Phosphorylation-mediated activation of mouse Xkr8 scramblase for phosphatidylserine exposure

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.