Xenopus oocyte germinal-vesicle breakdown induced by [Val12]Ras is inhibited by a cytosol-localized Ras mutant.
The GTPase-activating protein (GAP) has been postulated to function either as a negative regulator or as a possible target protein of Ras in mammalian cells and Xenopus oocytes. Ras must be localized in the plasma membrane of vertebrate cells to function, but GAP is localized in the cytosol. To test whether Ras function depends on a cytosolic factor such as GAP, we microinjected into Xenopus oocytes a form of Saccharomyces cerevisiae RAS1 ([Leu68]RAS1 terminated at residue 185, called [Leu68]RAS1(term.] that lacks the consensus membrane localization site, does not respond to GAP in a GTPase assay, but binds to GAP 100-fold more tightly than [Val12]Ras. [Leu68]RAS1(term.) alone did not stimulate oocyte germinal-vesicle breakdown. Instead, [Leu68]RAS1(term.) was observed to inhibit the action of insulin-like growth factor 1 or microinjected [Val12]Ras but not the action of progesterone as monitored by germinal-vesicle breakdown. Coinjection of purified mammalian GAP with [Leu68]RAS1(term.) reduced the inhibition of [Val12]Ras-stimulated germinal-vesicle breakdown. The results raise the possibility that a cytosolic factor is required for the action of [Val12]Ras in Xenopus oocytes and that this factor is either GAP or another protein with which GAP can compete for binding to [Leu68]RAS1(term.).
Gibbs
J B
JB
Department of Molecular Biology, Merck Sharp & Dohme Research Laboratories, West Point, PA 19486.
Schaber
M D
MD
Schofield
T L
TL
Scolnick
E M
EM
Sigal
I S
IS
D000818:Animals; D002478:Cells, Cultured; D003600:Cytosol / Q000502:physiology; D005260:Female; D020558:GTP Phosphohydrolases / Q000378:metabolism; D020690:GTPase-Activating Proteins; D011905:Genes, ras; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008962:Models, Theoretical; D009154:Mutation; D009865:Oocytes / Q000502:physiology* / Q000648:ultrastructure; D011506:Proteins / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras); D014633:Valine; D014982:Xenopus laevis; D020703:ras GTPase-Activating Proteins
D020690:GTPase-Activating Proteins; D008565:Membrane Proteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D020703:ras GTPase-Activating Proteins; D020558:GTP Phosphohydrolases; D016283:Proto-Oncogene Proteins p21(ras); D014633:Valine