Phosphorylation of CD45 by casein kinase 2. Modulation of activity and mutational analysis.
Phosphorylation of CD45 by casein kinase 2. Modulation of activity and mutational analysis.
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酪蛋白激酶 2 对 CD45 进行磷酸化。活性调节和突变分析。
DOI：
10.1074/jbc.274.11.7454
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Esselman,WJ
中科院分区：
文献类型：
--
作者：
Wang,Y;Guo,W;Liang,L;Esselman,WJ
CD45是一种受体型蛋白酪氨酸磷酸酶（PTP），是淋巴细胞抗原特异性刺激和增殖所必需的。本研究旨在确定淋巴细胞中磷酸化CD45的特定激酶的性质，并确定磷酸化对CD45 PTP活性的影响。通过结合免疫沉淀、免疫消耗和特异性抑制的凝胶激酶测定，确定了一个磷酸化CD45的主要细胞质淋巴细胞激酶是酪蛋白激酶2 （CK2）。对CK2共识位点的突变分析表明，CK2的靶点位于D2结构域19个氨基酸的酸性插入中，而965、968、969和973氨基酸的Ser - Ala突变取消了CK2对CD45的磷酸化。CK2磷酸化使CD45活性向磷酸化髓鞘碱性蛋白方向增加3倍，这种增加通过PP2A治疗是可逆的。在CK2位点Ser突变为Glu具有与磷酸化相同的效果，并且CD45的vmax也增加了两倍。体内分离的CD45被高度磷酸化，如果不事先用磷酸酶PP2A去磷酸化，则不能被CK2磷酸化。我们得出结论，CK2是一种主要的淋巴细胞激酶，负责CD45的体内磷酸化，CK2特定位点的磷酸化调节CD45 PTP活性。
CD45 is a receptor-type protein-tyrosine phosphatase (PTP) that is required for antigen-specific stimulation and proliferation in lymphocytes. This study was designed to determine the nature of specific kinases in lymphocytes that phosphorylate CD45 and to determine the effect of phosphorylation on CD45 PTP activity. A major cytoplasmic lymphocyte kinase that phosphorylated CD45 was identified as casein kinase 2 (CK2) by use of an in-gel kinase assay in combination with immunoprecipitation, immunodepletion, and specific inhibition. Mutational analysis of CK2 consensus sites showed that the target for CK2 was in an acidic insert of 19 amino acids in the D2 domain, and Ser to Ala mutations at amino acids 965, 968, 969, and 973 abrogated CK2 phosphorylation of CD45. CK2 phosphorylation increased CD45 activity 3-fold toward phosphorylated myelin basic protein, and this increase was reversible by PP2A treatment. Mutation of Ser to Glu at the CK2 sites had the same effect as phosphorylation and also tripled theVmaxof CD45. CD45 isolatedin vivowas highly phosphorylated and could not be phosphorylated by CK2 without prior dephosphorylation with phosphatase PP2A. We conclude that CK2 is a major lymphocyte kinase that is responsible forin vivophosphorylation of CD45, and phosphorylation at specific CK2 sites regulates CD45 PTP activity.