title: Regulation by phorbol ester and protein kinase C inhibitors, and by a protein phosphatase inhibitor (okadaic acid), of P-glycoprotein phosphorylation and relationship to drug accumulation in multidrug-resistant human KB cells.
abstract: Covalent modification by phosphorylation is a characteristic of the P-glycoproteins expressed in multidrug-resistant cells. This report describes analysis of P-glycoprotein phosphorylation in multidrug-resistant human KB-V1 cells and a study of the relationship of phosphorylation and drug accumulation. In isolated membranes, phosphorylation of P-glycoprotein by purified protein kinase C (PKC) was rapid, and time-dependent dephosphorylation was inhibited by okadaic acid, an inhibitor of type 1 and type 2A protein phosphatases. In 32P-labeled intact KB-V1 cells, P-glycoprotein phosphorylation was stimulated by both 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of PKC, and okadaic acid. Two-dimensional thin layer tryptic phosphopeptide maps indicated that the sites of phosphorylation were similar in control, TPA-treated, and okadaic acid-treated cells and that they corresponded to those phosphorylated by PKC in vitro. The protein kinase inhibitor staurosporine, and the PKC-selective inhibitors calphostin C and the alkyl-lysophospholipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine, inhibited P-glycoprotein phosphorylation in vitro and in intact cells. Drug accumulation assays demonstrated that in KB-V1 cells TPA caused a decrease, whereas staurosporine and calphostin C caused an increase, in accumulation of [3H]vinblastine. These compounds did not significantly alter [3H]vinblastine levels in drug-sensitive KB-3 cells. These results suggest that PKC is chiefly responsible for P-glycoprotein phosphorylation in KB-V1 cells, that membrane-associated protein phosphatases 1 and 2A are active in dephosphorylation of P-glycoprotein, and that phosphorylation of P-glycoprotein may be an important mechanism for modulation of drug-pumping activity.
authors.affiliation: Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia 30322.
authors.forename: T C
authors.initials: TC
authors.lastname: Chambers
authors.forename: B
authors.initials: B
authors.lastname: Zheng
authors.forename: J F
authors.initials: JF
authors.lastname: Kuo
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000470:Alkaloids / Q000494:pharmacology; D004351:Drug Resistance; D004988:Ethers, Cyclic / Q000494:pharmacology*; D006801:Humans; D007624:KB Cells / Q000187:drug effects; D008562:Membrane Glycoproteins / Q000378:metabolism* / Q000502:physiology; D009281:Naphthalenes; D019319:Okadaic Acid; D010449:Peptide Mapping; D010641:Phenotype; D010742:Phospholipid Ethers / Q000494:pharmacology; D010748:Phosphopeptides / Q000737:chemistry; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D010766:Phosphorylation; D011083:Polycyclic Compounds / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors* / Q000502:physiology; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014700:Verapamil / Q000494:pharmacology; D014747:Vinblastine / Q000493:pharmacokinetics*
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000470:Alkaloids; D004988:Ethers, Cyclic; D008562:Membrane Glycoproteins; D009281:Naphthalenes; D010742:Phospholipid Ethers; D010748:Phosphopeptides; D011083:Polycyclic Compounds; D019319:Okadaic Acid; C026659:edelfosine; D014747:Vinblastine; D014700:Verapamil; D011493:Protein Kinase C; D010749:Phosphoprotein Phosphatases; D019311:Staurosporine; C058819:calphostin C; D013755:Tetradecanoylphorbol Acetate