Purification and characterization of the major tyrosine protein kinase from the human promyelocytic cell line, HL60.
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 II phosphorylation activity. A gel-sizing experiment, labeling with [35S]ATP[gamma s] and autophosphorylation of the enzyme in the presence of [gamma-32P]ATP, 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 and fes oncogene products. Phosphorylation of angiotensin II and casein by this 40-kDa human promyelocytic kinase was stimulated by high ionic strength especially from class IA metal salts. The Km for ATP was 2 microM and the Vmax 3.1 nmol.min-1.mg-1 using angiotensin II 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.
Ernould
A P
AP
Département de Cancérologie Expérimentale, Institut de Recherches Servier, Suresnes, France.
Ferry
G
G
Barret
J M
JM
Genton
A
A
Boutin
J A
JA
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000804:Angiotensin II / Q000378:metabolism; D001667:Binding, Competitive; D015153:Blotting, Western; D002364:Caseins / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D015473:Leukemia, Promyelocytic, Acute / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008670:Metals / Q000494:pharmacology; D009994:Osmolar Concentration; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D014407:Tumor Cells, Cultured
D002364:Caseins; D008670:Metals; D000804:Angiotensin II; C022571:adenosine 5'-O-(3-thiotriphosphate); D008345:Manganese; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D011505:Protein-Tyrosine Kinases; D008274:Magnesium