Regulation of RhoA GTP hydrolysis by the GTPase-activating proteins p190, p50RhoGAP, Bcr, and 3BP-1.
The small GTP-binding protein RhoA becomes inactivated by hydrolyzing bound GTP to GDP through its intrinsic GTPase activity which is further stimulated by a family of Rho GTPase-activating proteins (GAPs). Here we have compared the kinetics of interaction between recombinant RhoA and the RhoGAP domains of p190, p50RhoGAP, Bcr, and 3BP-1. The intrinsic rate of GTP hydrolysis by RhoA is relatively slow when compared to other Rho-family GTPases such as Cdc42 or Rac1 with a rate constant of 0.022 min-1, which can be further stimulated at least 4000-fold by p190 or p50RhoGAP. The RhoGAP domains of Bcr and 3BP-1, which were thought to be inactive toward RhoA, are also found capable of stimulating the GTPase activity of RhoA in a dose-dependent manner. The supreme catalytic activities of p190 and p50RhoGAP toward RhoA reside mostly in their lower Km values (1.79 and 2.83 microM, respectively) which correlate well with their binding affinity for GMP-PNP-bound RhoA (2.18 and 2. 47 microM, respectively), in contrast with Bcr and 3BP-1 which interact with the activated RhoA with much higher Km (89 microM). However, the mechanisms of catalysis by p190 and p50RhoGAP are distinct in at least three aspects: (1) p50RhoGAP displays an effect of product inhibition by binding to the GDP-bound form of RhoA with a Kd of 6 microM in comparison with the Kd for p190 of 33 microM; (2) the Km of p190 increases drastically upon the increase of salt and Mg2+ concentrations, conditions under which only modest changes of Km for p50RhoGAP are observed; and (3) p50RhoGAP remains partially active toward the effector domain mutants of RhoA, Y34K, and T37A, whereas p190 is completely inactive toward Y34K and T37A. These results suggest that there exists a unique mechanism of functional interaction between RhoA and individual RhoGAP which involves distinct structural determinants of the small G-protein to cause the apparent differences in kinetic properties.
Zhang
B
B
Department of Biochemistry, University of Tennessee, Memphis, Tennessee 38163, USA.
Zheng
Y
Y
D002352:Carrier Proteins / Q000378:metabolism; D004268:DNA-Binding Proteins; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D009687:Nuclear Proteins / Q000378:metabolism; D015513:Oncogene Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D011505:Protein-Tyrosine Kinases; D011518:Proto-Oncogene Proteins; D051562:Proto-Oncogene Proteins c-bcr; D011994:Recombinant Proteins; D012097:Repressor Proteins; D020742:rhoA GTP-Binding Protein
C074560:ARHGAP35 protein, human; C513043:ARHGAP5 protein, human; C513041:Arhgap35 protein, mouse; C513042:Arhgap5 protein, mouse; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D009687:Nuclear Proteins; D015513:Oncogene Proteins; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; C094762:Sh3bp1 protein, mouse; C085379:rho GTPase-activating protein; D006160:Guanosine Triphosphate; D011505:Protein-Tyrosine Kinases; C496613:BCR protein, human; D051562:Proto-Oncogene Proteins c-bcr; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein