title: Rho isoform-specific interaction with IQGAP1 promotes breast cancer cell proliferation and migration.
abstract: We performed a proteomics screen for Rho isoform-specific binding proteins to clarify the tumor-promoting effects of RhoA and C that contrast with the tumor-suppressive effects of RhoB. We found that the IQ-motif-containing GTPase-activating protein IQGAP1 interacts directly with GTP-bound, prenylated RhoA and RhoC, but not with RhoB. Co-immunoprecipitation of IQGAP1 with endogenous RhoA/C was enhanced when RhoA/C were activated by epidermal growth factor (EGF) or transfection of a constitutively active guanine nucleotide exchange factor (GEF). Overexpression of IQGAP1 increased GTP-loading of RhoA/C, while siRNA-mediated depletion of IQGAP1 prevented endogenous RhoA/C activation by growth factors. IQGAP1 knockdown also reduced the amount of GTP bound to GTPase-deficient RhoA/C mutants, suggesting that IQGAP enhances Rho activation by GEF(s) or stabilizes Rho-GTP. IQGAP1 depletion in MDA-MB-231 breast cancer cells blocked EGF- and RhoA-induced stimulation of DNA synthesis. Infecting cells with adenovirus encoding constitutively active RhoA(L63) and measuring absolute amounts of RhoA-GTP in infected cells demonstrated that the lack of RhoA(L63)-induced DNA synthesis in IQGAP1-depleted cells was not due to reduced GTP-bound RhoA. These data suggested that IQGAP1 functions downstream of RhoA. Overexpression of IQGAP1 in MDA-MB-231 cells increased DNA synthesis irrespective of siRNA-mediated RhoA knockdown. Breast cancer cell motility was increased by expressing a constitutively-active RhoC(V14) mutant or overexpressing IQGAP1. EGF- or RhoC-induced migration required IQGAP1, but IQGAP1-stimulated migration independently of RhoC, placing IQGAP1 downstream of RhoC. We conclude that IQGAP1 acts both upstream of RhoA/C, regulating their activation state, and downstream of RhoA/C, mediating their effects on breast cancer cell proliferation and migration, respectively.
authors.affiliation: Department of Medicine and Cancer Center, University of California, San Diego, La Jolla, California 92093, USA.
authors.forename: Darren E
authors.initials: DE
authors.lastname: Casteel
authors.forename: Stephanie
authors.initials: S
authors.lastname: Turner
authors.forename: Raphaela
authors.initials: R
authors.lastname: Schwappacher
authors.forename: Hema
authors.initials: H
authors.lastname: Rangaswami
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Su-Yuo
authors.forename: Shunhui
authors.initials: S
authors.lastname: Zhuang
authors.forename: Gerry R
authors.initials: GR
authors.lastname: Boss
authors.forename: Renate B
authors.initials: RB
authors.lastname: Pilz
mesh_terms: D001667:Binding, Competitive; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation; D005260:Female; D006160:Guanosine Triphosphate / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D011485:Protein Binding; D017368:Protein Prenylation; D040901:Proteomics; D034622:RNA Interference; D020703:ras GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism*; D020741:rho GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*; D020743:rhoB GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*; D000075024:rhoC GTP-Binding Protein
chemical_list: C101203:IQ motif containing GTPase activating protein 1; D020703:ras GTPase-Activating Proteins; D006160:Guanosine Triphosphate; C066690:RHOC protein, human; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein; D020743:rhoB GTP-Binding Protein; D000075024:rhoC GTP-Binding Protein