title: Protein kinase C-mediated phosphorylation does not regulate drug transport by the human multidrug resistance P-glycoprotein.
abstract: P-glycoprotein (P-gp) is an active transporter that can confer multidrug resistance by pumping cytotoxic drugs out of cells and tumors. P-gp is phosphorylated at several sites in the "linker" region, which separates the two halves of the molecule. To examine the role of phosphorylation in drug transport, we mutated P-gp such that it could no longer be phosphorylated by protein kinase C (PKC). When expressed in yeast, the ability of the mutant proteins to confer drug resistance, or to mediate [3H]vinblastine accumulation in secretory vesicles, was indistinguishable from that of wild type P-gp. A matched pair of mammalian cell lines were generated expressing wild type P-gp and a non-phosphorylatable mutant protein. Mutation of the phosphorylation sites did not alter P-gp expression or its subcellular localization. The transport properties of the mutant and wild type proteins were indistinguishable. Thus, phosphorylation of the linker of P-gp by PKC does not affect the rate of drug transport. In light of these data, the use of agents that alter PKC activity to reverse multidrug resistance in the clinic should be considered with caution.
authors.affiliation: Imperial Cancer Research Laboratories, Nuffield Department of Clinical Biochemistry, Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom.
authors.forename: H R
authors.initials: HR
authors.lastname: Goodfellow
authors.forename: A
authors.initials: A
authors.lastname: Sardini
authors.forename: S
authors.initials: S
authors.lastname: Ruetz
authors.forename: R
authors.initials: R
authors.lastname: Callaghan
authors.forename: P
authors.initials: P
authors.lastname: Gros
authors.forename: P A
authors.initials: PA
authors.lastname: McNaughton
authors.forename: C F
authors.initials: CF
authors.lastname: Higgins
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001693:Biological Transport, Active; D002460:Cell Line; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004317:Doxorubicin / Q000493:pharmacokinetics; D004351:Drug Resistance; D005452:Fluoresceins / Q000493:pharmacokinetics; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D010766:Phosphorylation; D017354:Point Mutation; D011493:Protein Kinase C / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014162:Transfection; D014747:Vinblastine / Q000493:pharmacokinetics
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D005452:Fluoresceins; D009838:Oligodeoxyribonucleotides; C085925:calcein AM; D014747:Vinblastine; D004317:Doxorubicin; D011493:Protein Kinase C