IQGAP1 Integrates Ca2+/Calmodulin and B-Raf Signaling

Jian-Guo Ren,Zhigang Li,David B Sacks

doi:10.1074/jbc.m804626200

Jian-Guo Ren, Zhigang Li + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m804626200

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Abstract

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.

Highlights

Isoforms of the small GTP-binding protein Ras [2, 3]
Via its interaction with IQGAP1, Ca2ϩ/calmodulin is able to modulate the activity of these IQGAP1 binding partners. These data, combined with the observations that Ca2ϩ/calmodulin regulates MAPK signaling [13], led us to investigate whether IQGAP1 integrates Ca2ϩ/ calmodulin signaling with the MAPK cascade
Ca2ϩ Regulates the Binding of IQGAP1 to B-Raf in Vitro—We previously documented a direct interaction between IQGAP1 and B-Raf [33]

Summary

EXPERIMENTAL PROCEDURES

Materials—Lipofectamine 2000, tissue culture reagents, and Pfx polymerase were purchased from Invitrogen. In Vitro Binding Assays—Equal amounts of [35S]methioninelabeled IQGAP1 constructs or pure His-tagged IQGAP1 protein from insect cells were incubated for 3 h at 4 °C with 5 ␮g GST-B-Raf in 1 ml of buffer A (50 mM Tris-HCl, pH 7.4, 150 nM NaCl, 1 mM EGTA, 1% Triton X-100) containing 1 mM phenylmethylsulfonyl fluoride, protease inhibitor mixture, and 1 mM EGTA or 1 mM CaCl2. Anti-Myc monoclonal or anti-IQGAP1 polyclonal antibodies were incubated with protein G- or protein A-Sepharose beads, respectively, for 2 h at 4 °C, washed four times with buffer B and incubated for 3 h with at 4 °C with equal amounts of precleared protein lysate. Densitometry of ECL signals were analyzed with Un-scan-it software (Silk Scientific Corp.) Statistical analysis was performed by one way analysis of variance or Student’s t test with GraphPad Prism 4 (GraphPad software)

RESULTS

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DISCUSSION