PURIFICATION OF A MAJOR TYROSINE KINASE FROM RBL-2H3 CELLS PHOSPHORYLATING FC(EPSILON)RI GAMMA-CYTOPLASMIC DOMAIN AND IDENTIFICATION AS THEBTK TYROSINE KINASE
Dj. Price et al., PURIFICATION OF A MAJOR TYROSINE KINASE FROM RBL-2H3 CELLS PHOSPHORYLATING FC(EPSILON)RI GAMMA-CYTOPLASMIC DOMAIN AND IDENTIFICATION AS THEBTK TYROSINE KINASE, Biochimica et biophysica acta. Molecular cell research, 1265(2-3), 1995, pp. 133-142
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.