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A novel GTPase-activating protein for R-Ras
T Yamamoto1, T Matsui, M Nakafuku
1Division of Signal Transduction, Nara Institute of Science and Technology, Ikoma, Japan.
Abstract:
R-Ras, belonging to the Ras small GTP-binding protein superfamily, has been implicated in regulation of various cell functions such as gene expression, cell proliferation, and apoptotic cell death. In the present study, we purified an R-Ras-interacting protein with molecular mass of about 98 kDa (p98) from bovine brain cytosol by glutathione S-transferase (GST)-R-Ras affinity column chromatography. This protein bound to GTP gamma S (guanosine 5'-(3-O-thio)triphosphate, a nonhydrolyzable GTP analog).R-Ras but not to GDP.R-Ras, GTP gamma S.R-Ras with a mutation in the effector domain (R-RasA64), GTP gamma S.Ha-Ras, or GTP gamma S.RalA. We obtained a cDNA encoding p98 on the basis of its partial amino acid sequences. The predicted protein consists of 834 amino acids whose calculated mass, 95,384 Da, is close to the apparent molecular mass of p98. The amino acid sequence shows a high degree of sequence similarity to the entire sequence of Gap1m, one of the GTPase-activating proteins (GAP) for Ha-Ras. A recombinant protein consisting of the GAP-related domain of p98 fused to maltose-binding protein stimulated GTPase activity of R-Ras, and showed a weak effect on that of Ha-Ras but not that of Rap1 or Rho. These results clearly indicate that p98 is a novel GAP for R-Ras. Thus, we designated this protein as R-Ras GAP.
Insights
Researchers identified a novel GTPase-activating protein (GAP) for R-Ras, a key regulator in cell functions. This R-Ras GAP protein specifically binds to GTP-bound R-Ras, highlighting its role in cellular signaling pathways.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- R-Ras is a small GTP-binding protein involved in regulating gene expression, cell proliferation, and apoptosis.
- Understanding R-Ras regulation is crucial for deciphering its role in various cellular processes.
Purpose of the Study:
- To identify and characterize novel proteins that interact with R-Ras.
- To determine the function of an isolated R-Ras-interacting protein.
Main Methods:
- Affinity chromatography using glutathione S-transferase (GST)-R-Ras.
- GTP binding assays with various Ras proteins and GTP analogs.
- cDNA sequencing and recombinant protein expression.
- GTPase activity assays.
Main Results:
- An approximately 98 kDa protein (p98) was purified from bovine brain cytosol.
- p98 specifically bound to GTPγS-bound R-Ras, but not GDP-bound R-Ras or other Ras family members.
- The cDNA sequence predicted an 834-amino acid protein with high similarity to known GTPase-activating proteins (GAPs).
- A recombinant fragment of p98 stimulated the GTPase activity of R-Ras, confirming its GAP function.
Conclusions:
- The purified protein, p98, is a novel GTPase-activating protein (GAP) for R-Ras.
- This novel R-Ras GAP plays a significant role in modulating R-Ras signaling pathways.
- The discovery provides new insights into the regulatory mechanisms of R-Ras in cellular functions.