The identification and characterization of a GDP-dissociation inhibitor (GDI) for the CDC42Hs protein.
The ras-related protein, CDC42Hs, is a 22-kDa GTP-binding protein which is the human homolog of a Saccharomyces cerevisiae yeast-cell-division cycle protein. In attempting to isolate and biochemically characterize mammalian proteins capable of regulating various activities of CDC42Hs, we have identified an activity in bovine brain cytosol which effectively inhibits the dissociation of [3H]GDP from the platelet- or the Spodoptera frugiperda-expressed CDC42Hs protein. The purification of this activity was achieved by a series of steps which included ammonium sulfate fractionation, DEAE-Sephacel, Mono-Q, and Mono-S chromatographies. The purified CDC42Hs regulatory protein has an apparent molecular weight of 28,000, and cyanogen bromide-generated peptide sequences of this protein were identical to sequences from the carboxyl-terminal portion of rho-GDP-dissociation inhibitor (rho-GDI) (Fukumoto, Y., Kaibuchi, K., Hori, Y., Fujioka, H., Araki, S., Ueda, T., Kikuchi, A., and Takai, Y. (1990) Oncogene 5, 1321-1328). In addition, an Escherichia coli-expressed, glutathione S-transferase-rho-GDI fusion protein fully substitutes for the GDI which we have purified from bovine brain in its ability to inhibit GDP dissociation from CDC42Hs. These findings suggest either that a common regulatory protein (GDI) is capable of inhibiting GDP dissociation from the rho and CDC42Hs proteins or that these two GTP-binding proteins interact with GDI proteins of very similar structure. The purified brain GDI protein shows little ability to inhibit GDP dissociation from the E. coli-expressed CDC42Hs and is capable of only a very weak inhibition of the dissociation of [35S]guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) from the Spodoptera frugiperda-expressed CDC42. However, brain GDI very effectively inhibits the ability of the human dbl oncogene product to catalyze GDP dissociation from CDC42Hs. In addition to influencing guanine nucleotide association with CDC42Hs, the purified brain GDI protein also appears to catalyze the dissociation of CDC42Hs from the plasma membranes of human placenta and human epidermoid carcinoma (A431) cells. This effect by the GDI protein is observed whether the membrane-associated CDC42Hs is preincubated with GDP, GTP gamma S, or no guanine nucleotides, and occurs over a similar concentration range as that necessary for the inhibition of the intrinsic GDP dissociation.
Leonard
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Department of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853.
Hart
M J
MJ
Platko
J V
JV
Eva
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A
Henzel
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W
Evans
T
T
Cerione
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RA
D000595:Amino Acid Sequence; D000818:Animals; D001792:Blood Platelets / Q000378:metabolism*; D001921:Brain / Q000378:metabolism; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D003600:Cytosol / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D019204:GTP-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020730:Guanine Nucleotide Dissociation Inhibitors; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009036:Moths; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D020764:cdc42 GTP-Binding Protein; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors
D020730:Guanine Nucleotide Dissociation Inhibitors; D011994:Recombinant Proteins; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D006153:Guanosine Diphosphate; D019204:GTP-Binding Proteins; D020764:cdc42 GTP-Binding Protein