Phosphorylation-mediated activation of mouse Xkr8 scramblase for phosphatidylserine exposure

Area of Science:

• Cell Biology
• Biochemistry
• Molecular Biology

Background:

• Phosphatidylserine (PtdSer) exposure to the cell surface is a critical regulator of cellular processes, controlled by flippases and scramblases.
• Xkr8 is a scramblase known to activate during apoptosis, but its role in constitutive PtdSer exposure requires further elucidation.

Purpose of the Study:

• To investigate the regulatory mechanisms of Xkr8-mediated phosphatidylserine (PtdSer) exposure.
• To determine the influence of temperature, kinase/phosphatase activity, and flippase function on Xkr8 activity.

Main Methods:

• Utilized mouse Ba/F3 pro B cell line with exogenous Xkr8 expression.
• Performed experiments at varying temperatures (4 °C and 20 °C).
• Employed kinase and phosphatase inhibitors, Phos-tag PAGE, mass spectrometry, mutational analysis, and gene deletion of flippases.

Main Results:

• Xkr8-mediated PtdSer exposure was temperature-dependent (4 °C vs. 20 °C).
• Kinase inhibition and phosphatase activation affected PtdSer exposure, with three phosphorylation sites identified on Xkr8.
• Phosphomimic mutations altered Xkr8 sensitivity to inhibitors, and flippase deletion enabled constitutive PtdSer exposure.

Conclusions:

• Xkr8 scramblase activity and PtdSer exposure are modulated by phosphorylation and flippase activity.
• Phosphorylation can enhance Xkr8-mediated PtdSer exposure, while flippase activity counteracts this effect.
• These findings provide insights into the intricate regulation of PtdSer exposure in cellular signaling.