Laboratory of Biochemistry and Immunology, World Premier International Research Center, Immunology Frontier Research Center, Osaka University, Suita, 565-0871 Osaka, Japan.
Division of Cell Signaling, Fujii Memorial Institute of Medical Sciences, Tokushima University, 770-8503 Tokushima, Japan.
Proc Natl Acad Sci U S A. 2019 Feb 19;116(8):2907-2912. doi: 10.1073/pnas.1820499116. Epub 2019 Feb 4.
The exposure of phosphatidylserine (PtdSer) to the cell surface is regulated by the down-regulation of flippases and the activation of scramblases. Xkr8 has been identified as a scramblase that is activated during apoptosis, but its exogenous expression in the mouse Ba/F3 pro B cell line induces constitutive PtdSer exposure. Here we found that this Xkr8-mediated PtdSer exposure occurred at 4 °C, but not at 20 °C, although its scramblase activity was observed at 20 °C. The Xkr8-mediated PtdSer exposure was inhibited by a kinase inhibitor and enhanced by phosphatase inhibitors. Phosphorylated Xkr8 was detected by Phos-tag PAGE, and a mass spectrometric and mutational analysis identified three phosphorylation sites. Their phosphomimic mutation rendered Xkr8 resistant to the kinase inhibitor for PtdSer exposure at 4 °C, but unlike phosphatase inhibitors, it did not induce constitutive PtdSer exposure at 20 °C. On the other hand, when the flippase genes were deleted, the Xkr8 induced constitutive PtdSer exposure at high temperature, indicating that the flippase activity normally counteracted Xkr8's ability to expose PtdSer. These results indicate that PtdSer exposure can be increased by the phosphorylation-mediated activation of Xkr8 scramblase and flippase down-regulation.
磷脂酰丝氨酸（PtdSer）暴露于细胞表面受到翻转酶下调和 scramblase 激活的调节。Xkr8 已被鉴定为凋亡过程中激活的 scramblase，但在小鼠 Ba/F3 前 B 细胞系中外源表达会诱导 PtdSer 持续暴露。在这里，我们发现这种 Xkr8 介导的 PtdSer 暴露发生在 4°C，而不是 20°C，尽管在 20°C 观察到其 scramblase 活性。激酶抑制剂可抑制 Xkr8 介导的 PtdSer 暴露，而磷酸酶抑制剂可增强其活性。通过 Phos-tag PAGE 检测到磷酸化的 Xkr8，通过质谱分析和突变分析鉴定出三个磷酸化位点。它们的磷酸模拟突变使 Xkr8 在 4°C 时对激酶抑制剂介导的 PtdSer 暴露具有抗性，但与磷酸酶抑制剂不同，它不会在 20°C 时诱导 PtdSer 持续暴露。另一方面，当删除翻转酶基因时，Xkr8 在高温下诱导 PtdSer 持续暴露，表明翻转酶活性通常会抵消 Xkr8 暴露 PtdSer 的能力。这些结果表明，通过 Xkr8 scramblase 的磷酸化介导激活和翻转酶下调可以增加 PtdSer 暴露。
