Glass D B, Cheng H C, Kemp B E, Walsh D A
J Biol Chem. 1986 Sep 15;261(26):12166-71.
Synthetic peptides corresponding to the active domain of the heat-stable inhibitor protein of cAMP-dependent protein kinase (Cheng, H.-C., Kemp, B. E., Pearson, R. B., Smith, A. J., Misconi, L., Van Patten, S. M., and Walsh, D. A. (1986) J. Biol. Chem. 261, 989-992) were tested as inhibitors of cGMP-dependent protein kinase. The peptides themselves were not substrates. cGMP-dependent protein kinase activity was assayed using histone H2B and two synthetic peptide substrates. Consistent with previous observations of other peptide inhibitors of this enzyme (Glass, D. B. (1983) Biochem. J. 213, 159-164), the inhibitory peptides had no effect on the phosphorylation of histone H2B, but they competitively inhibited cGMP-dependent phosphorylation of the two peptide substrates. The parent inhibitor peptide, PKI(5-24)amide, and a series of analogs had Ki (or IC50) values for cGMP-dependent protein kinase in the range of 15-190 microM. In contrast to their effects on the cAMP-dependent protein kinase, the inhibitory peptides were substantially less potent with cGMP-dependent protein kinase, and potency was reduced by the presence of the NH2-terminal residues (residues 5-13). We conclude that the two protein kinases share a recognition of the basic amino acid cluster within the pseudosubstrate region of the peptide, but that the cGMP-dependent protein kinase does not recognize additional NH2-terminal determinants that make the inhibitor protein extremely potent toward the cAMP-dependent enzyme. Even- when tested at high concentrations and with peptide substrates, the native inhibitor protein did not inhibit cGMP-dependent protein kinase under assay conditions in which the peptides derived from it were inhibitory. Thus, the native inhibitor protein appears to have structural features which block interaction with the cGMP-dependent enzyme and enhance its selectivity for cAMP-dependent protein kinase.
对与环磷酸腺苷（cAMP）依赖性蛋白激酶的热稳定抑制蛋白活性结构域相对应的合成肽（Cheng, H.-C., Kemp, B. E., Pearson, R. B., Smith, A. J., Misconi, L., Van Patten, S. M., and Walsh, D. A. (1986) J. Biol. Chem. 261, 989 - 992）进行了测试，以确定其作为环磷酸鸟苷（cGMP）依赖性蛋白激酶抑制剂的效果。这些肽本身不是底物。使用组蛋白H2B和两种合成肽底物对cGMP依赖性蛋白激酶活性进行了测定。与先前对该酶其他肽抑制剂的观察结果一致（Glass, D. B. (1983) Biochem. J. 213, 159 - 164），抑制性肽对组蛋白H2B的磷酸化没有影响，但它们竞争性抑制了两种肽底物的cGMP依赖性磷酸化。母体抑制肽PKI(5 - 24)酰胺以及一系列类似物对cGMP依赖性蛋白激酶的Ki（或IC50）值在15 - 190微摩尔范围内。与它们对cAMP依赖性蛋白激酶的作用相反，抑制性肽对cGMP依赖性蛋白激酶的效力明显较低，并且由于NH2末端残基（残基5 - 13）的存在，效力降低。我们得出结论，这两种蛋白激酶在肽的假底物区域内共享对碱性氨基酸簇的识别，但cGMP依赖性蛋白激酶不识别使抑制蛋白对cAMP依赖性酶极具效力的额外NH2末端决定簇。即使在高浓度下用肽底物进行测试，天然抑制蛋白在其衍生肽具有抑制作用的测定条件下也不抑制cGMP依赖性蛋白激酶。因此，天然抑制蛋白似乎具有阻止与cGMP依赖性酶相互作用并增强其对cAMP依赖性蛋白激酶选择性的结构特征。
