title: Bacterial expression of the linker region of human MDR1 P-glycoprotein and mutational analysis of phosphorylation sites.
abstract: Phosphorylation may play a role in modulating multidrug resistance by P-glycoprotein (P-gp). The linker region between the two homologous halves of human P-gp harbors several serine residues which are phosphorylated by protein kinase C (PKC) in vitro. We used the glutathione S-transferase gene fusion system to express and purify a series of fusion proteins containing the relevant portion (residues 644-689) of the linker region of the human MDR1 gene product. The fusion proteins were subjected to in vitro phosphorylation and phosphopeptide mapping analysis to identify specific phosphorylation sites. On the basis of a mutational strategy in which individual serine residues were systematically replaced with nonphosphorylatable alanine residues, Ser-661 and Ser-667 were identified as major PKC sites and Ser-683 was identified as a minor PKC site. Ser-661 and Ser-667 were also found to be the primary sites of phosphorylation for a novel membrane-associated P-gp specific kinase isolated from the multidrug-resistant KB-V1 cell line. Individual phosphorylation sites were recognized independently of each other. These data show that the linker region of P-gp represents a target for multisite phosphorylation not only for PKC but also for the P-gp specific V1 kinase. Specific serine phosphorylation sites are identified, and evidence is presented that the V1 kinase has a specificity which overlaps, but is more restricted than, that of PKC. In addition, these studies also suggest that the use of GST fusion peptides may be applicable for the analysis of multisite and ordered protein phosphorylation in other systems.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock 72205, USA.
authors.forename: T C
authors.initials: TC
authors.lastname: Chambers
authors.forename: U A
authors.initials: UA
authors.lastname: Germann
authors.forename: M M
authors.initials: MM
authors.lastname: Gottesman
authors.forename: I
authors.initials: I
authors.lastname: Pastan
authors.forename: J F
authors.initials: JF
authors.lastname: Kuo
authors.forename: S V
authors.initials: SV
authors.lastname: Ambudkar
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000737:chemistry; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018076:DNA, Complementary; D010748:Phosphopeptides; D011993:Recombinant Fusion Proteins; D011493:Protein Kinase C