ROLE OF THE NOVEL PHOSPHOLYLATION IN TRANSPORT PUMPS

新型磷酸化在运输泵中的作用

基本信息

批准号：
08044047
负责人：
TANIGUCHI Kazuya
金额：
$ 5.89万
依托单位：
HOKKAIDO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

H/K-ATPase preparations (G_1) of pig stomach showed Ca dependent Ser-27 (k_<1/2>=0.6muM) phosphorylation and independent Tyr-10 and -7 phosphorylation of the catalytic subunit of H/K-ATPase preferentially. Addition of a detergent, 3-[(3-Cholamidepropyl)-dimethylammonio]-1-propane-sulfonate (CHAPS) or a specific PKC inhibitor abolished the Ca dependent phosphorylation. The addition of CHAPS permitted to phosphorylate synthetic copolymer substrates and N-terminal domains of alpha-chain of H/K-ATPase fused with maltose binding protein. Western blotting analysis and invitro kinase assay using fusion proteins and others showed that the Ser-kinase recognized with antibodies against PKCalpha and PKCgamma and the Tyr-kinase with that against N-terminal part of SRC but not against C-terminal part of SRC are responsible for these phosphorylation. The Tyr-kinase was neither recognized with antibody against Yes nor Lyn. Column chromatographic separation of CHAPS solubilized kinases and the reactvities of sample with antibodies indicated that the apparent molecular weight of the Ser kinase was 80 kDa and that of Tyrkinase was 60 kDa. Breakdown of phosphoserine of the fusion protein by G1 was biphasically inhibited by okadaic acid with little effect of Mg and Ca suggesting the presence of protein phosphatase-1 and 2A but neither -2B nor -2C.The presence of phosphotyrosine phosphatase, SHP-2 but not SHP-1 wasimmunologically detected. These data and others strongly suggested that conventional PKCs, a novel SRC-related Tyr-kinase and phosphatases present in near N-terminal membrane domain of H/K-ATPase are participating specific phosphorylation and dephosphorylation of Ser-27 and Tyr-10 and -7.

猪胃的H/k-ATPase制剂（G_1）表现出依赖性的SER-27（K_ <1/2> = 0.6MUM）磷酸化以及H/K-ATPase催化亚基的独立Tyr-10和-7磷酸化优先。添加洗涤剂3 - [（3-胆碱丙基）-Dimethymonio] -1-丙烷 - 磺酸盐（CHAPS）或特定的PKC抑制剂废除了CA依赖性磷酸化。允许添加与麦芽糖结合蛋白融合的h/k-ATPase的磷酸化合成共聚物底物和N末端结构域的添加。使用融合蛋白和其他的蛋白质印迹分析和Invitro激酶测定法表明，用针对PKCALPHA和PKCGAMMA的抗体识别的Ser-激酶以及与SRC的N端部分相对的Tyr-kinase，但不针对SRC的C-末端部分，负责这些磷酸化。 Tyr-kinase既没有对YES也没有抗体认可。 CHAPS溶解的激酶和样品与抗体的反应观念的柱色谱分离表明，Ser激酶的表观分子量为80 kDa，暴君的表观分子量为60 kDa。冈田酸对融合蛋白的磷酸蛋白分解受到双重抑制，而Mg和Ca的作用很小，表明存在蛋白质磷酸酶-1和2a，但既不是-2B也不是-2C。这些数据和其他数据强烈表明，H/K-ATPase的近N末端膜结构域中存在的常规PKC是一种与SRC相关的Tyr-激酶和磷酸酶，参与Ser-27和Tyr-10和-7的Ser-27和Tyr-10和-7的近末端膜结构域。