Posttranslational modification of Bcl-2 facilitates its proteasome-dependent degradation: Molecular characterization of the involved signaling pathway
Posttranslational modification of Bcl-2 facilitates its proteasome-dependent degradation: Molecular characterization of the involved signaling pathway
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DOI：
10.1128/mcb.20.5.1886-1896.2000
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发表时间：
2000-03-01
影响因子：
5.3
通讯作者：
Dimmeler, S
中科院分区：
文献类型：
--
作者：
Breitschopf, K;Haendeler, J;Dimmeler, S
促凋亡分子与抗凋亡分子的比例是影响细胞对细胞凋亡易感性的重要决定因素。因此，近端信号转导事件引起的抗凋亡蛋白水平的降低可能开启了细胞凋亡的途径。在内皮细胞中，肿瘤坏死因子α(TNF-α)诱导去磷酸化和随后泛素依赖的抗凋亡蛋白Bc l-2的降解。在这里，我们研究了不同假定的磷酸化位点在促进Bcl-2降解方面的作用。共有的蛋白激酶B/Akt位点或潜在的蛋白激酶C或环状AMP依赖的蛋白激酶位点的突变不会影响Bcl2的稳定性。相反，三个共有的丝裂原活化蛋白(MAP)激酶位点的失活导致了一种泛素化的Bcl-2蛋白，随后被26S蛋白酶体降解。Bcl2中这些位点的失活表明Ser87的去磷酸化似乎起到了主要作用。根据脉冲追逐研究，该位置的Ser到Ala的替换会导致50%的降解，而Ala替换Thr74会导致25%的降解。我们进一步证明，与肿瘤坏死因子-α孵育可诱导完整细胞中Bcl2 Ser87的去磷酸化。此外，MAP激酶触发了Bcl2的磷酸化，而在MAPKs特异性磷酸酶或MAPKs特异性抑制剂PD98059的存在下，观察到Bcl2的磷酸化水平降低。此外，我们还发现，氧化应激通过降低MAP的活性来介导肿瘤坏死因子-α刺激的蛋白水解酶对Bc l-2的降解。综上所述，这些结果证明了MAP激酶对Bcl2磷酸化的直接保护作用，以对抗内皮细胞和其他细胞的凋亡挑战。
The ratio of proapoptotic versus antiapoptotic Bcl-2 members is a critical determinant that plays a significant role in altering susceptibility to apoptosis. Therefore, a reduction of antiapoptotic protein levels in response to proximal signal transduction events may switch on the apoptotic pathway. In endothelial cells, tumor necrosis factor alpha (TNF-alpha) induces dephosphorylation and subsequent ubiquitin-dependent degradation of the antiapoptotic protein Bcl-2. Here, we investigate the role of different putative phosphorylation sites to facilitate Bcl-2 degradation. Mutation of the consensus protein kinase B/Akt site or of potential protein kinase C or cyclic AMP-dependent protein kinase sites does not affect Bcl-2 stability. In contrast, inactivation of the three consensus mitogen-activated protein (MAP) kinase sites leads to a Bcl-2 protein that is ubiquitinated and subsequently degraded by the 26S proteasome. Inactivation of these sites within Bcl-2 revealed that dephosphorylation of Ser87 appears to play a major role. A Ser to Ala substitution at this position results in 50% degradation, whereas replacement of Thr74 with Ala leads to 25% degradation, as assessed by pulse-chase studies. We further demonstrated that incubation with TNF-alpha induces dephosphorylation of Ser87 of Bcl-2 in intact cells. Furthermore, MAP kinase triggers phosphorylation of Bcl-2, whereas a reduction in Bcl-2 phosphorylation was observed in the presence of MAP kinase-specific phosphatases or the MAP kinase-specific inhibitor PD98059. Moreover, we show that oxidative stress mediates TNF-alpha-stimulated proteolytic degradation of Bcl-2 by reducing MAP kinase activity. Taken together, these results demonstrate a direct protective role for Bcl-2 phosphorylation by MAP kinase against apoptotic challenges to endothelial cells and other cells.