The Function of the p190 Rho GTPase-activating Protein Is Controlled by Its N-terminal GTP Binding Domain*
- From the ‡Center for Cell Signaling, University of Virginia, Charlottesville, Virginia 22908 and the§Program of Cell and Molecular Biology, University of Vermont, Burlington, Vermont 05405
Abstract
p190 is a GTPase-activating protein (GAP) for the Rho family of GTPases. The GAP domain of p190 is at the C terminus of the protein. At its N terminus, p190 contains a GTP binding domain of unknown significance. We have introduced a mutation (Ser36 → Asn) into this domain of p190 that decreased its ability to bind guanine nucleotide when expressed as a hemagglutinin (HA)-tagged protein in COS cells. In vitro,both the wild type and S36N mutant HA-p190 proteins showed similar GAP activities toward RhoA, but when expressed in NIH 3T3 fibroblasts only wild type p190 appeared able to function as a RhoGAP. Wild type HA-p190 induced a phenotype of rounded cells with long, beaded extensions similar to that seen when Rho function is disrupted by ADP-ribosylation. HA-p190(S36N), although expressed at a similar level to the wild type protein, had no discernible effect on the cells. The beaded extension phenotype induced by wild type HA-p190 required GAP function. A GAP-defective mutant, p190(R1283A), had no effect on cell morphology. Moreover, the beaded extension phenotype could be suppressed by co-expression of a gain-of-function Rho mutant, RhoA(G14V), or Rac mutant, Rac1(G12V). Activation of the Jun kinase (JNK) via muscarinic receptors was inhibited by wild type HA-p190, but JNK activity was enhanced by the S36N mutant. Co-expression of HA-p190 with a fragment containing only the mutated GTP binding domain partially inhibited the beaded extension phenotype, suggesting that it may sequester a factor required for p190 function. Taken together these data demonstrate that within the cell, the Rho/Rac GAP activity of p190 can be regulated by the N-terminal GTP binding domain.
Footnotes
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↵* This work was supported by Grant CA 38888 (to I. G. M.) from the National Cancer Institute, Dept. of Health and Human Services, and by a pilot grant (to D. A. L.) from the Lake Champlain Cancer Research Organization, Vermont.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵¶ To whom correspondence should be addressed. Tel.: 804-982-0074; Fax: 804-924-1236; E-mail: imacara{at}virginia.edu.
- Abbreviations:
- JNK
-
c-Jun N-terminal kinase
- GEF
-
guanine exchange factor
- GAP
-
GTPase-activating protein
- GBD
-
GTP binding domain
- PCR
-
polymerase chain reaction
- HA
-
hemagglutinin
- GFP
-
green fluorescent protein
- GDI
-
guanine dissociation inhibitor
- GST
-
glutathione S-transferase
- MOPS
-
4-morpholinepropanesulfonic acid
- PAGE
-
polyacrylamide gel electrophoresis.
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- Received July 14, 1998.
- Revision received September 25, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
