Abstract
RGC1 and RGC2 comprise a functional RalGAP complex (RGC) that suppresses RalA activity. The PI3-kinase/Akt signaling pathway activates RalA through phosphorylation-mediated inhibition of the RGC. Here we identify a novel phosphorylation-dependent interaction between 14-3-3 and the RGC. 14-3-3 binds to the complex through an Akt-phosphorylated residue, threonine 715, on RGC2. Interaction with 14-3-3 does not alter in vitro activity of the GTPase-activating protein complex. However, blocking the interaction between 14-3-3 and RGC2 in cells increases suppression of RalA activity by the RGC, suggesting that 14-3-3 inhibits the complex through a non-catalytic mechanism. Together, these data show that 14-3-3 negatively regulates the RGC downstream of the PI3-kinase/Akt signaling pathway.
Highlights
The RalGAP (GTPase-activating protein) complex (RGC) inhibits RalA activity and is negatively regulated by PI3-kinase/Akt signaling
Phosphorylation was increased by insulin in a dose-dependent manner that correlated with activation of Akt, which we previously showed catalyzes RGC2 phosphorylation on multiple residues [20] (Fig. 1C)
Both inhibitors blocked RGC2 detection by the phospho-14-3-3 binding site antibody (Fig. 1D). These data raised the possibility that 14-3-3 may recognize a residue or residues on RGC2 that are phosphorylated by Akt in response to insulin
Summary
The RalGAP (GTPase-activating protein) complex (RGC) inhibits RalA activity and is negatively regulated by PI3-kinase/Akt signaling. Blocking the interaction between 14-3-3 and RGC2 in cells increases suppression of RalA activity by the RGC, suggesting that 14-3-3 inhibits the complex through a non-catalytic mechanism Together, these data show that 14-3-3 negatively regulates the RGC downstream of the PI3-kinase/Akt signaling pathway. Inhibition of the RGC by phosphorylation allows for an insulin-stimulated increase in RalA activity in adipocytes, where this small GTPase plays a critical role in Glut exocytosis and glucose uptake [7, 20]. We report that Akt-catalyzed phosphorylation of RGC2 increases 14-3-3 binding to the RGC and that this interaction inhibits RGC function in cells These data establish a role for 14-3-3 in hormone-stimulated activation of RalA
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