PURIFICATION AND CHARACTERIZATION OF THE MAJOR TYROSINE PROTEIN-KINASE FROM THE HUMAN PROMYELOCYTIC CELL-LINE, HL-60

The major tyrosine protein kinase from HL60 (a human non-differentiate d promyelocytic cell line) has been purified almost to homogeneity as judged by silver-stained SDS/PAGE. The procedure involved four chromat ographic steps: DEAE-Sepharose, casein-agarose, cibacron-blue-agarose and hexyl-agarose. The purification resulted in more than 1000-fold en richment in angiotensin II phosphorylation activity. A gel-sizing expe riment, labeling with [S-35]ATP[gammas] and autophosphorylation of the enzyme in the presence of [gamma-P-32]ATP, all led to the identificat ion of a single protein species with a molecular mass of about 40 kDa. Western blot experiments showed that this protein does not belong to the src family and is not related to the abl and fes oncogene products . Phosphorylation of angiotensin II and casein by this 40-kDa human pr omyelocytic kinase was stimulated by high ionic strength especially fr om class IA metal salts. The K(m) for ATP was 2 muM and the V(max) 3.1 nmol . min-1 . mg-1 using angiotensin H as a substrate. The kinase re quires the presence of either Mn2+ or Mg2+ for full activity and utili zes ATP or dATP but not GTP as phosphate donor. Based on numerous bioc hemical observations, it was possible to demonstrate that kinase is di fferent from any other tyrosine protein kinases described in the liter ature. This 40-kDa protein was used as a molecular tool for testing so me tyrosine protein kinase inhibitors described in the literature. It is one of the rare tyrosine protein kinases purified from human cancer cells to date.