Purification and characterization of the major tyrosine protein kinase from the human promyelocytic cell line, HL60

Abstract

The major tyrosine protein kinase from HL60 (a human non-differentiated promyelocytic cell line) has been purified almost to homogeneity as judged by silver-stained SDS/PAGE. The procedure involved four chromatographic steps : DEAE-Sepharose, casein-agarose, cibacron-blue -agarose and hexyl-agarose. The purification resulted in more than 1000-fold enrichment in angiotensin I1 phosphorylation activity. A gel-sizing experiment, labeling with [35S]ATP [ys] and autophosphorylation of the enzyme in the presence of [Y-~~PIATP, all led to the identification 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 andfes oncogene products. Phosphorylation of angiotensin 11 and casein by this 40-kDa human promyelocytic kinase was stimulated by high ionic strength especially from class IA metal salts. The K, for ATP was 2 pM and the V,, 3.1 nmol min-' . mg-' using angiotensin fI as a substrate. The kinase requires the presence of either Mn2+ or Mg2+ for full activity and utilizes ATP or dATP but not GTP as phosphate donor. Based on numerous biochemical observations, it was possible to demonstrate that kinase is different from any other tyrosine protein kinases described in the literature. This 40-kDa protein was used as a molecular tool for testing some tyrosine protein kinase inhibitors described in the literature. It is one of the rare tyrosine protein kinases purified from human cancer cells to date.Protein phosphorylation plays a key role in the control of cellular communication [l]. Amongst the enzymes catalyzing the phosphorylation of other proteins (on specific amino acid residues), the tyrosine protein kinases (TPK) are known to be involved in the regulation of growth signal transmission and cellular transformation [2], according to their functional similarity to growth factor receptors and oncogene products [3]. A growing number of TPK(s) have been identified, mostly from molecular cloning of protein kinase genes rather than through enzyme purification [4]. TPK(s) can be classified in three categories [5][6][7]: (a) the transmembrane-receptor-associated TPK activity as found, for instance, in epidermal growth factor EGF, platelet growth factor PDGF and insulin receptors; (b) the TPK(s) linked to the inner face of the plasma membrane represented by the src family with src, yes, f g r , f m , lyn, hck, lck and blk oncogene products which are all myristoylated [8] and (c) the cytosolic TPK(s) with major representatives being abl and fes/fgr oncogene products.From a molecular pharmacological point of view, TPK(s) are new targets for inhibitors [9] To understand the cellular function of these TPKs one needs to characterize them on the basis of classical biochemistry studies (purification, molecular identification, kinetic data, effect of modulators, etc.). A major example was recently provided by Litwin et al. [24] and Cheng et al. [25], on a TPK specifically phosphorylating the p34cdc2 kinase ...

Tyrosine phosphorylation of cytoplasmic proteins in proliferating, differentiating, apoptotic HL-60 cells and blood neutrophils

“…Although phosphotyrosines represent only a small proportion of the total phosphoamino acid content of eukaryotic cells, specific tyrosine phosphorylations or dephosphorylations have been shown to play a key role in the regulation of cell differentiation [12][13][14][15][16][17][18]. Many protein tyrosine kinases (PTKs) are involved in growth control and are implicated in cell transformation [19][20][21][22][23], where protein tyrosine phosphatases (PTPs) have also been shown to have important regulatory functions [24,25]. Thus, the balance between the actions of PTKs and PTPs determine the levels of phosphorylation, which have appeared crucial for induction and maintenance of the differentiated phenotype of cells [26,27].…”