Rogatsky I, Trowbridge J M, Garabedian M J
Department of Microbiology and the Kaplan Comprehensive Cancer Center, New York University School of Medicine, New York, New York 10016, USA.
J Biol Chem. 1999 Aug 6;274(32):22296-302. doi: 10.1074/jbc.274.32.22296.
Both estradiol binding and phosphorylation regulate transcriptional activation by the human estrogen receptor alpha (ER). We have previously shown that activation of the cyclin A-CDK2 complex by overexpression of cyclin A leads to enhanced ER-dependent transcriptional activation and that the cyclin A-CDK2 complex phosphorylates the ER N-terminal activation function-1 (AF-1) between residues 82 and 121. Within ER AF-1, serines 104, 106, and 118 represent potential CDK phosphorylation sites, and in this current study, we ascertain their importance in mediating cyclin A-CDK2-dependent enhancement of ER transcriptional activity. Cyclin A overexpression does not enhance transcriptional activation by an ER derivative bearing serine-to-alanine changes at residues 104, 106, and 118. Likewise, the cyclin A-CDK2 complex does not phosphorylate this triple-mutated derivative in vitro. Individual serine-to-alanine mutations at residues 104 and 106, but not 118, decrease ER-dependent transcriptional enhancement in response to cyclin A. The same relationship holds for ER phosphorylation by cyclin A-CDK2 in vitro. Finally, enhancement of ER transcriptional activation by cyclin A is evident in the absence and presence of estradiol, as well as in the presence of tamoxifen, suggesting that the effect of the cyclin A-CDK2 on ER transcriptional activation is AF-2-independent. These results indicate that the enhancement of ER transcriptional activation by the cyclin A-CDK2 complex is mediated via the AF-1 domain by phosphorylation of serines 104 and 106. We propose that these residues control ER AF-1 activity in response to signals that affect cyclin A-CDK2 function.
雌二醇结合和磷酸化均调节人雌激素受体α（ER）的转录激活。我们之前已经表明，通过过表达细胞周期蛋白A激活细胞周期蛋白A-CDK2复合物会导致ER依赖性转录激活增强，并且细胞周期蛋白A-CDK2复合物会使ER N端激活功能1（AF-1）在82至121位残基之间发生磷酸化。在ER AF-1中，丝氨酸104、106和118代表潜在的CDK磷酸化位点，在本研究中，我们确定它们在介导细胞周期蛋白A-CDK2依赖性增强ER转录活性中的重要性。在104、106和118位残基处带有丝氨酸到丙氨酸变化的ER衍生物，细胞周期蛋白A的过表达不会增强其转录激活。同样，细胞周期蛋白A-CDK2复合物在体外不会使这种三重突变衍生物发生磷酸化。在104和106位残基处的单个丝氨酸到丙氨酸突变（而非118位）会降低细胞周期蛋白A诱导的ER依赖性转录增强。体外细胞周期蛋白A-CDK2对ER的磷酸化也存在相同关系。最后，无论有无雌二醇以及是否存在他莫昔芬，细胞周期蛋白A均可增强ER转录激活，这表明细胞周期蛋白A-CDK2对ER转录激活的作用不依赖于AF-2。这些结果表明，细胞周期蛋白A-CDK2复合物对ER转录激活的增强是通过丝氨酸104和106的磷酸化经由AF-1结构域介导的。我们提出，这些残基响应影响细胞周期蛋白A-CDK2功能的信号来控制ER AF-1的活性。
