Calmodulin modulates the interaction between IQGAP1 and Cdc42: Identification of IQGAP1 by nanoelectrospray tandem mass spectrometry

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.