Identification of RhoGAP22 as an Akt-Dependent Regulator of Cell Motility in Response to Insulin

Abstract

Insulin exerts many of its metabolic actions via the canonical phosphatidylinositide 3 kinase (PI3K)/Akt pathway, leading to phosphorylation and 14-3-3 binding of key metabolic targets. We previously identified a GTPase-activating protein (GAP) for Rac1 called RhoGAP22 as an insulin-responsive 14-3-3 binding protein. Insulin increased 14-3-3 binding to RhoGAP22 fourfold, and this effect was PI3K dependent. We identified two insulin-responsive 14-3-3 binding sites (pSer¹⁶ and pSer³⁹⁵) within RhoGAP22, and mutagenesis studies revealed a complex interplay between the phosphorylation at these two sites. Mutating Ser¹⁶ to alanine blocked 14-3-3 binding to RhoGAP22 in vivo, and phosphorylation at Ser¹⁶ was mediated by the kinase Akt. Overexpression of a mutant RhoGAP22 that was unable to bind 14-3-3 reduced cell motility in NIH-3T3 fibroblasts, and this effect was dependent on a functional GAP domain. Mutation of the catalytic arginine of the GAP domain of RhoGAP22 potentiated growth factor-stimulated Rac1 GTP loading. We propose that insulin and possibly growth factors such as platelet-derived growth factor may play a novel role in regulating cell migration and motility via the Akt-dependent phosphorylation of RhoGAP22, leading to modulation of Rac1 activity.

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Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.05583-11.

ACKNOWLEDGMENTS

We thank James Burchfield and Katarina Mele for assistance with microscopy and image analysis, respectively. Mouse p68RacGAP cDNA was a gift from Cam Patterson from the University of North Carolina. Double Akt knockout MEFs were a gift from Morris Birnbaum at University of Pennsylvania. CHO IR/IRS-1 cells were a gift from Morris White, Harvard Medical School. Plat-E cells were a gift from T. Kitamura from University of Tokyo. Mass spectrometric analysis for this work was carried out at the Bioanalytical Mass Spectrometry Facility, University of New South Wales (UNSW).

Mass spectrometric analysis for this work was supported in part by grants from the Australian Government Systemic Infrastructure Initiative and Major National Research Facilities Program (UNSW node of the Australian Proteome Analysis Facility) and by the UNSW Capital Grants Scheme. This work was supported by grants from the National Health and Medical Research Council of Australia and Diabetes Australia Research Trust (to D.E.J.). D.E.J. is an NHMRC senior principle research fellow.