THE P53 TUMOR SUPPRESSOR PROTEIN IS PHOSPHORYLATED AT SERINE-389 BY CASEIN KINASE-II
THE P53 TUMOR SUPPRESSOR PROTEIN IS PHOSPHORYLATED AT SERINE-389 BY CASEIN KINASE-II
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DOI：
10.1002/j.1460-2075.1990.tb07524.x
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发表时间：
1990-10-01
期刊：
影响因子：
11.4
通讯作者：
ECKHART, W
中科院分区：
文献类型：
--
作者：
MEEK, DW;SIMON, S;ECKHART, W
野生型小鼠p53的整个编码序列在λ噬菌体的PL启动子的控制下在大肠杆菌中表达。在SDS聚丙烯酰胺凝胶上，细菌p53蛋白具有与来自SV 3 T3细胞的p53相同的迁移率，并且在细菌裂解物中被三种P53特异性单克隆抗体识别，包括对野生型小鼠p53具有特异性的PAb 246。通过高度纯化的大鼠酪蛋白激酶II制剂使细菌p53的免疫沉淀物磷酸化：掺入的化学计量为1摩尔磷酸盐/摩尔p53。磷酸化残基通过磷酸肽图谱鉴定为丝氨酸389，这是体内p53磷酸化的主要位点。在SV 3 T3细胞裂解物中检测到p53（丝氨酸389）激酶活性;该活性在磷酸纤维素和Mono Q柱上与酪蛋白激酶II共纯化，并被肝素抑制。来自SV 3 T3细胞的p53-T抗原复合物的免疫沉淀物也具有相关的丝氨酸389激酶活性。丝氨酸389的磷酸化被肝素有效抑制，并被过量的未标记GTP淬灭。这些数据表明，p53是酪蛋白激酶II的生理底物，酪蛋白激酶II响应于有丝分裂原而被刺激，使核癌蛋白磷酸化，并且可能在细胞外信号转导至核中发挥作用。
The entire coding sequence of wild-type mouse p53 was expressed in Escherichia coli under control of the PL promoter of bacteriophage lambda. The bacterial p53 protein had identical mobility to p53 from SV3T3 cells on SDS polyacrylamide gels and was recognized in bacterial lysates by three P53-specific monoclonal antibodies, including PAb246 which is specific for wild-type mouse p53. Immunoprecipitates of the bacterial p53 were phosphorylated by a highly purified preparation of rat casein kinase II: the stoichiometry of incorporation was .apprx. 1 mol of phosphate per mol of p53. The phosphorylated residue was identified by phosphopeptide mapping as serine 389, which is a major site of p53 phosphorylation in vivo. p53 (serine 389) kinase activity was detected on lysates of SV3T3 cells; this activity co-purified with casein kinase II on phosphocellulose and Mono Q columns and was inhibited by heparin. Immunoprecipitates of the p53-T antigen complex from SV3T3 cells also had associated serine 389 kinase activity. Phosphorylation of serine 389 by this kinase potently inhibited by heparin and quenched by excess unlabelled GTP. The data indicate that p53 is a physiological substrate of casein kinase II, which is stimulated in response to mitogens, phosphorylates nuclear oncoproteins, and may play a role in the transduction of extracellular signals to the nucleus.