Phosphatidylinositol phosphatase activity of Phogrin regulates insulin secretion

描述（由申请方提供）：胰岛素储存在胰腺β细胞的致密核心囊泡（DCV）中。从囊泡释放胰岛素需要DCV膜与质膜的瞬时融合。这在一定程度上是由DCV中的磷脂和跨膜蛋白调节的。光凝蛋白（IA-2 β）是一种定位于分泌颗粒的跨膜蛋白酪氨酸磷酸酶样蛋白。这表明它在调节胰岛素分泌中起作用，但是Phogrin的潜在催化位点含有Asp代替预期的酪氨酸磷酸酶共有Ala，并且没有关于Phogrin的酶活性的报道。我已经获得了Phogrin能够使磷脂酰肌醇磷脂（PIP）（包括PI（3）P和PI（4，5）P2）去磷酸化的证据。结合PI（3）P和PI（4，5）P2的蛋白质结构域用于将特异性蛋白质定位于正确的囊泡隔室并改变结合蛋白质的活性，从而起到调节分泌囊泡的形成、释放和再循环的作用。我将测试的假设，Phogrin是一个PIPase，它发挥了作用，通过调节PIP水平的分泌囊泡和再循环内体调节胰岛素的释放。我将在四个具体目标中检验这个假设： 目标1.在体外表征全长光蛋白的PIPase活性。 目标二。测试Phogrin的PIPase活性的药理学抑制剂。 目标3：检验以下假设：PKA磷酸化的光蛋白用于在PM处使光蛋白磷酸化，使得其在胞吐和囊泡再循环期间不水解PM和内体中的PIP。 目标4。使用内源性磷酸化蛋白的敲低来检验内源性磷酸化蛋白是一种内源性磷酸化蛋白的假设。 DCV PI（4，5）P2含量的决定因素，并在促分泌素刺激的胞吐中起作用。 这些研究将为了解胰岛素分泌提供新的信息，并推动我们了解糖尿病中胰岛素释放分泌途径的哪些点可能被错误调节。此外，我希望获得的数据将指导我未来的临床治疗研究，以纠正胰岛素分泌的异常步骤。

DESCRIPTION (provided by applicant): Insulin is stored in dense core vesicles (DCVs) in pancreatic beta cells. Release of insulin from a vesicle requires transient fusion of the DCV membrane with the plasma membrane. This is regulated, in part, by phospholipids and transmembrane proteins in the DCV. Phogrin (IA-2beta) is a transmembrane protein tyrosine phosphatase-like protein that is localized to secretory granules. This has suggested that it plays a role in regulating insulin secretion, but a potential catalyitc site of Phogrin contains Asp in place of an expected tyrosine phosphatase consensus Ala, and no enzymatic activity for Phogrin has ever been reported. I have obtained evidence that Phogrin is able to dephosphorylate phosphatidylinositol phospholipids (PIPs), including PI(3)P and PI(4,5)P2. Protein domains that bind PI(3)P and PI(4,5)P2 serve to localize specific proteins to the correct vesicle compartment and to alter the activity of the bound proteins and thus, function to regulate the formation, release, and recycling of secretory vesicles. I will test the hypothesis that Phogrin is a PIPase and that it plays a role in regulating insulin release by regulating PIP levels in secretory vesicles and recycling endosomes. I will test this hypothesis in 4 specific aims: Aim 1. Characterize the PIPase activity of full length Phogrin in vitro. Aim 2. Test pharmacologic inhibitors of the PIPase activity of Phogrin. Aim 3. Test the hypothesis that phosphorylation of Phogrin by PKA serves to inactivate Phogrin at the PM so that it does not hydrolyze PIPs in the PM and endosome during exocytosis and vesicle recycling. Aim 4. Use knockdown of endogenous Phogrin to test the hypothesis that endogenous Phogrin is a determinant of DCV PI(4,5)P2 content and plays a role in secretagogue-stimulated exocytosis. These studies will provide new information toward the understanding of insulin secretion and push forward our knowledge as to what points of the insulin release secretory pathway may be mis-regulated in diabetes. Further, I expect to obtain data which will guide my future studies toward clinical treatments to correct aberrant steps in insulin secretion.