The function of the p190 Rho GTPase-activating protein is controlled by its N-terminal GTP binding domain.

Tatsis N, Lannigan DA, Macara IG
J Biol Chem. 1998 273 (51): 34631-8

PMID: 9852136 · DOI:10.1074/jbc.273.51.34631

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.

MeSH Terms (27)

3T3 Cells Amino Acid Sequence Amino Acid Substitution Animals Asparagine Binding Sites Calcium-Calmodulin-Dependent Protein Kinases Carbachol Conserved Sequence COS Cells DNA-Binding Proteins Enzyme Activation GTPase-Activating Proteins Guanine Nucleotide Exchange Factors Guanosine Triphosphate JNK Mitogen-Activated Protein Kinases Kinetics Mice Mitogen-Activated Protein Kinases Mutagenesis, Site-Directed Nuclear Proteins Phosphoproteins Receptors, Muscarinic Recombinant Proteins Repressor Proteins Serine Transfection

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