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.

Na^+，K^+ - ATPase通过AKAP/PKA复合物的磷酸化研究了三个主要唾液腺中A-激酶锚定蛋白（AKAP）/PKA复合物的表达，即腮腺（PG），下颌骨（SMG）和Subblane gland（slangual gland（Sland）， 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不是，提示Na^+，Akap/pKA的k^+ATPase磷酸化是PG在三个主要的唾液网格中特有的特征。Na^+/k^+/2cl^ - cotransporter（nkcc1）n-terminus n-terminus via camp/pka pathway1。 N-末端磷酸化大大更新了腮腺细胞。然而，我们在这里表明，直接在体外系统中直接通过PKA磷酸化N-末端。2。通过添加cAMP添加cAMP以透化acini而没有刺激β-肾上腺素受体。该结果表明，N-末端并未直接被PKA磷酸化，但是它通过PKA激活触发的PKA级联反应清楚地磷酸化。3。cAMP依赖于N-末端的磷酸化被包括将局部N-末端肽添加到包括Thr-208中的部分N-Terminus肽竞争性地阻断。该结果表明，NKCC1的功能受到N末端上THR-208的磷酸化的调节。4。我们进行了NKCC1基因的克隆，并制备了E.Coli，将其转化为基因。我们将在各种磷酸化位点（即N-末端和PKA共识位点）从THR上修改为ALA的质粒。