Abstract
Aggregation of the high affinity IgE receptors (Fc epsilon RI) on rat basophilic leukemia RBL-2H3 cells results in protein tyrosine phosphorylations. Previously we reported that there is prominent tyrosine phosphorylation of approximately 72-kDa proteins (pp72) and that the tyrosine kinase p72syk is one component of pp72. Here we studied further the relationship of p72syk to pp72. The aggregation of Fc epsilon RI induced the activation of p72syk which was parallel to its tyrosine phosphorylation. By in vitro kinase assay of immune complexes purified with anti-phosphotyrosine antibodies, p72syk was the major pp72 tyrosine kinase. However, by immunoblotting with anti-phosphotyrosine antibodies, p72syk was a minor component of pp72. The heterogeneous nature of pp72 was indicated by different studies. Under optimum conditions of one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, pp72 consisted of a heterogeneous group of 69-, 71-, and 72-kDa tyrosine-phosphorylated proteins. There were differences in the tyrosine phosphorylation of these proteins in cells activated in the absence of extracellular calcium or when stimulation was with the calcium ionophore A23187 or with phorbol myristate acetate. One of the proteins migrating at 69 kDa was p72syk. By two-dimensional gel electrophoresis pp72 was found to consist of multiple tyrosine-phosphorylated protens including 71-80-kDa proteins that associate with p53/56lyn. A 75-kDa tyrosine-phosphorylated protein, different from pp72, was identified as p75HS1 (SPY75). These results demonstrate the heterogeneous nature of the pp72 and that p72syk is activated after Fc epsilon RI aggregation.
Highlights
- - thatthere is prominent tyrosine phosphorylation of 15-30 s in the tyrosine phosphorylation of 72-kDa proteins 72-kDa proteins and that the tyrosine kinase
One dimensional sodium dodecyl sulfate-polyacrylamide geglroup is the Syk family of protein tyrosine kinases which inelectrophoresis, pp72 consisted of a heterogeneous cludes ZAP-70, a kinase involved in T cell receptor signaling group of69,71, and 72-kDa tyrosine-phosphorylated [23,24,25,26]. p72"yk,expressed in several hematopoietic cells, is typroteins
In the SDS-PAGE (8%gels), to separate the 50-80-kDa proteins optimally, thecurrent was stopped when the 49.5-kDa prestained rosine phosphorylation that was detectable within 30 s and reached a maximum at 10 min after stimulatio(nFig. lB)
Summary
Cells were stimulated with antigen for the indicated times (in min), solubilized in 1% Triton X-100 lysis buffer, and immunoprecipitated with anti-Syk antibodies (lanes I d ) or preimmune rabbit IgG (lane 6). Samples from the same washed immunoprecipitates were eluted, separated by SDS-PAGE (10% gel), and analyzed by immunoblotting with either anti-phosphotyrosine (upper bund) or anti-Sykl antibodies (lower bund). In the experiments to deplete p720k, cell lysates were first incubated for 2h a t 4 "C with either preimmune rabbitIgG or anti-Sykl antibodies coupled to Sepharose 4B not decrease the intensityof pp in anti-phosphotyrosine immunoblots (Fig. 2 A ). The anti-Sykl antibody completely depleted p72wk from these lysates of FceRI-activated cells (data not shown, seeFig. 6B).In 35Smetabolically labeled cells, there was only a slight decrease in the intensityof pp precipitated with anti-phosphotyrosine antibodies after depletion beads, and the supernatantswere used for immunoprecipitation. There are 72-kDa protein tyrosine kinase(s), called PTK72, activated after FceRI aggregation [6, 13].in vitro immune complex kinase assays were used to investigate the relationship ofp72"Yk to the FccRI-activated 72-kDa protein
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