Multiple mechanisms for the phosphorylation of C-terminal regulatory sites in rabbit muscle glycogen synthase expressed in COS cells.
Multiple mechanisms for the phosphorylation of C-terminal regulatory sites in rabbit muscle glycogen synthase expressed in COS cells.
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DOI：
10.1042/bj3130045
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
A. V. Skurat;P. Roach
中科院分区：
文献类型：
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作者：
A. V. Skurat;P. Roach
糖原合酶可通过糖原合酶激酶 3 催化的 C 端残基 Ser652（位点 4）、Ser648（位点 3c）、Ser644（位点 3b）和 Ser640（位点 3a）连续磷酸化而失活。在体外，糖原合酶激酶 3 的作用需要糖原合酶首先在 Ser656（位点 5）处被酪蛋白激酶 II 磷酸化。最近我们证明失活仅与位点 3a 和位点 3b 的磷酸化有关，并且在 COS 细胞中，这些位点的修饰可以通过独立于任何 C 端磷酸化的替代机制发生 [Skurat 和 Roach (1995) J. Biol.化学。 270、12491-12497]。为了解决这些机制，在糖原合酶中引入了多个 Ser-->Ala 突变，使得只有位点 3a 或位点 3b 保持完整。 Arg637-->Gln 的额外突变消除了位点 3a 的磷酸化，表明 Arg637 对于其相应蛋白激酶识别位点 3a 可能很重要。类似地，Pro645-->Ala 的额外突变消除了位点 3b 的磷酸化，表明可能涉及“脯氨酸导向”蛋白激酶。单独的 Arg637 突变不会激活糖原合酶，正如由于位点 3a 磷酸化丧失所预期的那样。相反，Arg637 的突变和位点 3b 处的 Ser-->Ala 替换是实质性激活所必需的。结果表明，位点 3a 和 3b 可以通过不同的蛋白激酶彼此独立地磷酸化。然而，位点 3b 的磷酸化可以通过酶（例如糖原合酶激酶 3）增强位点 3a 的磷酸化。
Glycogen synthase can be inactivated by sequential phosphorylation at the C-terminal residues Ser652 (site 4), Ser648 (site 3c), Ser644 (site 3b) and Ser640 (site 3a) catalysed by glycogen synthase kinase-3. In vitro, glycogen synthase kinase-3 action requires that glycogen synthase has first been phosphorylated at Ser656 (site 5) by casein kinase II. Recently we demonstrated that inactivation is linked only to phosphorylation at site 3a and site 3b, and that, in COS cells, modification of these sites can occur by alternative mechanisms independent of any C-terminal phosphorylations [Skurat and Roach (1995) J. Biol. Chem. 270, 12491-12497]. To address these mechanisms multiple Ser-->Ala mutations were introduced in glycogen synthase such that only site 3a or site 3b remained intact. Additional mutation of Arg637-->Gln eliminated phosphorylation of site 3a, indicating that Arg637 may be important for recognition of site 3a by its corresponding protein kinase(s). Similarly, additional mutation of Pro645-->Ala eliminated phosphorylation of site 3b, indicating a possible involvement of 'proline-directed' protein kinase(s). Mutation of Arg637 alone did not activate glycogen synthase as expected from the loss of phosphorylation at site 3a. Rather, mutation of both Arg637 and the Ser-->Ala substitution at site 3b was required for substantial activation. The results suggest that sites 3a and 3b can be phosphorylated independently of one another by distinct protein kinases. However, phosphorylation of site 3b can potentiate phosphorylation of site 3a, by an enzyme such as glycogen synthase kinase-3.