Fig. 4.

Phosphorylation sites of mXkr8 critical for the PtdSer exposure in Ba/F3 cells. (A, Upper) The amino acid sequences of the caspase recognition motif and its downstream region in mXkr8 of the six indicated animals were aligned. The amino acid residues that were identical in all animals are in red. The caspase recognition motifs are shadowed, while the putative phosphorylation sites are highlighted in red. (A, Lower) The three phosphorylation sites (Thr-356, Ser-361, and Thr-375) in mXkr8 were individually (T356A, S361A, T375A, T356D, S361D, or T375D) or together (S/T-3A or S/T-3D) mutated into Ala or Asp. The mutated residues are highlighted in yellow. (B) Localization of the Xkr8 mutants at the plasma membrane. The WT and the indicated mXkr8 mutants were fused to EGFP and expressed in DKO-BaF cells. The stable transformants were observed by confocal microscopy for GFP (green) and Hoechst 33342 (blue). (Scale bar: 10 μm.) (C) Kinase-independent PtdSer exposure by the phosphorylation-mimic mutations of mXkr8. DKO-BaF cells expressing WT, T356D, S361D, T375D, or S/T-3D mXkr8 were treated for 30 min at 37 °C with 10 μM STS (red) or DMSO vehicle (green) and then stained at 4 °C for 15 min with Cy5-annexin V. The annexin V staining profiles in the PI-negative and GFP-positive population are shown. (D) Effect of the Ala mutations on mXkr8-mediated constitutive PtdSer exposure in Ba/F3 cells. DKO-BaF transformants expressing the WT, T356A, S361A, T375A, or S/T-3A mXkr8 were stained at 4 °C for 15 min with Cy5-annexin V. The annexin V staining profiles in the PI-negative and GFP-positive population are shown. (E) Ca²⁺-dependent and temperature-sensitive PtdSer exposure by the phosphorylation-mimic mutation of mXkr8. (Upper) DKO-BaF cells expressing S/T-3D were pretreated with (red) or without (green) BAPTA-AM before being stained with Cy5-annexin V. (Lower) DKO-BaF cells expressing the WT or S/T-3D mutant mXkr8 were stained at 20 °C with Cy5-annexin V.