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

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Presentation transcript:

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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