Regulation of ion-transporters by A-kinase regulated via anchoring protein

通过锚定蛋白调节的 A 激酶对离子转运蛋白的调节

基本信息

批准号：
14571775
负责人：
KURIHARA Kinji
金额：
$ 2.18万
依托单位：
Meikai University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2005
项目状态：
已结题

项目摘要

Phosphorylation of Na^+, K^+-ATPase via AKAP/PKA complex1.We have examined the expression of A-kinase anchoring protein (AKAP)/PKA complex in the three major salivary glands, i.e., the parotid gland (PG), submandibular gland (SMG), and sublingual gland (SLG), of the rat. AKAP-150 mRNA and RII regulatory subunit of PKA were clearly detected in the PG, but they were hardly detectable in either the SMG or SLG.2.Incubation with [γ-^<32>P]ATP revealed that Na^+,K^+-ATPase in the PG membranes was quickly phosphorylated upon the addition of cAMP, whereas the ATPases in the membranes from SMG and SLG were not, suggesting phosphorylation of Na^+,K^+-ATPase by AKAP/PKA are characteristics specific to PG among the three major salivary glands.Phosphorylation of Na^+/K^+/2Cl^- cotransporter (NKCC1) N-terminus via cAMP/PKA pathway1.We have previously shown that Na^+,K^+,2Cl^--cotransport activity in parotid acinar cells is dramatically upregulated by N-terminus phosphorylation. However we showed here that N-terminus was not phosphorylated by PKA directly in vitro system.2.The phosphorylation of N-terminus was mimicked by the addition of cAMP to permeabilized acini without stimulation of β-adrenergic receptors. This result indicates that the N-terminus was not phosphorylated by PKA directly, however it was clearly phosphorylated by PKA cascade triggered by PKA activation.3.The cAMP dependent phosphorylation of the N-terminus was blocked competitively by the addition of partial N-terminus peptides including Thr-208 into permeabilized acini. This result indicated that functions of NKCC1 were regulated by the phosphorylation of Thr-208 on the N-terminus.4.We carried out cloning of NKCC1 gene and prepared E.Coli transfected its gene. We are going to prepare the plasmids modified to Ala from Thr at various phosphorylation sites, i.e. Thr-203, -208 and -221 in N-terminus and at PKA consensus site.

通过 AKAP/PKA 复合物磷酸化 Na^+、K^+-ATPase1。我们检测了 A-激酶锚定蛋白 (AKAP)/PKA 复合物在三个主要唾液腺（即腮腺 (PG)、在大鼠的下颌下腺（SMG）和舌下腺（SLG）中清楚地检测到AKAP-150 mRNA和PKA的RII调节亚基。 PG，但它们在 SMG 或 SLG 中几乎检测不到。2.与 [γ-^ 32 P]ATP 一起孵育表明，PG 膜中的 Na^+,K^+-ATPase 在添加后迅速磷酸化cAMP 的存在，而 SMG 和 SLG 膜中的 ATP 酶则不然，这表明 AKAP/PKA 对 Na^+,K^+-ATP 酶的磷酸化是 PG 特有的特征三个主要唾液腺之间。Na^+/K^+/2Cl^- 协同转运蛋白 (NKCC1) N 末端通过 cAMP/PKA 途径磷酸化1。我们之前已经表明 Na^+,K^+,2Cl^-- N 末端磷酸化显着上调腮腺腺泡细胞中的共转运活性，然而我们在此表明 N 末端在体外不直接被 PKA 磷酸化。 2.通过在不刺激β-肾上腺素受体的情况下向透化的腺泡中添加cAMP来模拟N-末端的磷酸化。该结果表明N-末端没有被PKA直接磷酸化，但它明显被PKA磷酸化。 PKA激活引发的级联反应。3.通过添加部分N端来竞争性阻断N端的cAMP依赖性磷酸化结果表明NKCC1的功能是通过N端Thr-208的磷酸化来调节的。4.我们对NKCC1基因进行了克隆并制备了转染该基因的大肠杆菌。准备在各个磷酸化位点将 Thr 修饰为 Ala 的质粒，即 Thr-203、-208 和 -221 N 末端和 PKA 共有位点。