Phosphorylation of Sites 3a and 3b (Ser640 and Ser644) in the Control of Rabbit Muscle Glycogen Synthase(*)
Phosphorylation of Sites 3a and 3b (Ser640 and Ser644) in the Control of Rabbit Muscle Glycogen Synthase(*)
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DOI：
10.1074/jbc.270.21.12491
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发表时间：
1995-05
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
糖原合成酶激酶-3通过四个COOH末端残基Ser652(第4位)、Ser648(3c位)、Ser644(3b位)和Ser640(3a位)的顺序磷酸化来灭活兔肌肉糖原合成酶。糖原合成酶-3只有在酪蛋白激酶II催化Ser656(第5位)的磷酸化后才能有效识别糖原合成酶。本研究特别讨论了3a和3b位在调节COS细胞表达的糖原合成酶中的作用。同时在同一蛋白质分子中3个a、b和c、4和5位的SerSerLysAla替换消除了包含这些位点的蛋白降解片段Arg634-Lys682中的32P标记。该突变酶(在氨基末端2位也有一系列→丙氨酸取代)的-/+葡萄糖-6-P活性比为∼0.8，与完全去磷酸化的酶相似。在3a或3b位点恢复丝氨酸残基恢复了Arg634-Lys682肽的标记，并导致活性比下降到0.4-0.6。当重新引入3a和3b两个位点时，该酶完全失活。因此，3a和3b位点足以灭活糖原合成酶，并协同作用控制活性。这项研究证明了3a和3b位点的磷酸化存在另一种机制，该机制不依赖于先前5位点的磷酸化。
Glycogen synthase kinase-3 inactivates rabbit muscle glycogen synthase by sequential phosphorylation of four COOH-terminal residues Ser652 (site 4), Ser648 (site 3c), Ser644 (site 3b), and Ser640 (site 3a). Effective recognition of glycogen synthase by glycogen synthase kinase-3 occurs only after the phosphorylation of Ser656 (site 5) catalyzed by casein kinase II. The present study addresses specifically the role of sites 3a and 3b in the regulation of glycogen synthase expressed in COS cells. Simultaneous Ser → Ala substitutions at sites 3 a, b and c, 4, and 5 in the same protein molecule eliminated 32P labeling in the proteolytic fragment Arg634-Lys682, which contains these sites. This mutant enzyme (which also had a Ser → Ala substitution at site 2 in the NH2 terminus) had a -/+ glucose-6-P activity ratio of ∼0.8, similar to that of totally dephosphorylated enzyme. Reinstating serine residues at either site 3a or site 3b restored labeling in the Arg634-Lys682 peptide and caused a decrease in the activity ratio to 0.4-0.6. When both sites 3a and 3b were reintroduced, there was complete inactivation of the enzyme. Thus, sites 3a and 3b are sufficient for the inactivation of glycogen synthase and act synergistically to control activity. This investigation demonstrates the existence of an alternate mechanism for the phosphorylation of sites 3a and 3b that does not depend on prior phosphorylation of site 5.