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BIOLOGIE MOLÉCULAIRE ET CELLULAIRE UdeM SMC6051/52 et BIM6026/27, et McGill 516-604D Activation cellulaire. Tyrosine et Ser/Thr Kinase (MAP kinases) Philippe P. Roux, Ph.D. Chaire de recherche en signalisation et protéomique Professeur agrégé, département de pathologie et biologie cellulaire Institut de recherche en immunologie et en cancérologie (IRIC) Université de Montréal [email protected] 16 janvier 2014, 16h-18h IRCM, salle André-Barbeau Topics for today’s lecture 1. PROTEIN PHOSPHORYLATION AND DATABASES 2. PROTEIN KINASES 3. CONTROL OF PROTEIN KINASE ACTIVITY BREAK 4. DETERMINANTS OF SUBSTRATE SPECIFICITY 5. IMPACT OF PHOSPHORYLATION ON PROTEIN FUNCTION 6. MAPK SIGNALLING PATHWAYS The basics of protein phosphorylation 2-3% of the genome is dedicated to phosphorylation and encodes protein kinases and phosphatases What percentage of the is dedicated ~ 500 genome kinase genes and 150 to phosphorylation? phosphatase genes in humans ~ 33 protein kinases (20 Tyr-K et 13 Ser/Thr-K) are mutated in human How many protein kinases diseases γ β α and protein phosphatases > 150 protein are kinases associated to there? cancer (overexpression, amplification, deletion, etc) Protein phosphorylation: important dates 1906: Discovery of the first phosphorylated protein (Vitellin) by Phoebus A. Levene 1933: With Fritz Lipmann, Levene discovers a phosphoserine in the protein casein 1954: First description of a kinase activity on casein 1955: Fischer & Krebs and Sutherland demonstrate that conversion between phosphorylase a and b results from one cycle of phosphorylation 1959: Identification of the first protein kinase, the phosphorylase kinase, by Fischer and Krebs 1968: Discovery of protein kinase A (PKA) by Krebs and characterization of the first kinase cascade (PKA->phosphorylase kinase->phosphorylase) 1978: Demonstration of a kinase activity associated with the product of the Src oncogene by Ray Erikson (Ser or Tyr kinase?) 1980: Discovery of tyrosine phosphorylation (by Src) by Tony Hunter 1981: Characterization of the protein phosphatase 2B (calcineurin) 1987: Discovery of the MAP kinases as the second kinase cascade 1991: Elucidation of the crystal structure of PKA by Susan Taylor 1992: Nobel prize in Physiology and Medicine to Krebs & Fischer 2001: FDA approval of Gleevec for the treatment of CML 2002: Description of the human kinome Nobel prize 1992 How many phosphorylation sites are there? If there are ~10,000 proteins per cell with an average length of 400 aa (~ 17% of which are Ser, Thr or Tyr), then there are ~700,000 potential phosphorylation sites for any given kinase (including hidden residues). Although protein kinases have relatively similar structures, even the most promiscuous can select their many substrates from among the theoretical 700,000 potential phosphorylation sites: For example, ERK2 Akt JAK B-Raf MEK1 phosphorylates only two substrates: ERK1 and ERK2 CaMK, CK2 and CDK phosphorylate hundreds of substrates Specificity is achieved using several mechanisms (we’ll get to that later!) many phosphosites are there? and the totalThen… number how of phosphorylation sites is probably closer to 250,000 Available phosphorylation databases 1. Phospho.ELM 8.2: contains 4,687 experimentally verified phosphorylated proteins from different species with 2,217 tyrosine, 14,518 serine and 2,914 threonine sites collected from scientific literature (Diella, et al., 2004; Diella, et al., 2008). 2. PhosphoSitePlus: is a web-based database to collect protein modification sites, including protein phosphorylation sites from scientific literature as well as high-throughput discovery programs. PhosphoSitePlus contains 73,017 phosphorylation sites (Hornbeck, et al., 2004). 3. PhosphoNET: PhosphoNET presently holds data on more than 26,000 phosphorylation sites in over 5350 human proteins that have been collected from the scientific literature and other reputable websites. 4. HPRD: HPRD currently contains information for 16,972 PTMs which belong to various categories with phosphorylation (10,858), dephosphorylation (3,118) and glycosylation (1,860) forming the majority of the annotated PTMs (Keshava Prasad, et al., 2009). 5. PHOSIDA: a phosphorylation site database, integrates thousands of high-confidence in vivo phosphosites identified by mass spectrometry-based proteomics in various species (Gnad, et al., 2007). 6. PhosphoPep v2.0: contains MS-derived phosphorylation data from 4 different organisms, including fly (Drosophila melanogaster), human (Homo sapiens), worm (Caenorhabditis elegans), and yeast (Saccharomyces cerevisiae) (Bodenmiller, et al., 2008). 7. Swiss-Prot knowledge base: for each protein annotation, the "Amino acid modifications" in the "Sequence annotation (Features)" section collected the post-translational modification information of proteins (Farriol-Mathis, et al., 2004). 8. dbPTM 2.0: integrates experimentally verified PTMs from several databases, and to annotate the predicted PTMs on Swiss-Prot proteins (Lee, et al., 2006). 9. SysPTM 1.1: provides a systematic and sophisticated platform for proteomic PTM research, equipped not only with a knowledge base of manually curated multi-type modification data, but also with four fully developed, in-depth data mining tools. (Li, et al., 2009). 10. PhosphoPOINT: is a comprehensive human kinase interactome and phospho-protein database, containing 4195 phospho-proteins with a total of 15,738 phosphorylation sites (Yang, et al., 2008). 11. NetworKIN 1.0: is a method for predicting in vivo kinase-substrate relationships, that augments consensus motifs with context for kinases and phosphoproteins. It's a great resource of phospho-regulatory network (Linding, et al., 2007; Linding, et al., 2008). http://www.uniprot.org/ http://www.phosphosite.org/ Topics for today’s lecture 1. PROTEIN PHOSPHORYLATION AND DATABASES 2. PROTEIN KINASES 3. CONTROL OF PROTEIN KINASE ACTIVITY BREAK 4. DETERMINANTS OF SUBSTRATE SPECIFICITY 5. IMPACT OF PHOSPHORYLATION ON PROTEIN FUNCTION 6. MAPK SIGNALLING PATHWAYS Protein kinases S. cerevisiae: 118 (1.9%) D. melanogaster: 236 (1.7%) C. elegans: 435 (2.3%) H. sapiens: 518 (1.7%) A. thaliana: 1049 (4.1%) The human kinomeThe (based on sequence homology only) kinome Protein kinases: 10 groups 134 families 201 sub-families 518 human genes coding for protein kinases - 478 eucaryotic PKs - 40 atypical PKs TK, tyrosine kinase; TKL, tyrosine kinase-like; STE, homologs of Ste7, Ste11, Ste20 kinases; CK1, casein kinase 1; AGC, containing PKA, PKG, PKC families; CAMK, calcium/calmodulin-dependent protein kinase; CMGC, containing CDK, MAPK, GSK3, CLK families; RGC, receptor guanylate cyclase; Other; Atypical Conservation of protein kinase sub-families during evolution From the 201 sub-families found in humans… 51 sub-families conserved in the four kinomes = 25% 144 sub-families conserved in metazoans (human, fly and worm) = 72% > 95% human kinases have an orthologue in the mouse 13 sub-families of kinases present only in humans (ex. Tie) Protein Tyrosine Kinases Tyrosine kinases • 90 human genes • 2 families of tyrosine kinases 1. receptor tyrosine kinases (58) 2. non-receptor tyrosine kinases (32) Non-receptor Receptor ProteinSerine/Threonine Serine/Threonine Kinases Kinases Protein Serine/Threonine Kinases • 352 human genes • 2 families of Ser/Thr kinases 1. receptor Ser/Thr kinases (12) 2. cytoplasmic Ser/Thr kinases (340) Ser/Thr and Tyr protein kinases have similar structures 5 β-sheets + 1 α-helix Domains I-IV: Orient and interact with the Mg-ATP complex that gives γ-phosphate N-term lobe ATP molecule Domain V: Links both lobes Domains VI-XI: Interact with substrate and initiate the transfer of phosphate C-term lobe Mostly α-helices Human CDK2 Conserved structure of Ser/Thr and Tyr kinase domains Typically 250-300 aa Phosphorylation site(s) Interacts with and orients ATP GxGxxG K E I II III Activation loop (T-loop) Hinge region IV V HRDxxxxN VIa N-terminal lobe VIb Act as a base acceptor (catalytic) DFG APE D VII IX VIII R X C-terminal lobe The HRDxxxxN motif in subdomain VIb varies between Ser/Thr and Tyr kinases: The consensus signature of Ser/Thr kinases: HRDLKxxN The consensus signature of Tyr kinases: HRDLAARN or HRDLRAAN The nine amino acids in red are invariable XI Domain structure of Ser/Thr and Tyr kinases There are 9 invariable amino acids (here in PKA) Gly52 Lys72 Glu91 Asp166 Asn171 Asp184 Glu208 Asp220 Arg280 The highly conserved residues are identified by an arrow. The region underlined corresponds to the activation loop, which often contains one or several phosphorylation sites (underlined in black). 3D localization of the 9 invariable amino acids in PKA Where are these residues located in the folded protein? Typical experimental mutations in protein kinases: • • Mutation of Lys in subdomain II (K to R) Mutations of Asp in subdomain IX (D to A) The (un)targeted cancer kinome 1970s Discovery of the first oncogene, vSrc, as an enzyme with Tyr kinase activity 1980s First potent but non-selective tools for protein kinase inhibition (staurosporine) 2001 Approval of the first kinase inhibitor, imatinib (Gleevec), for the treatment of CML by targeting BCR-Abl Since 2001, only nine small-molecule kinase inhibitors have been approved for cancer (Abl, PDGFR, cKit, EGFR and VEGFR family) How much do we know about protein kinases? What percentage of the kinome has been studied? 25-50% of the kinome remains unexplored!!! Federov et al. (2010) Nature Chem Biol Topics for today’s lecture 1. PROTEIN PHOSPHORYLATION AND DATABASES 2. PROTEIN KINASES 3. CONTROL OF PROTEIN KINASE ACTIVITY BREAK 4. DETERMINANTS OF SUBSTRATE SPECIFICITY 5. IMPACT OF PHOSPHORYLATION ON PROTEIN FUNCTION 6. MAPK SIGNALLING PATHWAYS Control of protein kinase activity 1. Regulated by ligands - EGFR dimerization 2. Regulation by second messengers - PKA, PKC and Ca2+/CaM kinases 3. Regulation by phosphorylation - Autophosphorylation (most kinases) - Upstream kinase (ERK, RSK, etc) 4. Regulation by regulatory subunits - CDKs are dependent on cyclins - PI3K requires regulatory subunit 5. Regulation by interaction prot-prot - Src and GSK3 intramolecular interactions 6. Regulation by synthesis/degradation - Mos, ERK3 Control of protein kinase activity 1. Regulated by ligands - EGFR dimerization 2. Regulation by second messengers - PKA, PKC and Ca2+/CaM kinases 3. Regulation by phosphorylation - Autophosphorylation (most kinases) - Upstream kinase (ERK, RSK, etc) 4. Regulation by regulatory subunits - CDKs are dependent on cyclins - PI3K requires regulatory subunit 5. Regulation by interaction prot-prot - Src and GSK3 intramolecular interactions 6. Regulation by synthesis/degradation - Mos, ERK3 Control of protein kinase activity Activation loop sequences 1. Regulated by ligands - EGFR dimerization 2. Regulation by second messengers - PKA, PKC and Ca2+/CaM kinases 3. Regulation by phosphorylation - Autophosphorylation (most kinases) - Upstream kinase (ERK, RSK, etc) 4. Regulation by regulatory subunits - CDKs are dependent on cyclins - PI3K requires regulatory subunit 5. Regulation by interaction prot-prot - Src and GSK3 intramolecular interactions 6. Regulation by synthesis/degradation - Mos, ERK3 PKA JNK Control of protein kinase activity 1. Regulated by ligands - EGFR dimerization 2. Regulation by second messengers - PKA, PKC and Ca2+/CaM kinases 3. Regulation by phosphorylation - Autophosphorylation (most kinases) - Upstream kinase (ERK, RSK, etc) 4. Regulation by regulatory subunits - CDKs are dependent on cyclins - PI3K requires regulatory subunit 5. Regulation by interaction prot-prot - Src and GSK3 intramolecular interactions 6. Regulation by synthesis/degradation - Mos, ERK3 The Regulation of Cyclin-Dependent Kinase (CDK) In the absence of cyclin, the C helix of CDK (also called the PSTAIRE helix) is rotated so as to move a crucial catalytic glutamate out of the active site (DeBondt et al., 1993). This is correlated with an inhibitory conformation of the activation loop. Cyclin binding reorients the PSTAIRE helix so as to place the glutamate within the active site (Jeffrey et al., 1995). The activation loop adopts a near-active conformation upon cyclin binding, and its subsequent phosphorylation further stabilizes the active form (Russo et al., 1996). Control of protein kinase activity 1. Regulated by ligands - EGFR dimerization 2. Regulation by second messengers - PKA, PKC and Ca2+/CaM kinases 3. Regulation by phosphorylation - Autophosphorylation (most kinases) - Upstream kinase (ERK, RSK, etc) 4. Regulation by regulatory subunits - CDKs are dependent on cyclins - PI3K requires regulatory subunit 5. Regulation by interaction prot-prot - Src and GSK3 intramolecular interactions 6. Regulation by synthesis/degradation - Mos, ERK3 The Regulation of Src Tyrosine Kinase In Src, intramolecular interactions between the phosphorylated tail and the SH2 domain, and between the SH2-kinase linker and the SH3 domain, stabilize inhibitory conformations of both helix C and the activation loop (Schindler et al., 1999; Xu et al., 1999). The conformation of C in the off state is quite similar to that seen in CDK. Disengagement of the SH2 domain by dephosphorylation of the tail (at Tyr527), combined with phosphorylation of the activation loop (at Tyr416), allows the C helix to move into an active conformation. Control of protein kinase activity 1. Regulated by ligands - EGFR dimerization 2. Regulation by second messengers - PKA, PKC and Ca2+/CaM kinases 3. Regulation by phosphorylation - Autophosphorylation (most kinases) - Upstream kinase (ERK, RSK, etc) 4. Regulation by regulatory subunits - CDKs are dependent on cyclins - PI3K requires regulatory subunit 5. Regulation by interaction prot-prot - Src and GSK3 intramolecular interactions 6. Regulation by synthesis/degradation - Mos, ERK3 Catalytically Inactive Protein Kinases Inactive protein kinases • ~ 50 kinase domains that are mising a catalytic residue and are predicted to be inactive • non-catalytic role: modulation, scaffold, other? • pathological roles (ex: Jak2, B-Raf, HER3) Schematic of Janus Kinase Structure Janus kinases comprise FERM, SH2, pseudokinase, and kinase domains. The FERM domain mediates receptor interactions. Both the FERM and pseudokinase domains regulate catalytic activity. Fig. 2 Involvement of the cytokine receptor-tyrosine kinase axis in MPN oncogenesis. The four main myeloid growth factor receptors involved in MPN pathogenesis are represented with their schematic principal downstream signaling involving the binding of JAK2, and the phosphorylation of PI3K, AKT, STATs, and MAPK (red arrows and brackets). The adaptor and E3 ubiquitinligase C-CBL down-regulates cKIT and JAK2 signaling. Red stars indicate the oncogenic mutations that occur in MPN resulting in a constitutive or enhanced downstream signaling (red) with modulation of transcription and protein levels for cell cycle, proliferation, and apoptosis-related factors. Protein Kinases as Therapeutic Targets Topics for today’s lecture 1. PROTEIN PHOSPHORYLATION AND DATABASES 2. PROTEIN KINASES 3. CONTROL OF PROTEIN KINASE ACTIVITY BREAK 4. DETERMINANTS OF SUBSTRATE SPECIFICITY 5. IMPACT OF PHOSPHORYLATION ON PROTEIN FUNCTION 6. MAPK SIGNALLING PATHWAYS Topics for today’s lecture 1. PROTEIN PHOSPHORYLATION AND DATABASES 2. PROTEIN KINASES 3. CONTROL OF PROTEIN KINASE ACTIVITY BREAK 4. DETERMINANTS OF SUBSTRATE SPECIFICITY 5. IMPACT OF PHOSPHORYLATION ON PROTEIN FUNCTION 6. MAPK SIGNALLING PATHWAYS Determinants of substrate specificity Factors that determines substrate specificity 1. Structure of the catalyticare cleft If protein kinases very similar… 2. Phosphoacceptor sites in substrates 3. Subcellular targeting 4. ERK2 Akt JAK B-Raf …what are the factors that will determine substrate Docking sites specificity? 1. Structure of the catalytic cleft – Tyr versus Ser/Thr ATP Hydroxyl side chain Complementary of cleft with substrate • Substrate is recognized by subdomains VIB and VIII • Depth of cleft provides some specificity (Tyr vs Ser/Thr) • Complementarity of residues in cleft in terms of hydrophobicity and charge Figure 1 | The catalytic clefts of Tyr kinases are deeper than those of Ser/Thr kinases and this determines their specificities for Tyr or Ser/Thr. a | The structure of the Tyr kinase domain of the insulin receptor (IRK) bound to a Tyr substrate peptide (Protein Data Bank (PDB) ID: 1IR3) and b | a modelled Ser substrate peptide. Unlike Ser, Tyr extends far enough into the catalytic cleft to be efficiently phosphorylated. c | The structure of the Ser/Thr kinase cyclin-dependent kinase-2 (CDK2) bound to a Ser substrate peptide (PDB ID: 1QMZ) and d | a modelled Tyr substrate peptide. Tyr is too large to fit into the catalytic cleft. Structures and modelled substrates were created using PyMol134. ATP is shown in red. Most of the peptide substrate is black, with hydroxyl sidechain oxygens shown in red. Ser versus Thr residues, is there a difference? The assumption is that there is little preference for one or another, but in fact, this assumption is incorrect… In proteins from humans, worm and yeast, the expected Ser:Thr ratio is: 1.5 : 1 - 8,5% of residues are Ser - 5,7% of residues are Thr - 3.0% of residues are Tyr However… • Partial acid hydrolysis and phosphoamino-acid analysis of 32P labelled cells gives a ratio of 9:1 pSer:pThr. • Recent global mass spectrometry studies of phosphorylation have What happens in reality? found the distribution of pSer, pThr and ptyr sites to be around 79.3%, 16.9% and 3.8%, respectively, or a 5:1 ratio of pSer:pThr. The reality is that most Ser/Thr kinases have a preference for phosphorylating Ser residues and Most Ser/Thr phosphatases show a striking bias towards dephosphorylating Thr residues. 2. Phosphoacceptor sites in substrates • Consensus phosphorylation sequences : sequences situated just before and after the phospho-acceptor residue (Ser, Thr, Tyr) • In most cases, four residues before (minus) and after (plus) the phosphorylation site determine specificity for the catalytic cleft • Complementarity of residues in cleft and substrate in terms of hydrophobicity and charge A method to determine the consensus phosphorylation sequence -5 -4 -3 -2 -1 0 +1 +2 +3 +4 Y-A-X-X-X-X-X-S/T-X-X-X-X-A-G-K-K(biotin) Array peptide library on streptavidin membrane Incubate with kinase and [γ-32P]-ATP Quench and wash membrane Expose to phosphor screen Scanning the human proteome for probable substrates Translate into probabilities for each position P G A C S T V I L M F Y W H K R Q N D E pT pY -5 0.63 1.55 0.62 0.82 0.72 0.56 0.47 0.57 0.35 0.58 0.62 0.55 0.63 0.91 1.70 7.18 0.61 0.53 0.18 0.22 0.38 0.71 -4 -3 1.15 0.80 1.19 0.20 0.96 0.26 0.94 0.36 1.33 0.37 0.88 0.39 0.92 0.32 0.60 0.38 0.77 0.24 1.22 0.42 0.82 0.38 0.78 0.35 0.97 0.28 0.96 0.37 1.75 0.67 2.17 13.58 1.06 0.33 0.79 0.15 0.31 0.07 0.42 0.09 0.45 0.04 0.66 0.03 -2 0.58 1.28 0.30 1.03 4.45 0.70 0.16 0.23 2.33 0.29 0.07 0.61 0.03 2.30 2.95 1.53 0.61 0.41 0.06 0.09 0.02 0.13 -1 2.68 1.33 1.18 0.19 1.31 0.71 0.63 0.41 0.65 0.85 0.81 1.11 0.77 2.03 1.22 0.93 0.83 1.28 0.69 0.39 0.17 0.46 +1 0.26 0.41 1.05 0.26 1.90 1.83 1.75 1.48 0.86 1.29 1.38 1.41 0.73 1.37 0.52 0.54 0.80 0.76 0.52 0.90 0.77 1.13 +2 1.18 1.48 1.26 0.15 1.59 0.95 0.21 0.35 0.80 0.78 0.72 1.95 0.12 2.01 0.93 1.31 0.99 1.55 1.22 0.44 1.34 0.84 +3 1.23 1.05 1.16 0.47 1.97 0.66 0.89 0.77 0.62 0.99 1.12 1.03 0.94 1.12 0.53 1.08 1.13 0.81 1.22 1.20 1.60 1.42 +4 1.69 1.14 0.67 0.43 2.23 1.12 0.85 0.63 0.61 0.95 0.85 0.60 0.68 1.74 0.92 0.89 1.60 0.77 1.18 0.76 1.02 1.00 Prediction of kinase-specific phosphorylation sites 1. ScanProsite: consists of documentation entries describing protein domains, families and functional sites as well as associated patterns and profiles to identify them (de Castro, et al., 2006; Hulo, et al., 2008). 2. ELM: is a resource for predicting functional sites in eukaryotic proteins (Puntervoll, et al., 2003). 3. PhosphoMotif Finder: contains known kinase/phosphatase substrate as well as binding motifs that are curated from the published literature. It reports the PRESENCE of any literature-derived motif in the query sequence (Amanchy, et al., 2007). 4. PREDIKIN 1.0: produces a prediction of substrates for serine/threonine protein kinases based on the primary sequence of a protein kinase catalytic domain (Brinkworth, et al., 2003). 5. ScanSite 2.0: searches for motifs within proteins that are likely to be phosphorylated by specific protein kinases or bind to domains such as SH2 domains, 14-3-3 domains or PDZ domains (Obenauer,et al., 2003). 6. NetPhosK 1.0: produces neural network predictions of kinase specific eukaryotic protein phosphoylation sites. Currently NetPhosK covers 17 kinases (Blom, et al., 2004). 7. GPS 2.1:The current version of GPS system. We renamed the tool as the Group-basedPrediction System. GPS 2.1 software was implemented in JAVA and could predict kinase-specific phosphorylation sites for 408 human Protein Kinases in hierarchy (Xue, et al., 2008). 8. KinasePhos 2.0: New version of kinase-specific phosphorylation site prediction tool that is based the sequenece-based amino acid coupling-pattern analysis and solvent accessibility as new features of SVM (support vector machine) (Wong, et al., 2007). 9. NetPhorest: is a non-redundant collection of 125 sequence-based classifiers for linear motifs in phosphorylation-dependent signaling. The collection contains both family-based and gene-specific classifiers (Miller, et al., 2008). http://scansite.mit.edu/ 3. Subcellular targeting Roles of scaffolding proteins 1. Specificity of signaling modules 2. Amplification of signal 3. Spatial restriction to certain substrates 4. Prevents unwanted phosphorylation AKAP mediated subcellular targeting Scaffolds within the MAPK pathways 4. Docking sites (the MAPK example) • Protein kinases often (but not always) form stable interactions with their substrates and regulators • Docking sites may simply function by increasing the local concentration of substrate around the kinase • Docking sites may also precisely align the substrate with the kinase catalytic domain • Also serves to activate or inhibit the kinase activity Conditional (phospho-dependent) docking sites • PLK1 Contains a polo-box binding domain (PBD) that binds to phosphorylated substrates that have the consensus S-pS/T-P/X. PLK1 may target substrates that have been previously phosphorylated by CDK1, a proline-directed kinase. • GSK3 Often requires a phosphorylated Ser residue at position +4 for efficient phosphorylation (priming). GSK3 is itself inactivated by a phosphorylation event in its Nterminus that loops back and inhibits the kinase by binding to a docking groove. Many docking sites have been included in scansite for general searches… http://scansite.mit.edu/ Topics for today’s lecture 1. PROTEIN PHOSPHORYLATION AND DATABASES 2. PROTEIN KINASES 3. CONTROL OF PROTEIN KINASE ACTIVITY BREAK 4. DETERMINANTS OF SUBSTRATE SPECIFICITY 5. IMPACT OF PHOSPHORYLATION ON PROTEIN FUNCTION 6. MAPK SIGNALLING PATHWAYS 1. Change of conformation: the MAP kinase ERK2 Phosphorylation-mediated conformational changes usually results from the creation of new hydrogen bonds between the phosphate groups and neighboring amino acid residues. Structural examination of phosphorylated proteins revealed two types of hydrogen bonds: - with the positively-charged guanidinium side chain of arginine residues - with the main-chain nitrogens of α-helices The hydrogen bondings induced by phosphorylation consequently alter the conformations of the target proteins, thereby modulating their functional properties. Dual phosphorylation of the activation loop segment residues (TEY) changes the conformation by three ways: 1. Orients the C helix 2. Promotes lobe closure (phospho-TEY associates with Arg residues in N-term lobe) 3. Organizes the C-term extension The Regulation of MAP Kinase by Phosphorylation of the Activation Segment. Phosphorylation of the activation segment creates a network of interactions that properly orient the C helix, promote lobe closure, and organize the Cterminal extension (shown in yellow) into a functionally important homodimerization interface (Zhang et al., 1994; Canagarajah et al., 1997; Khokhlatchev et al., 1998). 2. Steric hindrance: Isocitrate dehydrogenase Isocitrate dehydrogenase (IDH) is an enzyme which participates in the citric acid cycle. It catalyzes the third step of the cycle: the oxidative decarboxylation of isocitrate, producing alphaketoglutarate (α-ketoglutarate) and CO2 while converting NAD+ to NADH. This is a two-step process, which involves oxidation of isocitrate (a secondary alcohol) to oxalosuccinate (a ketone), followed by the decarboxylation of the carboxyl group beta to the ketone, forming alpha-ketoglutarate. isocitrate Phosphorylation blocks substrate binding to isocitrate dehydrogenase. A, Surface representation with isocitrate (blue) bound to the active site. B, Phosphorylation of serine 113 (yellow) blocks isocitrate binding. 3. Modification of protein interaction Table 2 | Phosphotyrosine-binding domains SH2 PTB pY N-P-X-pY Diverse RTK Signaling SH2 Domain SH2 domains contain a central anti-parallel b-sheet surrounded by two ahelices. The phosphopeptide generally binds as an extended b-strand that lies at right angles to the SH2 b-sheet. Conserved residues contribute to the hydrophobic core or are involved in pY recognition while more variable residues contribute to specific recognition of C-terminal residues. An invariant Arg residue in the SH2 domain coordinates the phosphate oxygens of pY and is essential for high affinity phosphopeptide binding. The figure shows the SH2 domain of v-src bound to a pYEEI peptide ligand. • non-catalytic module of ~ 100 amino acids homologous to c-Src • binding affinity for Tyr-P >> Tyr (Kd ~50-500 nM) • SH2 binds to specific phosphopeptide sequences (C-terminal) • autophosphorylation of RTKs acts as molecular ‘switch’ for signal transduction Topics for today’s lecture 1. PROTEIN PHOSPHORYLATION AND DATABASES 2. PROTEIN KINASES 3. CONTROL OF PROTEIN KINASE ACTIVITY BREAK 4. DETERMINANTS OF SUBSTRATE SPECIFICITY 5. IMPACT OF PHOSPHORYLATION ON PROTEIN FUNCTION 6. MAPK SIGNALLING PATHWAYS Protein Kinase Cascades MAP Kinase Signalling Pathways Extracellular stimuli Effectors The MAP Kinase Family ERK1 ERK2 ERK5 p38α p38β p38γ p38δ JNK1 JNK2 JNK3 ERK7 NLK ERK3 ERK4 GFLTEYVATRWYR IML217 180 GFLTEYVATRWYRAPEIML198 216 YFMTEYVATRWYRAPELML234 177 DEMTGYVATRWYRAPEIML195 177 EEMTGYVATRWYRAPEIML195 180 SEMTGYVVTRWYRAPEVIL198 177 AEMTGYVVTRWYRAPEVIL195 180 FMMTPYVVTRYYRAPEVIL198 180 FMMTPYVVTRYYRAPEVIL198 218 FMMTPYVVTRYYRAPEVIL236 172 QAVTEYVATRWYRAPEVLL190 283 RHMTQEVVTQYYRAPEILM301 186 GHLSEGLVTKWYRSPRLLL204 183 GYLSEGLVTKWYRSPRLLL201 199 Pleiotropic Functions of MAP Kinases Wnt signaling, HSC stroma embryo growth, lung function proliferation, angiogenesis proliferation, survival, senescence inflammation, development, cell cycle neural apoptosis, obesity, T-cell function DDR, autophagy Multiple Roles of ERK1/2 in Cell Proliferation Activation of the Ras-ERK1/2 Pathway in Cancer Regulation of ERK1/2 MAP Kinases by Mitogens Mitogens A-Raf B-Raf C-Raf MEK1 MEK2 ERK1 ERK2 ? > 160 cellular targets growth proliferation survival Activation of MAP Kinases by Phosphorylation Dual phosphorylation of the activation loop segment residues (TEY) changes the conformation by three ways: 1. Orients the C helix 2. Promotes lobe closure (phospho-TEY associates with Arg residues in N-term lobe) 3. Organizes the C-term extension Dephosphorylation and Inactivation of MAP Kinases Regulation of ERK1/2 Pathway by Feedback Loops Targeting the ERK1/2 Pathway in Cancer vemurafenib selumetinib Lessons from Raf Inhibition in Melanoma A 38-year-old man with BRAF-mutant melanoma and miliary, subcutaneous metastatic deposits. Photographs were taken (A) before initiation of PLX4032, (B) after 15 weeks of therapy with PLX4032, and (C) after relapse, after 23 weeks of therapy. Questions? Some general references... • Ubersax and Ferrell (2007) Nature Rev Mol Cell Biol 8:530-41 • Manning and Cantley (2007) Cell 129:1261-74 • Frame and Cohen (2001) Biochem J 359:1-16 • Manning et al. (2002) Science 539:1912-34 • Manning et al. (2002) Trends Biochem Sci 27:514-20 • Adams (2001) Chem Rev 101:2271-90