IgE receptor type I-dependent tyrosine phosphorylation of phospholipid scramblase.
To identify new effectors of IgE receptor (FcepsilonRI) signaling, we purified proteins from FcepsilonRI-stimulated RBL-2H3 rat mast cells on anti-phosphotyrosine beads and generated mouse monoclonal antibodies (mAb) against these proteins. Two mAbs bound to a protein that was identified as a new isoform of phospholipid scramblase (PLSCR) after screening an RBL-2H3 cDNA expression library. This isoform differed from PLSCR1 by the absence of an 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 phospholipids across the plasma membrane. Although rat PLSCR is a 37-kDa protein, anti-phosphotyrosine immunoblots revealed the presence of 37-49 kDa phosphoproteins 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 led to the identification of these phosphoproteins as phospho-PLSCR itself. Stimulation of RBL-2H3 cells upon FcepsilonRI engagement resulted in a dramatic increase in PLSCR tyrosine phosphorylation. A comparison of the relative amounts of phospho-PLSCR and nonphosphorylated PLSCR demonstrated that only a tiny fraction was thus modified, indicating a finely targeted involvement of PLSCR in FcepsilonRI signaling. Thus, this study reports the cloning of a new isoform of PLSCR, as well as the first observation that a member 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 the role of phospholipid scramblases and to the mechanisms involved in their regulation.
Pastorelli
C
C
INSERM U485, Institut Pasteur, 25 rue du Dr. Roux, 75015 Paris, France.
Veiga
J
J
Charles
N
N
Voignier
E
E
Moussu
H
H
Monteiro
R C
RC
Benhamou
M
M
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D018076:DNA, Complementary / Q000276:immunology; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D049294:Phospholipid Transfer Proteins; D010766:Phosphorylation; D051381:Rats; D017455:Receptors, IgE / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D018076:DNA, Complementary; D008565:Membrane Proteins; D049294:Phospholipid Transfer Proteins; D017455:Receptors, IgE; D014443:Tyrosine