Lohrum M, Scheidtmann K H
Institut für Genetik, Abt. Molekulargenetik, Universitat Bonn.
Oncogene. 1996 Dec 19;13(12):2527-39.
The tumor suppressor protein p53 is phosphorylated at multiple sites in the amino-terminal transactivation domain and at several sites in the carboxy-terminal region. Phosphorylation appears to modulate its DNA binding activity. Here we demonstrate that phosphorylation of p53 also modulates its transcriptional activity. Okadaic acid treatment of cells resulted in enhanced phosphorylation of p53 and concomitantly in enhanced transactivation of an mdm2 promoter-linked luciferase reporter gene. This effect was cell type specific, however, since transactivation was enhanced in rat and mouse fibroblasts but reduced in the human Saos-2 cell line. Moreover, the effect was dependent on the promoter. In rat cells transcription from the mdm2, waf1 (cip1) and bax gene promoters, and the artificial PG13 promoter was enhanced by okadaic acid treatment whereas that from the cyclin G promoter was reduced. When various phosphorylation site mutants of p53 were tested for transactivation of these promoters, they behaved differently. Amino-terminal mutants exhibited reduced transcriptional activities on mdm2, waf1 and cyclin G promoters but enhanced activities with bax and PG13 promoters. On the other hand, a mutant at the cdk phosphorylation site, A313, showed reduced activity with mdm2 and waf1 promoters but enhanced activity with the cyclin G promoter, and finally, mutant A390 exhibited enhanced activity on waf1 and bax promoters, but reduced activity on the cyclin G promoter. These results suggest that phosphorylation of p53 may have different effects on its transcriptional activity, depending on the cellular environment and the particular response element. Moreover, both, amino- and carboxy-terminal phosphorylation sites seem to be involved in modulating the DNA-binding and the transactivation activities.
肿瘤抑制蛋白p53在氨基末端反式激活结构域的多个位点以及羧基末端区域的几个位点被磷酸化。磷酸化似乎可调节其DNA结合活性。在此我们证明，p53的磷酸化还可调节其转录活性。用冈田酸处理细胞导致p53磷酸化增强，同时mdm2启动子连接的荧光素酶报告基因的反式激活增强。然而，这种效应具有细胞类型特异性，因为在大鼠和小鼠成纤维细胞中反式激活增强，而在人Saos-2细胞系中则降低。此外，这种效应取决于启动子。在大鼠细胞中，冈田酸处理可增强mdm2、waf1（cip1）和bax基因启动子以及人工PG13启动子的转录，而细胞周期蛋白G启动子的转录则降低。当测试p53的各种磷酸化位点突变体对这些启动子的反式激活时，它们的表现不同。氨基末端突变体在mdm2、waf1和细胞周期蛋白G启动子上表现出降低的转录活性，但在bax和PG13启动子上活性增强。另一方面，cdk磷酸化位点的突变体A313在mdm2和waf1启动子上活性降低，但在细胞周期蛋白G启动子上活性增强，最后，突变体A390在waf1和bax启动子上活性增强，但在细胞周期蛋白G启动子上活性降低。这些结果表明，p53的磷酸化对其转录活性可能有不同影响，这取决于细胞环境和特定的反应元件。此外，氨基末端和羧基末端的磷酸化位点似乎都参与调节DNA结合和反式激活活性。
