Transfection of Syk protein tyrosine kinase reconstitutes high affinity IgE receptor-mediated degranulation in a Syk-negative variant of rat basophilic leukemia RBL-2H3 cells.
Aggregation of the high affinity receptor for immunoglobulin E (Fc epsilon RI) on mast cells results in rapid tyrosine phosphorylation and activation of Syk, a cytoplasmic protein tyrosine kinase. To examine the role of Syk in the Fc epsilon RI signaling pathway, we identified a variant of RBL-2H3 cells that has no detectable Syk by immunoblotting and by in vitro kinase reactions. In these Syk-deficient TB1A2 cells, aggregation of Fc epsilon RI induced no histamine release and no detectable increase in total cellular protein tyrosine phosphorylation. However, stimulation of these cells with the calcium ionophore did induce degranulation. Fc epsilon RI aggregation induced tyrosine phosphorylation of the beta and gamma subunits of the receptor, but no increase in the tyrosine phosphorylation of phospholipase C-gamma 1 and phospholipase C-gamma 2 and no detectable increase in intracellular free Ca2+ concentration. By transfection, cloned lines were established with stable expression of Syk. In these reconstituted cells, Fc epsilon RI aggregation induced tyrosine phosphorylation of phospholipase C-gamma 1 and phospholipase C-gamma 2, an increase in intracellular free Ca2+ and histamine release. These results demonstrate that Syk plays a critical role in the early Fc epsilon RI-mediated signaling events. It further demonstrates that Syk activation occurs downstream of receptor phosphorylation, but upstream of most of the Fc epsilon RI-mediated protein tyrosine phosphorylations.
Zhang
J
J
Laboratory of Immunology, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892, USA.
Berenstein
E H
EH
Evans
R L
RL
Siraganian
R P
RP
D000818:Animals; D001491:Basophils / Q000502:physiology*; D002118:Calcium / Q000502:physiology; D004792:Enzyme Precursors / Q000502:physiology*; D006636:Histamine Release; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000378:metabolism; D015471:Leukemia, Basophilic, Acute; D008407:Mast Cells / Q000502:physiology*; D051966:Phospholipase C gamma; D019000:Phosphotyrosine / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000502:physiology*; D051381:Rats; D011940:Receptor Aggregation; D017455:Receptors, IgE / Q000502:physiology*; D015398:Signal Transduction; D000072377:Syk Kinase; D014162:Transfection; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000378:metabolism
D004792:Enzyme Precursors; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D017455:Receptors, IgE; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases; D000072377:Syk Kinase; C000606898:Syk protein, rat; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D002118:Calcium