Identification of the human platelet GTPase activating protein for the CDC42Hs protein.
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 approximately 3500-fold. The CDC42Hs-GAP activity appeared to correspond to a polypeptide with an apparent Mr of approximately 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 (CDC42HsVal-12) has a significantly reduced intrinsic GTPase activity (relative to the wild type CDC42HsGly-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.
Department of Biochemistry, Cellular and Molecular Biology, Cornell University, Ithaca, New York 14853.
M J
MJ
Hart
K
K
Shinjo
A
A
Hall
T
T
Evans
R A
RA
Cerione
D001792:Blood Platelets / Q000201:enzymology*; D002845:Chromatography; D003001:Cloning, Molecular; D004789:Enzyme Activation; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D011506:Proteins / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D020764:cdc42 GTP-Binding Protein; D020703:ras GTPase-Activating Proteins
D020690:GTPase-Activating Proteins; D008565:Membrane Proteins; D011506:Proteins; D011994:Recombinant Proteins; D020703:ras GTPase-Activating Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020764:cdc42 GTP-Binding Protein