Abstract：
The expression level of the tumor suppressor p53 is controlled by the E3 ubiquitin ligase MDM2 with a regulatory feedback loop, which allows p53 to upregulate its inhibitor MDM2. In this manuscript we demonstrated that p90RSK binds and phosphorylates MDM2 on serine 166 both in vitro and in vivo by kinase assay, immunoblot, and co-immunoprecipitation assay; this phosphorylation increases the stability of MDM2 which in turn binds p53, ubiquitinating it and promoting its degradation by proteasome. A pharmacological inhibitor of p90RSK, BI-D1870, decreases MDM2 phosphorylation, and restores p53 function, which in turn transcriptionally increases the expression of cell cycle inhibitor p21 and of pro-apoptotic protein Bax and downregulates the anti-apoptotic protein Bcl-2, causing a block of cell proliferation, measured by a BrdU assay and growth curve, and promoting apoptosis, measured by a TUNEL assay. Finally, an immunohistochemistry evaluation of primary thyroid tumors, in which p90RSK is very active, confirms MDM2 stabilization mediated by p90RSK phosphorylation.
摘要：
肿瘤抑制因子p53的表达水平由E3泛素连接酶MDM2控制，具有调节反馈环，这允许p53上调其抑制剂MDM2。在本手稿中，我们证明了p90RSK通过激酶测定在体外和体内结合并磷酸化丝氨酸166上的MDM2，免疫印迹,和共免疫沉淀测定；这种磷酸化增加了MDM2的稳定性，MDM2又结合p53，使其泛素化并促进其通过蛋白酶体降解。p90RSK的药理抑制剂，BI-D1870，减少MDM2磷酸化，并恢复p53功能，这反过来转录增加细胞周期抑制剂p21和促凋亡蛋白Bax的表达，并下调抗凋亡蛋白Bcl-2，导致细胞增殖阻断，通过BrdU测定和生长曲线测量，促进细胞凋亡，通过TUNEL测定法测量。最后,原发性甲状腺肿瘤的免疫组织化学评估，其中p90RSK非常活跃，证实了P90RSK磷酸化介导的MDM2稳定。
