Signal transduction Major definitions Signal transduction is a basic Paracrine signaling occurs between local cells where the signals elicit quick responses and last only a short amount of time due to the degradation of the paracrine ligands. Endocrine signaling occurs between distant cells and is mediated by hormones released from specific endocrine cells that travel to target cells, producing a slower, longlasting response. process in molecular cell biology involving the conversion of a signal from outside the cell to a functional change within the cell. ECB consider 4 general pathways of signal transduction: endocrine, paracrine, synaptic and contact dependent More generally, signaling can be either via diffused molecules or due to surfacesurface interactions Intracellular signaling Intracellular signaling molecules can relay, amplify, integrate and distribue the incoming signal. Participants of the intrecellular signaling cascades: extracellular signals (soluble ligands, other cell surface receptors) Receptors Adaptor proteins Second messengers (intracellular soluble ligands) Effector proteins Key proteins that control intracellular signaling act as molecular switches Alberts et al. ECB Alberts et al. ECB Kinases. Kinome Major definitions Kinases are enzymes that catalyze the transfer of the γ-phosphate of ATP (GTP) on a corresponding substrate Particuarly, protein kinases catalyze the transfer on protein alcohol (on Ser and Ther) and/or phenolic (on Tyr) groups Kinases are classified by sequence comparison of their catalytic domains, and in second turn by sequence similarity and domain structure outside of the catalytic domains and biological functions Human kinome is a set of all human kinases It includes about 500 protein kinase(518 according to Manning et al., 2002) and a lot of kinases specific to non-protenaceus substrates Most of kinase families are conserved in eukaryotes and particularly in multicellular eukaryotes (144 of 189 for PK) Classification Group Family Examples AGC 17 70 AGCI. Cyclic-nucleotideregulated PK PKA (cAMP-dependent PK) PKG (Cyclic GMP-dependent PK) AGCII. Diacylglycerolactivated/phsospholipiddependent PK PKC (protein kinase C) including Ca2+-dependent (cPKC) ,Ca2+indepdendent (nPKC), atypical (aPKC) AGCIII. Related to PKA and PKC RAC AGCIV. G-protein-coupledreceptor phosphorylating PK βARK1 and βARK1 (βadrenergic receptor kinases) , GRK5 and GRK6 Classification Group Family Examples CAMK 33 84 CAMKI. Ca2+/calmoduline regulated PK CAMKs EG2K (Elongation factor 2 kinase) Phosphorylase kinases (PhK-γM) Myosin light chain kinases (MLCK) MAP kinase-activated PK 2(MAPKAP2) CAMKII. AMPK family AMPK AMP-activated PK CMGCI. Cyclin-dependent kinases (CDKs) Cdc2, Cdk2 etc CMGCII. MAPK (or Erk) family Erk1, Erk2(extracellular signal regulated kinases, known as p44 MAPK and p42 MAPK) P38 (MPK2, HOG1-related protein) Jnk1, Jnk2 (SAPKs, stress-activated protein kinases) CMGC Classification Group Family Examples STE MAPK cascade families, 3 51 Ste7/MAP2K/MEK MEK1, MEK2 (MAP ERK Kinases) Ste11/MAP3K MEKK Ste20/MAP4K/Pak p21(CDC42/Rac) activated kinase Group Classification Tyrosine kinase group Family Examples 30 95 PTK1. Src Src (cellular homolog of Rous sarcoma virus oncoprotein) Yes ,Yrk, Fgr, Fyn, Lyn, Lck (Lymphoid T-cell protein-tyrosine kinase)Blk PTKII. Brk family Breast expressed PK PTKIII.Tec Tec (Tyrosine kinase expressed in hepatocellular carcinoma) Emt (Itk, Tsk, expressed in mainly in T-cells) PTKIV. Csk C terminal Src kinase; negative regulator of Src PTKVIII. Jak family Janus kinases (Jaks), Tyk2 )Transducer of interferon signals) PTKIX. Ack Ack CDC42Hs-associated kinase PTKX. Fak Fak Focal adhesion kinase Group Classification Family Tyrosine kinase 30 group Examples 95 PTKXI. EGFR family EGFR, HER2/c-neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4). PTKXII. Eph/Elk/Eck receptor family Eph (erythropoeitin-producing hepatoma PK) Eck (epithelial cell linase) Elk (Epl like kinase detected in brain) PTK. Tie Tie PTKXVI. Fibroblast growth factor receptor family FGFR1 (Flg, Cek1), FGFR2 (Bek, K-Sam) PTKXVII. Insulin receptor family InsR, IGF1R (insulin like growth factor receptor) PTKXXII. Hepatocyte growth factor receptor HGFR (Met), Sea, Ron, Stk (stem cell derived tyrosine kinase) Classification Group Family Examples TKL tyrosine kinase–like , 7 48 MLK mixed-lineage kinase LISK (LIMK/TESK) RGC IRAK interleukin-1 (IL-1) receptor– associated kinase], Raf Raf (cellular homolog of retroviral oncogene product) RIPK receptor-interacting protein kinase (RIP)], STRK activin and TGF-β receptors Receptor guanylate cyclase, 1 5 Platelet-derived growth factor receptor PDGFR, CSF1R (Colony stimulating factor 1 receptor) Classification Group Family Examples Others 37 106 Activin/TGFβ receptor family ActR 13 40 Atypical PKs Kinase domain structure Hanks and Hunter, 1995 Functional activities of the kinase Binding and orientation of ATP (GTP) as a complex with divalent cation Binding of a target protein Enzymatic transfer of the γphosphate group to the acceptor hydroxyl residue Catalytic kinase domain consists of 300-350 aa and is organized in 12 dubdomain The catalytic activity depends on cation (Mg2+ or Mn2+ ) binding, often on phosphorylaton within the VIII subdomain and sometimes on binding of a ligand or a catalytic subunit Figure 1. Two Views of the Structure of PKA [70] Scheeff ED, Bourne PE (2005) Structural Evolution of the Protein Kinase–Like Superfamily. PLoS Comput Biol 1(5): e49. doi:10.1371/journal.pcbi.0010049 Figure 1 Dendrogram of 491 ePK domains from 478 genes. G. Manning et al. Science 2002;298:1912-1934 Other kinases Enzyme Target Aminoglycoside 3′ and/or 5′′ hydroxyl of phosphotransferase APH(3′)- aminoglycoside antibiotics IIIa Functional role Antibiotic inactivation Choline kinase (CK) Phosphatidylcholine precursor Phosphatidylcholine synthesis Phosphoinositide 3 kinases (PI3Ks) Phosphatidylinositol (PI) or its forms PtdIns(3)P, PI(3,4,5)P3 Type IIβ phosphatidylinositol phosphatidylinositol 5phosphate kinase (PIPKIIβ) phosphate (PI5P) PI(4,5)P2 Figure 4. Proposed Phylogeny for the Kinase-Like Superfamily, Based on a Unified Bayesian Analysis Both the Sequence Alignment in Figure 3 and the Structural Character Matrix in Table 2 Scheeff ED, Bourne PE (2005) Structural Evolution of the Protein Kinase–Like Superfamily. PLoS Comput Biol 1(5): e49. doi:10.1371/journal.pcbi.001004 Regulatory GTP-binding proteins =regulatory GTPases=G-proteins Major classes of regulatory GTPbinding proteins Trimeric G- proteins associated with GTP—binding protein coupled receptors Small (monomeric) G proteins inactive active Heterotrimeric guanine-nucleotide binding proteins (G-proteins) G-proteins are peripheraly proteins of the alpha- subunit is myristoylated and can be palmytolated Gamma-subunitc is prenylated plasma membrane G-proteins provide signal coupling to seventransmembrane-surface-receptors known as G-protein couplde receptors (GPCR). G proteins are composed of monomers of alpha, beta, and gamma subunits. The alpha-subunit is a GTPase The beta- and gamma-subunits are tightly associated. Receptor phosphorylation upon signal binding mediate GDP/GTP exchange . The GTP bound alpha-subunit dissociate from beta- and gamma-subunits Dissociated subunits initiate cellular response by altering the activity of effectors G-protein coupled receptors (GPCRs) GPCRs IS the largest family of integral membrane proteins About 800 GPCR genes are identified in the human genome GPCRs are comprised of seven transmembrane a-spirals (TM), an extracellular N-terminus and an intracellular C-terminal domain. GPCR are activated by ligand binding that causes conformation changes Ligand-bound GPCR affects the G-protein alpha-subunit decreasing its affinity to GDP GDP/GTP exchange take place due to a Alberts et al., ESB decreased affinity to GDP and higher intracellular concentrations of GTP Approximately 30% of all current therapeutic agents acting directly on GPCRs GPCR ligands and effectors Ligands Effectors Hormones: epinephrine, acetylcholine, noradrenaline, dopamine, histamine Lipids: prostaglandins, leukotriens, Lysophosphatidic acid (LPA) Chemokines Regulatory peptides: thrombin, bombesin, bradykinin Nucleotides Adenylyl cyclase, PKA, PKC, Phospholiases (PLC), Rho GTPase, PI3K, ion channels Biological functions Cell proliferaion, differentiation, migration, angiogenesis, cancer G-protein functions The β-adrenergic receptor is the GPCR for the hormone epinephrine. epinephrine and glucagon binding activates β-adrenergic receptor β-adrenergic receptor stimulate GTP/GDP transition in a stimulatory G-protein (Gs with alpha subunit Gsa ). Gs activates adenylyl cyclases to switch on cyclic-AMP formation that results in PKA activation etc. GTP/GDP degradation stop the cascade GPCR regulatory proteins GPCR kinases (GRKs) and British Journal of Pharmacology Volume 165, Issue 6, pages 1717-1736, 22 FEB 2012 DOI: 10.1111/j.1476-5381.2011.01552.x http://onlinelibrary.wiley.com/doi/10.1111/j.14765381.2011.01552.x/full#f1 arrestins causes receptor desensitization (uncoupling) from hetereotrimeric Gproteins (fast recycling) or CME (slow recycling) Receptor activity-modifying proteins (RAMPS) modify the expression, and pharmacology of calcitonin receptor and calcitonin-like receptor (CRLR) Regulators of G-protein signalling (RGS) act as GTPase activating proteins Ion channels as integrators of G-protein mediated signaling: Sympathetic stimulation in the heart Noradrenalin intecrats with βAR, activates Gs Atsushi Inanobe , Yoshihisa Kurachi Biochimica et Biophysica Acta (BBA) - Biomembranes, Volume 1838, Issue 2, 2014, 521 - 531 increased heartbeat Hoe to stop and what happens if it isn’t gonna stop ? Figure 2 Cholera pathogenesis and cholera toxin action A, B (cholera toxin subunits); GM1 (GM1 ganglioside receptor); Gsa (G protein alfa subunit); AC (adenylate cyclase); Gi (G protein); cAMP (cyclic AMP); CFTR (cystic fibrosis transmembrane conductance regulator). Cholera toxin blocks Gsa in the GTP-bound state via a reaction of ADP-ribosylation Clemens, J. et al. (2011) New-generation vaccines against cholera Nat. Rev. Gastroenterol. Hepatol. doi:10.1038/nrgastro.2011.174 Small regulatory GTPases = small G proteins = small GTP binding proteins =Ras superfamily Small GTPAses are monomeric proteins GDI GTP-bound form is active, GDP-bound form is inactive Three types of regulatory proteins control small G-protein activity GAP- GTPase activating proteins increase its low intrinsic hydrolase activity to trasfser G-protein into an INACTIVE form GDI- GTPase dissociation inhibitors stabilize the GDP-bound, inactive state of G proteins GEF- guanine nucleotide exchange factors accelerate nucleotide exchange in response to cellular signals to transfer Gprotein into an ACTIVE form Small G-protein families Family Control of Ras Gene expression Rho Rab Gene expression; Cytosceleton rearrangements Vesicular transport Sar1/Arf Vesicular transport Ran Nuclear transport Cell cycle Second messengers Second messengers Small intracellular molecules that amplify incoming signal cAMP Products of PtdIns2P degradation: Ins3P and DAG cGMP Phospholipid signaling