Identification of a putative effector for Cdc42Hs with high sequence similarity to the RasGAP-related protein IQGAP1 and a Cdc42Hs binding partner with similarity to IQGAP2.
Cdc42 is a Ras-related GTP-binding protein that has been implicated in the regulation of the actin cytoskeleton and cell morphology. In this study, we have identified a protein with a molecular mass approximately 180 kDa from rabbit liver cytosol (designated p180), which binds preferentially to the GTP- and guanosine 5'-3-O-(thio)triphosphate-bound forms of Cdc42. Binding of p180 to GTP-bound Cdc42 maintains it in the GTP-bound state. Another cytosolic protein, with an apparent molecular mass of 175 kDa (p175), was also found to interact with Cdc42, but this association showed less dependence on guanine nucleotides. Both p180 and p175 were capable of binding to Rac1 but not to RhoA or Ha-Ras. The limit functional domain of the Cdc42-GAP protein did not compete with p180 or p175 for binding to Cdc42. However, the Cdc42-binding domain from mPAK-3, a member of the PAK (p21 activated kinase) family of serine/threonine kinases, competed with both proteins. The binding of p180 or p175 was inhibited by mutations of the putative effector loop of Cdc42. p180 and p175 also bound less effectively to a Cdc42/Ras chimera in which loop 8 from Ras was substituted for the predicted loop 8 in Cdc42 that includes a 13-amino acid insert present in all Rho family members but absent in Ras. Microsequencing of a p180 peptide revealed 92% identity with the human IQGAP1 protein, while two peptides derived from p175 were 89 and 100% identical to human IQGAP2. These findings identify IQGAP1 and IQGAP2 as a new class of target/effectors that utilize both regions of the switch I domain and an insert region distinct to Rho proteins for binding to Cdc42.
McCallum
S J
SJ
Department of Biochemistry, Veterinary Medical Center, Cornell University, Ithaca, New York 14853, USA.
Wu
W J
WJ
Cerione
R A
RA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D003600:Cytosol / Q000737:chemistry; D004587:Electrophoresis, Agar Gel; D019204:GTP-Binding Proteins / Q000378:metabolism*; D011905:Genes, ras; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000737:chemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins; D020703:ras GTPase-Activating Proteins; D020742:rhoA GTP-Binding Protein
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; C101203:IQ motif containing GTPase activating protein 1; C101002:IQGAP2 protein, human; D020703:ras GTPase-Activating Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein