Rho GDP-dissociation inhibitor (GDIa) inhibits glucose-stimulated insulin secretion (GSIS) in part by locking Rho GTPases in an inactive GDP-bound form. The onset of GSIS causes phosphorylation of GDIa at Ser174, a critical inhibitory site for GDIa, leading to the release of Rho GTPases and their subsequent activation. However, the kinase regulator(s) that catalyzes the phosphorylation of GDIa in islet β cells remains elusive. We propose that SAD-A, a member of AMP-activated protein kinase-related kinases that promotes GSIS as an effector kinase for incretin signaling, interacts with and inhibits GDIa through phosphorylation of Ser174 during the onset GSIS from islet β cells. Coimmunoprecipitation and phosphorylation analyses were carried out to identify the physical interaction and phosphorylation site of GDIa by SAD-A in the context of GSIS from INS-1 β cells and primary islets. We identified GDIa directly binds to SAD-A kinase domain and phosphorylated by SAD-A on Ser174, leading to dissociation of Rho GTPases from GDIa complexes. Accordingly, overexpression of SAD-A significantly stimulated GDIa phosphorylation at Ser174 in response to GSIS, which is dramatically potentiated by glucagonlike peptide-1, an incretin hormone. Conversely, SAD-A deficiency, which is mediated by short hairpin RNA transfection in INS-1 cells, significantly attenuated endogenous GDIa phosphorylation at Ser174. Consequently, coexpression of SAD-A completely prevented the inhibitory effect of GDIa on insulin secretion in islets. In summary, glucose and incretin stimulate insulin secretion through the phosphorylation of GDIa at Ser174 by SAD-A, which leads to the activation of Rho GTPases, culminating in insulin exocytosis.
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