Tyrosine phosphorylation of the scaffold protein IQGAP1 in the MET pathway alters function

Andrew C Hedman,Dean E Mcnulty,Zhigang Li,Laëtitia Gorisse,Roland S Annan,David B Sacks

doi:10.1074/jbc.ra120.015891

Abstract

IQGAP1 is a key scaffold protein that regulates numerous cellular processes and signaling pathways. Analogous to many other cellular proteins, IQGAP1 undergoes post-translational modifications, including phosphorylation. Nevertheless, very little is known about the specific sites of phosphorylation or the effects on IQGAP1 function. Here, using several approaches, including MS, site-directed mutagenesis, siRNA-mediated gene silencing, and chemical inhibitors, we identified the specific tyrosine residues that are phosphorylated on IQGAP1 and evaluated the effect on function. Tyr-172, Tyr-654, Tyr-855, and Tyr-1510 were phosphorylated on IQGAP1 when phosphotyrosine phosphatase activity was inhibited in cells. IQGAP1 was phosphorylated exclusively on Tyr-1510 under conditions with enhanced MET or c-Src signaling, including in human lung cancer cell lines. This phosphorylation was significantly reduced by chemical inhibitors of MET or c-Src or by siRNA-mediated knockdown of MET. To investigate the biological sequelae of phosphorylation, we generated a nonphosphorylatable IQGAP1 construct by replacing Tyr-1510 with alanine. The ability of hepatocyte growth factor, the ligand for MET, to promote AKT activation and cell migration was significantly greater when IQGAP1-null cells were reconstituted with IQGAP1 Y1510A than when cells were reconstituted with WT IQGAP1. Collectively, our data suggest that phosphorylation of Tyr-1510 of IQGAP1 alters cell function. Because increased MET signaling is implicated in the development and progression of several types of carcinoma, IQGAP1 may be a potential therapeutic target in selected malignancies.

Highlights

IQGAP1 is a protein scaffold that participates in signaling pathways, such as the mitogen-activated protein kinase (MAPK) [1, 2] and phosphoinositide-3-kinase (PI3K)/AKT [3, 4] pathways, and modulates the cytoskeleton via the small GTPases, including CDC42 and RAC1 [5, 6]
Samples were analyzed by SDS-PAGE, and Western blots were probed with anti-IQGAP1 and anti-phosphotyrosine antibodies
These findings suggest that IQGAP1 is tyrosine-phosphorylated in cells, but under tight control by tyrosine phosphatases

Summary

Introduction

IQGAP1 is a protein scaffold that participates in signaling pathways, such as the mitogen-activated protein kinase (MAPK) [1, 2] and phosphoinositide-3-kinase (PI3K)/AKT [3, 4] pathways, and modulates the cytoskeleton via the small GTPases, including CDC42 and RAC1 [5, 6]. No tyrosine phosphorylation of immunoprecipitated IQGAP1 was detected in control cells. To identify the specific tyrosine residue(s) that are phosphorylated, we immunoprecipitated endogenous IQGAP1 from vanadate- and control-treated MDA-MB-231 cells and analyzed the appropriate SDS-PAGE bands (Fig. 1B) by MS.

Results

Conclusion