BUDDING AND FISSION YEAST CASEIN KINASE-I ISOFORMS HAVE DUAL-SPECIFICITY PROTEIN-KINASE ACTIVITY
BUDDING AND FISSION YEAST CASEIN KINASE-I ISOFORMS HAVE DUAL-SPECIFICITY PROTEIN-KINASE ACTIVITY
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DOI：
10.1091/mbc.5.8.877
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发表时间：
1994-08-01
影响因子：
3.3
通讯作者：
KURET, J
中科院分区：
文献类型：
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作者：
HOEKSTRA, MF;DHILLON, N;KURET, J
我们检查了酿酒酵母 Hrr25p 和粟酒裂殖酵母 Hhp1、Hhp2 和 Cki1 蛋白激酶亚型的活性和底物特异性。这四种基因产物是酪蛋白激酶I（CKI）的同种型，这些蛋白激酶的序列预测它们是蛋白丝氨酸/苏氨酸激酶。然而，这四种蛋白激酶中的每一种，当在大肠杆菌中以活性形式表达时，都会被抗磷酸酪氨酸抗体识别。 P-32 标记蛋白质的磷酸氨基酸分析显示丝氨酸、苏氨酸和酪氨酸残基发生磷酸化。大肠杆菌产生的 Hhp1、Hhp2 和 Cki1 形式在酪氨酸上进行自磷酸化，并且 Hhp1 和 Hhp2 都能够磷酸化酪氨酸蛋白激酶合成肽底物聚合物聚 E(4)Y(1)。对粟酒裂殖酵母细胞的免疫复合物蛋白激酶测定表明，含有 Hhp1 的沉淀物与蛋白酪氨酸激酶活性相关，并且这些免疫沉淀物中存在的 Hhp1 在酪氨酸残基上被磷酸化。虽然 Ser/Thr 磷酸酶对 Hhp1 和 Hhp2 的去磷酸化对比活性影响不大，但 Hhp1 和 Hhp2 的酪氨酸去磷酸化导致聚 E(4)Y(1) 和酪蛋白的 Km 增加 1.8 至 3.1 倍。这些数据表明，来自两种不同酵母的四种不同的 CKI 亚型具有蛋白酪氨酸激酶活性并编码双特异性蛋白激酶。
We have examined the activity and substrate specificity of the Saccharomyces cerevisiae Hrr25p and the Schizosaccharomyces pombe Hhp1, Hhp2, and Cki1 protein kinase isoforms. These four gene products are isotypes of casein kinase I (CKI), and the sequence of these protein kinases predicts that they are protein serine/threonine kinases. However, each of these four protein kinases, when expressed in Escherichia coli in an active form, was recognized by anti-phosphotyrosine antibodies. Phosphoamino acid analysis of P-32-labeled proteins showed phosphorylation on serine, threonine, and tyrosine residues. The E. coli produced forms of Hhp1, Hhp2, and Cki1 were autophosphorylated on tyrosine, and both Hhp1 and Hhp2 were capable of phosphorylating the tyrosine-protein kinase synthetic peptide substrate polymer poly-E(4)Y(1). Immune complex protein kinases assays from S. pombe cells showed that Hhp1-containing precipitates were associated with a protein-tyrosine kinase activity, and the Hhp1 present in these immunoprecipitates was phosphorylated on tyrosine residues. Although dephosphorylation of Hhp1 and Hhp2 by Ser/Thr phosphatase had little effect on the specific activity, tyrosine dephosphorylation of Hhp1 and Hhp2 caused a 1.8-to 3.1-fold increase in the Km for poly-E(4)Y(1) and casein. These data demonstrate that four different CKI isoforms from two different yeasts are capable of protein-tyrosine kinase activity and encode dual-specificity protein kinases.