Identification of the human platelet GTPase activating protein for the CDC42Hs protein

M. J. Hart, K. Shinjo, A. Hall, T. Evans, R. A. Cerione

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Abstract

The CDC42Hs protein appears to be an isoform of the ras-related GTP-binding protein G25K and is an apparent human homolog of the Saccharomyces cerevisiae cell-division-cycle protein, CDC42Sc. In this study, we report the identification of a GTPase-activating protein (GAP) for CDC42Hs from human platelets (designated from here on as CDC42Hs-GAP). The CDC42Hs-GAP activity was solubilized from platelet membranes, recovered through successive chromatography steps (the final step being Mono-Q chromatography), and purified ~3500-fold. The CDC42Hs-GAP activity appeared to correspond to a polypeptide with an apparent M(r) of ~25,000. The GTPase activities of the purified human platelet CDC42Hs, the Escherichia coli-recombinant CDC42Hs, and the Spodoptera frugiperda-recombinant GTP-binding proteins are all stimulated by the CDC42Hs-GAP to identical extents, which indicates that the recombinant CDC42Hs proteins are as effective as the native human platelet protein in coupling to the GAP. However, a mutant form of the E. coli-recombinant CDC42Hs which contains a valine residue at position 12 (CDC42Hs(Val-12)) has a significantly reduced intrinsic GTPase activity (relative to the wild type CDC42Hs(Gly-12)) which is not stimulated by the CDC42Hs-GAP. The CDC42Hs-GAP also does not stimulate the GTPase activities of the ras or rap GTP-binding proteins; however, it is capable of a weak stimulation of the GTPase activity of mammalian rho. Based on the apparent similarities in the molecular size of the CDC42Hs- and rho-GAPs (i.e. 25-30 kDa), and the cross-reactivity of rho with the CDC42Hs-GAP, it seems likely that the CDC42Hs- and rho-GAPs will constitute a specific subclass of the ras-related GAP superfamily.

Original languageEnglish (US)
Pages (from-to)20840-20848
Number of pages9
JournalJournal of Biological Chemistry
Volume266
Issue number31
Publication statusPublished - Jan 1 1991

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Hart, M. J., Shinjo, K., Hall, A., Evans, T., & Cerione, R. A. (1991). Identification of the human platelet GTPase activating protein for the CDC42Hs protein. Journal of Biological Chemistry, 266(31), 20840-20848.