BPS 502 Chapter 15 • Cell Communication • Signaling through enzymelinked cell surface receptors Reading Assignment MBC, Chapter 15 Levitzki A. 2003, EGF Receptor as a Therapeutic Target. Lung Cancer, 41: S9-S14. Objectives • Be able to describe and give examples of each of the six classes of enzyme-linked receptors discussed. • Understand the importance of these signaling pathways in disease states, especially in cancer. • Be able to discuss the various means of inhibition of these signaling pathways in clinical use and in various phases of clinical trials. •Understand and describe the signaling pathways that depend on regulated proteolysis. Enzyme-Linked Cell-Surface Receptors • They respond to extracellular signaling proteins called growth factors that promote growth, proliferation, differentiation or cell survival Enzyme-Linked Cell-Surface Receptors • They act as local mediators at low concentrations (10- 9–10-11 M). •The response can be slow (several hours) due to the number of intracellular steps that are required leading to changes in gene expression. •Effects may be direct and rapid acting on the cytoskeleton to control cell movement and shape changes. •Abnormalities in these fundamental signaling events can lead to cancer. Six Classes of enzyme-linked receptors identified to date 1. Receptor tyrosine kinases – phosphorylate specific tyrosines on specific intracellular signaling proteins. (EGFR) 2. Tyrosine-kinase-associated receptors – these enzymes associate with intracellular proteins that have tyrosine kinase activity. (Cytokine/Jak-Stat) 3. Receptor-like tyrosine phosphatases – remove phosphate groups from tyrosines of specific intracellular proteins. 4. Receptor serine/threonine kinases – phosphorylate specific serines or threonines on associated gene regulatory proteins. (TGF-b/Smad) 5. Receptor guanylyl cyclases – directly catalyze the production of cyclic GMP in the cytosol. (Natriuretic peptides receptor) 6. Histidine-kinase-associated receptors- activate a 2-component signaling pathway whereby the kinase phosphorylates itself on histidine (autophosphorylation) and then immediately transfers the phosphate to a second intracellular protein. Only occurs in yeasts and plants involved in chemotaxis. Like G-Protein Coupled Receptors, Enzyme-linked receptors are transmembrane proteins with the ligand-binding domain on the outer surface and a cytosolic domain that either has intrinsic enzymatic activity or associates with an enzyme. 1. Receptor Tyrosine Kinases Activation of enzyme-linked receptors occurs by oligomerization of the receptor. This causes reorientation of the receptor chains, autophosphorylation, stimulating the kinase activity and creating docking sites for intracellular proteins. Three ways signaling proteins can cross-link receptors: (A) PDGF is a dimer and cross-links two receptors together. (B) FGF is a monomer and can bind in clusters to proteoglycans enabling the ligands to cross-link the FGF receptor (C) Ephrins are membrane-bound ligands and cluster prior to binding thus causing receptor cross-linkng. Phosphorylated Tyrosines Serve as Docking Sites for Proteins with SH2 domains Some examples: Phospholipase C-g (enzyme) Src (relay signaling protein) PI-3’-kinase All share a highly conserved phosphotyrosine-binding domain, either SH2 or SH3 domains (Src homology region) or PTB (phosphotyrosine bindng) domains These proteins act as adaptor proteins to broadcast signals along multiple pathways. Hypothetical Signaling Pathway Using Modular Domains Binding of SH2-Containing intracellular signaling proteins to an activated PDGF receptor X-ray crystal structure of an SH2 domain that acts as a plug-in molecule recognizing phosphotyrosines and adjacent amino acid side chains. Ras proteins belong to the large Ras superfamily of monomeric GTPases -Ras proteins contain a covalently linked lipid group that anchors the protein to the cytoplasmic face of the plasma membrane -There are multiple Ras proteins, each acting in distinct cell types -Ras functions as a switch, cycling between 2 conformational states -mutations of the ras gene promote the development of cancer, 30% of human tumors have a ras activation mutation. Guanine nucleotide exchange factor (GEF) activates Ras GTPase-activating proteins (GAP) inactivates Ras Activation of Ras by an activated receptor tyrosine kinase Grb-2 is an adaptor protein linking receptor tyrosine kinase to Ras via the GEF called Sos. This is only one way of activating Ras. Activation of ras is short-lived because it is inactivated by phosphatases and GAPS. Once activated, Ras activates other signaling proteins to relay the signal downstream along several pathways. One of the pathways activated by Ras is the MAP Kinase Pathway MAPK pathway consists of a kinase cascade of no fewer than three enzymes: Raf MEK MAP kinase (ERK) is phosphorylated by MEK on both a threonine and a tyrosine. ERK then travels to the nucleus phosphorylating proteins and activating immediate early genes, G1 cyclins are among them. The Mitogen-activated Protein Kinase (MAPK) Cascades SIGMA-ALDRICH At least 5 parallel MAP kinase modules can operate in a mammalian cell. These modules are activated by different kinds of cell stress such as UV irradiation, heat shock, osmotic stress and stimulation by inflammatory cytokines. The MAP kinase pathway stimulates cell division through the action of mitogens. To prolliferate, growth factors stimulate proliferation. PI-3-kinase activation mainly stimulates cell growth. Ras can also help to activate PI-3’-kinase. Ras Effectors and Downstream Pathways Macaluso et al., 2002, J Cell Physiol., 192:125-130 EGF Receptor Signal Transduction Pathway SIGMA-ALDRICH Strategy to prevent cross-talk between MAPK Signaling Pathways: Separate pathways are organized by scaffold proteins in budding yeast and in mammalian cells Phosphatidylinostitol 3-kinase (PI-3’-Kinase) Phosphorylates Inositol Phospholipids Rather than Proteins PI-3’-Kinase catalyzes the phosphorylation of inositol phospholipids at the 3 position of the inositol ring The 2 lipids in orange can serve as docking sites for signaling proteins with Plecstrin Homology (PH) domains Inositol phospholipid phosphatases (PTEN) remove the 3 position phosphate. PTEN is a tumor suppressor gene, mutations result in prolonged growth signaling. These mutations are found in various cancers including endometrial, some ovarian and some breast cancers. PI 3-K stimulates cell survival and growth by activating protein kinase B (PKB or Akt) One example of transmission of the proliferation signal: 1. 2. 3. 4. A survival signal activates a RTK, with recruits and activates PI 3’Kinase. PI 3’K produces the phosphorylated inositol phospholipids which serve as docking sites for 2 serine/threonine kinases PKB and PDK1 PKB is then further phosphorylated and activated by PDK1. Activated PKB dissociated from the membrane, and phosphorylates BAD, releasing a death inhibitory protein resulting in inhibition of apoptosis. The result is continued proliferation. Five parallel intracellular signaling pathways activated by G-protein-linked receptors, receptor tyrosine kinases, or both 2. Tyrosine-Kinase-associated Receptors Depend on Cytoplasmic Tyrosine Kinases for Their Activity •These receptors lack intrinsic tyrosine kinase activity and rely on cytoplasmic tyrosine kinases. •The largest family of cytoplasmic TK receptors is the Src family (Src, Yes, Fgr, Fyn, Lck, Lyn, Hck, Blk). •These proteins contain SH2 and SH3 domains and are located on the cytoplasmic side of the plasma membrane. •Integrin receptors interact with focal adhesion kinase (FAK) that is a cytoplasmic TK. •Cytokine receptors are stably associated with a class of cytoplasmic TKs called Jaks. The Jak-STAT signaling pathway activated by a-interferon Cytokines are secreted by cells in response to a viral infection and bind to neighboring cells to induce proteins that increase resistance to virus. All cytokine receptors are associated with one or more Jaks (4 types) Janus kinases (Jaks) Signal transducers and activators of transcription (STATs). 7 known STATs 3. Some Protein Tyrosine Phosphatases Act as Cell-Surface Receptors Protein tyrosine phosphatases (PTPs) remove selected phosphotyrosines on a subset of tyrosine-phosphorylated proteins. Exhibit high degree of substrate selectivity. These enzyme ensure that the tyrosine phosphorylations are short-lived and are responsible for regulating the intensity of the signal. There are about 30 known PTPs and occur as both transmembrane and cytoplasmic forms. Protein Tyrosine Phosphatases The role of receptor-like tyrosine phosphatases is not yet clearly understood. They are thought to act as receptors, but their ligands have not been identified. Some have been shown to display features of celladhesion. Some can activate receptors on neighboring cells. 4. Receptor Serine/Threonine Protein Kinase Smad-dependent signaling pathway activated by TGF-b Transforming growth factor b (TGFb) consists of a large number of structurally related, secreted, dimeric proteins. They mediate a wide range of biological functions in animals: proliferation, differentiation, ECM production, cell death, tissue repair and immune regulation. They act through receptor serine/threonine kinases type I and type II Smad family members are directly phosphorylated by the type I receptor, and moves to the nucleus to direct gene transcription The size and location of protein kinases 5. Receptor Guanylyl Cyclases •Single pass transmembrane proteins with an extracellular binding site for a signal molecule and intracellular guanylyl cyclase catalytic domain. •Binding of ligand activates the cyclase domain to produce cyclic GMP, which in turn binds to and activates a cyclic GMP-dependent protein kinase (PKG). •PKG phosphorylates specific proteins on serine or threonine. •Some signaling molecules that use these receptor types include natriuretic peptides (NPs) (regulate salt and water balance and dilate blood vessels). 6. Histadine-Kinase Associated Receptors Involved in Bacterial Chemotaxis •Dimeric transmembrane proteins that bind specific attractants and repellants •The cytoplasmic tails of the receptors are stably associated with an adapter protein CheW and a histidine kinase, CheA – couples the receptor to the flagellar motor. •Binding of a repellent activate CheA causing autophosphorylation on a histidine, and immediate transfer of the phosphate to an aspartic acid on a messenger protein, CheY. •CheY dissociates from the receptor, diffuses through the cytosol, and causes the flagellar motor to rotate clockwise and the bacterium tumbles. •This type of signaling is used in yeast and plants, but not found in animals. What are the opportunities for therapeutic applications? Modes of EGFR Inhibition A. Levitzki. 2003. Lung Cancer, 41: S9-S14 Iressa/Gefitinib (EGFR Inhibitor) • Used to treat non-small-cell lung cancer (NSCLC) – 85% of all lung cancers • Works in 25% of cases in Japan, but only effective in 10% of U.S. patients. • Suspected a genetic factor in efficacy • Has variable effects based on a single gene Science. 2004 Jun 4;304(5676):1458-61 EGFR Mutations in Lung Cancer: Correlation with Clinical Response to Gefitinib Therapy J.Guillermo Paez,1,2 *Pasi A.Ja ¨nne,1,2 *Jeffrey C.Lee,1,3 * Sean Tracy,1 Heidi Greulich,1,2 Stacey Gabriel,4 Paula Herman,1 Frederic J.Kaye,5 Neal Lindeman,6 Titus J.Boggon,1,3 Katsuhiko Naoki,1 Hidefumi Sasaki,7 Yoshitaka Fujii,7 Michael J.Eck,1,3 William R.Sellers,1,2,4 † Bruce E.Johnson,1,2 † Matthew Meyerson 1,3,4 † SCIENCE VOL 304 4 JUNE 2004 Convergence of Results With Different Approaches Lynch et al.: • Sequenced the entire coding region of the EGFR from 9 patients treated with gefitinib with clinical response. • 8/9 mutations found in responders; 0/7 in nonresponders Paez et al.: • Sequenced the exons of 47 TK activation loop domain 58 lung cancers from Japanese patients and 61 from U.S. patients. • Narrowed search to EGFR in 119 cases of NSCLC • Mutation found in 5/5 responders; 0/4 nonresponders Both Studies Reveal Mutations in the EGRF Tyrosine Kinase Domain -either small in frame deletions or amino acied substitutions -Cluster around the ATP binding pocket -activating mutations Table 1. Protein kinase inhibitors in clinical trials Genentech.com Genentech.com Data from Phase III clinical trials of Herceptin Genentech.com Table 2. Antibody protein kinase inhibitors in clinical trials Overview of the PI3K/Akt Pathway Compounds that Directly Inhibit the PI3K/Akt Pathway Signaling Pathways that Depend on Regulated Proteolysis: -Notch -Wnt -NF-kB The Notch Receptor Protein is Widely Used in Animal Development Notch genes encode heterodimeric transmembrane receptors that regulate differentiation, proliferation and apoptosis. Mammals have 4 known Notch genes and 2 families of Notch ligands: Delta and Jagged. Both Notch and Delta are single-pass transmembrane proteins that require proteolytic processing to function. Well known for its role in nerve cell production in Drosophila Deregulated expression of Notch receptors, ligands and downstream targets is described in many cancers Activation of Notch by Proteolytic Cleavage 1. Full-length Notch is cleaved in the trans-golgi by a furin and is transported to the plasma membrane. 2. Notch binds delta that is displayed on an adjacent cell triggers the next 2 proteolytic cleavages. 3. extracellular cleavage mediated by TACE leaving 12 amino acids. 4. Cleavage mediated by presenilin that releases the Notch tail to migrate to the nucleus. Notch binds to the transcriptional repressor CSL and activates gene transcription. Wnt Proteins Bind to Frizzled Receptors and Inhibits b-Catenin Degradation Wnt proteins are secreted glycoproteins that act as mediators to control many aspects of development. Wnt binds to the Frizzled receptor (family of 7 transmembrane proteins resembling G-proteins. Signaling is mediated by the cytoplasmic protein dishevelled. Dishevelled acts by regulating the proteolysis of a multifunctional protein, b-catenin, which functions in cell-cell adhesion and as a latent gene regulatory protein. In the absence of a wnt signal, bcatenin is phosphorylated in a degradation complex and is degraded in the proteosome. In the presence of Wnt, in association with a co-receptor protein LRP and leads to inhibition of b-catenin phosphorylation and degradation. As a result, the unphosphorylated bcatenin accumulates in the nucleus and displaces the repressor protein Groucho. Wnt target genes are expressed including c-myc , a powerful stimulator of cell growth and proliferation APC (adenamatous polyposis coli) gene mutations occur in 80% of benign colon polyps that lead to cancer. Wnt ligand overexpression is documented in many tumors including breast. Multiple Stress and Proinflammatory Stimuli Act Through an NF-kB-Dependent Signaling Pathway Two cytokines that are important in inducing the inflammatory response are TNF-a and interleukin-1 (IL-1). These cytokines bind to cell-surface receptors and activate NF-kB. There are 5 NF-kB proteins in mammals (RelA, RelB, c-Rel, NF-kB1, NF-kB2). These form a variety of homo- and heterodimers each activating a specific set of genes. Inhibitory proteins IkB, bind tightly to the dimers and hold them in an inactive state. TNF-a signals lead to recruitment of several intracellular signaling proteins (RIP, TRADD, TRAF2) that in turn activate a specific serine/threonine kinase, IkB kinase. This releases the IkB exposing the nuclear localization signal of NF-kB and it translocates to the nucleus.