BIOLOGIE MOLÉCULAIRE ET CELLULAIRE (SMC6052/BIM6028, et EXMD604) Activation cellulaire. Tyrosine et Ser/Thr Kinase (MAP kinases)

BIOLOGIE MOLÉCULAIRE ET CELLULAIRE (SMC6052/BIM6028, et EXMD604) Activation cellulaire. Tyrosine et Ser/Thr Kinase (MAP kinases) Philippe P. Roux, Ph.D. Chaire de recherche du Canada en signalisation cellulaire et protéomique Professeur agrégé, département de pathologie et biologie cellulaire Chercheur principal, Institut de recherche en immunologie et en cancérologie (IRIC) Université de Montréal philippe.roux@umontreal.ca 12 janvier 2017, 16h-18h IRCM

Topics for today s lecture 1. PROTEIN PHOSPHORYLATION 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 γ β α 2-3% of the genome is dedicated to phosphorylation and encodes protein What percentage of the kinases and phosphatases genome is dedicated to ~ 500 kinase phosphorylation? genes and 150 phosphatase genes in humans ~ 33 protein kinases (20 Tyr-K et 13 Ser/Thr-K) How many are mutated protein human kinases diseases and protein phosphatases > 150 protein kinases are there? associated to 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 describing how reversible phosphorylation works as a switch to activate proteins and regulate various cellular processes 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: Some examples, MEK1 phosphorylates only two substrates: ERK1 and ERK2 CaMK, CK2 and CDK phosphorylate hundreds of substrates ERK2 JAK Akt B-Raf Specificity is achieved using several mechanisms (we ll get to that later!) and the total Then number how of phosphorylation many phosphosites sites is probably are there? closer to 250,000

Evolutionarily conserved sites are more likely to be functional Landry et al. (2009) Trends Genet

An explanation for the evolution of activating phosphorylation sites Ancestor (A) Phosphorylation of a surface-exposed serine residue in an active protein (yellow) would be expected to decrease the protein s activity (gray). (B) Structures of phosphoserine, phosphothreonine, aspartic acid, and glutamic acid. Carbon atoms are represented in green, phosphorus atoms in magenta, and oxygen atoms in red. Ancestor (C) Phosphorylation of a serine or threonine residue could conditionally restore an important electrostatic interaction originally mediated by an acidic amino acid, thereby activating the protein. Pearlman et al. (2011) Cell

An explanation for the evolution of activating phosphorylation sites Pearlman et al. (2011) Cell

The human kinome (based The on kinome sequence homology, not function) 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/calmodulindependent protein kinase; CMGC, containing CDK, MAPK, GSK3, CLK families; RGC, receptor guanylate cyclase; Other; Atypical 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%)

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

Protein Serine/Threonine Kinases 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 a similar structure 5 β-sheets + 1 α-helix N-term lobe ATP molecule Domains I-IV: Orient and interact with the Mg-ATP complex that gives γ-phosphate Domain V: Links both lobes C-term lobe Domains VI-XI: Interact with substrate and initiate the transfer of phosphate Human CDK2 Mostly α-helices

Conserved structure of Ser/Thr and Tyr kinase domains Typically 250-300 aa Phosphorylation site(s) Interacts with and orients ATP αc-helix Hinge region Activation loop (T-loop) Act as a base acceptor (catalytic) GxGxxG K E HRDxxxxN DFG APE D R I II III IV V VIa VIb VII VIII IX X XI N-terminal lobe C-terminal lobe The nine amino acids in red are invariable

3D localization of the 9 invariable amino acids There are 9 invariable amino acids (here in PKA) Gly 52 Lys 72 Glu 91 Asp 166 Asn 171 Asp 184 Glu 208 Asp 220 Arg 280 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)

Control of protein kinase activity 1. Regulated by ligands - EGFR dimerization 2. Regulation by second messengers - PKA, PKC and Ca 2+ /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 Ca 2+ /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.

Determinants of substrate specificity Factors that determines substrate specificity 1. Structure of the catalytic cleft If protein kinases are very similar 2. Phosphoacceptor sites in substrates what are the factors that will determine substrate specificity? 3. Subcellular targeting 4. Docking sites ERK2 Akt JAK B-Raf

1. Structure of the catalytic cleft Tyr versus Ser/Thr Substrate is recognized by subdomains VIB and VIII Depth of cleft provides some specificity (Tyr vs Ser/Thr) ATP Hydroxyl side chain Complementary of cleft with substrate 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.

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 S/T-P-X-K R-R-X-S/T-Ψ

2. Phosphoacceptor sites in substrates

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 prolinedirected kinase. Barr et al. (2004) Nature Rev

1. Change of conformation: 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 three types of hydrogen bonds: - With the positively-charged guanidinium side chain of arginine residues - With the positively-charged cationic ammonium side chain of lysine residues - With the main-chain nitrogens of α-helices Dual phosphorylation of the activation loop segment residues (TEY) changes the conformation by three ways: 1. Orients the αc helix 2. Promotes lobe closure (phospho-tey associates with Arg residues in N-term lobe) 3. Organizes the C-term extension

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 CO 2 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 (positive and negative) SH2 Domain Table 2 Phosphotyrosine-binding domains SH2 py Diverse PTB N-P-X-pY RTK Signaling 14-3-3 proteins

Potential outcome of phosphorylation on biological function Changes in protein conformation, steric hindrance and interacting partners can affect a wide range of biological functions, including: Subcellular localization Protein stability Enzymatic activity Protein-protein interaction Intramolecular interaction etc The functional consequences of phosphorylation can be addressed using phosphorylation site mutants Serine/Threonine to alanine (S/T to A), or Tyrosine to phenylalanine (Y to F) - The goal is to prevent phosphorylation of the protein Serine/Threonine/Tyrosine to aspartic/glutamic acids - The goal is to mimic phosphorylation by adding a negative charge - Generally works for conformational changes and disruption of interactions - Does not usually work when phosphorylation creates a docking site

MAPK signalling pathways Extracellular stimuli Effectors

RHMTQEVVTQYYRAPEILM FMMTPYVVTRYYRAPEVIL FMMTPYVVTRYYRAPEVIL FMMTPYVVTRYYRAPEVIL EEMTGYVATRWYRAPEIML DEMTGYVATRWYRAPEIML AEMTGYVVTRWYRAPEVIL SEMTGYVVTRWYRAPEVIL GFLTEYVATRWYR IML YFMTEYVATRWYRAPELML QAVTEYVATRWYRAPEVLL GHLSEGLVTKWYRSPRLLL ERK1 ERK2 ERK5 ERK3 ERK4 ERK7 NLK p38α p38β p38γ p38δ JNK1 JNK2 JNK3 180 198 186 183 204 201 199 216 177 177 177 180 180 180 218 172 283 217 234 195 195 195 198 198 198 236 190 301 GFLTEYVATRWYRAPEIML GYLSEGLVTKWYRSPRLLL The MAPK family Activation loop

Pleiotropic functions of the different MAPKs Wnt signaling, HSC stroma embryo growth, lung function proliferation, angiogenesis proliferation, survival, senescence DDR, autophagy Human kinome inflammation, development, cell cycle Neural apoptosis, obesity, T-cell function

Multiple roles of ERK1/2 in cell proliferation Cell growth Cell cycle progression

Regulation of ERK1/2 by mitogens and growth factors Mitogens A-Raf B-Raf C-Raf MEK1 MEK2 ERK1 ERK2? > 160 cellular targets growth proliferation survival

Negative regulation of ERK1/2 pathway by feedback loops

Activation of the Ras/ERK pathway in cancer

Targeting the ERK1/2 pathway in cancer vemurafenib selumetinib

Lessons from B-Raf (V600E) inhibition in Melanoma Before treatment After 15 weeks on PLX4032 After 23 weeks on PLX4032 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.

Mechanisms of resistance to BRAF inhibitors Tentori et al. (2013) Trends Pharm Sci

The (un)targeted cancer kinome 1970s 1980s Discovery of the first oncogene, vsrc, as an enzyme with Tyr kinase activity 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, less than 15 small-molecule kinase inhibitors have been approved for cancer treatment (Abl, PDGFR, ckit, EGFR and VEGFR family)

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