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Xenopus oocyte germinal-vesicle breakdown induced by [Val12]Ras is inhibited by a cytosol-localized Ras mutant
J B Gibbs1, M D Schaber, T L Schofield
1Department of Molecular Biology, Merck Sharp & Dohme Research Laboratories, West Point, PA 19486.
Abstract:
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.).
Insights
Ras signaling in Xenopus oocytes may require a cytosolic factor. This study suggests GTPase-activating protein (GAP) or a competing protein influences Ras function, impacting oocyte development.
Area of Science:
- Cellular signaling and molecular biology
- Ras protein function and regulation
- Xenopus oocyte development
Background:
- Ras GTPase-activating protein (GAP) is implicated as a regulator or target of Ras proteins in mammalian cells and Xenopus oocytes.
- Ras requires plasma membrane localization for function in vertebrate cells, while GAP is primarily cytosolic.
- The role of cytosolic factors in Ras-mediated signaling remains unclear.
Purpose of the Study:
- To investigate whether Ras function in Xenopus oocytes depends on cytosolic factors like GAP.
- To determine the interaction between a modified yeast Ras protein, a cytosolic factor, and Ras signaling pathways.
Main Methods:
- Microinjection of a modified Saccharomyces cerevisiae RAS1 protein ([Leu68]RAS1(term.)) lacking a membrane localization site into Xenopus oocytes.
- Assessing the effect of [Leu68]RAS1(term.) on oocyte germinal-vesicle breakdown stimulated by insulin-like growth factor 1, progesterone, or microinjected [Val12]Ras.
- Evaluating the impact of co-injecting mammalian GAP with [Leu68]RAS1(term.) on Ras-stimulated signaling.
Main Results:
- [Leu68]RAS1(term.) alone did not induce germinal-vesicle breakdown but inhibited signaling by insulin-like growth factor 1 and [Val12]Ras.
- The inhibitory effect of [Leu68]RAS1(term.) was specific, not affecting progesterone-induced germinal-vesicle breakdown.
- Co-injection of GAP with [Leu68]RAS1(term.) partially reversed the inhibition of [Val12]Ras-stimulated germinal-vesicle breakdown.
Conclusions:
- Ras action in Xenopus oocytes may necessitate a cytosolic factor.
- The results suggest that GAP, or another protein that competes with GAP for binding to [Leu68]RAS1(term.), could be this required cytosolic factor.
- This study provides evidence for a role of cytosolic components in regulating Ras signaling pathways in oocytes.