Modulation of guanine nucleotides bound to Ras by oncogenes, growth factors

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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

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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)

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Ras Structure in GTP Bound Form
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The Ras Pathway
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Role of GAP in activating Ras
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Signaling Downstream of Ras
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Convergence of Pathways
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Writing the First Chapter
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Guanine Nucleotides bound to Ras in Oncogene
Transformed Cells
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Quantitation of Guanine Nucleotides bound to Ras
in Quiescent PDGF stimulated NIH3T3 Cells
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Dose Dependence of PDGF Activation of Ras Guanine
Nucleotide State
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Changes in Guanine Nucleotides Complexed to Ras
after PDGF Stimulation
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Ras Activation by Growth Factors
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Overexpression of GAP
V8
V11
GAP4
GAP1
GAP3
GAP4 w/ Antibody
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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
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Effect of GAP on Guanine Nucleotides bound to Ras
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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

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• Tentative PDGF leads to tyrosine phosphorylation
  of GAP that in turn stimulates Ras

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Problems with the Paper

• Language
• Westerns
• Graphs without error bars!

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