Biochemical comparisons of the Saccharomyces cerevisiae Bem2 and Bem3 proteins. Delineation of a limit Cdc42 GTPase-activating protein domain.
The Bem2 and Bem3 proteins, which appear to play roles in the regulation of bud site formation in Saccharomyces cerevisiae, show striking homology to a number of proteins that compose a family of GTPase-activating proteins (GAPs) for the rho-subgroup of ras-related GTP-binding proteins. These members include human platelet GAP for Cdc42Hs (the human homolog of a S. cerevisiae GTP-binding protein that regulates bud site assembly), the break point cluster region protein, the brain protein chimerin, the 85-kDa regulatory subunit ( p85) of the phosphatidylinositol 3-kinase, and the ras-GAP-binding protein (p190). A fusion protein composed of the glutathione S-transferase protein and the rho-GAP homology region of Bem3 (designated GST-Bem3) stimulates the GTPase activity of the wild-type Cdc42Hs protein (Cdc42HsGly-12), but has no stimulatory effect on a GTPase-defective mutant (Cdc42HsVal-12), whereas a GST-Bem2 fusion protein does not stimulate the GTPase activity of either form of Cdc42Hs. We have compared the ability of GST-Bem3 to serve as a GAP for Cdc42Hs relative to other members of the rho-GAP subfamily and found the following order of potency: human platelet Cdc42Hs GAP > p190 > Bem3 > break point cluster region protein, whereas p85, like Bem2, shows no GAP activity or any ability to bind to the GTP-bound form of Cdc42Hs. We have taken advantage of the functional specificity exhibited by Bem3 (versus Bem2) in using Bem2/Bem3 chimeras, as well as different deletion mutant versions of the Bem3 protein, to delineate the limits of a functional Cdc42 GAP domain. The results of this study indicate that the carboxyl-terminal approximately 224 amino acids (which contain three regions of homology to the other members of the rho-GAP family) represent a "limit GAP." The first two appear to be important for binding to Cdc42Hs and for partial GAP activity.[1]References
- Biochemical comparisons of the Saccharomyces cerevisiae Bem2 and Bem3 proteins. Delineation of a limit Cdc42 GTPase-activating protein domain. Zheng, Y., Hart, M.J., Shinjo, K., Evans, T., Bender, A., Cerione, R.A. J. Biol. Chem. (1993) [Pubmed]
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