Purification of a major tyrosine kinase from RBL-2H3 cells phosphorylating Fc epsilon RI gamma-cytoplasmic domain and identification as the Btk tyrosine kinase.
Immunoglobulin E high affinity receptor-mediated signal transduction in mast cells results in a number of protein tyrosine kinases being activated as very early events in the process leading to degranulation. Some 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 by 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 molecular weight 79 kDa which we designated cd gamma kinase. The kinase was purified to near homogeneity by Heparin-agarose, Mono Q, and CM Sepharose chromatographies. The yield of enzyme was approx. 200 micrograms/10(9) cells. We characterized this kinase by its ability to phosphorylate both the cd gamma peptide (Km = 0.2 mM) and the cytoplasmic fragment of the Band III protein. The cd gamma kinase was distinguished from syk by inability to be precipitated by anti-syk antiserum and by partial peptide mapping. Cd gamma kinase was also distinguished from syk by cd gamma peptide and Band III substrate specificity. We identified the cd gamma kinase by Western blotting and by partial phosphopeptide mapping as Btk, the B-cell tyrosine kinase found to be defective in X-linked agammaglobulinemia.
Price
D J
DJ
Procept Inc., Cambridge, MA 02139.
Kawakami
Y
Y
Kawakami
T
T
Rivnay
B
B
D000595:Amino Acid Sequence; D000818:Animals; D001402:B-Lymphocytes / Q000201:enzymology*; D015471:Leukemia, Basophilic, Acute / Q000201:enzymology / Q000473:pathology; D008213:Lymphocyte Activation; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010455:Peptides / Q000138:chemical synthesis; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000302:isolation & purification*; D051381:Rats; D017455:Receptors, IgE / Q000737:chemistry / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D010455:Peptides; D017455:Receptors, IgE; D011505:Protein-Tyrosine Kinases