A novel mammalian Ras GTPase-activating protein which has phospholipid-binding and Btk homology regions.

Abstract

We have previously purified a novel GTPase-activating protein (GAP) for Ras which is immunologically distinct from the known Ras GAPs, p120GAP and neurofibromin (M. Maekawa, S. Nakamura, and S. Hattori, J. Biol. Chem. 268:22948-22952, 1993). On the basis of the partial amino acid sequence, we have obtained a cDNA which encodes the novel Ras GAP. The predicted protein consists of 847 amino acids whose calculated molecular mass, 96,369 Da, is close to the apparent molecular mass of the novel Ras GAP, 100 kDa. The amino acid sequence shows a high degree of similarity to the entire sequence of the Drosophila melanogaster Gap1 gene. When the catalytic domain of the novel GAP was compared with that of Drosophila Gap1, p120GAP, and neurofibromin, the highest degree of similarity was again observed with Gap1. Thus, we designated this gene Gap1m, a mammalian counterpart of the Drosophila Gap1 gene. Expression of Gap1m was relatively high in brain, placenta, and kidney tissues, and it was expressed at low levels in other tissues. A recombinant protein consisting of glutathione-S-transferase and the GAP-related domain of Gap1m stimulated GTPase of normal Ras but not that of Ras having valine at the 12th residue. Expression of the same region in Saccharomyces cerevisiae suppressed the ira2- phenotype. In addition to the GAP catalytic domain, Gap1m has two domains with sequence closely related to those of the phospholipid-binding domain of synaptotagmin and a region with similarity to the unique domain of Btk tyrosine kinase. These results clearly show that Gap1m is a novel Ras GAP molecule of mammalian cells.

The Drosophila Bruton’s Tyrosine Kinase (Btk) Homolog Is Required for Adult Survival and Male Genital Formation

“…Among these four families, the Bruton's tyrosine kinase (Btk) family is unique in that the kinases in this family have extended N-terminal sequences that are called collectively the pleckstrin homology (PH) domain (17,18). This domain is known to bind the ␤␥ subunits of heterotrimeric G proteins as well as membrane lipids such as phosphatidylinositol-4,5-bisphosphate (16,25,27,29). Another feature unique to the Btk family resides in the restricted localization of its members in mammalian tissues.…”

Reduced expression of neurofibromin in human meningiomas

“…2 can be explained by the presence of maltoside-susceptible GAP activity that is distinct from neurofibromin. It is not known, however, whether other p21 ras-specific GAPs are susceptible to inhibition by maltoside (Maekawa et al, 1994;Weisbach et al, 1994).…”

“…2 and no. 18), their interpretation is complicated by recent identification of other p21 ras-specific GAP proteins in the brain tissue (Maekawa et al, 1994;Weisbach et al, 1994;Baba et al, 1995). At present, it is not known whether these proteins are expressed in meningiomas and whether their GAP activity is susceptible to inhibition by maltoside.…”

“…To eliminate the possibility that other p21 ras-specific GAP molecules (Maekawa et al, 1994;Weisbach et al, 1994;Baba et al, 1995), besides neurofibromin and p120 GAP, contribute to the total and maltoside-sensitive GAP activity of meningioma lysates, neurofibromin and p120 GAP were immunoprecipitated from the soluble fraction of meningiomas, and the GAP activity of immunoprecipitated proteins was determined in p21 ras GTPase assay.…”

“…The NFl gene encodes a 2818 amino acid protein, designated neurofibromin. A 360 amino acid region of neurofibromin shows significant homology to the catalytic domain of the mammalian p21 ras-specific 120-kDa GTPase-activating protein (p120 GAP), yeast equivalents IRAI and IRA2 proteins and recently identified mammalian p21 ras-specific GAPs (Ballester et al, 1990;Maekawa et al, 1994;Weisbach et al, 1994;Baba et al, 1995). This GAP-related domain (GRD) of neurofibromin, as well as the full-length neurofibromin, can negatively regulate p21 ras in vitro by stimulating its weak intrinsic GTPase activity (Xu et al, 1990;Golubic et al, 1992).…”