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 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)
Ras Structure in GTP Bound Form
The Ras Pathway
Role of GAP in activating Ras
Signaling Downstream of Ras
Convergence of Pathways
Writing the First Chapter…
Guanine Nucleotides bound to Ras in Oncogene Transformed Cells
Quantitation of Guanine Nucleotides bound to Ras in Quiescent & PDGF stimulated NIH3T3 Cells
Dose Dependence of PDGF Activation of Ras Guanine Nucleotide State
Changes in Guanine Nucleotides Complexed to Ras after PDGF Stimulation
Ras Activation by Growth Factors
Overexpression of GAP GAP4 GAP1 GAP3 V8 V11 GAP4 w/ Antibody
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
Effect of GAP on Guanine Nucleotides bound to Ras
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
Tentative: PDGF leads to tyrosine phosphorylation of GAP that in turn stimulates Ras
Problems with the Paper Language Westerns Graphs without error bars!
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