Diabetes Research Institute, University of Miami L. Miller School of Medicine, Miami, Florida 33136, USA.
J Biol Chem. 2012 Aug 17;287(34):28932-42. doi: 10.1074/jbc.M112.389452. Epub 2012 Jun 20.
We have previously demonstrated a role for Nephrin in glucose stimulated insulin release (GSIR). We now hypothesize that Nephrin phosphorylation is required for GSIR and that Dynamin influences Nephrin phosphorylation and function. MIN6-C3 Nephrin-deficient pancreatic beta cells and human islets were transfected with WT-Nephrin or with a mutant Nephrin in which the tyrosine residues responsible for SH2 domain binding were substituted with phenylalanine (3YF-Nephrin). GSIR and live images of Nephrin and vesicle trafficking were studied. Immunoprecipitation experiments and overexpression of WT-Dynamin or dominant negative Dynamin mutant (K44A-Dynamin) in WT-Nephrin, 3YF-Nephrin, or Nephrin siRNA-transfected cells were utilized to study Nephrin-Dynamin interaction. In contrast to WT-Nephrin or to single tyrosine mutants, 3YF-Nephrin did not positively affect GSIR and led to impaired cell-cell contacts and vesicle trafficking. K44A-Dynamin prevented the effect of Nephrin on GSIR in the absence of protein-protein interaction between Nephrin and Dynamin. Nephrin gene silencing abolished the positive effects of WT-Dynamin on GSIR. The effects of protamine sulfate and vanadate on Nephrin phosphorylation and GSIR were studied in MIN6 cells and human islets. WT-Nephrin phosphorylation after glucose occurred at Tyr-1176/1193 and resulted in improved GSIR. On the contrary, protamine sulfate-induced phosphorylation at Tyr-1176/1193/1217 was associated with Nephrin degradation and impaired GSIR. Vanadate, which prevented Nephrin dephosphorylation after glucose stimulation, improved GSIR in human islets and MIN6 cells. In conclusion, Dynamin-dependent Nephrin phosphorylation occurs in response to glucose and is necessary for Nephrin-mediated augmentation of GSIR. Pharmacological modulation of Nephrin phosphorylation may thus facilitate pancreatic beta cell function.
我们之前已经证明 Nephrin 在葡萄糖刺激胰岛素释放（GSIR）中起作用。我们现在假设 Nephrin 磷酸化是 GSIR 所必需的，并且 Dynamin 影响 Nephrin 磷酸化和功能。MIN6-C3 Nephrin 缺陷型胰腺β细胞和人胰岛被转染 WT-Nephrin 或 Tyr 残基负责 SH2 结构域结合的突变体 Nephrin（3YF-Nephrin）。研究了 GSIR 和 Nephrin 和囊泡运输的实时图像。利用免疫沉淀实验和 WT-Dynamin 或显性负性 Dynamin 突变体（K44A-Dynamin）在 WT-Nephrin、3YF-Nephrin 或 Nephrin siRNA 转染细胞中的过表达，研究了 Nephrin-Dynamin 相互作用。与 WT-Nephrin 或单个 Tyr 突变体相反，3YF-Nephrin 不能积极影响 GSIR，导致细胞-细胞接触和囊泡运输受损。在 Nephrin 和 Dynamin 之间没有蛋白质-蛋白质相互作用的情况下，K44A-Dynamin 阻止了 Nephrin 对 GSIR 的影响。Nephrin 基因沉默消除了 WT-Dynamin 对 GSIR 的积极影响。在 MIN6 细胞和人胰岛中研究了鱼精蛋白硫酸盐和钒酸盐对 Nephrin 磷酸化和 GSIR 的影响。葡萄糖后 WT-Nephrin 在 Tyr-1176/1193 处发生磷酸化，导致 GSIR 改善。相反，鱼精蛋白硫酸盐诱导的 Tyr-1176/1193/1217 磷酸化与 Nephrin 降解和 GSIR 受损有关。钒酸盐可防止葡萄糖刺激后 Nephrin 去磷酸化，改善人胰岛和 MIN6 细胞中的 GSIR。总之，Dynamin 依赖性 Nephrin 磷酸化是对葡萄糖的反应，是 Nephrin 介导的 GSIR 增强所必需的。Nephrin 磷酸化的药理学调节可能因此促进胰腺β细胞功能。
