A Novel GTPase-activating Protein for R-Ras

Takaharu Yamamoto,Takeshi Matsui,Masato Nakafuku,Akihiro Iwamatsu,Kozo Kaibuchi

doi:10.1074/jbc.270.51.30557

Takaharu Yamamoto, Takeshi Matsui + Show 3 more

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https://doi.org/10.1074/jbc.270.51.30557

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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.

Highlights

Accumulating evidence indicates that Ras (Ha-Ras, Ki-Ras, N-Ras) serve as downstream molecules for tyrosine kinase-type receptors such as epidermal growth factor receptor, as well as Src family members
A protein with mass of about 98 kDa (p98) was detected in the 200 mM NaCl-eluate from GTP␥S1⁄7GST-R-Ras affinity column but not from glutathione S-transferase (GST) or GDP1⁄7GST-R-Ras affinity column (Fig. 1A). p98 was not detected in the eluate of GTP␥S1⁄7GST-R-RasA64 affinity column
In vitro translated protein migrated with an apparent size of about 98 kDa, which was the same size as native p98, and co-migrated with native p98 purified from GTP␥S1⁄7GST-R-Ras affinity column

Summary

A Novel GTPase-activating Protein for R-Ras*

(Received for publication, August 14, 1995, and in revised form, October 3, 1995). Takaharu Yamamoto‡, Takeshi Matsui‡, Masato Nakafuku‡, Akihiro Iwamatsu§, and Kozo Kaibuchi‡**. 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. 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␥S (guanosine 5؅-(3-O-thio)triphosphate, a nonhydrolyzable GTP analog)1⁄7R-Ras but not to GDP1⁄7R-Ras, GTP␥S1⁄7RRas with a mutation in the effector domain (R-RasA64), GTP␥S1⁄7Ha-Ras, or GTP␥S1⁄7RalA. To understand the specific functions of R-Ras, we attempted to identify proteins that interact with R-Ras in the present study, and have purified an R-Ras-interacting protein with molecular mass of about 98 kDa by GST-R-Ras affinity column chromatography, cloned its cDNA, determined its primary structure, and identified it as a novel R-Ras GAP

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DISCUSSION