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.
Joyal
J L
JL
Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
Annan
R S
RS
Ho
Y D
YD
Huddleston
M E
ME
Carr
S A
SA
Hart
M J
MJ
Sacks
D B
DB
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
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