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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H/K-ATPase制剂(G_1)优先表现出依赖钙的Ser-27(k_&lt；1/2&gt；=0.6muM)磷酸化和H/K-ATPase催化亚基Tyr-10和-7的非依赖性磷酸化。加入去污剂3-[(3-Cholamidepropyl)-dimethylammonio]-1-propane-sulfonate(CHAPS)或特定的蛋白激酶C抑制剂可阻断钙依赖的磷酸化。允许添加CHAPS来磷酸化合成共聚底物和H/K-ATPase与麦芽糖结合蛋白融合的阿尔法链的N-末端结构域。Western blotting分析和体外融合蛋白等分析表明，抗PKCalpha和PKCGamma抗体识别的丝氨酸激酶和抗SRC N-末端而不是SRC C-末端的Tyr-Kinase负责这些磷酸化。酪氨酸激酶既不能被抗Yes抗体识别，也不能被Lyn抗体识别。CHAPS溶解的激酶的柱层析分离和样品与抗体的反应性表明，丝氨酸激酶的表观分子量为80 kDa，酪氨酸激酶的表观分子量为60 kDa。冈田酸对融合蛋白中磷酸丝氨酸的降解被冈田酸两相抑制，而镁和钙的作用很小，提示融合蛋白中存在蛋白磷酸酶-1和2 A，但不存在蛋白-2B和-2 C。这些数据和其他数据有力地表明，存在于H/K-ATPase近N端膜结构域的新的SRC相关酪氨酸激酶和磷酸酶参与了Ser-27和Tyr-10和-7的特异性磷酸化和去磷酸化。