2024 Tyrosine Kinase Receptors x1.pdf

RECEPTOR TYROSINE KINASES Molecular Pharmacology & Drug Discovery 3310 Fiona Pixley 2024 2023 CANCER LECTURES Tumour cells have acquired 7(8) deadly capabilities:Independence from growth signals Insensitivity to anti-growth signals Able to evade apoptotic signals Become immortal Develop own blood su...

RECEPTOR TYROSINE KINASES Molecular Pharmacology & Drug Discovery 3310 Fiona Pixley 2024 2023 CANCER LECTURES Tumour cells have acquired 7(8) deadly capabilities:Independence from growth signals Insensitivity to anti-growth signals Able to evade apoptotic signals Become immortal Develop own blood supply Evading immune destruction Able to invade and grow in distant sites Able to evade the immune system 2024 - the role of phosphorylation in cancer pathogenesis & how to target it Hanahan & Weinberg, 2000/11 LECTURE 1 OUTCOMES From this lecture, you should be able to:Explain the role of phosphorylation as an regulatory on/off switch in signal transduction & intracellular signalling Outline the enzymatic mechanism of protein kinases Describe how receptors, either containing their own intrinsic kinase domain or linked to a cytoplasmic kinase, transduce EC to IC signals Describe the structure and function of receptor tyrosine kinases (kinase activity is intrinsic to, i.e. part of, the receptor) Outline the RTK activation and signalling paradigm (examples help) Describe the structure/function of linked kinase receptors (no intrinsic kinase) Outline the importance of RTKs in cancer pathogenesis KINASE-LINKED RECEPTORS are quite different to ion channels, GPCRs & nuclear receptors:One transmembrane domain Intracellular enzyme/kinase domain: intrinsic (part of ICD) or linked to a cytoplasmic or non-receptor kinase Kinases phosphorylate their substrates Phosphorylation regulates function → on/off switch mechanism PROTEIN KINASES & PHARMACOLOGY Phosphorylation - regulatory switch for many processes receptors, enzymes, ion channels and transporters, TFs…… Receptor tyrosine kinases - small in number but involved in many diseases, esp. cancer Currently the pharmaceutical industry’s most popular drug target SIGNALLING & Ligand PHOSPHORYLATION Multicellular organisms need cell-to-cell communication mechanisms: extracellular molecule - ligand cell surface molecule - receptor 1. 2. 3. 4. receptor activation → phosphorylation phosphorylation of intracellular proteins activation of signalling pathways activation of target functions Phosphorylation at many steps switches intracellular protein activity off or on Kinase Receptor Intracellular signalling pathways P P P P P Target organelles metabolic enzymes gene P regulatory proteins Metabolism Gene expression P cytoskeletal proteins Cell motility PHOSPHORYLATION Commonest type of protein post-translational modification (PTM) The MAJOR mechanism for regulation of protein activity ~30% of cellular proteins are modified by phosphorylation Kinases phosphorylate & phosphatases dephosphorylate OH CH2 ATP ADP Protein kinase Protein Protein phosphatase O P CH2 P Active Protein P Inactive Pi Phosphorylation - switch, turns protein activity “on” or “off” Turnover is very rapid → allows rapid response to stimuli WHY IS PROTEIN PHOSPHORYLATION SO IMPORTANT? Regulates signals for: division growth metabolism differentiation motility organelle trafficking membrane transport muscle contraction immunity learning and memory WHAT ARE PROTEIN KINASES? What’s their structure and how do they work? PROTEIN KINASES Large gene family (>500) - ~2% of genome Enzymes (kinase - ‘move’/add PO4 group) Transfer γ-phosphate from ATP to protein Highly conserved kinase domain Phosphorylate specific residues - Ser (S), Thr (T) & Tyr (Y) Two main structural/functional groups: S/T kinases Y kinases Cell Signaling Technology PROTEIN KINASE STRUCTURE Highly conserved catalytic domain for ALL KINASES :~250 amino acid domain divided into two lobes: N C β-sheet N-ter lobe α-helical C-ter lobe P ATP - sits in inter-lobular cleft Substrate - also fits into cleft } Highly conserved residues in cleft catalyse ATPγ PO4 transfer to S, T or Y Catalytic cleft/pocket depth: shallow (S/T) deep (Y) Substrate’s amino acids close to S, T or Y residue determine specificity TYROSINE KINASES Cell Signaling Technology …phosphorylate tyrosine residues on target proteins TYROSINE KINASES Transducers → convert extracellular into intracellular signals First step in intracellular signalling Two main receptor types: True receptor tyrosine kinases (RTKs): intrinsic kinase within intracellular domain (pink) Receptors linked to tyrosine kinases: cytokine receptors (interleukins, G-CSF, GM-CSF, erythropoietin, others) have no kinase domain - linked to cytoplasmic tyrosine kinases (blue)→ “binary RTKs” Cell Signaling Technology TYROSINE PHOSPHORYLATION Much less common (~0.05%) than serine phosphorylation (S, ~90%) or threonine phosphorylation (T, ~10%) …but linked to cancer → unregulated tyrosine phosphorylation drives most cancers Tyrosine kinases (Y)→ 0.3% of genome but 30% of oncogenes Oncogenes versus proto-oncogenes: oncogenes cause cancer when mutated or over-expressed their normal cellular counterparts, proto-oncogenes, regulate cell growth, proliferation, survival, migration etc. (i.e. hallmarks of cancer) KINASE RECAP What do kinases do? γ _______________________________________ How does phosphorylation affect the substrate protein? _______________________________________ What % of phosphorylation events are tyrosine? _______________________________________ α β TYROSINE KINASES AS ONCOGENES TKs are primary regulators of proliferation, survival, migration, angiogenesis → i.e. first responders Deregulation of TKs - on/off switch is stuck in “on” → constitutive activation of signals Evading immune destruction e.g. retroviral oncogenes (v-src, v-erbB, v-fms) are hijacked genes → mutations → deregulation normal gene - proto-oncogene e.g. c-src, c-erbB, → regulated TK activity & controlled signalling RECEPTOR TYROSINE KINASES (RTKS) How do RTKs convert an extracellular signal into an intracellular response? Structure suggests function Classified by extracellular domains 90 Tyrosine Kinases → 58 receptors (RTK) → 20 RTK Families I II III... All RTKs have a very similar molecular architecture: Extracellular domain for ligand binding - variable & modular One transmembrane domain Intracellular tyrosine kinase (TK) domain Regulatory regions: juxta-membrane domain C-terminal domain kinase insert (III-VI, XIV) Lemmon & Schlessinger Cell 2010 31 RTKs are either mutated or over-expressed in human cancers RTK STRUCTURE GUIDES FUNCTION Similar molecular architecture: Extracellular domain - ligand binding TM domain - single α helix Intracellular tyrosine kinase (TK) domain Extracellular Intracellular Tyrosine (Y) residues - autophosphorylated Juxta-membrane and C-ter regulatory regions Mechanism of activation is highly conserved Downstream signalling mechanisms also conserved SIGNALS RTKS ARE ACTIVATED BY LIGANDS RTKs transduce many signals: Growth factors Cytokines Hormones EGF Ligands RTK ligand examples:Insulin EGF (epidermal growth factor) VEGF (vascular endothelial growth factor) CSF-1 (macrophage colony stimulating factor) RTK ACTIVATION PARADIGM Without its ligand, basal kinase activity of RTK monomers is very low Ligand addition triggers ligand-mediated dimerisation of RTKs (1 + 1 = 2) Close apposition of 2 RTK molecules activates intrinsic kinase domains Kinase domains phosphorylate tyrosine (Y) residues within the RTK in a very specific way: 1. Activation loop Y on the partner RTK, i.e. in trans 2. Regulatory Ys to remove kinase inhibition 3. Signalling Ys to provide docking sites for downstream proteins ACTIVATION PARADIGM EXAMPLE s s s s s s CSF-1 Homodimer s s s s s s s s s s P Y544 Y559 P CSF-1R SFKs K614 P P P Y697 P Y706 P Y721 P s s s s s s s s s s s s s s s s PI3K, PLCγ2, Socs1 P P P P Ub Ub P P Y921 P Y974 P dephosphorylation ubiquitylation Grb2, Mona, Socs1 Y807 P Activation Loop P proliferation survival differentiation motility down regulation Grb2 Cbl Ub Ub Ub Ub Ub Ub P P Switch mechanism: rapid transient RTK ACTIVATION - STEP BY STEP 1. 2. 3. 4. 5. Ligand - CSF-1 (homodimer) binds two CSF-1R (RTK) monomers CSF-1R monomers dimerise - brings their kinase domains together Kinase domains trans-autophosphorylate Y807 on the other CSF-1R Activation loop Y phosphorylation → ↑↑ kinase activity Phosphorylation of additional Y residues on RTK - 7 Ys in CSF-1R DOWNSTREAM SIGNALLING & INACTIVATION 6. pY residues on RTK are docking sites for proteins containing pY binding domains (SH2, PTB - next lecture) 7. Docked proteins are then phosphorylated on Y by RTK 8. Activation of multiple downstream pathways (next lecture) 9. Phosphatases → remove phosphates & turn signals off 10. Ubiquitylation → tags RTKs leading to internalisation & degradation NON-RECEPTOR TYROSINE KINASES (NON-RTKS) …are not receptors - how are they activated? associate with receptors that lack a tyrosine kinase domain when receptors bind their ligand → dimerisation receptor dimerisation → associated non-RTKs come together → TK domain activation TYROSINE KINASE-LINKED RECEPTORS = = RECEPTOR + ASSOCIATED NON-RTK Many GF/cytokine receptors lack an intrinsic kinase domain Rely on non-covalently associated non-receptor TKs (non-RTK) Same basic paradigm: L P P P P P Ligand binding Receptor dimerisation Autophosphorylation/activation of non-RTK Phosphorylation /activation of substrates P Tyrosine kinase domain P Signal 1. 2. 3. 4. P SH3 SH2 Kinase-like TK Domain SFKs JAKs RTK ACTIVATION PARADIGM https://www.youtube.com/watch?v=LT_ws4Xvj7M PROPAGATION OF RTK SIGNALS SH2 P Y Substrates of RTKs include: RTKs & non-RTKs themselves - 1° substrates Downstream signalling substrates Enzymes (PI3K, PLC, SHP1) Adaptor molecules (Grb2, Shc, Nck) Docking proteins (IRS1 & IRS2, Dok1) Structural proteins (paxillin, cadherin, integrins) RTK-substrate (and substratedownstream molecule) interactions are mediated by a series of discrete protein-protein interaction domains PTB P Y SH3 P-X-X-P lipid Next lecture PH WHAT DO RTKS DO? Key roles of RTKs/binary TKs → transduce signals to fundamental pathways :- proliferation migration metabolism differentiation/development cell survival …which is why RTKs are so dangerous when their tight regulatory control is lost INSULIN RECEPTOR (INS-R) SIGNALLING Glucose transporter recruitment to plasma membrane Insulin receptor (Ins-R) monomer is cleaved into α & β subunits bound by S-S bridges - heterodimeric sub-units 2 sub-units (already covalently dimerised) Insulin binds to α subunits → activates TK domain (β subunit) INSULIN RECEPTOR (INS-R) SIGNALLING Glucose P transporter recruitment to plasma membrane P P Tyrosine kinase activation → phosphorylation of Ins-R in trans Docking & phosphorylation of pY binding substrates Signal propagation to target proteins: GLUT-4 recruitment to membrane Anabolic effects on fuel storage enzymes RTKS & CANCER! RTKs signal to:proliferation migration metabolism differentiation/development cell survival EGFR/HER/ERBB FAMILY OF RTKS EGFR = epidermal growth factor receptor (aka HER/ErbB) 4 HER/ErbB receptors 1. 2. 3. 4. EGFR/HER1/ErbB1 HER2/ErbB2 HER3/ErbB3 HER4/ErbB4 Form homodimers & heterodimers, e.g. EGFR-EGFR & EGFR-HER2 HER2 can’t bind ligands → must heterodimerise to activate kinase domain HER3 kinase domain is inactive → must heterodimerise to become phosphorylated and activate downstream signalling 7 ligands → high (EGF, TGFα) & low affinity (AREG) - full vs partial agonists EGFR/HER/ERBB SIGNALLING Activates many signalling pathways: Ras/Raf/MAPK PI3K/AKT JAK/STAT (PLCγ & Nck) which regulate: proliferation survival migration/invasion Next Lecture in epithelial cells RTK SIGNALLING & DISEASE Phosphorylation is a switch mechanism for signal transduction inside cells Transient state: switched off much of the time switched on only when required then rapidly switched off again Constitutive activation of RTKs → dysfunction/disease Cancer RTK inactivation → dysfunction/disease (inactivation is not as common as constitutive activation) Third Lecture KINASE-LINKED RECEPTORS Which of the following statements about protein tyrosine kinases is CORRECT: A. Tyrosine kinases remove phosphate groups from tyrosine residues B. RTKs convert extracellular signals to intracellular signals through activation of the intracellular kinase domain C. All 90 tyrosine kinases are single pass membrane spanning RTKs D. RTKs are activated in the absence of ligand - ligand binding amplifies the signal E. RTKs signal to cell proliferation but not to differentiation or motility FURTHER READING Lipsick J, 2019. A history of cancer research: tyrosine kinases. Cold Spring Harbor Perspectives in Biology 11: a035592 Lemmon MA & Schlessinger J, 2010. Cell signaling by receptor tyrosine kinases. Cell 141: 1117-1134. Yamaoka T et al., 2018. Receptor tyrosine kinase-targeted cancer therapy. International Journal of Molecular Sciences 19: 3491