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Ras-GTP-MAPK Signaling Cascade: Cellular Response Activation

This text explains the activation of the Ras-GTP-MAPK signaling cascade, a crucial pathway in cell communication. It describes the role of signal molecules, activated receptors, and the phosphorylation cascade that leads to cellular response. The text also covers the activation and inactivation of various proteins involved in this pathway.

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Ras-GTP-MAPK Signaling Cascade: Cellular Response Activation

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  1. Signal molecule Activated Ras-GTP A G-Protein Receptor Inactive MAPKKK 1 Active MAPKKK 1 Inactive MAPKK 2 ATP Phosphorylation cascade P ADP Active MAPKK 2 PP P Inactive MAPK 3 ATP P ADP Active MAPK 3 PP i Inactive protein ATP P ADP Active protein Cellular response PP P  i BCOR 011 Cell Communication II Lect 19 And they tell 2 friends And they tell 2 friends And they tell 2 friends… Chapt 11 10/17/05 i Protein Phosphatases P

  2. Lecture Outline • Finish Trimeric G-Protein: Phospholipase C • -DAG, IP3 second messengers • Protein Kinase C, Ca++ release • Nitric Oxide, cGMP activation • - consequences of failing to inactivate • Tyrosine Kinase Receptor • - RAS G-protein, MAP Kinase Cascade • - Cell Cycle Control, Gene Control • Internal Receptors – Steroid Receptor • - Gene Regulation

  3. TWO subclasses of trimeric G-protein-activated signal transduction pathways: A. target protein adenylate cyclase cAMP-> PKA B. target protein phospholipase C

  4. target effector enzyme is Phospholipase C PLC cleaves a membrane phospholipid (Phoshatidyl inositol) to two 2nd Messengers: Inositol-1,4,5-Trisphosphate (InsP3) & Diacylglycerol (DAG)

  5. DAG Lipid Soluble InsP3 Water Soluble PIP2

  6. DAG Activates Protein Kinase C (Starts Cascade) InsP3 Ligand for ER ligand- gated Ca++ channels  Ca++ levels

  7. 2 3 1 4 5 6 A signal molecule binds to a receptor, leading to activation of phospholipase C. DAG functions as a second messenger in other pathways. Phospholipase C cleaves a plasma membrane phospholipid called PIP2 into DAG and IP3. EXTRA- CELLULAR FLUID Signal molecule (first messenger) G protein DAG GTP PIP2 G-protein-linked receptor Phospholipase C IP3 (second messenger) IP3-gated calcium channel Endoplasmic reticulum (ER) Various proteins activated Cellularresponse Ca2+ Ca2+ (second messenger) The calcium ions activate the next protein in one or more signaling pathways. IP3 quickly diffuses through the cytosol and binds to an IP3– gated calcium channel in the ER membrane, causing it to open. Calcium ions flow out of the ER (down their con- centration gradient), raising the Ca2+ level in the cytosol. Figure 11.12

  8. Response: Protein Kinase C phosphorylates target proteins (ser & thr) cell growth regulation of ion channels cytoskeleton increases cell pH Protein secretion Ca++ Binds & activates calmodulin Calmodulin-binding proteins activated (kinases & phosphatases)

  9. Shut Off • - remember, signal needs to be transient • Must shut off cascade: • removal of ligand, • (self)-hydrolysis of GTP, • remove IP3, protein phosphatases, Ca++ ion pumps

  10. Direct activation - signal: nitric oxide (NO) It’s a gas! lipid soluble, binds directly to activate enzyme made by: endothelial cells (line blood vessels) NO synthase target: guanylyl cyclase GTP->cGMP response: relaxes smooth muscle vessels dilate, blood flow +NO arginine citrulline

  11. Shut off by cGMP phosphodiesterase target

  12. Nitroglycerine– taken to relieve angina 1977 –Ferid Murad Nitroglycerine acts to elict release of NO, relaxes cardiac muscle 1998 Nobel Prize – Murad, Furchgott, Ignarro

  13. Nitro glycerine Shut off By cGMP phosphodiesterase VIAGRA

  14. Sildenafil citrate (VIAGRA)

  15. cell-surface receptors a. ion-channel-linked b. G-protein-linked • Protein-kinase associated • (enzyme-linked)

  16. Mitogen Activated Protein-Kinase associated receptors receptor has enzymatic activity -only when ligand binds ligand: allosteric effector -> conformational change receptor tyrosine kinases Growth & differentiation control of cell cycle control of gene expression bad news when messed up - cancer

  17. Growth factor Reception Receptor Phosphorylation cascade Transduction CYTOPLASM Inactive transcription factor Active transcription factor Response P DNA Gene mRNA NUCLEUS Figure 11.14 Growth Factor “Mitogen-activated” Signaling Cascade

  18. EGFR – epidermal growth factor receptor • Monomer receptor • 1 transmembrane • segment • Ligand binds – • receptors dimerize • Activates Tyr kinase • autophosphorylate • Scaffold to Bind/activate • Target proteins EGF – growth factor or mitogen • GDP/GTP exchange • Activate Ras G-protein “Mitosis-generator”

  19. Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Signal-binding site Signalmolecule Signal molecule Helix in the Membrane Receptor tyrosine kinases Tyr Tyr Tyr Tyr Tyrosines Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Receptor tyrosinekinase proteins(inactive monomers) Dimer CYTOPLASM Figure 11.7 Activatedrelay proteins Cellularresponse 1 P P Tyr P Tyr P P P P Tyr Tyr P Cellularresponse 2 P P P Tyr Tyr P 6 ATP 6 ADP Activated tyrosine- kinase regions (unphosphorylated dimer) Fully activated receptor tyrosine-kinase (phosphorylated dimer) Inactiverelay proteins

  20. Receptor tyrosine kinases can activate ras ras is a monomeric G-protein “molecular switch”

  21. Signal molecule A relay molecule activates protein kinase 1. MAP Kinase Kinase Kinase Receptor Activated relay molecule 4 1 5 3 2 MAP Kinase Kinase Kinase Inactive protein kinase 1 Active protein kinase 1 transfers a phosphate from ATP to an inactive molecule of protein kinase 2, thus activating this second kinase. Active protein kinase 1 MAP Kinase Kinase Active protein kinase 2 then catalyzes the phos- phorylation (and activation) of protein kinase 3. Inactive protein kinase 2 ATP MAP Kinase Kinase Phosphorylation cascade P ADP Active protein kinase 2 PP P i Enzymes called protein phosphatases (PP) catalyze the removal of the phosphate groups from the proteins, making them inactive and available for reuse. Finally, active protein kinase 3 phosphorylates a protein (pink) that brings about the cell’s response to the signal. Inactive protein kinase 3 MAP Kinase ATP P ADP Active protein kinase 3 MAP Kinase PP P i Inactive protein ATP P ADP Active protein Cellular response PP P  i Ras-GTP A phosphorylation cascade Figure 11.8

  22. Ras activation sets off aphosphorylation cascade Mitogen Activated Protein Kinases MAPKs 10 100 MAPKKK MAPKK 1,000 Controls: -Transcription Factors -Translation Factors -Cell Division MAPK 100,000

  23. How do you turn it off? GTPase (GTP->GDP + P) phosphatases molecular switch on internal timer If timer broken – on all the time

  24. PROBLEMS IN CANCER: - broken ras – won’t shut off • Broken receptor – thinks ligand there • even when it isn’t • broken MAPK – on all the time, even when • not phosphorylated RESULT: continuous signal for cell to divide

  25. Specific signal transduction cascades: • receptor-mediated cell-surface • receptor-mediated intracellular Lipid soluble things: steroid hormones

  26. EXTRACELLULAR FLUID Hormone (testosterone) Steroid Hormone Plasma membrane The steroid hormone testosterone passes through the plasma membrane. Lipid soluble Crosses membranes Testosterone binds to a receptor protein in the cytoplasm, activating it. Receptor protein Binds intracellular receptor In cytosol Hormone- receptor complex The hormone- receptor complex enters the nucleus and binds to specific genes. steroid receptor Complex Changes shape Releases from tether protein Travels to Nucleus The bound protein stimulates the transcription of the gene into mRNA. DNA mRNA steroid receptor Binds DNA Turns genes ON NUCLEUS New protein The mRNA is translated into a specific protein. CYTOPLASM Figure 11.6

  27. The Specificity of Cell Signaling • The different combinations of proteins in a cell • Give the cell great specificity in both the signals it detects and the responses it carries out Same hormone can give different responses in different cells

  28. Signalmolecule Receptor Relaymolecules Pathway “cross-talk” Response 3 Response 2 Response 1 Cell B. Pathway branches, leading to two responses Cell A. Pathway leads to a single response Figure 11.15 Activationor inhibition Response 4 Response 5 Cell C. Cross-talk occurs between two pathways Cell D. Different receptor leads to a different response

  29. Specific pathways 1. Cell-surface receptor mediated a. ion-channel-linked b. trimeric G-protein-linked (i) adenylyl cyclasecAMP ->protein kinase A (ii) phospholipase C InsP3, DAG, Ca++, protein kinase C Direct activation - NO c. protein kinase-associated (enzyme-linked) Receptor tyrosine kinase monomeric G-protein (ras), MAPKs 2. Intracellular receptors – steroid hormones, dioxin

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