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 t hat phosphorylation may regulate the drug transport activity of P-glyc oprotein. Domain mapping, utilizing a combination of cyanogen bromide digestion and immunoblot analysis, was used to reveal the major phosph orylation sites in murine mdr1b P-glycoprotein. After labeling of J7.V 1-1 cells with [P-32]P(i), or labeling membranes with [gamma-P-32]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 lin ker 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, i dentified serine 669 as the single site of phosphorylation. Comparison of two-dimensional tryptic phosphopeptide maps prepared from syntheti c peptide and P-glycoprotein, both of which were phosphorylated in vit ro 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-dir ected 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 c onductance 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 co nsensus phosphorylation sites. Since the R domain is believed to be in volved in the regulation of cystic fibrosis conductance regulator func tion by phosphorylation, it is possible that the linker region plays a similar regulatory role in P-glycoprotein function.