High-affinity IgE receptor-mediated stimulation of rat basophilic leukemia (RBL-2H3) cells induces early and late protein-tyrosine phosphorylations.
We reported previously that stimulation of RBL-2H3 cells through the high-affinity IgE receptor resulted in tyrosine phosphorylation of a 72-kDa protein (pp72) that was coupled to signal transduction. In the present study, although pp72 tyrosine phosphorylation was induced only by antigen triggering, stimulation of RBL-2H3 cells by either antigen or the calcium-ionophore A23187 led to increased tyrosine phosphorylation of a 110-kDa protein (pp110). This tyrosine phosphorylated protein was also observed when RBL-2H3 cells were transfected with the G protein-coupled m3 muscarinic receptor and then stimulated to secrete with carbachol. In contrast to tyrosine phosphorylation of pp72, antigen-induced pp110 tyrosine phosphorylation required extracellular calcium, was absent in cells depleted of protein kinase C, and was detected between 1 and 5 min after stimulation. The protein-tyrosine kinase inhibitor genistein blocked both histamine release and tyrosine phosphorylation induced by A23187. Altogether, the data suggest a role for pp110 in secretion. However, protein kinase C activation induced pp110 tyrosine phosphorylation but not histamine release demonstrating that pp110 tyrosine phosphorylation alone is not sufficient for degranulation. We conclude that tyrosine phosphorylation of pp72 is associated with the early steps of IgE receptor-generated signaling, whereas pp110 tyrosine phosphorylation occurs secondary to calcium influx and protein kinase C activation.
Laboratory of Immunology, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892.
M
M
Benhamou
V
V
Stephan
K C
KC
Robbins
R P
RP
Siraganian
D000818:Animals; D000944:Antigens, Differentiation, B-Lymphocyte / Q000378:metabolism*; D001491:Basophils / Q000378:metabolism*; D001692:Biological Transport; D015153:Blotting, Western; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D019833:Genistein; D006636:Histamine Release / Q000187:drug effects; D007073:Immunoglobulin E / Q000378:metabolism; D007529:Isoflavones / Q000494:pharmacology; D015471:Leukemia, Basophilic, Acute; D008407:Mast Cells / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D051381:Rats; D011961:Receptors, Fc / Q000378:metabolism*; D017455:Receptors, IgE; D015398:Signal Transduction; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism*
D000944:Antigens, Differentiation, B-Lymphocyte; D007529:Isoflavones; D011506:Proteins; D011961:Receptors, Fc; D017455:Receptors, IgE; D007073:Immunoglobulin E; D000001:Calcimycin; D014443:Tyrosine; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D011493:Protein Kinase C; D002118:Calcium