Flotow H, Graves P R, Wang A Q, Fiol C J, Roeske R W, Roach P J
Department of Biochemistry, Indiana University School of Medicine, Indianapolis 46223.
J Biol Chem. 1990 Aug 25;265(24):14264-9.
Phosphorylation of rabbit muscle glycogen synthase by cyclic AMP-dependent protein kinase has been shown to enhance subsequent phosphorylation by casein kinase I (Flotow, H., and Roach, P. J. (1989) J. Biol. Chem. 264, 9126-9128). In the present study, synthetic peptides based on the sequences of the four phosphorylated regions in muscle glycogen synthase were used to probe the role of substrate phosphorylation in casein kinase I action. With all four peptides, prior phosphorylation significantly stimulated phosphorylation by casein kinase I. A series of peptides was synthesized based on the NH2-terminal glycogen synthase sequence PLSRTLS7VSS10LPGL, in which phosphorylation at Ser7 is required for modification of Ser10 by casein kinase I. The spacing between the P-Ser and the acceptor Ser was varied to have 1, 2, or 3 intervening residues. The peptide with a 2-residue spacing (-S(P)-X-X-S-) was by far the best casein kinase I substrate. When the P-Ser residue at Ser7 was replaced with P-Thr, the resulting peptide was still a casein kinase I substrate. However, substitution of Asp or Glu residues at Ser7 led to peptides that were not phosphorylated by casein kinase I. Phosphorylation of one of the other peptides showed that Thr could also be the phosphate acceptor. From these results, we propose that there are substrates for casein kinase I for which prior phosphorylation is a critical determinant of protein kinase action. In these instances, an important recognition motif for casein kinase I appears to be -S(P)/T(P)-Xn-S/T- with n = 2 much more effective than n = 1 or n = 3. Thus, casein kinase I may be involved in hierarchal substrate phosphorylation schemes in which its activity is controlled by the phosphorylation state of its substrates.
环磷酸腺苷依赖性蛋白激酶对兔肌肉糖原合酶的磷酸化作用已被证明可增强随后酪蛋白激酶I的磷酸化作用（弗洛托，H.，和罗奇，P.J.（1989年）《生物化学杂志》264卷，9126 - 9128页）。在本研究中，基于肌肉糖原合酶四个磷酸化区域序列的合成肽被用于探究底物磷酸化在酪蛋白激酶I作用中的作用。对于所有这四种肽，预先磷酸化显著刺激了酪蛋白激酶I的磷酸化作用。基于氨基末端糖原合酶序列PLSRTLS7VSS10LPGL合成了一系列肽，其中酪蛋白激酶I对Ser10的修饰需要Ser7的磷酸化。磷酸化的丝氨酸（P - Ser）与受体丝氨酸之间的间隔变化为有1、2或3个间隔残基。具有2个残基间隔（-S(P)-X - X - S-）的肽是迄今为止最好的酪蛋白激酶I底物。当Ser7处的P - Ser残基被P - Thr取代时，所得肽仍是酪蛋白激酶I底物。然而，在Ser7处替换为天冬氨酸或谷氨酸残基导致肽不被酪蛋白激酶I磷酸化。对其他一种肽的磷酸化表明苏氨酸也可以是磷酸受体。从这些结果，我们提出存在酪蛋白激酶I的底物，对于它们来说预先磷酸化是蛋白激酶作用的关键决定因素。在这些情况下，酪蛋白激酶I的一个重要识别基序似乎是 -S(P)/T(P)-Xn - S/T-，其中n = 2比n = 1或n = 3有效得多。因此，酪蛋白激酶I可能参与分级底物磷酸化方案，其中其活性由其底物的磷酸化状态控制。
