3. 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

4. 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)

5. Ras Structure in GTP Bound Form

6. The Ras Pathway

7. Role of GAP in activating Ras

8. Signaling Downstream of Ras

9. Convergence of Pathways

10. Writing the First Chapter…

11. Guanine Nucleotides bound to Ras in Oncogene Transformed Cells

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

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

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

15. Ras Activation by Growth Factors

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

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

18. Effect of GAP on Guanine Nucleotides bound to Ras

19. 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

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

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

22. Over to Parima…