Characterization of the interactions between the small GTPase Cdc42 and its GTPase-activating proteins and putative effectors. Comparison of kinetic properties of Cdc42 binding to the Cdc42-interactive domains.
The small GTPase Cdc42 interacts with multiple factors to transduce diverse intracellular signals. The factors that preferentially recognize the GTP-bound, active state of Cdc42 include a panel of GTPase-activating proteins (GAPs), the Cdc42/Rac interactive binding (CRIB) motif-containing molecules, and the RasGAP domain containing IQGAP1 and IQGAP2. In the present study, we have determined the kinetic parameters underlying the functional interactions between the Cdc42-binding domains of some of these factors and Cdc42 by monitoring the continuous release of gammaPi and have compared the ability of the domains to bind to Cdc42. The catalytic efficiencies (Kcat/Km) of the GAP domains of Bcr, 3BP-1, and p190 on Cdc42 are found to be 60-, 160-, and over 500-fold less than that of Cdc42GAP, respectively, and the differences are due, to a large part, to differences in Km. The Km values of the GAP domains compare well to the binding affinity to the guanylyl imidodiphosphate-bound Cdc42, suggesting a rapid equilibrium reaction mechanism. The affinity of the Cdc42-binding domains of the CRIB motif of Wiskott-Aldrich Syndrome protein and p21(cdc42/rac)-activated kinase 1, and the RasGAP-related domain of IQGAP1, which all inhibit the intrinsic rate of GTP hydrolysis of Cdc42, are found to be 4, 0.7, and 0.08 microM, respectively. These quantitative analysis provide insight that Cdc42GAP functions as an effective negative regulator of Cdc42 by fast, relatively tight binding to the GTP-bound Cdc42, whereas IQGAP1 interacts with Cdc42 as a putative effector with over 10-fold higher affinity than the CRIB domains and GAPs, and suggest that various GAPs and effectors employ distinct mechanism to play roles in Cdc42-mediated signaling pathways.
Zhang
B
B
Department of Biochemistry, University of Tennessee, Memphis, Tennessee 38163, USA.
Wang
Z X
ZX
Zheng
Y
Y
D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism*; D004789:Enzyme Activation; 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; D010750:Phosphoproteins / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D012097:Repressor Proteins; D020764:cdc42 GTP-Binding Protein; D054462:p21-Activated Kinases; D020703:ras GTPase-Activating Proteins; D020666:ras-GRF1; D018909:src Homology Domains
C074560:ARHGAP35 protein, human; C513043:ARHGAP5 protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; C101203:IQ motif containing GTPase activating protein 1; C101002:IQGAP2 protein, human; D009687:Nuclear Proteins; D010750:Phosphoproteins; D011506:Proteins; C513039:RASGRF1 protein, human; D012097:Repressor Proteins; D020703:ras GTPase-Activating Proteins; D020666:ras-GRF1; C085379:rho GTPase-activating protein; D006160:Guanosine Triphosphate; C515911:PAK3 protein, human; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020764:cdc42 GTP-Binding Protein