Department of Pharmacology, University of California at San Diego, La Jolla, CA 92037, USA.
Division of Biochemistry, Department of Biochemistry and Molecular Biology, Graduate School of Medicine, Kobe University, Kobe 650-0017, Japan.
Sci Signal. 2019 Jan 1;12(562):eaat6662. doi: 10.1126/scisignal.aat6662.
Atypical protein kinase C (aPKC) isozymes are unique in the PKC superfamily in that they are not regulated by the lipid second messenger diacylglycerol, which has led to speculation about whether a different second messenger acutely controls their function. Here, using a genetically encoded reporter that we designed, aPKC-specific C kinase activity reporter (aCKAR), we found that the lipid mediator sphingosine 1-phosphate (S1P) promoted the cellular activity of aPKC. Intracellular S1P directly bound to the purified kinase domain of aPKC and relieved autoinhibitory constraints, thereby activating the kinase. In silico studies identified potential binding sites on the kinase domain, one of which was validated biochemically. In HeLa cells, S1P-dependent activation of aPKC suppressed apoptosis. Together, our findings identify a previously undescribed molecular mechanism of aPKC regulation, a molecular target for S1P in cell survival regulation, and a tool to further explore the biochemical and biological functions of aPKC.
非典型蛋白激酶 C（aPKC）同工酶在 PKC 超家族中是独特的，因为它们不受脂质第二信使二酰基甘油的调节，这导致人们猜测是否有不同的第二信使急性控制它们的功能。在这里，我们使用我们设计的一种遗传编码报告器，即 aPKC 特异性 C 激酶活性报告器（aCKAR），发现脂质介体 1-磷酸鞘氨醇（S1P）促进了 aPKC 的细胞活性。细胞内 S1P 直接与 aPKC 的纯化激酶结构域结合，并解除自动抑制约束，从而激活激酶。计算机模拟研究确定了激酶结构域上的潜在结合位点，其中一个通过生化方法进行了验证。在 HeLa 细胞中，S1P 依赖性 aPKC 激活抑制细胞凋亡。总之，我们的研究结果确定了 aPKC 调节的一个先前未描述的分子机制，鉴定了 S1P 在细胞存活调节中的一个分子靶标，以及进一步探索 aPKC 的生化和生物学功能的工具。
