HSS2305C Fall 2024 Lecture 17 PDF

HSS2305: Molecular Mechanisms of Disease Lecture 17 – Signal Transduction: RTKs and MAPKs November 11, 2024 Section C00 Fall 2024 Prof. Alex Green Studying Tips from your Peers Briefly review lecture slides before lectu...

HSS2305: Molecular Mechanisms of Disease Lecture 17 – Signal Transduction: RTKs and MAPKs November 11, 2024 Section C00 Fall 2024 Prof. Alex Green Studying Tips from your Peers Briefly review lecture slides before lecture “FLASHCARDS!!!!!!” Make flash cards after each lecture Anki - https://apps.ankiweb.net/ “It's a pain to get used to, but once you've got it going it's great.” Practice them daily! Watch the videos in the lectures/on Youtube Annotate slides Use active recall methods “Use analogies or relate a complicated topic with something in my personal life that's easy to remember” “Blurting method” Draw/Write everything you know on a white board/apper Correct/Add info from slides in different ink Study Erase Repeat CELL SIGNALLING Receptors Receptor types include: a) a) G-protein coupled receptors (GPCRs) b) Receptor protein-tyrosine kinases (RTKs) c) Ligand gated channels b) d) allow or conduct passage of ion d) Steroid hormone receptors nuclear receptors c) e) Specialized receptors i.e. B-and T-cell receptors immune response to generate diversity in immune response CELL SIGNALLING G-COUPLED PATHWAYS cAMP GMP IP3 & DAG Ligand binds GPCR Light causes conformational Ligand binds GPCR glucagon change in rhodopsin Epinephrine epinephrine Acetylcholine (GPCR) Serotonin Activates G-protein Activates G-protein Activates G-protein (alpha subunit Gs/Gi) (alpha subunit Gt) (alpha subunit Gq) Triggers activation or Triggers activation of cGMP Triggers activation of inhibition of adenylyl cyclase phosphodiesterase phospholipase C-beta (effector) (effector) (effector) ATP à cAMP cGMP à GMP PIP2 à IP3 & DAG Increase/decrease in Decrease in cytosolic cGMP IP3 - release Ca2+ from cytosolic cAMP which alters closes cation (Na+) intracellular stores activation of Protein Kinase channels causing A (PKA) membrane hyperpolarization Ca2+ and DAG - activation of Protein Kinase C (PKC) Cell Signalling signal transduction 2 major routes of message transmission 1. Generation of an intracellular second messenger via an effector enzyme Second messengers activate/inactivate target proteins 2. Recruit signaling proteins to their intracellular domains to initiate a protein activated cascade CELL SIGNALLING Receptors Receptor types include: a) a) G-protein coupled receptors (GPCRs) b) Receptor protein-tyrosine kinases (RTKs) c) Ligand gated channels b) d) allow or conduct passage of ion d) Steroid hormone receptors nuclear receptors c) e) Specialized receptors i.e. B-and T-cell receptors immune response to generate diversity in immune response RECEPTOR PROTEIN- TYROSINE KINASES (RTKS) RECEPTOR PROTEIN- TYROSINE KINASES (RTKS) HUMAN HEALTH AND DISEASE https://www.youtube.com/watch?v=0mKZRAt0GZg https://www.youtube.com/watch?v=6KiLt2yt5xI HER2-positive breast cancer: breast cancer that tests positive for human epidermal growth factor receptor 2 (HER2) – promotes growth of cancer cells  RECEPTOR PROTEIN- TYROSINE KINASES (RTKS) Transmembrane receptor proteins spans the plasma membrane just once Intracellular domain has tyrosine protein kinase activity Common Ligands Insulin Vascular Endothelial Growth Factor (VEGF) Platelet-Derived Growth Factor (PDGF) Epidermal Growth Factor (EGF) Fibroblast Growth Factor (FGF) Example of effects on skin (different for other organs):  RECEPTOR PROTEIN- TYROSINE KINASES (RTKS) 1 2 3 4 Step 1 and 2: ligand binding à receptor dimerization 1. Ligand mediated dimerization - 1 ligand binds to both receptor proteins at the same time (i.e. PDGF) 2. Receptor mediated dimerization - 2 ligands bind 2 receptor proteins (i.e. EGF) RECEPTOR PROTEIN- TYROSINE KINASES (RTKS) 1 2 3 4 Step 3: RTK phosphorylates itself à trans-autophosphorylation Phosphate groups added to tyrosine residues on intracellular domain of receptor RECEPTOR PROTEIN- TYROSINE KINASES (RTKS) 1 2 3 4 Step 4: Inactive relay proteins recruited to the receptor where they become phosphorylated à activated à cellular response Specialized domains (i.e SH2-Src homology region 2, PTB - phosphotyrosine-binding) ~ 100 aa long that can interact with phosphotyrosine RECEPTOR PROTEIN-TYROSINE KINASES (RTKS) ENDING THERESPONSE A cell must be able to stop responding to a signal For growth factor receptors, failure to do so could lead to uncontrolled mitosis à cancer RECEPTOR PROTEIN-TYROSINE KINASES (RTKS) ENDING THERESPONSE RTKs undergo autophosphorylation therefore destruction or endocytosis is required to terminate signal Receptor mediated endocytosis à quickly engulfing and destroying or recycling the ligand-receptor complex Mediated by CBL protein à targeted for ubiquitination à internalization à degraded in lysosome Those RTKs that are not ubiquitinated are recycled  RECEPTOR PROTEIN-TYROSINE KINASES (RTKS) RELAYPROTEINS Variety of relay proteins exist Adaptor proteins Function as a linkers in a signaling complex (i.e. Grb2) Docking proteins Acts as a docking station for several signaling proteins (i.e. Insulin receptor substrate: IRS) Transcription factors Once activated by receptor translocate to nucleus and alters gene transcription (i.e. STAT) Enzymes Signalling enzymes activated by receptor à perform function within the cell kinases (i.e. PI3K), phosphatases (i.e. shp2), lipases (i.e. PLCγ) RECEPTOR PROTEIN- TYROSINE KINASES (RTKS) 1 2 3 4 A variety of different relay proteins à different cellular responses RECEPTOR PROTEIN- TYROSINE KINASES (RTKS) 3 major signalling pathways are triggered: 1. PLCγ à IP3/DAG 2. PIP3 à AKT 3. Ras à MAPK  RECEPTOR PROTEIN- TYROSINE KINASES (RTKS) 1.PLCγ àIP3/DAG 3 major signalling pathways are triggered 1. PLCγ à IP3/DAG 2. PIP3 à AKT 3. Ras à MAPK  RECEPTOR PROTEIN-TYROSINE KINASES (RTKS) IP3 AND DAG SIGNALLING 1.PLCγ àIP3/DAG Example ligands: Platelet-derived growth factor (PDGF) Epidermal growth factor (EGF) Phospholipase C-γ (PLC- γ) Vascular Endothelial growth factor (VEGF) binds activated RTK via SH2 domain Tyrosine phosphorylation increases PLC-γ activity, stimulating the cleavage of PIP2 à IP3 & DAG  RECEPTOR PROTEIN-TYROSINE KINASES (RTKS) IP3 AND DAG SIGNALLING GCPR à IP3 & DAG RTKs à IP3 & DAG Ligand binds GPCR Ligand binds RTK Activates G-protein (alpha subunit Gq) Activates RTK- autophosphorylation Triggers activation of Phospholipase Phosphorylates/activates Cβ (PLCβ) Phospholipase Cγ (PLCγ) PIP2 à DAG, IP3 PIP2 à DAG, IP3 IP3 - release Ca2+ from intracellular IP3 - release Ca2+ from intracellular stores stores Ca2+ and DAG activation of Protein Ca2+ and DAG activation of Protein Kinase C (PKC) Kinase C (PKC) RECEPTOR PROTEIN- TYROSINE KINASES (RTKS) 2.PIP3 àAKT Another example diagram but integrating insulin signaling: Insulin receptor substrate 1 Insulin/ 3 major signalling IRS1 pathways are triggered 1. PLCγ à IP3/DAG 2. PIP3 à AKT 3. Ras à MAPK  RECEPTOR PROTEIN-TYROSINE KINASES (RTKS) 2.PIP3 àAKT Activated RTK phosphorylates IRS docking protein Recruitment of PI3 kinase to membrane Example ligands: PI3K phosphorylates lipids in plasma Vascular Endothelial Growth Facotor (VEGF) membrane (PIP2 à PIP3) Epidermal growth factor (EGF) Insulin PDK1, PDK2 and Akt recruited to the membrane Akt phosphorylated on Thr308 and Ser473 à conformational change and activation PIP3 PIP2  RECEPTOR PROTEIN-TYROSINE KINASES (RTKS) 2.PIP3 àAKT  RECEPTOR PROTEIN-TYROSINE KINASES (RTKS) 2.PIP3 àAKT https://www.youtube.com/watch?v=W3ZASYwqjNc https://www.youtube.com/watch?v=Z9xm7aqHW-o – “192-Obesity & Insulin Resistance”  RECEPTOR PROTEIN- TYROSINE KINASES (RTKS) 3.Ras àMAPK 3 major signalling pathways are triggered 1. PLCγ à IP3/DAG 2. PIP3 à AKT 3. Ras à MAPK  RECEPTOR PROTEIN-TYROSINE KINASES (RTKS) 3. Ras àMAPK Mitogen-activated protein kinases (MAPK) are a superfamily of protein kinases that can phosphorylate serine/threonine on proteins in the cell Mitogen- something that promotes growth (mit—osis) They regulate cell functions including proliferation, gene expression, differentiation, mitosis, cell survival, and apoptosis Different combinations of MAPKs are used depending on the extracellular signal provided  RECEPTOR PROTEIN-TYROSINE KINASES (RTKS) 3.Ras àMAPK MAPKs typically form multi-tiered pathways Stimulus à MAPKKK (MAP3K) à MAPKK (or MEK or MAP2K)à MAPK à Response In mammals, three major MAPK pathways have been identified: ERK p38 JNK RECEPTOR PROTEIN-TYROSINE KINASES (RTKS) 3.Ras àMAPK Mitogen activated protein kinase kinase kinase (MAP3K) Mitogen activated protein kinase kinase (MAP2K) Mitogen activated protein kinase (MAPK) RECEPTOR PROTEIN-TYROSINE KINASES (RTKS) 3.Ras àMAPK  RECEPTOR PROTEIN-TYROSINE KINASES (RTKS) RAS / MAPK SIGNALLING 3.Ras àMAPK RAS – MAP Kinase cascade: Turned on in response to wide variety of signals Key role in regulating cell proliferation and differentiation https://www.youtube.com/watch?v=oDj DUUhGVsI - role for MAPK cascade in cancer  RECEPTOR PROTEIN-TYROSINE KINASES (RTKS) Summary: 3.Ras àMAPK https://youtu.be/r7GoZ9vFCY8 1) 2) 3) 4) Step 1: Binding of growth factor leads to auto-phosphorylation of receptor Step 2: recruitment of Grb2 (adaptor protein) and SOS proteins to receptor Step 3: recruitment of Ras protein and replacement of GDP à GTP Ras = G protein (Guanosine nucleotide binding protein) with GTPase activity Step 4: Activated GTP-Ras binds to and phosphorylates Raf protein à activated Raf = MAP Kinase Kinase Kinase (MAPKKK)  RECEPTOR PROTEIN-TYROSINE KINASES (RTKS) 3.Ras àMAPK 5) Step 5: Activated Raf phosphorylates MEK 6) protein -> activation MEK = MAP Kinase Kinase (MAPKK) Step 6: Activated MEK phosphorylates ERK ½ proteins -> activation ERK ½ = MAP Kinases (MAPK) These proteins can phosphorylate > 160 proteins! 7) Step 7: Activated ERK ½ moves into nucleus and phosphorylates transcription factors -> activation -> altered gene expression Fos and Jun are commonly activated TFs Genes turned on -> cell proliferation (i.e. Cyclin D1, growth factors, cytokines etc.) RECEPTOR PROTEIN-TYROSINE KINASES (RTKS) GCPR à MAPK signalling RTKs à MAPK Signalling Ligand binds GPCR Ligand binds RTK Activates G-protein à 2nd messenger Activates RTK- autophosphorylation à activates a kinase (i.e. PKA, PKC) activation of Ras (related in structure to the G⍺ protein – and is also a GTPase) Triggers activation of MAPKKK Triggers activation of Raf (MAPKKK) Triggers activation of MAPKK Triggers activation of MEK (MAPKK) Triggers activation of ERK (MAPK) à Triggers activation of ERK (MAPK) à Response Response RECEPTOR PROTEIN-TYROSINE KINASES (RTKS) G COUPLED PROTEIN RECEPTORS GPCR-MAPKSIGNALLING G COUPLED PROTEIN RECEPTORS GPCR-MAPK SIGNALLING G COUPLED PROTEIN RECEPTORS GPCR-MAPKSIGNALLING DAG  G COUPLED PROTEIN RECEPTORS GPCR-MAPK SIGNALLING Ligand binds to GPCR Activates G protein a) Activates Gαs Activated AC generates cAMP cAMP activates PKA PKA phsophosylates Rap-1 à activates B-Raf b) Activates Gαq Activated PLCβ cleaves PIP2à IP3, DAG Ca2+ release, activation of PKC PKC phosphorylates and activates Raf-1 B-Raf (A) or Raf-1(B) activates MEK à activates ERK à alters gene trasncription MAPK SIGNALLING ERK as MAPK Response to growth factors cell proliferation, survival, and differentiation JNK and p38 as MAPK Response to inflammatory cytokines and cellular stress (e.g., ultraviolet irradiation) inflammation and cell death (apoptosis) CELL SIGNALLING CONVERGENCE, DIVERGENCEAND CROSS-TALK Convergence : signals from a variety of unrelated receptors, each binding their own ligand, converge to activate a common effector CELL SIGNALLING CONVERGENCE, DIVERGENCEAND CROSS-TALK Divergence : signals from the same ligand/receptor activate a variety of different effectors leading to different responses CELL SIGNALLING CONVERGENCE, DIVERGENCEAND CROSS-TALK Cross-talk : signals are passed back and forth between different pathways Next Lecture Wednesday Nov. 13, at 2:30 pm In STE G0103 Cell Proliferation and Apoptosis