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Differential regulation of rasGAP and neurofibromatosis gene product activities
1Department of Molecular Biology, Cetus Corporation, Emeryville, California 94608.
Abstract:
The ras-encoded p21ras proteins bind GTP very tightly, but catalyse hydrolysis to GDP very slowly. In humans, two genes encode proteins that stimulate this GTPase activity (GAP, or GTPase-activating proteins), one of relative molecular mass 120,000, referred to as p120-GAP, and another NF1-GAP, which is encoded by the neurofibromatosis type-1 gene. Both GAPs are widely expressed in mammalian tissues. Here we show that although they will both bind oncogenic mutants of p21ras, neither will stimulate their GTPase activity. NF1-GAP binds to the p21ras proteins up to 300 times more efficiently than p120-GAP. The two GAPs are inhibited to different extents by certain lipids: micromolar concentrations of arachidonate, phosphatidate and phosphatidylinositol-4,5-bisphosphate affect only NF1-GAP. This inhibition does not compete with p21ras, and lipid-inactivated NF1-GAP can still bind p21ras. We used the detergent dodecyl maltoside, which inhibits only NF1-GAP, to distinguish between the two activities in cell extracts and found both types present together in several mammalian cell lines. In contrast, GAP activity in extracts of Xenopus oocytes was not affected by dodecyl maltoside. By these criteria, the mammalian cells contain both GAP activities and the oocytes have only p120-like GAP activity. These results indicate that more than one GAP regulates p21ras in the same cell.
Insights
Two GTPase-activating proteins (GAPs) regulate p21ras proteins in human cells. NF1-GAP binds p21ras more efficiently but is inhibited by lipids, unlike p120-GAP.
Area of Science:
- Molecular Biology
- Cell Signaling
- Biochemistry
Background:
- p21ras proteins bind GTP tightly but hydrolyze it slowly.
- Two human genes encode GTPase-activating proteins (GAPs): p120-GAP and NF1-GAP (neurofibromatosis type-1 gene).
- Both GAPs are widely expressed in mammalian tissues.
Purpose of the Study:
- To investigate the differential regulation of p21ras by p120-GAP and NF1-GAP.
- To characterize the binding affinities and GTPase-stimulating activities of GAPs towards oncogenic p21ras mutants.
- To determine the effects of lipids and detergents on GAP activity and to differentiate GAP activities in cell extracts.
Main Methods:
- Binding assays to compare NF1-GAP and p120-GAP affinity for p21ras mutants.
- GTPase activity assays to assess the stimulation by GAPs.
- Lipid inhibition assays using arachidonate, phosphatidate, and phosphatidylinositol-4,5-bisphosphate.
- Detergent (dodecyl maltoside) inhibition assays to distinguish GAP activities in cell extracts from mammalian cells and Xenopus oocytes.
Main Results:
- Both p120-GAP and NF1-GAP bind oncogenic p21ras mutants, but neither stimulates their GTPase activity.
- NF1-GAP exhibits up to 300-fold higher binding efficiency to p21ras than p120-GAP.
- Specific lipids inhibit NF1-GAP but not p120-GAP, without affecting p21ras binding.
- Dodecyl maltoside inhibits NF1-GAP but not p120-GAP, allowing differentiation of activities.
- Mammalian cell extracts contain both GAP activities, while Xenopus oocyte extracts possess only p120-like GAP activity.
Conclusions:
- Multiple GAPs, including p120-GAP and NF1-GAP, coexist and regulate p21ras signaling within the same mammalian cell.
- Differential regulation by GAPs is influenced by lipid interactions and protein expression levels.
- The findings highlight the complexity of p21ras pathway regulation and suggest distinct roles for different GAPs.