Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center at San Antonio, San Antonio, Texas.
State Key Laboratory of Biomembrane and Membrane Biotechnology, School of Medicine, Tsinghua University, Beijing, China.
Endocrinology. 2018 Aug 1;159(8):3036-3047. doi: 10.1210/en.2017-03243.
Rho GDP-dissociation inhibitor (GDIα) 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 GDIα at Ser174, a critical inhibitory site for GDIα, leading to the release of Rho GTPases and their subsequent activation. However, the kinase regulator(s) that catalyzes the phosphorylation of GDIα 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 GDIα 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 GDIα by SAD-A in the context of GSIS from INS-1 β cells and primary islets. We identified GDIα directly binds to SAD-A kinase domain and phosphorylated by SAD-A on Ser174, leading to dissociation of Rho GTPases from GDIα complexes. Accordingly, overexpression of SAD-A significantly stimulated GDIα 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 GDIα phosphorylation at Ser174. Consequently, coexpression of SAD-A completely prevented the inhibitory effect of GDIα on insulin secretion in islets. In summary, glucose and incretin stimulate insulin secretion through the phosphorylation of GDIα at Ser174 by SAD-A, which leads to the activation of Rho GTPases, culminating in insulin exocytosis.
Rho GDP 解离抑制剂 (GDIα) 通过将 Rho GTPases 锁定在非活性 GDP 结合形式中来部分抑制葡萄糖刺激的胰岛素分泌 (GSIS)。GSIS 的起始导致 GDIα 在 Ser174 处发生磷酸化，这是 GDIα 的关键抑制位点，导致 Rho GTPases 的释放及其随后的激活。然而，胰岛 β 细胞中催化 GDIα 磷酸化的激酶调节剂仍不清楚。我们提出，SAD-A 是 AMP 激活的蛋白激酶相关激酶家族的成员，作为肠促胰岛素信号的效应激酶促进 GSIS，它在胰岛 β 细胞的 GSIS 起始时通过 Ser174 磷酸化与 GDIα 相互作用并抑制 GDIα。共免疫沉淀和磷酸化分析用于鉴定 SAD-A 在 INS-1 β 细胞和原代胰岛中 GSIS 时 GDIα 的物理相互作用和磷酸化位点。我们确定 GDIα 直接与 SAD-A 激酶结构域结合，并被 SAD-A 在 Ser174 处磷酸化，导致 Rho GTPases 从 GDIα 复合物中解离。因此，SAD-A 的过表达显著刺激了 GSIS 时 GDIα 在 Ser174 处的磷酸化，这一过程被肠促胰岛素激素胰高血糖素样肽-1 显著增强。相反，通过短发夹 RNA 转染在 INS-1 细胞中介导的 SAD-A 缺乏显著减弱了内源性 GDIα 在 Ser174 处的磷酸化。因此，SAD-A 的共表达完全阻止了 GDIα 对胰岛胰岛素分泌的抑制作用。总之，葡萄糖和肠促胰岛素通过 SAD-A 在 Ser174 处对 GDIα 的磷酸化刺激胰岛素分泌，导致 Rho GTPases 的激活，最终导致胰岛素的胞吐作用。
