IQGAP1 integrates Ca2+/calmodulin and B-Raf signaling

Ca2+ and calmodulin modulate numerous cellular functions, ranging from muscle contraction to the cell cycle. Accumulating evidence indicates that Ca2+ 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 Ca2+ and calmodulin with B-Raf signaling. In vitro analysis reveals that Ca2+ promotes the direct binding of IQGAP1 to B-Raf. This interaction is inhibited by calmodulin in a Ca2+-regulated manner. Epidermal growth factor (EGF) is unable to stimulate B-Raf activity in fibroblasts treated with the Ca2+ ionophore A23187. In contrast, chelation of intracellular free Ca2+ concentrations ([Ca2+](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, Ca2+ regulates the association of B-Raf with IQGAP1 in cells. Increasing [Ca2+](i) with Ca2+ ionophores significantly reduces co-immunoprecipitation of B- Raf and IQGAP1, whereas chelation of Ca2+ enhances the interaction. Consistent with these findings, increasing and decreasing [Ca2+](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 Ca2+ and calmodulin signaling to B-Raf function.