ROLE OF PROTEIN PHOSPHATASES IN INSULIN SECRETION

描述：（来自申请人的摘要）虽然大多数现有研究 由于一直专注于蛋白激酶，因此对蛋白激酶知之甚少。 蛋白磷酸酶在生理胰岛素中的调节和作用 胰腺β细胞的分泌物。某些催化剂的活性 中间代谢酶受其磷酸化的影响 status.例如，乙酰辅酶A羧化酶（ACC），一种脂肪生成酶， 由磷酸化（非活性）-去磷酸化（活性）调节。这里 研究人员描述了一种新的胞质冈田酸敏感的 （PP 2A样）PPase，使β细胞中的ACC去磷酸化并激活。 这种PPase的活性通过其C-末端亮氨酸的甲基化而增强。 此外，甲基化形式通过以下方式转化为其无活性的脱甲基化形式： 一种胞质丝氨酸酯酶样酶，也定位于β细胞中。 生理浓度的谷氨酸和镁刺激羧基 甲基化以及这种PPase的催化活性， 增加ACC活性。谷氨酸和镁的作用是特定的， ACC-磷酸酶，但不是ACC-激酶。葡萄糖促胰岛素浓度 或谷氨酰胺刺激该PPase的羧基甲基化， 细胞更重要的是，这种ACC-PPase的活性在胰岛中被消除， 来自GK大鼠，NIDDM模型，与来自对照的胰岛相比， Wistar大鼠使用分离的大鼠胰岛和纯β（HIT-T15和INS-1）细胞， 研究人员建议研究这种PPase的调节，从而 ACC，在3个细胞水平（即亚细胞组分、完整胰岛和 重组系统中的纯化蛋白质）。调查人员还将净化 这种PPase使用微囊藻毒素-琼脂糖亲和柱，以确定变化 在各种实验条件下，PP 2A的（A，B和C）亚基组成 条件下，以建立羧基甲基化之间的联系， C亚基与PP 2A卤酶的组装。调查人员将 对PP 2 Ac的甲基化和脱甲基化酶进行了表征，并对PP 2 Ac的甲基化和脱甲基化酶进行了研究。 ACC与其推定PPase相互作用。使用类似的方法， 研究人员将检查糖尿病患者ACC-磷酸酶的异常， GK胰岛，并研究这种缺陷是否可以通过治疗糖尿病逆转。 注射胰岛素或根皮苷的大鼠。拟议的研究应建立联系， ACC激活与分离β-胰岛素的生理性胰岛素分泌之间的关系 细胞这些研究还将确定基因座，是因果关系的， 观察到ACC-磷酸酶和ACC激活信号级联的缺陷， 糖尿病β细胞，导致胰岛素分泌异常， NIDDM模型。

DESCRIPTION: (From applicant's abstract) While a majority of extant studies have focused since on the protein kinases, very little is known of the regulation and roles of protein phosphatases (PPases) in physiologic insulin secretion from the pancreatic beta cell. The catalytic activity of certain enzymes of intermediary metabolism is influenced by their phosphorylation status. For example, acetyl CoA carboxylase (ACC), a lipogenic enzyme, is modulated by phosphorylation (inactive)- dephosphorylation (active). Here in the investigators describe a novel cytosolic, okadaic acid-sensitive (PP2A-like) PPase that dephosphorylates and activates ACC in the beta cell. Activity of this PPase is augmented by methylation at its C-terminal leucine. Further, the methylated form is converted to its inactive, demethylated form by a cytosolic serine esterease-like enzyme, also localize in the beta cell. Physiologic concentrations of glutamate and magnesium stimulate the carboxyl methylation as well as the catalytic activity of this PPase and concomitantly increase ACC activity. Effects of glutamate and magnesium are specific for ACC-phosphatase, but not ACC-kinase. Insulinotropic concentrations of glucose or glutamine stimulate the carboxyl methylation of this PPase in intact beta cells. More importantly, activity of this ACC-PPase is abolished in islet derived from the GK rat, a model for NIDDM, compared to islets from the control Wistar rats. Using isolated rat islets and pure beta (HIT-T15 and INS-1) cells, the investigators propose to study the regulation of this PPase, and thereby ACC, at 3 cellular levels (i.e. subcellular fractions, intact islets, and purified proteins in reconstituted systems). The investigators will also purify this PPase using microcystin -sepharose affinity columns to determine changes in the (A, B and C) subunit composition of PP2A under various experimental conditions, to establish a link between the carboxyl methylation of the C-subunit and the assembly of PP2A haloenzyme. The investigators will characterize the methylating and demethylating enzymes of PP2 Ac and study the interaction of ACC with its putative PPase. Using similar approaches, the investigators will examine the abnormalities in ACC-phosphatase in the diabetic GK islet and investigate if such a defect is reversed by treatment of diabetic rats with insulin or phlorizin. The proposed studies should establish a link between ACC activation and physiologic insulin secretion from isolated beta cells. These studies will also identify the locus that is causal to the observed defect in ACC-phosphatase and ACC activation signaling cascade in the diabetic beta cell, leading to abnormal insulin secretion demonstrable in this model for NIDDM.