N-benzyladriamycin-14-valerate (AD198) induces apoptosis through protein kinase C-delta-induced phosphorylation of phospholipid scramblase 3.
N-benzyladriamycin-14-valerate (AD198) induces apoptosis through protein kinase C-delta-induced phosphorylation of phospholipid scramblase 3.
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DOI：
10.1158/0008-5472.can-05-1688
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发表时间：
2005-11
期刊：
影响因子：
11.2
通讯作者：
Yongwen He;Jihua Liu;D. Durrant;Hung-Sheng Yang;T. Sweatman;L. Lothstein;Ray M. Lee
中科院分区：
文献类型：
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作者：
Yongwen He;Jihua Liu;D. Durrant;Hung-Sheng Yang;T. Sweatman;L. Lothstein;Ray M. Lee
磷脂乱序酶3（PLS 3）是在线粒体形态、功能和凋亡反应中起关键作用的酶。在细胞凋亡期间，活化的蛋白激酶C-δ（PKC-δ）易位到线粒体并磷酸化PLS 3。在这里，我们利用一种PKC-δ激活剂，一种靶向细胞核的蒽环类药物N-苄基阿霉素-14-戊酸酯（AD 198），来研究PKC-δ对PLS 3磷酸化的机制。PLS 3的过表达增强了AD 198诱导细胞凋亡的敏感性，而小干扰RNA下调PLS 3则降低了AD 198诱导细胞凋亡的敏感性。PKC-δ的过表达，但不是激酶缺陷的PKC-δ，和AD 198处理增强PLS 3的苏氨酸磷酸化。将磷酸化的苏氨酸定位到PLS 3的Thr 21。Thr 21突变为丙氨酸并不影响PLS 3的线粒体定位，但在体外通过PKC-δ和体内AD 198诱导的PLS 3磷酸化消除了苏氨酸磷酸化。与野生型PLS 3的表达相比，细胞中PLS 3（T21 A）的表达不能增强AD 198诱导的细胞凋亡。使用苄氧基羰基-Val-Ala-Asp-（OMe）氟甲基酮和环孢菌素A，我们还表明AD 198诱导的PLS 3磷酸化发生在半胱天冬酶激活的上游，并且独立于线粒体通透性转换。这些研究证实，AD 198激活的PKC-δ诱导线粒体PLS 3在Thr 21处的磷酸化，并且PLS 3是PKC-δ在AD 198诱导的细胞凋亡中的关键下游效应物。
Phospholipid scramblase 3 (PLS3) is an enzyme that plays a critical role in mitochondrial morphology, functions, and apoptotic response. During apoptosis, activated protein kinase C-delta (PKC-delta) translocates to mitochondria and phosphorylates PLS3. Here, we utilize an extranuclear-targeted anthracycline N-benzyladriamycin-14-valerate (AD198), a PKC-delta activator, to investigate the mechanism of PLS3 phosphorylation by PKC-delta. Overexpression of PLS3 enhanced, whereas down-regulation of PLS3 by small interfering RNA decreased, the sensitivity of AD198-induced apoptosis. Overexpression of PKC-delta, but not the kinase-defective PKC-delta, and AD198 treatment enhanced threonine phosphorylation of PLS3. The phosphorylated threonine was mapped to Thr21 of PLS3. Mutation of Thr21 to alanine did not affect mitochondrial localization of PLS3 but abolished threonine phosphorylation by PKC-delta in vitro and AD198-induced PLS3 phosphorylation in vivo. Expression of PLS3(T21A) in cells could not enhance AD198-induced apoptosis compared with expression of the wild-type PLS3. Using benzyloxycarbonyl-Val-Ala-Asp-(OMe) fluoromethyl ketone and cyclosporine A, we also showed that AD198-induced PLS3 phosphorylation occurs upstream of caspase activation and independent of mitochondrial permeability transition. These studies establish that AD198-activated PKC-delta induces phosphorylation of mitochondrial PLS3 at Thr21 and that PLS3 is a critical downstream effector of PKC-delta in AD198-induced apoptosis.