The anti-ganglioside monoclonal antibody AA4 induces protein tyrosine phosphorylations, but not degranulation, in rat basophilic leukemia cells.
The monoclonal antibody AA4 (mAb AA4) recognizes novel alpha-galactosyl derivatives of GD1b on rat basophilic leukemia (RBL-2H3) cells. The binding of mAb AA4 induced protein tyrosine phosphorylations without histamine release. Several of the same proteins including Fc epsilon RI beta, Fc epsilon RI gamma, p72syk, and phospholipase C-gamma 1 were tyrosine-phosphorylated by mAb AA4 binding and by the activation of the high affinity IgE receptor, Fc epsilon RI. There was also activation of the p53/56lyn and p72syk protein-tyrosine kinases, but compared to direct Fc epsilon RI activation, mAb AA4 did not result in increased tyrosine phosphorylation of pp105-115 or pp125FAK, and the receptor subunits (Fc epsilon RI beta and Fc epsilon RI gamma) were more heavily phosphorylated. Furthermore, the time course of the phosphorylations with mAb AA4 was slower than that induced by Fc epsilon RI aggregation. By immunofluorescence, the tyrosine-phosphorylated proteins after mAb AA4 stimulation were localized in patches at the cell membrane and in areas of cell-cell contact, whereas after Fc epsilon RI activation, there was a reticular cytoplasmic pattern. There were no protein tyrosine phosphorylations either when Fc epsilon RI was saturated with IgE or when F(ab')2 fragments of mAb AA4 were used, although the F(ab')2 fragments still induced morphological changes. There was also coprecipitation of the beta and gamma subunits of Fc epsilon RI with the anti-ganglioside antibody. These data strongly suggest the involvement of Fc epsilon RI in the antiganglioside-induced protein tyrosine phosphorylations. Moreover, phosphorylations of these proteins including the beta and gamma chains of Fc epsilon RI and activation of p53/56lyn and p72syk did not result in histamine release.
Laboratory of Immunology, NIDR, National Institutes of Health, Bethesda, Maryland 20892.
W D
WD
Swaim
K
K
Minoguchi
C
C
Oliver
M M
MM
Hamawy
H
H
Kihara
V
V
Stephan
E H
EH
Berenstein
R P
RP
Siraganian
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001491:Basophils / Q000187:drug effects / Q000378:metabolism*; D002240:Carbohydrate Sequence; D002451:Cell Compartmentation; D015550:Cell Degranulation; D005455:Fluorescent Antibody Technique; D005732:Gangliosides / Q000276:immunology / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism; D015471:Leukemia, Basophilic, Acute; D008969:Molecular Sequence Data; D051966:Phospholipase C gamma; D010750:Phosphoproteins / Q000302:isolation & purification; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D017455:Receptors, IgE / Q000378:metabolism; D015398:Signal Transduction; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000378:metabolism
D000911:Antibodies, Monoclonal; D005732:Gangliosides; D007527:Isoenzymes; D010750:Phosphoproteins; D017455:Receptors, IgE; D011505:Protein-Tyrosine Kinases; D010738:Type C Phospholipases; D051966:Phospholipase C gamma