title: The function of the p190 Rho GTPase-activating protein is controlled by its N-terminal GTP binding domain.
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.
authors.affiliation: Center for Cell Signaling, University of Virginia, Charlottesville, Virginia 22908, USA.
authors.forename: N
authors.initials: N
authors.lastname: Tatsis
authors.forename: D A
authors.initials: DA
authors.lastname: Lannigan
authors.forename: I G
authors.initials: IG
authors.lastname: Macara
mesh_terms: D016475:3T3 Cells; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001216:Asparagine; D001665:Binding Sites; D019556:COS Cells; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology; D017124:Conserved Sequence; D004268:DNA-Binding Proteins; D004789:Enzyme Activation; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D006160:Guanosine Triphosphate / Q000378:metabolism*; D048031:JNK Mitogen-Activated Protein Kinases; D007700:Kinetics; D051379:Mice; D020928:Mitogen-Activated Protein Kinases; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012097:Repressor Proteins; D012694:Serine; D014162:Transfection
chemical_list: C513041:Arhgap35 protein, mouse; C513042:Arhgap5 protein, mouse; D004268:DNA-Binding Proteins; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D009687:Nuclear Proteins; D010750:Phosphoproteins; D011976:Receptors, Muscarinic; D011994:Recombinant Proteins; D012097:Repressor Proteins; D012694:Serine; D001216:Asparagine; D006160:Guanosine Triphosphate; D002217:Carbachol; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases