Protein kinase C phosphorylates P-glycoprotein in multidrug resistant human KB carcinoma cells.
Studies were undertaken to identify the protein kinase(s) responsible for P-glycoprotein phosphorylation in multidrug-resistant (KB-V1) human carcinoma cells and to elucidate the functional role of phosphorylation. P-glycoprotein migrated on sodium dodecyl sulfate gels with apparent Mr 150,000 and is termed P150. When KB-V1 membrane vesicles were incubated with [gamma-32P] ATP, P150 was phosphorylated by an endogenous kinase that exhibited properties of membrane-inserted protein kinase C (PKC). Both membrane-bound P150 and purified P150 served as effective substrates for highly purified rat brain PKC which incorporated approximately 0.6 mol of phosphate/mol of P150. Enzyme assays showed that KB-V1 cells exhibit 4-fold higher PKC activity compared with the drug-sensitive KB-3 cell line. The basal phosphorylation of P150 observed in 32P-labeled cells was increased 2-fold by phorbol ester (PMA) treatment and reduced 30% by treatment with the isoquinolinsulfonamide H-7. Phosphopeptide maps of partially digested P150, phosphorylated either in vitro with PKC or in intact 32P-labeled control or PMA-stimulated cells, were indistinguishable from one another. Drug accumulation assays revealed that PMA treatment of KB-V1 cells significantly reduced [3H]vinblastine accumulation induced by verapamil or by tetrandrine. The results suggest that PKC is primarily responsible for P150 phosphorylation in KB-V1 cells and that phosphorylation may play a modulatory role in the drug transport process.
Chambers
T C
TC
Department of Biological Chemistry, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania 19486.
McAvoy
E M
EM
Jacobs
J W
JW
Eilon
G
G
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000255:Adenosine Triphosphate / Q000378:metabolism; D000470:Alkaloids / Q000494:pharmacology; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D044182:Benzylisoquinolines; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D004351:Drug Resistance / Q000235:genetics*; D006801:Humans; D007624:KB Cells; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000302:isolation & purification / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014700:Verapamil / Q000494:pharmacology; D014747:Vinblastine / Q000378:metabolism*
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000470:Alkaloids; D000972:Antineoplastic Agents, Phytogenic; D044182:Benzylisoquinolines; D008562:Membrane Glycoproteins; D009363:Neoplasm Proteins; D010710:Phosphates; C009438:tetrandrine; D014747:Vinblastine; D000255:Adenosine Triphosphate; D014700:Verapamil; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate