IgE receptor type I-dependent tyrosine phosphorylation of phospholipid scramblase

To identify new effecters of IgE receptor (Fc epsilon RI) signaling, we pur ified proteins from Fc epsilon RI-stimulated RBL-2H3 rat mast cells on anti -phosphotyrosine beads and generated mouse monoclonal antibodies (mAb) agai nst these proteins. Two mAbs bound to a protein that was identified as a ne w isoform of phospholipid scramblase (PLSCR) after screening an RBL-2H3 cDN A expression library. This isoform differed from PLSCR1 by the absence of a n exon 3-encoded sequence and by an insert coding six QGPY(P/A)GP repeats. The PLSCR family of proteins is responsible for a redistribution of phospho lipids across the plasma membrane, Although rat PLSCR is a 37-kDa protein, anti-phosphotyrosine immunoblots revealed the presence of 37-49 kDa phospho proteins in the material immunoprecipitated with either anti-PLSCR mAb but not with unrelated monoclonal or polyclonal antibodies. Depletion of PLSCR resulted in the absence of these phosphoproteins. Additional experiments le d to the identification of these phosphoproteins as phospho-PLSCR itself. S timulation of RBL-2H3 cells upon Fc epsilon RI engagement resulted in a dra matic increase in PLSCR tyrosine phosphorylation. A comparison of the relat ive amounts of phospho-PLSCR and nonphosphorylated PLSCR demonstrated that only a tiny fraction was thus modified, indicating a finely targeted involv ement of PLSCR in Fc epsilon RI signaling. Thus, this study reports the clo ning of a new isoform of PLSCR, as well as the first observation that a mem ber of the PLSCR family is a target for tyrosine kinases and is involved in signaling by an immune receptor. These findings open new perspectives on t he role of phospholipid scramblases and to the mechanisms involved in their regulation.