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 phosphorylat ed in membrane vesicle preparations in vitro only when coexpressed wit h PKC alpha, and phosphorylation was Ca2+-dependent and inhibited by t he PKC inhibitor Ro 31-8220. PGP and PKC alpha were tightly associated in membrane vesicles and were coimmunoprecipitated with antibodies ag ainst either PGP or PKC alpha. Photoaffinity labeling of membrane vesi cles with [H-3]azidopine indicated that drug binding to PGP was slight ly increased in the presence of PKC alpha. In contrast, PGP ATPase act ivity 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 result s indicate that PKC alpha in a positive regulator of PGP ATPase activi ty and suggest that this mechanism may account for the increased multi drug resistance observed in MDR1-expressing cells when PKC alpha activ ity is elevated.