Ceramide-induced apoptosis by translocation, phosphorylation, and activation of protein kinase Cδ in the Golgi complex
Ceramide-induced apoptosis by translocation, phosphorylation, and activation of protein kinase Cδ in the Golgi complex
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DOI：
10.1074/jbc.m312350200
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发表时间：
2004-03-26
影响因子：
4.8
通讯作者：
Saito, N
中科院分区：
文献类型：
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作者：
Kajimoto, T;Shirai, Y;Saito, N
蛋白激酶C（PKC）是一种钙/磷脂依赖性蛋白激酶，是细胞凋亡等多种细胞反应的关键酶。然而，PKC在细胞凋亡中的功能作用尚未阐明。在这项研究中，我们专注于参与PKC δ在神经酰胺诱导的HeLa细胞凋亡，并检查特定的PKC亚型在凋亡事件的时空激活的重要性。神经酰胺诱导的细胞凋亡被PKC δ特异性抑制剂rottlerin抑制，也被小干扰RNA敲低内源性PKC δ表达所阻断。神经酰胺诱导PKC δ易位到高尔基复合体，并通过酶铰链区Tyr（311）和Tyr（332）的磷酸化激活PKC δ。非磷酸化的PKC δ（突变体Y311F和Y332F）可以易位到高尔基复合体中响应神经酰胺，这表明酪氨酸磷酸化是不必要的易位。然而，神经酰胺未能激活缺乏C1B结构域的PKC δ，其不能移位到高尔基复合体，但可以通过酪氨酸磷酸化激活。这些发现表明，神经酰胺转运PKC δ的高尔基复合体和PKC δ被激活的酪氨酸磷酸化的车厢。此外，我们利用小干扰RNA对PKC δ的种特异性敲低，研究了PKC δ中Tyr（311）和Tyr（332）的磷酸化在神经酰胺诱导的细胞凋亡中的意义，发现Tyr（311）和Tyr（332）的磷酸化是神经酰胺诱导的细胞凋亡所必需的。我们在这里证明，PKC δ的靶向机制，其激活和易位到高尔基复合体的双重调节，是神经酰胺诱导的凋亡事件的关键。
Protein kinase C (PKC), a Ca2+/phospholipid-dependent protein kinase, is known as a key enzyme in various cellular responses, including apoptosis. However, the functional role of PKC in apoptosis has not been clarified. In this study, we focused on the involvement of PKCdelta in ceramide-induced apoptosis in HeLa cells and examined the importance of spatiotemporal activation of the specific PKC subtype in apoptotic events. Ceramide-induced apoptosis was inhibited by the PKCdelta-specific inhibitor rottlerin and also was blocked by knockdown of endogenous PKCdelta expression using small interfering RNA. Ceramide induced the translocation of PKCdelta to the Golgi complex and the concomitant activation of PKCdelta via phosphorylation of Tyr(311) and Tyr(332) in the hinge region of the enzyme. Unphosphorylatable PKCdelta (mutants Y311F and Y332F) could translocate to the Golgi complex in response to ceramide, suggesting that tyrosine phosphorylation is not necessary for translocation. However, ceramide failed to activate PKCdelta lacking the C1B domain, which did not translocate to the Golgi complex, but could be activated by tyrosine phosphorylation. These findings suggest that ceramide translocates PKCdelta to the Golgi complex and that PKCdelta is activated by tyrosine phosphorylation in the compartment. Furthermore, we utilized species-specific knockdown of PKCdelta by small interfering RNA to study the significance of phosphorylation of Tyr(311) and Tyr(332) in PKCdelta for ceramide-induced apoptosis and found that phosphorylation of Tyr(311) and Tyr(332) is indispensable for ceramide-induced apoptosis. We demonstrate here that the targeting mechanism of PKCdelta, dual regulation of both its activation and translocation to the Golgi complex, is critical for the ceramide-induced apoptotic event.