Identification of the major phosphorylation domain of murine mdr1b P-glycoprotein. Analysis of the protein kinase A and protein kinase C phosphorylation sites.
P-glycoprotein is phosphorylated in cells, and it has been suggested that phosphorylation may regulate the drug transport activity of P-glycoprotein. Domain mapping, utilizing a combination of cyanogen bromide digestion and immunoblot analysis, was used to reveal the major phosphorylation sites in murine mdr1b P-glycoprotein. After labeling of J7.V1-1 cells with [32P]Pi, or labeling membranes with [gamma-32P]ATP and either protein kinase A or protein kinase C, it was found that the majority of the label was contained within a single cyanogen bromide fragment (amino acid 627-682) that encompassed the majority of the linker region. The in vitro protein kinase C phosphorylation sites within this fragment were analyzed by a combination of fast atom bombardment mass spectrometry (FABMS) and two-dimensional phosphopeptide mapping. FABMS analysis of a protein kinase C-phosphorylated synthetic peptide, corresponding to a segment of the linker region of P-glycoprotein, identified serine 669 as the single site of phosphorylation. Comparison of two-dimensional tryptic phosphopeptide maps prepared from synthetic peptide and P-glycoprotein, both of which were phosphorylated in vitro with protein kinase C, revealed that serine 669 was also the major phosphorylation site in the intact glycoprotein. The in vitro protein kinase A phosphorylation site was identified as serine 681 by site-directed mutagenesis. Inspection of the gene organization and the deduced amino acid sequence of mdr1b P-glycoprotein revealed that the linker region, although shorter than the R domain (55 versus 241 amino acids), fits the operational definition of the R domain of cystic fibrosis conductance regulator. Like the R domain, the linker region is encoded by a single exon, is highly charged with alternating acidic and basic side chains, and contains several protein kinase A/protein kinase C consensus phosphorylation sites. Since the R domain is believed to be involved in the regulation of cystic fibrosis conductance regulator function by phosphorylation, it is possible that the linker region plays a similar regulatory role in P-glycoprotein function.
Orr
G A
GA
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461.
Han
E K
EK
Browne
P C
PC
Nieves
E
E
O'Connor
B M
BM
Yang
C P
CP
Horwitz
S B
SB
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004351:Drug Resistance; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014747:Vinblastine / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D014747:Vinblastine; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C