Flotow H, Roach P J
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis 46223.
J Biol Chem. 1991 Feb 25;266(6):3724-7.
Sites phosphorylated by casein kinase I have been characterized by the presence of acidic amino acids NH2-terminal to the modified residue. Recently, phosphoserine was shown to be a particularly effective determinant for casein kinase I action when present in the motif -S(P)-X-X-S- (Flotow, H., Graves, P. R., Wang, A., Fiol, C. J., Roeske, R. W., and Roach, P. J. (1990) J. Biol. Chem. 265, 14264-14269). Nonetheless, nonphosphorylated substrates for casein kinase I are well documented. In this study, we examined the efficacy of Asp and Glu residues as determinants of casein kinase I action using synthetic peptide substrates. Peptides with runs of Asp residues in the motif Dn-X-X-S- were substrates for casein kinase I. Peptides with n = 3 or 4 were the most effective substrates, much better than n = 2. The peptide with n = 1, a single Asp residue, was a very poor substrate. A block of 4 Glu residues was a little less effective as a substrate determinant than 4 Asp residues in an otherwise identical peptide. The most effective substrate, with the motif -D-D-D-D-X-X-S-, was specific for casein kinase I and was not detectably phosphorylated by cyclic AMP-dependent protein kinase, casein kinase II, glycogen synthase kinase 3, or phosphorylase kinase and thus will be useful for the specific assay of casein kinase I. This peptide was nonetheless significantly worse as a substrate than peptides in which casein kinase I action was determined by phosphoserine in the -3 position. Still, the fact that Asp or Glu residues can specify a casein kinase I substrate suggests that acidic character has a role in substrate selection by this protein kinase.
酪蛋白激酶I磷酸化的位点其特征是在修饰残基的氨基末端存在酸性氨基酸。最近，当丝氨酸磷酸化存在于基序-S(P)-X-X-S-中时，被证明是酪蛋白激酶I作用的一个特别有效的决定因素（弗洛托，H.，格雷夫斯，P.R.，王，A.，菲奥尔，C.J.，罗斯克，R.W.，和罗奇，P.J.（1990年）《生物化学杂志》265，14264 - 14269）。尽管如此，酪蛋白激酶I的非磷酸化底物也有充分的文献记载。在本研究中，我们使用合成肽底物研究了天冬氨酸和谷氨酸残基作为酪蛋白激酶I作用决定因素的效力。在基序Dn-X-X-S-中含有多个天冬氨酸残基的肽是酪蛋白激酶I的底物。n = 3或4的肽是最有效的底物，比n = 2的要好得多。n = 1的肽，即单个天冬氨酸残基，是非常差的底物。在其他方面相同的肽中，由4个谷氨酸残基组成的片段作为底物决定因素的效力比4个天冬氨酸残基略低。最有效的底物，其基序为-D-D-D-D-X-X-S-，对酪蛋白激酶I具有特异性，未被环磷酸腺苷依赖性蛋白激酶、酪蛋白激酶II糖原合酶激酶3或磷酸化酶激酶检测到磷酸化，因此将可用于酪蛋白激酶I的特异性检测。然而，该肽作为底物的效果仍明显不如酪蛋白激酶I作用由-3位的磷酸丝氨酸决定的肽。尽管如此，天冬氨酸或谷氨酸残基可以确定酪蛋白激酶I底物这一事实表明，酸性特征在该蛋白激酶的底物选择中起作用。
