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 (pSer 16 and pSer 395 ) within RhoGAP22, and mutagenesis studies revealed a complex interplay between the phosphorylation at these two sites. Mutating Ser 16 to alanine blocked 14-3-3 binding to RhoGAP22 in vivo, and phosphorylation at Ser 16 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.

ARHGAP22 Localizes at Endosomes and Regulates Actin Cytoskeleton

“…By using this antibody, we have analyzed endogenous ARHGAP22 expression in various cell types. Among cell lines tested, we found that mouse C2C12 myoblasts express endogenous ARHGAP22 protein [14]. We therefore determined localization of endogenous ARHGAP22 in C2C12 cells.…”

FilGAP and its close relatives: a mediator of Rho–Rac antagonism that regulates cell morphology and migration

“…Hence future experiments must dissect and account for all the GAPs in a given cell to define the total contribution they make to the differential regulation of GTPase activity. ARHGAP25 and ARHGAP22 genes are expressed in all tissues, but spleen and peripheral leucocytes have the highest level of ARHGAP25 products [61], whereas vascular endothelial cells have the highest levels of the ARHGAP22 gene products [55,68,69]. Podocytes are particularly enriched in FilGAP, up-regulating its expression ∼70-fold when they differentiate in situ [24,60].…”

“…Mutation of Ser 402 to alanine reduced FilGAP activity in cells, but only modestly compared with the substitution of all six sites [24,26]. If this hypothesis is true, phosphorylation of FilGAP could regulate turnover of Rac through associations with other cellular components and/or by facilitating dissociation from FLNa under force [69,72] (see below). More likely is that phosphorylation of FilGAP leads to differential targeting of the RacGAP, either altering membrane binding or by exposing a new partner interaction.…”

“…However, phosphorylation of multiple serine and threonine residues in the connecting regions between the GAP and CC domain has been detected by MS analysis in certain cells (at http://www.phosphosite.org/) [73]. Other phosphorylation sites have also been implicated in 14-3-3 binding [69]. Other phosphorylation sites have also been implicated in 14-3-3 binding [69].…”

“…Interestingly, phosphorylation of the conserved Ser 395 of ARHGAP22-encoded proteins is downstream of Akt, and promotes binding to 14-3-3 protein. Mutation of phosphorylation sites, including Ser 395 , disrupts binding to 14-3-3, alters the GAP activity of ARHGAP22 products, and reduces cell motility [69]. Mutation of phosphorylation sites, including Ser 395 , disrupts binding to 14-3-3, alters the GAP activity of ARHGAP22 products, and reduces cell motility [69].…”