PURIFICATION OF A MAJOR TYROSINE KINASE FROM RBL-2H3 CELLS PHOSPHORYLATING FC(EPSILON)RI GAMMA-CYTOPLASMIC DOMAIN AND IDENTIFICATION AS THEBTK TYROSINE KINASE

Immunoglobulin E high affinity receptor-mediated signal transduction i n mast cells results in a number of protein tyrosine kinases being act ivated as very early events in the process leading to degranulation. S ome of these, such as the src kinases and the syk kinase, are known to be involved in this receptor-associated activation. In this paper we describe the search for other activation-associated tyrosine kinases b y the ability to phosphorylate a cytoplasmic domain peptide of the Fc epsilon RI gamma-subunit. In utilizing a purification step previously used to isolate the 72 kDa syk kinase, we detected another kinase of m olecular weight 79 kDa which we designated cd gamma kinase. The kinase was purified to near homogeneity by Heparin-agarose, Mono Q, and CM S epharose chromatographies. The yield of enzyme was approx. 200 mu g/10 (9) cells. We characterized this kinase by its ability to phosphorylat e both the cd gamma peptide (K-m = 0.2 mM) and the cytoplasmic fragmen t of the Band III protein. The cd gamma kinase was distinguished from syk by inability to be precipitated by anti-syk antiserum and by parti al peptide mapping. Cd gamma kinase was also distinguished from syk by cd gamma peptide and Band III substrate specificity. We identified th e cd gamma kinase by Western blotting and by partial phosphopeptide ma pping as Btk, the B-cell tyrosine kinase found to be defective in X-li nked agammaglobulinemia.