Characterization of a membrane-associated protein kinase of multidrug-resistant HL60 cells which phosphorylates P-glycoprotein.
Cells containing increased levels of the membrane phosphoprotein P-glycoprotein exhibit a multidrug-resistant phenotype. In the present study we have analyzed protein kinases capable of phosphorylating P-glycoprotein in membranes of HL60 cells isolated for resistance to vincristine. Analysis of this system demonstrates that in isolated membranes the protein kinase inhibitor staurosporine greatly reduces P-glycoprotein phosphorylation. In contrast, the kinase inhibitor H-7 does not affect this reaction. Fractionation of solubilized membrane proteins from sensitive and resistant cells on DEAE-cellulose reveals a major protein kinase (PK-1) which exhibits optimal activity in the presence of Mn2+ and histone H1. This enzyme fraction does not contain detectable levels of protein kinase C or cAMP-dependent protein kinase. PK-1 phosphorylation of two endogenous proteins is, however, greatly enhanced in the presence of phosphatidylserine or phosphatidyl-inositol. In reaction mixtures containing Mg2+ or Mn2+ in the absence of phospholipid, PK-1 from resistant cells phosphorylates an endogenous protein of 180 kilodaltons (P180), which exhibits an electrophoretic mobility identical to P-glycoprotein. In parallel experiments with PK-1 from sensitive cells there is no detectable phosphorylation of a P180 protein. P180 phosphorylated by PK-1 from resistant cells is immunoprecipitated by antibody against P-glycoprotein. Additional studies demonstrate that PK-1 is capable of phosphorylating specific synthetic peptides which correspond to the sequence of P-glycoprotein. Peptide phosphorylation occurs at both serine and threonine residues. These studies thus identify a novel membrane-associated protein kinase in HL60 cells which is capable of phosphorylating P-glycoprotein. This enzyme may have an important role in regulating levels of multidrug resistance.
Staats
J
J
Division of Biology, Kansas State University, Manhattan 66506.
Marquardt
D
D
Center
M S
MS
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D004351:Drug Resistance; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D015473:Leukemia, Promyelocytic, Acute; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000378:metabolism*; D010455:Peptides / Q000138:chemical synthesis; D010743:Phospholipids / Q000494:pharmacology; D010766:Phosphorylation; D010879:Piperazines / Q000494:pharmacology; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism*; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured / Q000201:enzymology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000596:Amino Acids; D007546:Isoquinolines; D008562:Membrane Glycoproteins; D009363:Neoplasm Proteins; D010455:Peptides; D010743:Phospholipids; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D011494:Protein Kinases