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Modulation of guanine nucleotides bound to Ras by oncogenes, growth factors & GTPase activating protein

Modulation of guanine nucleotides bound to Ras by oncogenes, growth factors & GTPase activating protein. JB Gibbs, MS Marshall, EM Scolnick, RA Dixon, & US Vogel JBC, Nov 1990. Ras. Monomeric G protein (~ 20 KDa in mammalian cells) Membrane bound

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Modulation of guanine nucleotides bound to Ras by oncogenes, growth factors & GTPase activating protein

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  1. Modulation of guanine nucleotides bound to Ras by oncogenes, growth factors & GTPase activating protein JB Gibbs, MS Marshall, EM Scolnick, RA Dixon, & US VogelJBC, Nov 1990

  2. Ras • Monomeric G protein (~ 20 KDa in mammalian cells) • Membrane bound • Linked to various pathways- Tyrosine kinase receptors • Protoncogene in normal cells • Mutation leads to “constitutionally switched on” state – leads cancer • Regulator of Gunaosine Triphosphate (GTP) • Mutation may occur in Ras or in GAP gene (NFL-1)

  3. Ras Structure in GTP Bound Form

  4. The Ras Pathway

  5. Role of GAP in activating Ras

  6. Signaling Downstream of Ras

  7. Convergence of Pathways

  8. Writing the First Chapter…

  9. Guanine Nucleotides bound to Ras in Oncogene Transformed Cells

  10. Quantitation of Guanine Nucleotides bound to Ras in Quiescent & PDGF stimulated NIH3T3 Cells

  11. Dose Dependence of PDGF Activation of Ras Guanine Nucleotide State

  12. Changes in Guanine Nucleotides Complexed to Ras after PDGF Stimulation

  13. Ras Activation by Growth Factors

  14. Overexpression of GAP V8 V11 GAP4 GAP1 GAP3 GAP4 w/ Antibody

  15. Tyrosine Phosphorylation of GAP in response to PDGF Stimulation ~3, w/PT V8 w/ PDGF ~1, w/ GAP Ab GAP4 PDGF Stimulated V8 Unstimulated GAP4 Unstimulated

  16. Effect of GAP on Guanine Nucleotides bound to Ras

  17. Conclusions • In response to PDGF & other chemokines, %GTP bound to Ras increases • With respect to PDGF, this effect is dose dependent • The reaction kinetics shows an initial increase, followed by a decrease in %bound GTP • In GAP overexpressing cell lines, PDGF stimulation leads to tyrosine phosphorylation of GAP

  18. Tentative: PDGF leads to tyrosine phosphorylation of GAP that in turn stimulates Ras

  19. Problems with the Paper • Language • Westerns • Graphs without error bars!

  20. Over to Parima…

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