Studies on the substrate specificity of Ca2+/calmodulin-dependent protein kinase kinase alpha.
Studies on the substrate specificity of Ca2+/calmodulin-dependent protein kinase kinase alpha.
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Ca2/钙调蛋白依赖性蛋白激酶激酶α底物特异性的研究。
DOI：
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发表时间：
1997
期刊：
影响因子：
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通讯作者：
H. Fujisawa
中科院分区：
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作者：
S. Okuno;T. Kitani;H. Fujisawa
已知Ca 2 +/钙调蛋白依赖性蛋白激酶（CaM-激酶）激酶a分别通过Thr 196和Thr 177的磷酸化激活CaM-激酶IV和I，但在CaM-激酶IV的许多磷酸化位点中仅能磷酸化Thr 196 [Kitani，T.，Okuno，S.，Fujisawa，H. 121，804-810]，表明其高度的底物特异性。在本研究中，使用各种蛋白质和合成肽作为底物，以解决其生理功能的CaM-激酶激酶a的底物特异性进行了检查。在许多蛋白质和合成肽中，包括几种已知作为各种蛋白激酶的良好底物的蛋白质和合成肽，只有CaM-激酶IV和I以及含有CaM-激酶IV的Thr 196或CaM-激酶I的Thr 177周围序列的肽被CaM-激酶α显著磷酸化，而热变性（在60 ℃下5分钟）的CaM-激酶IV和I不被磷酸化。含有钙调蛋白激酶IV或I磷酸化位点的肽作为钙调蛋白激酶a的底物活性远低于天然钙调蛋白激酶IV或I。因此，钙调素激酶激酶a表现出高度的底物特异性，不仅识别特定的氨基酸序列，而且识别钙调素激酶IV和I的天然构象。
Ca2+/calmodulin-dependent protein kinase (CaM-kinase) kinase a, which is known to activate CaM-kinases IV and I by phosphorylation of Thr196 and Thr177, respectively, can only phosphorylate Thr196 among many phosphorylation sites of CaM-kinase IV [Kitani, T., Okuno, S., and Fujisawa, H. (1997) J. Biochem. 121, 804-810], indicating its high degree of substrate specificity. In the present study, the substrate specificity of CaM-kinase kinase a was examined using various proteins and synthetic peptides as substrates as a means to address its physiological function. Among a number of proteins and synthetic peptides, including several known as good substrates for various protein kinases, only CaM-kinases IV and I and peptides containing the sequence surrounding Thr196 of CaM-kinase IV or Thr177 of CaM-kinase I were significantly phosphorylated by CaM-kinase kinase alpha, while the heat-denatured (at 60 degrees C for 5 min) CaM-kinases IV and I were not phosphorylated. Peptides containing the phosphorylation site of CaM-kinase IV or I were far less active as substrates for CaM-kinase kinase a than were native CaM-kinase IV or I. Thus, CaM-kinase kinase a showed a high degree of substrate specificity, recognizing not only specific amino acid sequences but also the native conformation of CaM-kinases IV and I.