proteins with RGS domains also act as G protein-regulated effectors. These include activators of the Rho family of monomeric GTP-binding proteins (see below) and GPCR kinases, which are feedback regulators of GPCR function. The second group of G protein GAPs are phospholipase C-βs. These enzymes are effectors that are stimulated by both Gαq and by Gβγ, but they also act as Gq GAPs, probably to control output kinetics. 14.22 Heterotrimeric G proteins are controlled by a regulatory GTPase cycle 619 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 620 Single photon responses of GAP-deficient mice Current (pA) 0.50 knockout heterozygous wild-type 0.25 0.00 0 2 Time (s) 4 Light flash FIGURE 14.27 G protein GAPs can accelerate signal termination upon removal of agonist, and often do not act as inhibitors during the response to receptor. The figure shows the electrical response of a mouse photoreceptor (rod) cell to a single photon of light. In mice that lack RGS9, the GAP for the photoreceptor G protein Gt, the signal is prolonged for many seconds because hydrolysis of GTP bound to Gt is slow. In wild-type or heterozygous mice, hydrolysis takes place in about 15 milliseconds, and the decay of the signal is much faster. Note that the maximal output is similar in wild-type and mutant mice, indicating that the GAP does not act as an inhibitor in rod cells. In humans, genetic loss of RGS9 leads to severe loss of vision that is particularly marked in bright light. Reproduced from Chen et al. Nature. 2000. 403:557–560. Permission also granted by Ching-Kang Jason Chen, Virginia Commonwealth University. While the GTPase cycle described in Figure 14.26 is general, it is highly simplified. Interactions among receptor, Gα, Gβγ, GAP, and effector are frequently simultaneous and often demonstrate complex cooperative interactions. For example, Gβγ inhibits the release of GDP (to minimize spontaneous activation), promotes the exchange catalyst activity of the receptor, inhibits GAP activity, and helps initiate receptor phosphorylation that leads to desensitization. The other components can be nearly this multifunctional. In addition, inputs from other proteins can alter the dynamics of the GTPase cycle at several points. The core G protein module is, thus, functionally versatile as a signal processor in addition to being versatile in the scope of its targets. 620 CHAPTER 14 Principles of cell signaling 14.23 Small, monomeric GTPbinding proteins are multiuse switches Key concepts • Small GTP-binding proteins are active when bound to GTP and inactive when bound to GDP. • GDP/GTP exchange catalysts known as GEFs (guanine nucleotide exchange factors) promote activation. • GAPs accelerate hydrolysis and deactivation. • GDP dissociation inhibitors (GDIs) slow spontaneous nucleotide exchange. Monomeric GTP-binding proteins, which are encoded by about 150 genes in animals, modulate a wide variety of cellular processes including signal transduction, organellar trafficking, intra-organellar transport, cytoskeletal assembly, and morphogenesis. The small GTP-binding proteins that most clearly function in signal transduction are the Ras and Ras-related proteins (Ral, Rap) and the Rho/Rac/Cdc42 proteins, about 10-15 in all. They are usually about 20-25 kDa in size and are homologous to the GTP-binding domains of Gα subunits. The regulatory activities of the small GTPbinding proteins are controlled by a GTP binding and hydrolysis cycle like that of the heterotrimeric G proteins, with similar regulatory inputs. They are activated by GTP, and hydrolysis of bound GTP to GDP terminates activation. GDP/GTP exchange catalysts, known as GEFs (guanine nucleotide exchange factors, functionally analogous to GPCRs) promote activation, and GAPs accelerate hydrolysis and consequent deactivation. In addition, GDP dissociation inhibitors (GDIs) slow spontaneous nucleotide exchange and activation to dampen basal activity, an activity shared by Gβγ subunits for the heterotrimeric G proteins. While the underlying biochemical regulatory events are essentially identical for monomeric and heterotrimeric G proteins, monomeric G proteins use the basic GTPase cycle in additional ways. Signal output by heterotrimeric G proteins and many monomeric G proteins is usually thought to reflect a balance of their active (GTP-bound) and inactive (GDP-bound) states in a rapidly turning-over GTPase cycle. GEFs favor formation of more active G protein, and GAPs favor the inactive state. In contrast, probably an equal number of the monomeric G proteins behave as acute on-off switches. Upon binding GTP, they initiate a process (regulation, recruitment of other pro- 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 621 teins). They then maintain this activity, sometimes for many seconds or minutes, until they are acted upon by a GAP. For example, the monomeric G protein Ran regulates nucleocytoplasmic trafficking of protein and RNA in both directions, cooperating with carrier proteins known as karyopherins (see 5.15 The Ran GTPase controls the direction of nuclear transport). In the nucleus, high Ran GEF activity promotes GTP binding. Nuclear Ran-GTP then binds import karyopherins to drive dissociation of newly arrived cargo and promote return of the karyopherin to the cytoplasm. It also binds export karyopherins to permit binding of outgoing cargo. Outside the nucleus, high Ran GAP activity promotes GTP hydrolysis. Cytoplasmic Ran-GDP dissociates from both the export karyopherins to allow dissociation of outgoing cargo and from the import karyopherins to allow them to bind cargo for import. Thus, for monomeric G proteins such as Ran, each phase of the GTPase cycle determines a specific, coupled step in a parallel regulatory cycle. A second major difference between the monomeric and the heterotrimeric G proteins is the structures of the GEFs, GAPs, and GDIs. Both GEFs and GAPs for monomeric GTP-binding proteins are structurally heterogeneous (although some clearly related families are evident). In addition, mechanisms for regulating these GEFs and GAPs are equally diverse. They include phosphorylation by protein kinases; allosteric regulation by heterotrimeric and/or monomeric G proteins, by second messengers and by other regulatory proteins; subcellular sequestration or recruitment to scaffolds; and assorted other mechanisms. The Ras proteins were the first small GTPbinding proteins to be discovered. They were identified as oncogene products because they cause malignant growth if they are either overexpressed or persistently activated by mutation; they are among the most commonly mutated genes in human tumors. Several viral ras genes figure prominently as oncogenes. Mammalian cells contain three ras genes (H, N, and K). They may share inputs and outputs to varying extents, and they can compensate for each other in some genetic screens. It has been difficult to assign unique functions to the individual Ras proteins. Inputs to the Ras proteins are diverse and speak to the importance of Ras proteins as a crucial node in signaling. Ras GEFs and GAPS are regulated by both receptor and nonreceptor Tyr kinases through direct phosphorylation and by recruitment of the regulators to the plasma membrane. Other Ras has three main effectors Function Effector Target Protein kinase cascade Raf MAPK Lipid kinase PI 3-kinase Akt Exchange factor RalGDS Exocyst cytoplasmic serine/threonine kinases also converge on Ras activation. Rap1, another member of the Ras family, may also fit directly into this network because it is suspected of competing with Ras proteins for protein kinase targets; in vivo it can suppress the oncogenic activity of Ras. Rap1 is regulated independently, however, and acts on independent signaling pathways as well. One of its GAPs is stimulated by the Gi class of G proteins, for example, and its several GEFs are stimulated by Ca2+, diacylglycerol, and cAMP. Ras proteins generally regulate cell growth, proliferation, and differentiation by modulating the activities of multiple effector proteins. The best known and best studied Ras effector is the protein kinase Raf, which initiates a MAPK cascade. FIGURE 14.28 shows well established Ras effectors. Rho, Rac, and Cdc42 are related monomeric GTP-binding proteins that are involved in generating signals that affect cell morphology. Each class of proteins regulates its own array of effectors and is controlled by separate groups of GEFs, GAPs, and GDIs. Effectors regulated by this family include phospholipases C and D, multiple protein and lipid kinases, proteins that nucleate or reorganize actin filaments, and components of the neutrophil oxygen activating system, among others (see 8.14 Small G proteins regulate actin polymerization). 14.24 FIGURE 14.28 Ras-GTP binds to many proteins. Three well established effectors include Raf, PI 3-kinase, and RalGDS. Activation of these effectors activates a MAPK pathway, increases PI 3-kinase activity, and promotes assembly of a protein complex involved in exocytosis of secretory vesicles. Protein phosphorylation/ dephosphorylation is a major regulatory mechanism in the cell Key concepts • Protein kinases are a large protein family. • Protein kinases phosphorylate Ser and Thr, or Tyr, or all three. • Protein kinases may recognize the primary sequence surrounding the phosphorylation site. • Protein kinases may preferentially recognize phosphorylation sites within folded domains. 14.24 Protein phosphorylation/dephosphorylation is a major regulatory mechanism in the cell 621 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 622 Protein phosphorylation is the most common form of regulatory posttranslational modification. It occurs in all organisms, and it is estimated that about one-third of proteins in animals are at some time phosphorylated. Phosphorylation can stimulate or inhibit the catalytic activity of an enzyme, the affinity with which a protein binds other molecules, its subcellular localization, its ability to be further covalently modified, or its stability. Single phosphorylations may cause 500-fold or greater changes in activity, and proteins are often phosphorylated on multiple residues in complex and interacting patterns. Most protein phosphorylation in eukaryotes, and essentially all in animals, is catalyzed by protein kinases; dephosphorylation is catalyzed by phosphoprotein phosphatases. Both classes of enzymes are controlled by diverse mechanisms. In addition, proteins are often phosphorylated by multiple protein kinases, resulting in the generation of a range of activity states. This complexity allows inputs from different signaling pathways to be integrated into the resulting activity of the target. In bacteria, plants, and fungi, an additional protein phosphorylating system known as twocomponent signaling is vital. The protein ki- Protein kinases are two substrate enzymes SUBSTRATE 2 (Mg 2+ . ATP) SUBSTR A TE 1 ( Pro te in SER) H N H C O C CH 2 N H C O Mg 2+ O- O- P O + OH O- O Adenine O- P O P O O O CH 2 (Mg 2+ . Trip h osp h ate) Rib ose P ROTEIN KIN ASE PR O DU CT 1 ( Pho s p ho ryla ted p ro t ein ) H N H C O O- C CH 2 P O- N H C O O P RODUCT 2 (Mg 2+ . ADP) Mg 2+ O- O- P O O O Adenine OP O CH 2 O ( Mg 2+ . Dip h osp h ate) Rib FIGURE 14.29 Protein kinases transfer the -phosphoryl group from ATP to serine, threonine, or tyrosine residues in protein substrates. 622 CHAPTER 14 Principles of cell signaling nases involved in two-component signaling are unrelated to the eukaryotic protein kinase superfamily and phosphorylate aspartate residues rather than serine, threonine, or tyrosine. Protein kinases transfer a phosphoryl group from ATP to Ser, Thr, and Tyr residues of protein substrates to form chemically stable phosphate esters, as shown in FIGURE 14.29. In animals, the distribution of phosphate among these three amino acid residues is uneven: ~90%-95% is on Ser, 5%-8% on Thr, and less than 1% on Tyr residues. The human genome contains approximately 500 genes that encode protein kinases, and many protein kinase mRNAs undergo alternative splicing. This makes the protein kinase gene superfamily one of the largest functional gene groups. The number and diversity of these enzymes emphasize the great and varied uses of protein kinases to regulate cellular functions. Although some protein kinases have a limited tissue and/or developmental distribution, many are ubiquitously expressed. Protein kinases are grouped according to their residue specificity. Protein kinases that phosphorylate Ser will usually also recognize Thr, hence the name protein Ser/Thr kinase. Multicellular organisms have protein Tyr kinases, which only recognize Tyr. Dual specificity protein kinases can phosphorylate Ser, Thr, and Tyr in the appropriately restricted substrate conformational context and are generally the most selective of the protein kinases. The analysis of the kinomes of several organisms has led to a more elaborate grouping derived from sequence relationships, shown in FIGURE 14.30, that also reflects to some extent on regulatory mechanisms and substrate specificity. For example, the AGC group is named for its founding members, cAMP-dependent protein kinase (PKA), cyclic GMP-dependent protein kinase (PKG), Ca2+, and phospholipiddependent protein kinase (PKC). These protein kinases are regulated by second messengers and prefer substrates that contain basic residues near the phosphorylation site. In addition to substrate specificity for amino acid residues, most protein kinases are also selective for local sequence surrounding the substrate site. Screening strategies have resulted in methods to predict if proteins contain consensus substrate sites for a wide variety of protein kinases. Antibodies can be used to identify and roughly quantitate protein phosphorylation at specific sites in proteins. Beyond local recognition, protein kinases may display marked substrate selectivity among similar proteins based on overall 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 623 Human kinome tree three-dimensional structure, for example, or among proteins that have been differentially covalently modified by phosphorylation or ubiquitination. In animal cells, some protein kinases are hormone receptors that span the plasma membrane. Some protein kinase receptors are protein serine/threonine kinases, such as the transforming growth factor- (TGF-)receptor, but the majority are protein tyrosine kinases, including receptors for insulin, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and other regulators of cell growth and differentiation. Other protein kinases are intrinsically soluble intracellular enzymes, although they may bind to one or more organellar membranes. FIGURE 14.30 The protein kinases in the human genome can be grouped according to sequence relationships that reveal seven major branches. The tyrosine kinases are contained within one major branch. The others are Ser/Thrspecific or dual specificity, and are named for the best described members: AGC from PKA, PKG, and PKC; CAMK from the calcium, calmodulin-dependent kinases; CMGC from CDKs, MAPKs, GSK3, Clks; CK1 from casein kinase 1; STE from Ste20, Ste11, and Ste7, the MAP4K, MAP3K, and MAP2K in the yeast mating pathway; and TKL, the Tyr kinase-like enzymes. Reproduced with permission from G. Manning, et al. 2002. Science. 298: 1912-1934. © 2002 AAAS. Photo courtesy of Gerard Manning, Salk Institute, and reprinted with permission of Cell Signaling Technology, Inc. (www.cellsignal.com). X-ray crystallographic structures of protein kinases have revealed a wealth of information about their mechanism of activation. The conserved minimum catalytic core of a protein kinase contains about 270 amino acids, yielding a minimum molecular mass of about 30,000 Da. Within this core, there are two folded domains that form the active site at their interface, as shown in FIGURE 14.31. One or both of the conserved lysine (Lys) or aspartate (Asp) residues that are required for phosphoryl transfer are frequently mutated to disrupt kinase activity. A sequence near the active site, referred to as the activation loop, often undergoes a conformational rearrangement to generate active forms of the protein kinases and is the most common site of regulatory phosphorylation in 14.24 Protein phosphorylation/dephosphorylation is a major regulatory mechanism in the cell 623 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 624 ERK2 inactive and active conformations INACTIVE (ERK2) ACTIVE (ERK2-P2) N N terminal terminal domain domain Thr183 Thr183 Thr183 Thr183 Tyr185 Tyr185 Tyr185 Tyr185 C C terminal terminal domain domain FIGURE 14.31 The structures of unphosphorylated, inactive MAPK ERK2 and phosphorylated, active ERK2 are compared. ERK2 has a typical protein kinase structure. The smaller N-terminal domain is composed primarily of strands and the larger C-terminal domain is primarily -helical. The active site is formed at the interface of the two domains. The activation loop emerges from the active site and is refolded following phosphorylation of the Tyr and Thr residues, inducing the repositioning of active site residues. ATP (not shown) binds in the interior of the active site; productive binding of protein substrates to the surface of the C-terminal domain is also facilitated by the reorganization of the activation loop. Structures generated from Protein Data Bank files 1ERK and 2ERK. Ligand His P His P His Asp P Asp ADP ATP Response regulator Asp P H2O P His phosphorylation Transfer of phosphate to Asp: response regulator active Response regulator deactivated FIGURE 14.32 The basic two-component system is composed of a signal-activated histidine kinase, referred to as a sensor, and an effector protein, the response regulator, that is activated when it is phosphorylated on an aspartate residue by the sensor. The activity of the response regulator is terminated when the aspartyl-phosphate is hydrolyzed. 624 CHAPTER 14 Principles of cell signaling 14.25 Two-component protein phosphorylation systems are signaling relays Key concepts • Two-component signaling systems are composed of sensor and response regulator components. • Upon receiving a stimulus, sensor components undergo autophosphorylation on a histidine (His) residue. • Transfer of the phosphate to an aspartyl residue on the response regulator serves to activate the regulator. Two-component signaling systems Sensor/ Histidine kinase the protein kinase family. There are unique inserts on the surface of protein kinases that generate specificity in localization, interaction with other regulatory molecules, and recognition of substrates. These landmarks allow both classification and genetic manipulation of protein kinases. Protein kinases have evolved numerous and diverse regulatory mechanisms to complement their number and multiple functions. These mechanisms include allosteric activation and inhibition by lipids, soluble small molecules and other proteins; activating and inhibitory phosphorylation and other covalent modifications, including proteolysis; and binding to scaffolds and adaptors to enhance activity or limit nonspecific activities. Many such inputs may regulate a single protein kinase in a complex combinatoric code. Further, multiple protein kinases that act sequentially, such as in a protein kinase cascade (see Figure 14.38), can create uniquely complex signaling patterns. Prokaryotes, plants, and fungi share an alternative mechanism for regulatory phosphorylation and dephosphorylation known as two-component signaling. FIGURE 14.32 shows a typical twocomponent system. In this system, the receptor, referred to as a sensor, responds to a stimulus by catalyzing its own phosphorylation on a His residue. Sensors include chemoattractant receptors in bacteria, a regulator of osmolarity in fungi, light-sensitive proteins, the receptor for the plantripening hormone ethylene, and other receptors for diverse environmental, hormonal, and metabolic signals. The mammalian mitochondrial dehydrogenase kinases are related in sequence to the bacterial histidine kinases, although the mammalian enzymes phosphorylate serine or threonine residues, not histidine. The phosphorylated sensor next transfers its covalently bound phos- 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 625 phate to an aspartyl residue on a second protein known as a response regulator. Response regulators initiate cellular responses, usually by binding to other cytoplasmic proteins and allosterically regulating their activities. Although all two-component systems follow this same general pattern, their structures and precise reaction pathways vary enormously. Some two-component systems are composed of only one protein (sensor and response regulator in a single polypeptide chain). Others are composed of a sensor protein and two aspartylphosphorylated proteins, in which the first or the second may display response regulatory activity. Finally, two-component systems usually lack conventional protein phosphatases. Hydrolysis of the aspartyl-phosphate bond may be spontaneous or regulated by the response regulator itself. 14.26 Pharmacological inhibitors of protein kinases may be used to understand and treat disease Key concepts • Protein kinase inhibitors are useful both for signaling research and as drugs. • Protein kinase inhibitors usually bind in the ATP binding site. Many inhibitors have been developed for basic research purposes to explore the functions of protein kinases. The importance of these enzymes in disease processes has also made them targets of drug screening projects yielding inhibitors for many protein kinases. The majority of pharmacological inhibitors of protein kinases compete with ATP binding. Because of the huge number of ATP-binding proteins in a cell, there are inevitable concerns about inhibitor specificity not only with respect to the other protein kinases but also to the other proteins that bind nucleotides. This problem has been mitigated with variable success through chemical library screening, structure-based modification of lead compounds, and inhibitor testing against panels of protein kinases. Many inhibitors with actions on PKA or PKCs, for example, have effects on several other members of the AGC family. Although pharmacological inhibitors with effects on PKA abound, the most selective are derived from the naturally occurring small inhibitory protein known as PKI or the Walsh inhibitor. In vitro and cell-based screens have identified much more selective inhibitors for MAP2Ks in the ERK1/2 pathway. These inhibitors have fewer known protein kinase cross reactivities, probably due to the fact that they do not bind in the ATP site. Among inhibitors that have progressed in the clinic, compounds developed against the EGF receptor and certain other protein tyrosine kinases have had considerable success. 14.27 Phosphoprotein phosphatases reverse the actions of kinases and are independently regulated Key concepts • Phosphoprotein phosphatases reverse the actions of protein kinases. • Phosphoprotein phosphatases may dephosphorylate phosphoserine/threonine, phosphotyrosine, or all three. • Phosphoprotein phosphatase specificity is often achieved through the formation of specific protein complexes. Protein phosphorylation is reversed by phosphoprotein phosphatases. These enzymes display distinct specificities and modes of regulation. Phosphoprotein phosphatases can be considered in two broad groups based on their specificity and sequence relationships: protein-serine/threonine phosphatases and protein-tyrosine phosphatases. Most protein-serine/threonine phosphatases are regulated by association with other proteins. Targeted localization is the major determinant of substrate specificity. Phosphoprotein phosphatase 1 (PP1) associates with a variety of regulatory subunits that specifically direct it to relevant organelles. One subunit (known as the G subunit), for example, specifies association with glycogen particles. The interaction with this subunit is itself regulated by phosphorylation. Small protein inhibitors can suppress PP1 activity. Phosphoprotein phosphatase 2A (PP2A) is composed of a catalytic subunit, a scaffolding subunit, and one of a large number of regulatory subunits. The regulatory subunit modulates activity and localization of the phosphatase. Some viruses alter the behavior of the cells they infect by interfering with phosphatase activity. For example, cells transformed with the SV40 virus express a viral protein known as small t anti- 14.27 Phosphoprotein phosphatases reverse the actions of kinases and are independently regulated 625 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 626 gen. Small t displaces the regulatory subunit from PP2A and alters the activity and the subcellular localization of the phosphatase. In addition, natural toxins such as okadaic acid, calyculin, and microcystin inhibit PP2A and PP1 to varying extents both in vitro and in intact cells. Another major protein-serine/threonine phosphatase, called calcineurin (also known as phosphoprotein phosphatase 2B), is regulated by Ca2+-calmodulin (see 14.15 Ca2+ signaling serves diverse purposes in all eukaryotic cells) and plays essential roles in cardiac development and T cell activation, among other events. The major mechanism of action of the immunosuppressants cyclosporin and FK506 is to inhibit calcineurin. The protein tyrosine phosphatases (PTPs) are cysteine-dependent enzymes that utilize a conserved Cys-Xaa-Arg motif to hydrolyze phosphoester bonds in their substrates. The PTPs are encoded by over 100 genes in humans and are classified in four subfamilies: the phosphotyrosine-specific phosphatases, the Cdc25 phosphatases, the dual specificity phosphatases (DSPs), and the low molecular weight phosphatases. Thirty-eight of the PTPs are highly selective for phosphotyrosine residues within substrates. Some of the phosphotyrosine-selective phosphatases are transmembrane proteins, whereas others are membrane associated. The most obvious function of the PTPs is to reverse the functions of tyrosine kinases; however, some have primary functions in transducing tyrosine kinase signals. For example, the protein tyrosine phosphatase SHP2 (also known as SHPTP2), binds to certain tyrosine kinase receptors through its SH2 domain and is itself tyrosine phosphorylated, thereby creating a binding site for the SH2 domain-containing adaptor protein, Grb2, which leads to activation of Ras (see 14.32 MAPKs are central to many signaling pathways). The Cdc25 phosphatases recognize cyclindependent kinase (CDK) family members as substrates and play a critical role in increasing CDK activity at key junctures of the cell cycle (see Figure 14.39 and 11.4 The cell cycle is a cycle of CDK function). Similar to the dual specificity kinases, the dual specificity phosphatases are specific for a restricted number of substrates. A number of DSPs dephosphorylate MAPKs; these DSPs are called MAP kinase phosphatases, or MKPs. Several of these have been implicated in MAPK nuclear entry and exit. Some MKPs are encoded by early response genes, whose 626 CHAPTER 14 Principles of cell signaling products are active near the initiation of the cell cycle (see 11.7 Entry into cell cycle and S phase is tightly regulated). Substrates of other PTP family members, such as the tumor suppressor PTEN, include phosphoinositides, which are phosphorylated derivatives of the glycerolipid phosphatidylinositol that serve as second messengers (see 14.16 Lipids and lipid-derived compounds are signaling molecules). Removal of the phosphate group inactivates the second messenger. It remains unclear whether members of this group work exclusively on phophoinositides or also on protein tyrosine phosphate. 14.28 Covalent modification by ubiquitin and ubiquitinlike proteins is another way of regulating protein function Key concepts • Ubiquitin and related small proteins, may be covalently attached to other proteins as a targeting signal. • Ubiquitin is recognized by diverse ubiquitin binding proteins. • Ubiquitination can cooperate with other covalent modifications. • Ubiquitination regulates signaling in addition to its role in protein degradation. An important mechanism for control of protein function is through covalent modification with small proteins of the ubiquitin family. Ubiquitin is one of a family of proteins referred to as ubiquitin-like (Ubl) proteins. Ubiquitin itself is highly conserved among species, suggesting the functional importance of all of its 76 residues. In addition to the long-established role of ubiquitin in initiating protein degradation, ubiquitin modification also has a variety of functions in signal transduction. Ubl proteins are conjugated to the substrate protein by an isopeptide bond between an amino group on the substrate, usually from a Lys side chain, and the C-terminal Gly residue of the processed Ubl protein. E1, E2, and E3 proteins are required to catalyze conjugation to Ubl proteins (see Biochem 4.3 Ubiquitin attachment to substrates requires multiple enzymes). Several Ubl proteins may be attached to one substrate, often by serial formation of a polyubiquitin chain. Mono- and polyubiquitination both change the 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 627 protein’s behavior to induce downstream signals. Monoubiquitination is a significant regulatory modification in vesicular trafficking and DNA repair. For example, the monoubiquitinated form of the FANCD2 protein becomes associated with the repair protein BRCA1 at sites of DNA repair. Modification by the Ubl protein SUMO has roles in nuclear transport, transcription, and cell cycle progression. Polyubiquitin chains are formed when the Lys residues of ubiquitin itself, particularly K48 and K63, are ubiquitinated. Addition of polyubiquitin with a K48 linkage generally directs proteins to the proteasome for degradation, whereas conjugation to polyubiquitin chains with a K63 linkage promotes signal transmission, not proteolysis. Protein-bound ubiquitin is recognized by a variety of ubiquitin binding domains, including UIM (ubiquitin-interacting motif), UBA (ubiquitin association), and certain zinc finger domains. Such domains have the capacity to act as receptors for ubiquitin within modified proteins. Activation of the transcription factor NF-κB occurs by a mechanism dependent on modification both by the addition of Ubl proteins and phosphorylation. This fascinating example of regulation by ubiquitin is depicted in FIGURE 14.33. Prior to stimulation, NF-κB is retained in the cytoplasm in an inactive form by binding to its inhibitor, IκB. Phosphorylation of IκB by the IκB kinase (IKK) complex promotes its recognition by a multisubunit E3 ligase, which directs its ubiquitination and subsequent proteasomal degradation. Destruction of IκB allows NF-κB to move to the nucleus to mediate changes in transcription. IκB can be stabilized in response to certain signals through covalent attachment of the Ubl, SUMO. Sumoylation occurs on the same Lys residues that must be conjugated to ubiquitin to achieve IκB degradation. Thus, SUMO attachment stabilizes IκB and attenuates NF-κB action. This is one of numerous examples of crosstalk between Ubl conjugates. A key regulatory event in NF-κB signaling is activation of the IKK complex. IKK is itself regulated by ubiquitination and phosphorylation. The cytokine interleukin-1β (IL-1) causes association of adaptor proteins with its receptor to create a receptor activation complex. The interleukin-1β receptor activation complex recruits another adaptor complex containing TRAF6. A phosphorylation event releases a TRAF6 complex from the receptor activation complex into the cytoplasm. TRAF6 contains a RING domain, and is an E3 ubiquitin ligase that catalyzes formation of Modification with Ubl proteins plays multiple roles in IL-1 β signaling IL-1 β CYTOPLASM TRAF6 TRAF6 TRAF6 TRAF6 K63 ubiquitination K48 ubiquitination ADP TRAF6 TRAF6 TAB2 TAK1 ATP Complex formation Degradation ATP ADP IKK IKK Nemo NUCLEUS DNA FIGURE 14.33 Activation of NF-B involves steps dependent on the interaction of proteins attached to ubiquitin through ubiquitin-binding proteins, competition by sumoylation, phosphorylation, and ubiquitin-mediated protein degradation. K63 polyubiquitin chains on the protein kinase TAK1. Polyubiquitinated TAK1 can then recruit TAB2 and TAB3, which are adaptor proteins with conserved zinc finger domains. These particular zinc finger domains bind to polyubiquitinated TAK1 and enhance its activity. TAK1, thus activated, phosphorylates and activates IKK, which then phosphorylates IκB, targeting it for degradation. Thus, ubiquitin-binding domains, such as the TAB2 and TAB3 zinc fingers, may selectively recognize K63 polyubiquitin chains to promote signal transmission. Naturally occurring small molecules may control ubiquitin ligase activity directly. Auxin (indole 3-acetic acid) is a plant hormone that regulates development by promoting the transcription of a large number of genes. Rather than 14.28 Covalent modification by ubiquitin and ubiquitin-like proteins is another way of regulating protein function 627 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 628 stimulating transcription factors, however, auxin accelerates the degradation of several specific transcriptional repressors. The auxin receptor is in fact a ubiquitin ligase complex that targets the auxin-regulated transcriptional repressors for proteolysis. F-box proteins account for all of the auxin binding activity in plant extracts. 14.29 The Wnt pathway regulates cell fate during development and other processes in the adult Key concepts • Seven transmembrane-spanning receptors may control complex differentiation programs. • Wnts are lipid-modified ligands. • Wnts signal through multiple distinct receptors. • Wnts suppress degradation of -catenin, a multifunctional transcription factor. Wnt pathways function during embryonic development and in the adult in morphogenesis, body patterning, axis formation, proliferation, and cell motility. The classical Wnt signaling mechanism was uncovered largely through studies of Drosophila and Xenopus development, as well as by analyzing genetic alterations in cancer. Wnt proteins are unusual extracellular ligands. In addition to carbohydrate, they contain covalently bound palmitate that is essential for their biological activity. Wnts transduce signals by binding to multiple distinct receptors. The most significant are members of the Frizzled family of seven-transmembrane-spanning receptors. Wnts regulate the stability of β-catenin, which either is rapidly degraded or, in response to Wnt, is stabilized to enter the nucleus and induce transcription by interacting with TCF (T-cell factor). Genes induced include c-jun, cyclin D1, and many others. The coordinated activities of the protein kinases glycogen synthase kinase 3 (GSK3) and casein kinase 1(CK1), the scaffolding proteins axin and adenomatous polyposis coli (APC), and the protein disheveled (DSH) are key to β-catenin stability. In the absence of Wnt, phosphorylation of β-catenin by CK1 and GSK3 promotes its ubiquitination and subsequent destruction by the proteasome. Axin and APC are required for phosphorylation of β-catenin by GSK3. In contrast to most seven transmembrane- 628 CHAPTER 14 Principles of cell signaling spanning receptors, the Frizzled family has not yet been shown to have significant functions mediated by a heterotrimeric G protein, and G proteins may not be central to this pathway. Instead a proximal step in signaling by Frizzled involves binding to DSH, which inactivates the β-catenin destruction mechanism. Mutations that cause changes in the amounts of components of the classical pathway are common in a wide variety of cancers. Both Wnts and β-catenin may be viewed as protooncogenes. APC is a tumor suppressor and is mutated in the majority of human colorectal cancers, for example. Either too little or too much axin can also disrupt Wnt signaling, and axin, like APC, is a tumor suppressor. Wnts utilize additional signaling mechanisms. The receptor proteins Lrp5/6 (which are related to the low-density lipoprotein receptor) are Wnt receptors and also bind axin. Wnts bind to tyrosine kinase receptors to influence axon guidance and to other proteins that inhibit their function. Through DSH, Wnts can regulate the JNK MAPK pathway and Rho family G proteins to control planar cell polarity. Certain Wnts increase intracellular calcium to activate calciumdependent signaling pathways. 14.30 Diverse signaling mechanisms are regulated by protein tyrosine kinases Key concepts • Many receptor protein tyrosine kinases are activated by growth factors. • Mutations in receptor tyrosine kinases can be oncogenic. • Ligand binding promotes receptor oligomerization and autophosphorylation. • Signaling proteins bind to the phosphotyrosine residues of the activated receptor. A large group of protein tyrosine kinases are receptors that span the plasma membrane and bind extracellular ligands, as shown in FIGURE 14.34. The receptors are generally activated by growth factors whose normal physiological functions are to promote growth, proliferation, development, or maintenance of differentiated properties. This group includes receptors for insulin, epidermal growth factor (EGF), and platelet derived growth factor (PDGF). These receptors both control the activities of many other protein kinases of all families and directly regulate other classes of signaling proteins. 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 629 Because of their physiologic roles as growth regulators, mutations that activate receptor tyrosine kinases are often oncogenic. For example, the oncogene erbB results from the mutational loss of the extracellular ligand-binding domain of a kinase closely related to the EGF receptor. This mutation causes constitutive activation of the protein kinase domain. Point mutations that affect the transmembrane domain can also cause oncogenic activation, as is found in the EGF receptor-related neu/HER2 oncogene (see 13.8 Cell growth and proliferation are activated by growth factors). Receptor tyrosine kinases are diverse both in their extracellular ligand-binding domains and, with the exception of a conserved tyrosine protein kinase domain, their intracellular regulatory regions. These receptors usually have one membrane span per monomer but some, such as the insulin receptor, which is a disulfidebonded heterotetramer, have two. Ligand binding to receptor tyrosine kinases favors receptor oligomerization and enhances kinase activity leading to increased Tyr phosphorylation of the intracellular domain of the receptor and of associated molecules. These tyrosine-phosphorylated motifs create docking sites for additional signal transducers and adaptors. A comparison of the PDGF and insulin receptors reveals common themes and a range of behaviors of receptor tyrosine kinases. The two PDGF receptors are monomeric receptor tyrosine kinases. The insulin receptor exists in two alternatively spliced forms each of which is a heterotetramer of two and two subunits. In each case, the receptor isoforms utilize some unique signaling mechanisms. PDGF and insulin each stimulate the kinase activity of their receptors, causing oligomerization and autophosphorylation. Seven or more sites are phosphorylated on the PDGF receptor, and each phosphotyrosine residue generates a binding site for one or more SH2 domain-containing proteins as illustrated in FIGURE 14.35. The PDGF receptor binds PI 3-kinase, p190 Ras GAP, phospholipase C-, Src (which may catalyze additional Tyr phosphorylation of the receptor), and the SHP2 tyrosine phosphatase which itself binds the adaptor Grb2 (see 14.32 MAPKs are central to many signaling pathways). With the exception of Src, all of these proteins are also receptor substrates. Thus, substrates are recruited to the receptor as a consequence of specific interactions of substrate SH2 domains with receptor phosphotyrosine producing changes in activities and distributions of numerous intracellular signal Receptor protein tyrosine kinase families Kinase inserts KINASE DOMAINS EGF receptor Insulin receptor PDGF receptor FGF receptor FIGURE 14.34 The monomeric tyrosine kinase receptors consist of a globular extracellular domain that binds ligand, a single transmembrane span, and a globular intracellular region containing the protein kinase domain. The intracellular regions contain additional sequences preceding, following, and, in the case of the PDGF and FGF receptor groups, inserted into the protein kinase domain. These regions contain sites of tyrosine phosphorylation-dependent interactions. The insulin receptor is encoded by a single gene. The precursor is proteolyzed into and subunits, which are disulfide bonded to each other. Disulfide bonds also link two subunits, yielding an obligate heterotetramer. Activation of the PDGF receptor leads to many outputs PDGF PDGF receptors Src p190 RasGAP p110 p85 PI 3-kinase Shp2 CYTOPLASM PLC- γ Grb2 SOS FIGURE 14.35 PDGF binds to its receptor and induces receptor autophosphorylation. The autophosphorylated receptor binds target proteins that contain SH2 domains. 14.30 Diverse signaling mechanisms are regulated by protein tyrosine kinases 629 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 630 transducers. This array of signaling events leads to increased proliferation of connective tissue during development and in wound healing. Autophosphorylation also occurs on the insulin receptor to stabilize the active state and to generate a smaller number of binding sites, as illustrated in FIGURE 14.36. A key event is the Tyr phosphorylation of insulin receptor substrate (IRS) proteins, notably IRS1, on as many as a dozen sites. IRS1 takes over interactions with several signaling effectors that, in the case of PDGF, bind directly to the receptor. Among these targets is PI 3-kinase which leads to activation of Akt-2 and several essential metabolic actions of insulin (see 14.16 Lipids and lipid-derived compounds are signaling molecules). IRS proteins are also phosphorylated by serine/threonine protein kinases to modulate their signaling capability. Tyr phosphorylation often enhances the enzymatic activity of the associated proteins. Other proteins gain enhanced function primarily as a consequence of greater proximity to targets achieved by binding through their SH2 domains to phosphotyrosine sites either on the receptors or IRS adaptors. The precise actions of the many tyrosine kinase receptors are determined by the overlapping sets of signal transducers with which they interact, as well as by detailed differences in amounts of signal transducers, adaptor accessory proteins and receptor expression patterns (see Figure 14.43). Insulin signaling through IRS1 Insulin Insulin receptor PIP 2 PIP 3 p85 p110 PI 3-kinase IRS1 CYTOPLASM Akt signaling FIGURE 14.36 Insulin binding to its receptor causes activation of the receptor tyrosine protein kinase and autophosphorylation. The receptor kinase also phosphorylates IRS1, a large adaptor with many potential phosphorylation sites. IRS1 is an essential intermediate in insulin action. PI 3-kinase binds to IRS1 via the SH2 domain within its p85 subunit. Akt and PDK1 bind to PIP3 produced by activated PI 3-kinase so that PDK1 can phosphorylate and activate Akt (see Figure 14.18). 630 CHAPTER 14 Principles of cell signaling 14.31 Src family protein kinases cooperate with receptor protein tyrosine kinases Key concepts • Src is activated by release of intrasteric inhibition. • Activation of Src involves liberation of modular binding domains for activation-dependent interactions. • Src often associates with receptors, including receptor tyrosine kinases. The first protein tyrosine kinase to be discovered was Src, which was identified as the transforming entity in the Rous sarcoma virus. Src is the prototype of a number of related enzymes, the Src family kinases. It participates in signaling pathways regulated by numerous cell surface receptors, including those that lack their own kinase domain (see in 14.34 Diverse receptors recruit protein tyrosine kinases to the plasma membrane). Src is bound to the plasma membrane via an N-terminal myristoyl group. In the inactive state, Src is phosphorylated on Tyr527, Cterminal to its catalytic domain, by CSK (C-terminal Src kinase). The structure and regulation of Src is depicted in FIGURE 14.37. Phosphorylation of Tyr527 causes it to bind to its own SH2 domain. The SH2 and SH3 domains suppress the kinase activity through interactions on the surface of the protein. The SH3 domain binds to an SH3 binding site distant from the active site. Activation of Src by dephosphorylation of Tyr527 causes its SH2 to dissociate; this causes a conformational change in the SH3 domain to dissociate it from the binding site. Viral isolates of Src are often truncated prior to Tyr527, which increases their activity. Conformational changes in the kinase domain resulting from dissociation of the SH3 promote Src autophosphorylation on Tyr416 in its activation loop and further increase protein kinase activity. An important consequence of the interaction of Src with its own SH2 and SH3 domains is that these domains cannot bind anything else when in the autoinhibited state; therefore, other interactions are promoted when the SH2 and SH3 domains are released from their associations with the Src kinase domain. The heterologous interactions of the SH2 and SH3 domains contribute to Src localization and signaling. 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 631 Structure and regulation of Src INACTIVE ACTIVE KINASE DOMAIN SH3 SH2 P P Tyr527 FIGURE 14.37 The structures of inactive and active Src are compared. The inactive protein is autoinhibited by binding to its own SH2 and SH3 domains. The SH2 domain binds to phosphorylated Tyr527. The SH3 domain binds to a noncanonical SH3-binding motif on the opposite side of the kinase domain active site. In contrast to the steric inhibition of PKA caused by its R subunit, inhibition of Src by its SH2 and SH3 domains is allosteric. In the active structure the SH2 and SH3 domains are not bound to the kinase domain and are available for heterologous interactions. Structures generated from Protein Data Base files 1FMK and 1Y57. 14.32 MAPKs are central to many signaling pathways Key concepts • MAPKs are activated by Tyr and Thr phosphorylation. • The requirement for two phosphorylations creates a signaling threshold. • The ERK1/2 MAPK pathway is usually regulated through Ras. Mitogen-activated protein kinases (MAPKs) are present in all eukaryotes. They are among the most common multifunctional protein kinases mediating cellular regulatory events in response to many ligands and other stimuli. MAPKs are activated by protein kinase cascades consisting of at least three protein kinases acting sequentially, as illustrated in FIGURE 14.38. Activation of a MAPK is catalyzed by a MAPK kinase (MAP2K), which is itself activated by phosphorylation by a MAPK kinase kinase (MAP3K). MAP3Ks are activated by a variety of mechanisms including phosphorylation by MAP4Ks, oligomerization, and binding to activators such as small G proteins. MAP2Ks are activated by phosphorylation on two Ser/Thr residues; MAP2Ks then activate MAPKs by dual phosphorylation on Tyr and Thr residues (Figure 14.30). Each MAP2K phosphorylates a limited set of MAPKs and few or no other substrates. The great specificity of MAP2Ks is one means of insulating MAPKs from activation by inappropriate signals. Both Tyr and Thr phosphorylations are required for maximum MAPK enzymatic activity. Studies on the MAPK ERK2 led to an understanding of the events induced by phosphorylation that are important for increased activity. Conformational changes include refolding of the activation loop to improve substrate positioning and realignment of catalytic residues; this is most obvious in the repositioning of helix C, which contains a Glu involved in phosphoryl transfer. Amplification occurs moving down the cascade from the MAP3K to the MAP2K step because the MAP2Ks are much more abundant than the MAP3Ks. The MAP2K to MAPK step may also amplify the signal if the MAPK is present in excess of the MAP2K. In addition, the phosphorylation of a MAPK by a MAP2K on a 14.32 MAPKs are central to many signaling pathways 631 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 632 MAPK pathways G e ne ric G Protein S. cerevisiae Mammals Gβγ R as Rac/Cdc42 Rac ? MAP4K Ste20p PAK/PKC Ste20 family Ste20 family ? MAP3K Ste11p R af many many MEKK2 MEKK3 MAP2K Ste7p MEK1 MEK2 MEK4 MEK7 MEK3 MEK6 MEK5 MAPK Fus3p ERK1 ERK2 JNK1 JNK2 JNK3 p38α p38β p38γ p38δ small or heterotrimer ERK5 Major targets: Transcription factor 14.33 Ste12p Protein kinase Output c-Jun ATF2 Elk-1 Rsk Mating MEF2 MEF2 MAPKAPK2 Rsk Proliferation Development Differentiation (and other processes) FIGURE 14.38 MAPK pathways can be regulated by a diverse group of upstream regulatory mechanisms that often include adaptors, small G proteins, and MAP4Ks. These molecules impinge on the activities of MAP3Ks. MAP3Ks regulate one or more MAP2Ks depending on localization and scaffolding. The MAP2Ks display great selectivity for a single MAPK type. MAPKs have overlapping and unique substrates and participate in signaling cascades leading to many cellular responses. Tyr and a Thr residue creates cooperative activation of the MAPK; this is another mechanism, in addition to those described for PKA and calmodulin, to introduce a threshold and apparently cooperative behavior into the pathway over a narrow range of input signal. This multistep cascade provides multiple sites for modulatory inputs from other pathways. Stabilized interactions between components are also important. MAP2Ks, as well as MAPK substrates and MAPK phosphatases, generally contain a basic/hydrophobic docking motif that interacts with acidic residues and binds in a hydrophobic groove on the MAPK catalytic domain. Additional components including scaffolds are necessary for the efficient activation of MAPK cascades in cells and usually have addi- 632 tional functions. Several scaffolds have been identified that bind to two or more components for each of the three major MAPK cascades, the ERK1/2, JNK1-3, and p38 α, β, γ, and δ cascades. The ERK1/2 pathway is regulated by most cell surface receptors, including receptors that employ tyrosine kinases, GPCRs, and others. The PDGF receptor, like most receptor systems, activates the ERK1/2 cascade through Ras. PDGF stimulates autophosphorylation of its receptor and the subsequent association of effectors with its cytoplasmic domain (see 14.30 Diverse signaling mechanisms are regulated by protein tyrosine kinases). In response to PDGF, ERK1/2 promotes cell proliferation and differentiation by phosphorylation of membrane enzymes, proteins involved in determining cell shape and motility, and also by concentrating in the nucleus to phosphorylate regulatory factors that control transcription. CHAPTER 14 Principles of cell signaling Cyclin-dependent protein kinases control the cell cycle Key concepts • The cell cycle is regulated by cyclin-dependent protein kinases (CDKs). • Activation of CDKs involves protein binding, dephosphorylation, and phosphorylation. Cell division is regulated positively and negatively by factors that stimulate proliferation and inputs that monitor cell state. The sum of these factors is integrated in the regulation of cyclindependent protein kinases (CDKs). CDKs are protein serine/threonine kinases that are major regulators of cell cycle progression. Most CDKs are regulated both by kinases and phosphatases and by association with other proteins called cyclins. Cyclins are synthesized and degraded every cell cycle. Because most CDKs are dependent upon cyclin binding for activation, the timing of the synthesis and degradation of individual cyclins determines when a CDK will function. The most notable noncycling member of the CDK family is Cdk5, which is highly expressed in terminally differentiated neurons. Cdk5 binds the non-cyclin protein p35 as its activating subunit. We will briefly examine the regulation of Cdc2, a major CDK in both mammals and yeast. The first step in regulation of Cdc2 is the association with cyclin. A second step required for activation of Cdc2 is phosphorylation of a Thr 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 633 CDKs require cyclin binding for activation Tyr15 Tyr15 Lys33 Lys33 Glu51 Glu51 Cdk2 Cdk2 Cyclin Cyclin A A FIGURE 14.39 The view of the crystal structure of CDK2 bound to cyclin A shows residues in the ATP binding site. The enlargement on the right shows the interaction between Lys33 and Glu51, catalytic residues that interact with ATP to promote phosphoryl transfer. Tyr15 is phosphorylated in inactive forms of CDK2. A phosphoryl group on Tyr15 inhibits CDK activity by interfering with ATP binding. Structure generated from Protein Data Bank file 1JST. residue in its activation loop by another CDK type kinase. In spite of its association with cyclin, this form of Cdc2 is not yet active due to inhibitory phosphorylation of Tyr and Thr residues in the ATP binding pocket. Release of inhibition by dephosphorylation of the residues in the ATP pocket is catalyzed by the Cdc25 family of phosphoprotein phosphatases, resulting in activation of Cdc2. The proximity of the Tyr residue to catalytic residues is shown in FIGURE 14.39. The complexity of activation of CDKs makes possible the imposition of cell cycle checkpoints. For more on CDKs and cyclins see 11.4 The cell cycle is a cycle of CDK function. 14.34 Diverse receptors recruit protein tyrosine kinases to the plasma membrane Key concepts • Receptors that bind protein tyrosine kinases use combinations of effectors similar to those used by receptor tyrosine kinases. • These receptors often bind directly to transcription factors. Many receptors act through protein tyrosine kinases, but their cell surface receptors lack kinase activity. Instead, these receptors act by recruiting and activating protein tyrosine kinases at the plasma membrane. In this group of receptors are integrins, which are key molecules involved in cell adhesion, growth hormone receptors, and receptors that mediate inflammatory and immune responses. While their structures vary enormously, their mechanisms of action are related. Integrins are receptors whose major function is to attach cells to the extracellular matrix. They also mediate some interactions with proteins on other cells, as depicted in FIGURE 14.40. Ligands for integrins include a number of extracellular matrix proteins, such as fibronectin, as well as cell surface proteins that cooperate in cell-cell interactions. Integrin ligation provides cells with information about their environment that influences cell behavior. Ligation of integrins initiates signals that control cell programs, including cell cycle entry, proliferation, survival, differentiation, changes in cell shape, and motility, as well as fine-tuning responses to other ligands. For more details on integrins see 15.13 Most integrins are receptors for extracellular matrix proteins and 15.14 Integrin receptors participate in cell signaling. 14.34 Diverse receptors recruit protein tyrosine kinases to the plasma membrane 633 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 634 Integrin signaling ECM INTEGRINS Paxillin Talin Src Vinculin Crk CAS FAK Crk ILK p85 PI 3Kinase p110 Grb2 Talin Vinculin Tensin SOS CYTOPLASM Actin filament FIGURE 14.40 Integrins bind to an array of cytoplasmic proteins to regulate the cytoskeleton and intracellular signaling pathways. The associated cytoskeletal elements include actin filaments and focal adhesion proteins -actinin, vinculin, paxillin, and talin. Signaling molecules include the focal adhesion kinase FAK; the adaptors Cas, Crk and Grb2; Src and CSK (see 14.31 Src family protein kinases cooperate with receptor protein tyrosine kinases), PI 3-kinase (see 14.16 Lipids and lipid-derived compounds are signaling molecules); and the Ras exchange factor SOS. Stimulation of GTP binding of Ras by SOS leads to activation of the MAPK pathway (see 14.32 MAPKs are central to many signaling pathways). Talin and α-actinin are among cytoskeletal proteins that interact directly with certain integrin subunits. These cytoskeletal proteins link integrins to complex cytoskeletal structures known as focal adhesions. Focal adhesions connect the cytoskeleton to signal transduction cascades that communicate states of cellular attachment to the regulation of cellular responses. Focal adhesion complexes contain the focal adhesion kinase FAK, which is activated by integrin ligation. Autophosphorylation of FAK recruits signaling proteins containing SH2 domains, especially the p85 subunit of PI 3-kinase and Src family protein kinases. The signaling molecules associated with the integrin-bound cytoskeletal proteins, whether focal adhesions or other structural complexes, mediate the diverse actions of integrins. The association of cytoskeletal proteins with integrin receptors also causes functional changes to the receptors. Signals that act over a distance, such as hormones, can also employ nonreceptor tyrosine kinases to transmit their message inside a cell. Growth hormone (GH) is a protein hormone secreted by the anterior pituitary gland that regulates bone growth, fat metabolism, and other 634 CHAPTER 14 Principles of cell signaling cellular growth phenomena. Absence of growth hormone results in short stature, whereas hypersecretion causes acromegaly, a form of gigantism. The GH receptor is a member of the cytokine receptor family, which includes receptors for prolactin, erythropoietin, leptin, and interleukins. All these receptors display similar biochemical functions, such as association with members of the JAK/TYK family of protein tyrosine kinases, but select for different but overlapping sets of cytoplasmic signaling proteins. Signal transduction by the GH receptor provides a model for receptors that lack enzymatic function and act as agonist-promoted scaffolds for intracellular signaling proteins. FIGURE 14.41 shows the structure of growth hormone bound to the extracellular domain of its receptor. The majority of binding energy comes from only a small number of residues in the binding interface. Inside the cell, signaling by the GH receptor depends significantly on its association with the cytoplasmic tyrosine protein kinase Janus kinase 2 (JAK2). FIGURE 14.42 shows that JAK2 binds to a proline-rich region of the receptor. Ligand binding induces receptor dimerization, which then promotes activation of JAK2 through intermolecular autophosphorylation. GH signaling is thus mediated primarily by inducing Tyr phosphorylation. In addition to JAK2 autophosphorylation, the receptor itself becomes Tyr phosphorylated. As is true for receptor tyrosine kinases, Tyr phosphorylation of the growth hormone receptor creates binding sites for signaling proteins that contain phosphotyrosine-binding domains. Primary targets are transcription factors known as signal transducers and activators of transcription, or STATs. STATs contain SH2 domains and bind Tyr-phosphorylated motifs on the growth hormone receptor. While receptor bound, STATs are Tyr phosphorylated by JAK2 and then released to travel to the nucleus to mediate changes in transcription. The growth hormone receptor and the associated JAK2 also activate other signaling pathways. For example, the adaptor Shc is Tyr phosphorylated by JAK2. Engagement of Shc leads to activation of Ras and the ERK1/2 MAPK pathway. Adaptors specialized for insulin-signaling pathways, insulin receptor substrates (IRS) 1, 2, and 3, are also growth hormone targets, perhaps reflecting the ability of growth hormone to induce certain insulin-like metabolic actions. Feedback circuits are also engaged during GH signaling. The growth hormone receptor complex binds the adaptor SH2-B, which has a 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 635 Growth hormone structure Growth hormone signaling is transduced by JAK2 Growth hormone Growth hormone receptor hGH Dimerization JAK2 JAK2 STAT JAK2s bind and phosphorylate receptor ΔΔ G STAT CYTOPLASM (kcal/mol) STATs bind and are phosphorylated > 1.5 0.5 to 1.5 -0.5 to 0.5 < -0.5 untested NUCLEUS FIGURE 14.41 Proteins often interact over a large surface area. Growth hormone binding to its receptor is an example of the energy of binding coming primarily from a small number of the contacts between the two proteins, creating an interaction hot spot. The complex of growth hormone bound to the growth hormone receptor-binding domain determined by crystallography has been peeled apart in this figure to show the binding energy associated with residues in the binding interface from each protein determined by mutagenesis and binding studies. Fewer than half of the residues in the interface contribute the majority of binding energy. Reproduced with permission from T. Clackson and J. A. Wells. 1995. Science. 267: 383–386. © AAAS. Photos courtesy of Tim Clackson, ARIAD Pharmaceuticals, Inc. stimulatory effect on growth hormone signaling. On the other hand, suppressors of cytokine signaling (SOCS proteins) are among the genes whose transcription is induced by growth hormone. As the name indicates, SOCS proteins inhibit cytokine signaling in some if not all cases by inhibiting the activity of JAK2. SOCS proteins contain an SH2 domain that facilitates their binding either to phosphorylated JAK2 or cytokine receptors. The mechanism of signaling inhibition may differ among SOCS proteins because some require the GH receptor to interfere with JAK2 signaling. SOCS-1, on the other hand, binds directly to the JAK2 activation loop and does not require a receptor to inhibit JAK2 activity. This mechanism may be particularly important in GH signaling because, in contrast to the ligand-in- Phosphorylated STATs bind DNA FIGURE 14.42 The growth hormone receptor binds to JAK2. Many GH signals are mediated by Tyr phosphorylation of the receptor by JAK2, which creates binding sites for signaling molecules with SH2 domains, notably STATs. STATs then enter the nucleus to cause changes in gene transcription. duced down regulation mechanisms controlling many receptors, the GH receptor is degraded in a ligand-independent manner. Receptors for cytokines also act by recruiting tyrosine kinases. The cytokines—signaling proteins that modulate inflammation and cell growth and differentiation—include interleukins, leukemia inhibitory factor, oncostatin M, cardiotrophin-1, cardiotrophin-like cytokine, and ciliary neurotrophic factor (CNTF). Each cytokine binds a unique receptor, but each receptor binds a transmembrane protein called gp130. Mechanisms of signaling by gp130 involve interactions with tyrosine kinases of the JAK/TYK types and transcription factors in the STAT family. This mechanism is similar to those employed by the growth hormone receptor. 14.34 Diverse receptors recruit protein tyrosine kinases to the plasma membrane 635 39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 636 Receptor signaling pathways PDGF Insulin Growth hormone IL-1β TGF-β PDGF receptor Insulin receptor GH receptor IL-1β receptor TGF -β type II receptor Adaptor/ subunit SHP2/Grb2 IRS1 Transducer SOS/Ras PI 3-kinase Kinase cascade MAPK Akt2 Ternary complex factors FOXO Ligand Receptor Transcription factor complex gp130 JAK JAK Type I receptor STATs STATs SMADs FIGURE 14.43 Major signaling cascades controlled by PDGF, insulin, TGF-, IL1, and growth hormone are compared. Each receptor either contains or interacts with a protein kinase that associates with or recruits a transducer. The transducer regulates downstream effectors either directly or through an intermediate protein kinase cascade. The effectors shown are transcriptional regulators. Phosphorylation by the transducer or kinase cascade activates all of the effectors except the FOXO proteins, which may be excluded from the nucleus by phosphorylation. The table only shows snapshots of much more complex signaling networks controlled by these ligands. Many of these and other intermediates serve multiple ligands. For example, IRS proteins also contribute to growth hormone and IL-1 signaling, and MAPK pathways are regulated by all of these ligands. T cell receptor signaling MHC Antigen TCR CD3 CD3 Lck ZAP-70 ITAM After binding of the TCR to the MHC-antigen complex, Lck phosphorylates ITAMS FIGURE 14.44 The T cell receptor (TCR) is a multisubunit receptor. It is phosphorylated on activation motifs or ITAMs by Lck, or a related Src family protein kinase. The phosphorylated residues create binding sites for another tyrosine protein kinase ZAP-70. ZAP-70 then recruits other signaling molecules to the complex including phospholipase C, PI 3-kinase, and a Ras exchange factor to activate downstream signaling pathways. 636 CHAPTER 14 Principles of cell signaling Unlike many cytokine receptors in this class, the CNTF receptor does not itself span the membrane. Instead, it is glycosyl phosphoinositol (GPI)-linked to the outer face of the plasma membrane. The GPI linkage is a covalent bond, and the receptor can be released into the extracellular fluid by a specific phospholipase. The freed receptor may interact with membranes of other cells to induce signals. The use of a common signal transducing subunit, gp130, suggests that unique mechanisms exist to create ligand-specific responses; under some circumstances competition by the ligand binding subunits for interaction with the gp130 signal transducer may influence signaling outcomes. FIGURE 14.43 illustrates some parallels in signaling pathways initiated by receptors with associated or intrinsic protein kinases. The last receptor type we will discuss takes the concept of the specific and common subunits even further. The complex multiprotein T cell receptor (TCR) is found uniquely on T lymphocytes and is responsible for the ability of these cells to recognize and respond to specific antigens. The TCR, illustrated in FIGURE 14.44, is composed of eight subunits that can be described as an assembly of four dimers, αβ, γε, δε, and ζζ. The specificity of antigen recognition is determined by the α and β subunits, which are different for each cell. The remaining subunits are invariant in TCRs. The CD3 complex γ, δ, and ε subunits are similar in sequence to one another. The ζ chain, unlike the other subunits, appears on certain other cell types and may be a component of other receptors, such as the Fc receptor, which binds a portion of certain immunoglobulins. A motif called the immunoreceptor tyrosine-based activation motif, or ITAM, which features closely spaced pairs of Tyr residues, is key to signaling by the TCR. The CD3 subunits each contain one ITAM and the ζ chain contains three ITAMs, for a total of ten motifs in each TCR. Engagement of the TCR causes the Src family kinases Lck and Fyn to phosphorylate the pairs of Tyr residues in the ITAMs. The ITAMs then bind the tandem SH2 domains of the protein tyrosine kinase ζ-chain-associated protein of 70 kDa (ZAP-70), which becomes activated by Src. Tyr phosphorylation sites on ZAP-70 bind to other adaptors and signaling molecules, and Tyr phosphorylation by ZAP-70 activates additional signal transducers. The sum of these events leads to the downstream responses of T cells to antigen engagement, which include cell cycle progression and the elaboration of cytokines such as interleukin-2. 39057_ch14_cellbio.qxd 14.35 8/28/06 5:11 PM Page 637 What’s next? New signaling proteins and new regulatory interactions seem to show up every day. The challenge now is to understand how cells organize these proteins and their individual interactions to create adaptable information-processing networks. How do cells use simple chemical reactions to sort and integrate multiple simultaneous inputs and then direct this information to diverse effector machinery? How do they interpret the inputs in the context of their growth and metabolic activities? In principle, three areas of research have to contribute to allow us to understand integrative cellular signaling. First, we need real-time, noninterfering biosensors to measure intracellular signaling reactions. Most current sensors use combinations of fluorescent moieties and signal-binding protein domains to provide fast optical readouts. For many pathways, several reactions can be monitored within cells over subsecond time scales. We need more, better, and faster sensors and sensors that can report with singlecell and subcellular resolution. Genetically encoded sensors will be complemented by synthetic molecules. Our ability to manipulate signaling networks is also improving dramatically but still falls short. We can manipulate signaling networks by overexpression, knockout, and knockdown of genes, but signaling pathways are wonderfully adaptive and frequently circumvent our best efforts to control them. We still need chemical regulators that can act promptly in cells. Structure-based design of such regulatory molecules will be vital. Last, our ability to analyze the behavior of signaling networks depends on our ability to measure and interpret signaling quantitatively. It is ironic but true that really complex systems cannot be described without explicit quantitative models for how they work. Computational modeling and simulation of signaling networks requires both better theoretical understanding of network dynamics and better algorithmic implementation. The goal is to understand how cells think. 14.36 Summary Signal transduction encompasses mechanisms used by all cells to sense and react to stimuli in their environment. Cells express receptors that recognize specific extracellular stimuli, includ- ing nutrients, hormones, neurotransmitters, and other cells. Upon receptor binding, signals are converted to well-defined intracellular chemical or physical reactions that change the activities and the organization of protein complexes within cells. The changes directed by the stimuli lead to altered cell behavior. The behavior of the cell is determined then by its intracellular state and the integrated information from extracellular stimuli so that the appropriate responses are achieved. The basic biochemical components and processes of signal transduction are conserved throughout biology. Families of proteins are used in a variety of ways for many different physiological purposes. Cells often use the same series of signaling proteins to regulate multiple processes, such as transcription, ion transport, locomotion, and metabolism. Signaling pathways are assembled into signaling networks to allow the cell to coordinate its responses to multiple inputs with its ongoing functions. It is now possible to discern conserved reaction sequences in and between pathways in signaling networks that are analogous to devices within the circuits of analog computers: amplifiers, logic gates, feedback and feed-forward controls, and memory. References 14.1 Introduction Review Sauro, H. M. and Kholodenko, B. N., 2004. Quantitative analysis of signaling networks. Prog. Biophys. Mol. Biol. v. 86 p. 5–43. 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