Department of Psychiatry, Osaka University Graduate School of Medicine, Osaka, Japan.
Psychogeriatrics. 2011 Jun;11(2):90-7. doi: 10.1111/j.1479-8301.2011.00355.x.
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).
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.
Recombinant XIAP was phosphorylated at Ser(87) 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 Ser(87) . Pulse chase experiments revealed that, when phosphorylated at Ser(87) , 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 µg/ml etoposide.
The findings of the present study indicate a role for PKC, through phosphorylation of XIAP at Ser(87) 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.
已有研究表明，多种蛋白激酶参与阿尔茨海默病（AD）神经元的凋亡性丢失。虽然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 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需阐明。本研究通过分析 X 连锁凋亡抑制蛋白（XIAP）的磷酸化，报告了 PKC 在应激条件下通过磷酸化 XIAP 促进细胞存活的可能作用。
体外实验中，用依赖磷酸化的抗 XIAP 抗体通过 PKC 对重组 XIAP 的活性进行孵育，证实 XIAP 在丝氨酸 87 位的磷酸化。PKC 激活剂佛波醇 12-肉豆蔻酸 13-醋酸盐（PMA）处理 SH-SY5Y 细胞也证实了 XIAP 在该位点的磷酸化增加。制备 XIAP 丝氨酸 87 位被丙氨酸取代的突变体构建体，并转染细胞。转染野生型或突变型 XIAP 后，用依托泊苷诱导凋亡时，用 PMA 处理细胞，通过计数活细胞和死细胞来评估细胞活力。
体外实验中，PKC 使 XIAP 在丝氨酸 87 位磷酸化，PKC 激活剂佛波醇 12-肉豆蔻酸 13-醋酸盐（PMA）处理 XIAP 转染的 SH-SY5Y 细胞诱导 XIAP 在丝氨酸 87 位磷酸化。脉冲追踪实验表明，当 XIAP 在丝氨酸 87 位磷酸化时，野生型 XIAP 比丝氨酸 87 位丙氨酸取代的 XIAP 更稳定，后者降解更快。重要的是，PKC 在该位点对 XIAP 的磷酸化使在 25μg/ml 依托泊苷诱导的凋亡条件下，细胞存活率增加约 2.5 倍。
本研究的结果表明，PKC 通过 XIAP 在丝氨酸 87 位的磷酸化及其稳定性在应激条件下的细胞存活中发挥作用，并进一步证实 PKC 在调节神经元内稳态方面的重要性，而 AD 可能会损害神经元内稳态。
