Identification of the in vivo phosphorylation sites for acidic-directed kinases in murine mdr1b P-glycoprotein.
P-glycoprotein, the multidrug resistance transporter, is phosphorylated in vivo and the major phosphorylation domain has been identified as the linker region (amino acids 629-686). The linker region is a highly charged segment of the transporter in which the negative and positive amino acid side chains are spatially segregated. Both of these charged domains contain several consensus phosphorylation sites for protein kinases. Three of the consensus phosphorylation sites for basic-directed kinases in murine mdr1b P-glycoprotein are utilized in vivo and have been identified as serines 665, 669, and 681. Mutagenesis of all the consensus basic-directed kinase phosphorylation sites in the linker region of human MDR1 P-glycoprotein did not alter the ability of the mutated transporter to confer the multidrug resistance phenotype in stably transfected cell lines. These studies would suggest that phosphorylation/dephosphorylation within the basic domain of the linker region is not directly involved in regulation of drug transporter activity. We now report that the linker region of mdr1b P-glycoprotein is also phosphorylated in vivo within the acidic domain (amino acids 631-658). These sites have been mapped using casein kinase II, a prototypic acidic-directed kinase, and a recombinant mdr1b linker region peptide (amino acids 621-687). Electrospray mass spectrometry demonstrated that casein kinase II could introduce up to five phosphates into the recombinant peptide. Two-dimensional phosphopeptide mapping indicated that all the phosphates were contained in a tryptic peptide consisting of amino acids 631-658. Phosphopeptide mapping of in vivo labeled P-glycoprotein, isolated from either J7.V1-1, a murine vinblastine-resistant cell line, or HeLa cells stably transfected with mdr1b P-glycoprotein cDNA, revealed that this tryptic peptide was phosphorylated in both proteins.
Glavy
J S
JS
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Horwitz
S B
SB
Orr
G A
GA
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D011494:Protein Kinases