ACTIVATION OF PROTEIN-TYROSINE KINASE P72(SYK) BY FC-EPSILON-RI AGGREGATION IN RAT BASOPHILIC LEUKEMIA-CELLS P72(SYK) IS A MINOR COMPONENT BUT THE MAJOR PROTEIN-TYROSINE KINASE OF PP72

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 similar to 72-kDa proteins (pp72) and that the tyrosine kinase p72(syk) is one component of pp72. Here we studied further the relationship of p72(syk) to pp72. The aggregation of Fc epsilon RI induced the activation of p72(syk) which was parallel to its tyrosine phosphorylation. By in vitro kinase assay of immune complexes purified with anti-phosphotyrosine antibodies, p72(syk) was the major pp72 tyrosine kinase. However, by immunoblotting with anti-phosphotyrosine antibodies, p72(syk) 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 p72(syk). By two-dimensional gel electrophoresis pp72 was found to consist of multiple tyrosine-phosphorylated proteins including 71-80-kDa proteins that associate with p53/56(lyn). A 75-kDa tyrosine-phosphorylated protein, different from pp72, was identified as p75(HS1) (SPY75). These results demonstrate the heterogeneous nature of the pp72 and that p72(syk) is activated after Fc epsilon RI aggregation.