IQGAP1 integrates Ca2+/calmodulin and B-Raf signaling

CA(2+) and Calmodulin modulate numerous cellular functions, ranging from muscle contraction to the cell cycle. Accumulating evidence indicates that CA(2+) and Calmodulin regulate the MAPK signaling pathway at multiple positions in the cascade, but the molecular mechanism underlying these observations is poorly defined. We previously documented that IQGAP1 is a scaffold in the MAPK cascade. IQGAP1 binds to and regulates the activities of ERK, MEK, and B-Raf. Here we demonstrate that IQGAP1 integrates CA(2+) and Calmodulin with B-Raf signaling. In vitro analysis reveals that CA(2+) promotes the direct binding of IQGAP1 to B-Raf. This interaction is inhibited by Calmodulin in a CA(2+)-regulated manner. Epidermal growth factor (EGF) is unable to stimulate B-Raf activity in fibroblasts treated with the CA(2+) ionophore A23187. In contrast, chelation of intracellular free CA(2+) concentrations ([CA(2+)](i)) significantly enhances EGF-stimulated B-Raf activity, an effect that is dependent on IQGAP1. Incubation of cells with EGF augments the association of B-Raf with IQGAP1. Moreover, CA(2+) regulates the association of B-Raf with IQGAP1 in cells. Increasing [CA(2+)](i) with CA(2+) ionophores significantly reduces co-immunoprecipitation of B-Raf and IQGAP1, whereas chelation of CA(2+) enhances the interaction. Consistent with these findings, increasing and decreasing [CA(2+)](i) increase and decrease, respectively, co-immunoprecipitation of Calmodulin with IQGAP1. Collectively, our data identify a previously unrecognized mechanism in which the scaffold protein IQGAP1 couples CA(2+) and Calmodulin signaling to B-Raf function.