Evidence that Ser87 of BimEL is phosphorylated by Akt and regulates BimEL apoptotic function
Evidence that Ser87 of BimEL is phosphorylated by Akt and regulates BimEL apoptotic function
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DOI：
10.1074/jbc.m505546200
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发表时间：
2006-01-13
影响因子：
4.8
通讯作者：
Howe, PH
中科院分区：
文献类型：
--
作者：
Qi, XJ;Wildey, GM;Howe, PH
BIM是细胞死亡的Bcl-2相互作用介体，是BH3-only促凋亡蛋白家族的成员。最近的研究表明，Bim的凋亡活性可以通过翻译后机制来调节，其中ERK磷酸化是Bim泛素化和蛋白酶体降解的信号。在这篇报道中，我们研究了导致Bim磷酸化的信号通路在依赖白介素3(IL3)的B细胞系BA/F3细胞中。IL-3刺激诱导Bim(EL)的磷酸化，Bim(EL)是Bim在细胞中表达的主要亚型之一，通过Western blotting至少形成三到四条对磷酸酶消化敏感的条带，证明了这一点。BimEL多个磷酸化物种的出现与Akt的激活有关，而与ERK的激活无关。PI3K抑制剂LY294002可阻断IL-3刺激的Akt活性，并部分阻断BimEL的磷酸化。体外激酶分析表明，重组Akt能直接磷酸化GST-Bim(EL)融合蛋白，并将Bim(EL)结构域中的Akt磷酸化位点确定为Ser(87)。进一步，我们证明了细胞因子刺激促进了BimEL与14-3-3蛋白的结合。最后，我们发现Ser(87)的突变显著增加了Bim(EL)的凋亡能力。我们认为Bim(EL)的Ser(87)是Akt靶向抑制Bim(EL)促凋亡功能从而促进细胞存活的重要调控位点。
Bim, the Bcl-2 interacting mediator of cell death, is a member of the BH3-only family of pro-apoptotic proteins. Recent studies have demonstrated that the apoptotic activity of Bim can be regulated through a post-translational mechanism whereby ERK phosphorylation serves as a signal for Bim ubiquitination and proteasomal degradation. In this report, we investigated the signaling pathways leading to Bim phosphorylation in Ba/F3 cells, an interleukin-3 (IL3)-dependent B-cell line. IL-3 stimulation induced phosphorylation of Bim(EL), one of the predominant isoforms of Bim expressed in cells, at multiple sites, as evidenced by the formation of at least three to four bands by Western blotting that were sensitive to phosphatase digestion. The appearance of multiple, phosphorylated species of BimEL correlated with Akt, and not ERK, activation. The PI3K inhibitor, LY294002, blocked IL-3-stimulated Akt activity and partially blocked BimEL phosphorylation. In vitro kinase assays showed that recombinant Akt could directly phosphorylate a GST-Bim(EL) fusion protein and identified the Akt phosphorylation site in the Bim(EL) domain as Ser(87). Further, we demonstrated that cytokine stimulation promotes BimEL binding to 14-3-3 proteins. Finally, we show that mutation of Ser(87) dramatically increases the apoptotic potency of Bim(EL). We propose that Ser(87) of Bim(EL) is an important regulatory site that is targeted by Akt to attenuate the pro-apoptotic function of Bim(EL), thereby promoting cell survival.