Hardy T A, Roach P J
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis 46202-5122.
J Biol Chem. 1993 Nov 15;268(32):23799-805.
The budding yeast Saccharomyces cerevisiae expresses two isoforms of glycogen synthase, of which glycogen synthase-2 (GS-2) appears to be the most important determinant of glycogen accumulation (Farkas, I., Hardy, T. A., Goebl, M. G., and Roach, P. J. (1991) J. Biol. Chem. 266, 15602-15607). Partial proteolysis of purified yeast glycogen synthase activated the enzyme, mimicking the effects of dephosphorylation. The cleavage was localized to the COOH terminus of the molecule and trypsin treatment released 32P from enzyme labeled in vivo with 32P or in vitro by cyclic AMP-dependent protein kinase. Similarly, when cells were labeled with 32P, no radioactivity was incorporated into a mutant form of GS-2 truncated at residue 643 while the wild type enzyme was phosphorylated at both Ser and Thr residues. The 9 Ser and Thr residues COOH-terminal to position 643 were mutated individually to Ala, and the GS-2 mutants were expressed from a low copy plasmid in yeast that lacked functional chromosomal copies of the two glycogen synthase genes. Mutations at Ser-650, Ser-654, and Thr-667 resulted in significant activation of yeast glycogen synthase and elevation in the level of accumulated glycogen as compared with wild type. Likewise, expression of the truncated GS-2 resulted in hyperactive enzyme and the overaccumulation of glycogen. None of the other Ser or Thr mutations substantially affected glycogen synthase activity and glycogen storage. We conclude that Ser-650, Ser-654, and Thr-667 are regulatory phosphorylation sites in vivo. However, in vitro, cyclic AMP-dependent protein kinase modified Ser residue(s) COOH-terminal to position 659, and so the identity of the physiological GS-2 kinases is unclear. Yeast strains bearing glc7 and gac1 mutations are defective in genes encoding type 1 protein phosphatase components and are impaired in their ability to accumulate glycogen. Expression of the truncated GS-2 in these strains restored glycogen accumulation, as did the presence of GS-2 mutated at Ser-650, Ser-654, or Thr-667. These data are consistent with the hypothesis that type 1 phosphatase regulates GS-2 by controlling its phosphorylation state.
出芽酵母酿酒酵母表达两种糖原合酶同工型，其中糖原合酶-2（GS-2）似乎是糖原积累的最重要决定因素（法卡斯，I.，哈迪，T.A.，戈布尔，M.G.，和罗奇，P.J.（1991年）《生物化学杂志》266，15602 - 15607）。纯化的酵母糖原合酶经部分蛋白酶解后被激活，模拟了去磷酸化的作用。切割定位在分子的COOH末端，用胰蛋白酶处理可从体内用32P标记或体外经环磷酸腺苷依赖性蛋白激酶标记的酶中释放出32P。同样，当细胞用32P标记时，在第643位残基处截短的GS-2突变体形式没有放射性掺入，而野生型酶在丝氨酸和苏氨酸残基处都被磷酸化。第643位残基COOH末端的9个丝氨酸和苏氨酸残基分别突变为丙氨酸，GS-2突变体由低拷贝质粒在缺乏两个糖原合酶基因功能性染色体拷贝的酵母中表达。与野生型相比，丝氨酸-650、丝氨酸-654和苏氨酸-667处的突变导致酵母糖原合酶显著激活以及积累的糖原水平升高。同样，截短的GS-2的表达导致酶活性过高和糖原过度积累。其他丝氨酸或苏氨酸突变均未对糖原合酶活性和糖原储存产生实质性影响。我们得出结论，丝氨酸-650、丝氨酸-654和苏氨酸-667是体内的调节性磷酸化位点。然而，在体外，环磷酸腺苷依赖性蛋白激酶修饰第659位残基COOH末端的丝氨酸残基，因此生理GS-2激酶的身份尚不清楚。携带glc7和gac1突变的酵母菌株在编码1型蛋白磷酸酶组分的基因方面存在缺陷，并且在积累糖原的能力上受损。在这些菌株中截短的GS-2的表达恢复了糖原积累，在丝氨酸-650、丝氨酸-654或苏氨酸-667处突变的GS-2的存在也恢复了糖原积累。这些数据与1型磷酸酶通过控制其磷酸化状态来调节GS-2的假设一致。
