猪胃H⁺,K⁺-ATP酶α链中的丝氨酸-27、酪氨酸-10和酪氨酸-7作为内在和外在蛋白激酶的钙依赖性可磷酸化位点。

Ser-27, Tyr-10 and Tyr-7 in the alpha-chain of pig stomach H+,K(+)-ATPase as Ca(2+)-dependent phosphorylatable sites by intrinsic and extrinsic protein kinases.

作者信息

Togawa K, Kaya S, Shimada A, Imagawa T, Mårdh S, Corbin J, Kikkawa U, Taniguchi K

机构信息

Graduate School of Science, Hokkaido University, Sapporo, Japan.

出版信息

Biochem Biophys Res Commun. 1996 Oct 23;227(3):810-5. doi: 10.1006/bbrc.1996.1589.

DOI:10.1006/bbrc.1996.1589

PMID:8886014

Abstract

When pig stomach membrane H+,K(+)-ATPase preparations were incubated with [gamma-32P]ATP, Mg2+ and Ca2+, reversible phosphorylation of specific Tyr and Ser residues in the N-terminal alpha-chain of H+,K(+)-ATPase occurred without any detectable phosphorylation in other regions of the alpha-chain. Mild tosylphenylalanyl chloromethyl ketone trypsin treatment followed by reverse-phase column chromatography yielded three radioactive peptide peaks. The first peak contained both Tyr10(32P) and Tyr7(32P) and the second peak contained Tyr10(32P). The third peak contained Ser27(32P) which was also obtained after trypsin treatment of partially purified H+,K(+)-ATPase preparations phosphorylated with protein kinase-C + Ca2+ or protein kinase-A. This is the first demonstration of Ca2(+)-dependent phosphorylation of the alpha-chain of H+,K(+)-ATPase by protein kinases.

摘要

当猪胃黏膜H⁺,K⁺-ATP酶制剂与[γ-³²P]ATP、Mg²⁺和Ca²⁺一起温育时，H⁺,K⁺-ATP酶N端α链中特定的酪氨酸（Tyr）和丝氨酸（Ser）残基发生可逆磷酸化，而α链的其他区域未检测到任何磷酸化。用轻度甲苯磺酰苯丙氨酰氯甲基酮处理胰蛋白酶，然后进行反相柱色谱，得到三个放射性肽峰。第一个峰同时含有Tyr10(³²P)和Tyr7(³²P)，第二个峰含有Tyr10(³²P)。第三个峰含有Ser27(³²P)，在用蛋白激酶-C + Ca²⁺或蛋白激酶-A磷酸化的部分纯化的H⁺,K⁺-ATP酶制剂经胰蛋白酶处理后也能得到该峰。这是首次证明蛋白激酶对H⁺,K⁺-ATP酶α链的Ca²⁺依赖性磷酸化。