Protein kinase C stabilizes X-linked inhibitor of apoptosis protein (XIAP) through phosphorylation at Ser87 to suppress apoptotic cell death
Protein kinase C stabilizes X-linked inhibitor of apoptosis protein (XIAP) through phosphorylation at Ser87 to suppress apoptotic cell death
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DOI：
10.1111/j.1479-8301.2011.00355.x
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发表时间：
2011-06-01
期刊：
影响因子：
2
通讯作者：
Takeda, Masatoshi
中科院分区：
文献类型：
--
作者：
Kato, Kiyoko;Tanaka, Toshihisa;Takeda, Masatoshi
背景：多种蛋白激酶参与了阿尔茨海默病（AD）的神经元凋亡。尽管一些研究支持蛋白激酶C（PKC）在淀粉样前体蛋白加工以及tau蛋白磷酸化中的作用，但PKC在凋亡性神经元死亡中的直接作用仍有待澄清。在本研究中，我们报告的PKC在细胞生存的可能作用，在应激条件下，通过磷酸化的X-连锁的凋亡抑制蛋白（XIAP）。方法：磷酸化的XIAP在Ser 87的蛋白质印迹分析确认采用磷酸化依赖性抗XIAP抗体孵育后，重组XIAP与活性PKC在体外。用PKC激活剂佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）处理的SH-SY 5 Y细胞中，也证实了XIAP在该位点的磷酸化增加。制备其中Ser 87被Ala取代的突变XIAP构建体，并转染至细胞。野生型或突变型XIAP转染后，细胞活力进行了评估，通过计数活细胞和死细胞处理与PMA在依托泊苷诱导apoptosis.Results：重组XIAP磷酸化Ser 87由PKC在体外和治疗的XIAP转染的SH-SY 5 Y细胞与PKC激活剂，佛波酯12-肉豆蔻酸酯13-乙酸酯（PMA）诱导XIAP磷酸化Ser 87。脉冲追踪实验表明，当在Ser 87磷酸化时，野生型XIAP比具有Ser 87 Ala取代的XIAP更稳定，后者降解更快。重要的是，在由25 μ g/ml依托泊苷诱导的凋亡条件下，通过PKC在该位点磷酸化XIAP使细胞存活率显著增加高达约2.5倍。本研究的结果表明，PKC通过XIAP在Ser 87的磷酸化及其稳定化，在应激条件下的细胞存活中的作用，并加强了PKC在调节神经元稳态中至关重要的观点，这可能在AD中受损。
Background: Multiple protein kinases have been shown to be involved in the apoptotic neuronal loss of Alzheimer's disease (AD). Although some studies support the role of protein kinase C (PKC) in amyloid precursor protein processing as well as in tau phosphorylation, a direct role for PKC in apoptotic neuronal death remains to be clarified. In the present study, we report on the possible role of PKC in cell survival during conditions of stress through phosphorylation of the X-linked inhibitor of apoptosis protein (XIAP).Methods: Phosphorylation of XIAP at Ser87 was confirmed by western blot analysis employing phosphorylation dependent anti-XIAP antibody after incubation of recombinant XIAP with active PKC in vitro. And increased phosphorylation of XIAP at the site was also confirmed in SH-SY5Y cells treated with PKC activator, phorbol 12-myristate 13-acetate (PMA). A mutant XIAP construct in which Ser87 was substituted by Ala, was prepared, and transfected to cells. After the transfection of wild or mutant XIAP, cells viability was evaluated by counting living and dead cells treated with PMA during etoposide-induced apoptosis.Results: Recombinant XIAP was phosphorylated at Ser87 by PKC in vitro and treatment of XIAP-transfected SH-SY5Y cells with a PKC activator, phorbol 12-myristate 13-acetate (PMA) induced phosphorylation of XIAP at Ser87. Pulse chase experiments revealed that, when phosphorylated at Ser87, wild-type XIAP is more stable than XIAP with a Ser87Ala substitution, which is degraded faster. Importantly, the phosphorylation of XIAP at the site by PKC significantly increased cell survival up to approximately 2.5 times under the condition of apoptosis induced by 25 mu g/ml etoposide.Conclusion: The findings of the present study indicate a role for PKC, through phosphorylation of XIAP at Ser87 and its stabilization, in cell survival under conditions of stress and lend strength to the idea that PKC is crucial in regulating neuronal homeostasis, which may be impaired in AD.