Manganese-phospholipid-stimulated protein kinase activity of human leukemic cells.
A protein kinase system with unusual characteristics was noted in extracts of HL-60 cells using endogenous proteins as substrates. This system exhibited a cation preference for manganese at an optimal concentration of 0.5 mM. Moderate activity was detectable with magnesium at an optimal concentration of 5.0 mM, but calcium was inactive. Activity was markedly stimulated by phospholipid, with phosphatidylglycerol and phosphatidylinositol exhibiting greater activity than phosphatidylserine. In isolated plasma membranes, the major substrate of this system was a 73-kDa protein, while cytoplasmic extracts exhibited larger amounts of a 42-kDa substrate. 73-kDa phosphorylating activity of membranes was comparably active at 0 and 31 degrees C, although in cytosol activity was greater at 31 degrees C. No 73-kDa protein phosphorylation was observed in the presence of Ca2+, Mg2+, and phosphatidylserine. Phosphoamino acid analysis of the 73-kD band revealed phosphothreonine and phosphoserine. The 42-kDa substrate was distinguishable from actin by two-dimensional gel electrophoresis, which disclosed that both major substrates were highly basic in the isoelectric focusing dimension. Protamine and histones (H2B greater than H1 greater than H3) exhibited phospholipid-stimulated phosphorylation in the presence of Mn2+, but phosvitin, casein, and vinculin were not substrates. High levels of phosphorylative activity involving the 73-kDa substrate were noted in nuclear extracts. Complex patterns of increase of this activity were noted in both cytosol and nuclear extracts following induction of differentiation with dimethyl sulfoxide, retinoic acid, or phorbol 12-myristate 13-acetate. This study thus demonstrated the presence of a previously undescribed type of protein kinase activity which exhibited alterations during leukemic cellular differentiation.
D002454:Cell Differentiation; D002460:Cell Line; D002855:Chromatography, Thin Layer; D006657:Histones / Q000378:metabolism; D006801:Humans; D007525:Isoelectric Focusing; D015470:Leukemia, Myeloid, Acute / Q000201:enzymology*; D008345:Manganese / Q000378:metabolism*; D008970:Molecular Weight; D010743:Phospholipids / Q000378:metabolism*; D011479:Protamines / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*
D006657:Histones; D010743:Phospholipids; D011479:Protamines; D008345:Manganese; D011494:Protein Kinases