Regulation of phosphorylation of Thr-308 of Akt, cell proliferation, and survival by the B55α regulatory subunit targeting of the protein phosphatase 2A holoenzyme to Akt
Regulation of phosphorylation of Thr-308 of Akt, cell proliferation, and survival by the B55α regulatory subunit targeting of the protein phosphatase 2A holoenzyme to Akt
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DOI：
10.1074/jbc.m709585200
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发表时间：
2008-01-25
影响因子：
4.8
通讯作者：
Chiang, Chi-Wu
中科院分区：
文献类型：
--
作者：
Kuo, Yi-Chun;Huang, Kai-Yun;Chiang, Chi-Wu
Akt 是一种蛋白丝氨酸/苏氨酸激酶，参与多种细胞过程的调节。 Akt 在调节残基 Thr-308 和 Ser-473 处的磷酸化导致其完全激活。人们早就知道蛋白磷酸酶 2A (PP2A) 可以负向调节 Akt 活性。 PP2A 全酶由结构亚基 (A)、催化亚基 (C) 和可变调节亚基 (B) 组成。在这里，我们报告了将 PP2A 全酶靶向 Akt 的特定 B 调节亚基的鉴定。我们通过前淋巴细胞 FL5.12 细胞中的免疫共沉淀分析发现 PP2A AB55C 全酶与 Akt 存在内源关联。 Akt 显示与 NIH3T3 细胞中异位表达的 B55 α 亚基相关。使用体外 Pulldown 分析证实了 B55 α 亚基和 Akt 之间的直接相互作用。有趣的是，我们发现，在 FL5.12 和 NIH3T3 细胞中，B55 α 亚基的过表达显着损害了 Akt Thr-308 的磷酸化，但程度较轻。与此同时，这些细胞中 B55 α 的过度表达大大降低了 Akt 底物子集（包括 FoxO3a）的磷酸化。沉默 B55 α 表达可显着增加 FL5.12 细胞和 NIH3T3 细胞中 Thr-308 处的磷酸化，但不增加 Ser-473 处的磷酸化。一致地，在体外去磷酸化测定中，PP2A AB55 α C 全酶优先去磷酸化磷酸-Thr-308，而不是磷酸-Ser-473。此外，B55 α 过表达会延缓 NIH3T3 细胞的增殖，而 B55 α 表达的敲低会增加 FL5.12 细胞在白细胞介素 3 剥夺后的存活率。总之，我们的数据表明，PP2A 全酶对 Akt 的 B55 α 依赖性靶向选择性调节 Akt Thr-308 磷酸化，从而调节细胞增殖和存活。
Akt is a protein serine/threonine kinase that is involved in the regulation of diverse cellular processes. Phosphorylation of Akt at regulatory residues Thr-308 and Ser-473 leads to its full activation. The protein phosphatase 2A ( PP2A) has long been known to negatively regulate Akt activity. The PP2A holoenzyme consists of the structural subunit ( A), catalytic subunit ( C), and a variable regulatory subunit ( B). Here we report the identification of the specific B regulatory subunit that targets the PP2A holoenzyme to Akt. We found endogenous association of PP2A AB55C holoenzymes with Akt by co-immunoprecipitation analyses in pro-lymphoid FL5.12 cells. Akt was shown to associate with ectopically expressed B55 alpha subunit in NIH3T3 cells. The direct interaction between B55 alpha subunit and Akt was confirmed using in vitro pulldown analyses. Intriguingly, we found that overexpression of B55 alpha subunit significantly impaired phosphorylation at Thr-308, but to a lesser extent at Ser-473 of Akt in both FL5.12 and NIH3T3 cells. Concomitantly, phosphorylation of a subset of Akt substrates, including FoxO3a, was substantially decreased by B55 alpha overexpression in these cells. Silencing of B55 alpha expression markedly increased phosphorylation at Thr-308 but not at Ser-473 in both FL5.12 cells and NIH3T3 cells. Consistently, PP2A AB55 alpha C holoenzymes preferentially dephosphorylated phospho-Thr-308 rather than phospho-Ser-473 in in vitro dephosphorylation assays. Furthermore, B55 alpha overexpression retarded proliferation of NIH3T3 cells, and knockdown of B55 alpha expression increased survival of FL5.12 cells upon interleukin-3 deprivation. Together, our data demonstrate that B55 alpha-dependent targeting of the PP2A holoenzyme to Akt selectively regulates Akt phosphorylation at Thr-308 to regulate cell proliferation and survival.