title: Calmodulin modulates the interaction between IQGAP1 and Cdc42. Identification of IQGAP1 by nanoelectrospray tandem mass spectrometry.
abstract: Calmodulin regulates numerous fundamental metabolic pathways by binding to and modulating diverse target proteins. In this study, calmodulin-binding proteins were isolated from normal (Hs578Bst) and malignant (MCF-7) human breast cell lines with calmodulin-Sepharose and analyzed by SDS-polyacrylamide gel electrophoresis. A protein that migrated at approximately 190 kDa bound to calmodulin in the presence of Ca2+ and was the only calmodulin-binding protein detected in the absence of Ca2+. This 190-kDa protein was identified as IQGAP1 by nanoelectrospray mass spectrometry and collision-induced dissociation tandem mass spectrometry. IQGAP1 coimmunoprecipitated with calmodulin from lysates of MCF-7 cells. Moreover, overlay with 125I-calmodulin confirmed that IQGAP1 binds directly to calmodulin. Analysis of the functional effects of the interaction revealed that Ca2+/calmodulin disrupted the binding of purified IQGAP1 to the Ras-related protein Cdc42 in a concentration-dependent manner. These data clearly identify IQGAP1 as the predominant calmodulin-binding protein in Ca2+-free breast cell lysates and reveal that calmodulin modulates the interaction between IQGAP1 and Cdc42.
authors.affiliation: Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: J L
authors.initials: JL
authors.lastname: Joyal
authors.forename: R S
authors.initials: RS
authors.lastname: Annan
authors.forename: Y D
authors.initials: YD
authors.lastname: Ho
authors.forename: M E
authors.initials: ME
authors.lastname: Huddleston
authors.forename: S A
authors.initials: SA
authors.lastname: Carr
authors.forename: M J
authors.initials: MJ
authors.lastname: Hart
authors.forename: D B
authors.initials: DB
authors.lastname: Sacks
mesh_terms: D002147:Calmodulin / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D011485:Protein Binding; D014407:Tumor Cells, Cultured; D020764:cdc42 GTP-Binding Protein; D020703:ras GTPase-Activating Proteins
chemical_list: D002147:Calmodulin; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; C101203:IQ motif containing GTPase activating protein 1; D020703:ras GTPase-Activating Proteins; D019204:GTP-Binding Proteins; D020764:cdc42 GTP-Binding Protein