Modulation of P-glycoprotein by protein kinase C alpha in a baculovirus expression system.
The modulation of P-glycoprotein by protein kinase C alpha (PKC alpha) was examined in a baculovirus expression system. PGP was phosphorylated in membrane vesicle preparations in vitro only when coexpressed with PKC alpha, and phosphorylation was Ca(2+)-dependent and inhibited by the PKC inhibitor Ro 31-8220. PGP and PKC alpha were tightly associated in membrane vesicles and were coimmunoprecipitated with antibodies against either PGP or PKC alpha. Photoaffinity labeling of membrane vesicles with [3H]azidopine indicated that drug binding to PGP was slightly increased in the presence of PKC alpha. In contrast, PGP ATPase activity was increased by PKC alpha as well as by verapamil, but only PKC-stimulated activity in the presence of verapamil was inhibited by Ro 31-8220. Mutation of serine-671 to asparagine in the linker region of PGP abolished PKC alpha-stimulated ATPase activity, and also inhibited to a lesser degree verapamil-stimulated ATPase activity. These results indicate that PKC alpha in a positive regulator of PGP ATPase activity and suggest that this mechanism may account for the increased multidrug resistance observed in MDR1-expressing cells when PKC alpha activity is elevated.
Ahmad
S
S
Department of Pharmacology, Georgetown University Medical Center, Washington, D.C. 20007.
Safa
A R
AR
Glazer
R I
RI
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000345:Affinity Labels; D000818:Animals; D001386:Azides; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D004095:Dihydropyridines; D004351:Drug Resistance / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009036:Moths; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000345:Affinity Labels; D001386:Azides; D002352:Carrier Proteins; D018076:DNA, Complementary; D004095:Dihydropyridines; D007527:Isoenzymes; D008562:Membrane Glycoproteins; C041130:azidopine; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D000251:Adenosine Triphosphatases