PKC-dependent phosphorylation of p27 at T198 contributes to p27 stabilization and cell cycle arrest
PKC-dependent phosphorylation of p27 at T198 contributes to p27 stabilization and cell cycle arrest
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DOI：
10.4161/cc.20003
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发表时间：
2012-04-15
期刊：
影响因子：
4.3
通讯作者：
Viglietto, Giuseppe
中科院分区：
文献类型：
--
作者：
De Vita, Fernanda;Riccardi, Miriam;Viglietto, Giuseppe
在这篇文章中，我们提出了实验证据表明，PKC磷酸化p27在T198在体外和体内，导致p27稳定和细胞周期停滞在MCF-7和HeLa细胞。我们的研究结果表明：（1）在体外激酶试验中，重组PKC α、β II、δ、eta和θ亚型磷酸化在大肠杆菌中表达的野生型重组p27蛋白;（2）过继表达的PKCa和d在HEK-293细胞中在T198处磷酸化转染的和内源性p27，（3）转染的和内源性p27的T198磷酸化被PKC激活剂[佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）]增加并被PKC抑制剂抑制（Rottlerin A，G06976，Calphostin C），（4）与增加的T198磷酸化平行，PMA诱导HeLa细胞中p27蛋白的稳定，而PKC抑制剂诱导p27稳定性的降低，最后，（5）PMA诱导的p27上调是PMA激活PKC诱导HeLa和MCF-7细胞生长停滞所必需的。这些结果表明，PKC-p27在T198磷酸化诱导的依赖性上调代表了一种介导PMA促进的生长停滞的机制，并提供了不同PKC同种型在控制细胞周期进程中发挥作用。
In this manuscript, we present experimental evidence that PKCs phosphorylate p27 at T198 in vitro and in vivo, resulting in p27 stabilization and cell cycle arrest in MCF-7 and HeLa cells. Our findings indicate that (1) recombinant PKC alpha, beta II, delta, eta and theta isoforms phosphorylate, in in vitro kinase assays, wild-type recombinant p27 protein expressed in E. coli and wildtype p27 protein immunoprecpitated from transfected HEK-293 cells but not the T198A mutant, (2) adoptive expressed PKCa and d phosphorylate both transfected and endogenous p27 at T198 in HEK-293 cells, (3) T198 phosphorylation of transfected and endogenous p27 is increased by PKC activators [Phorbol 12-myristate 13-acetate (PMA)] and suppressed by PKC inhibitors (Rottlerin A, G06976, Calphostin C), (4) in parallel with increased T198 phosphorylation, PMA induces stabilization of p27 protein in HeLa cells, whereas PKC inhibitors induce a decrease in p27 stability and, finally, (5) PMA-induced p27 upregulation is necessary for growth arrest of HeLa and MCF-7 cells induced by PKC activation by PMA.Overall, these results suggest that PKC-dependent upregulation of p27 induced by its phosphorylation at T198 represents a mechanism that mediates growth arrest promoted by PMA and provide novel insights on the ability of different PKC isoforms to play a role in controlling cell cycle progression.