Identification of the major phosphorylation domain of murine mdr1b P-glycoprotein

Analysis of the protein kinase A and protein kinase C phosphorylation sites

George A. Orr, Edward K H Han, Paul C. Browne, Edward Nieves, Brigid M. O'Connort, Chia-Ping H. Yang, Susan Band Horwitz

Research output: Contribution to journalArticle

57 Citations (Scopus)

Abstract

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 mdrlb P-glycoprotein. After labeling of J7.V1-1 cells with [32P]Pi, or labeling membranes with [γ-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.

Original languageEnglish (US)
Pages (from-to)25054-25062
Number of pages9
JournalJournal of Biological Chemistry
Volume268
Issue number33
StatePublished - Nov 25 1993

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Phosphorylation
P-Glycoprotein
Cyclic AMP-Dependent Protein Kinases
Protein Kinase C
Serine
Fast Atom Bombardment Mass Spectrometry
Phosphopeptides
Cyanogen Bromide
Amino Acids
Cystic Fibrosis
Labeling
Mass spectrometry
Atoms
Mutagenesis
Peptides
Site-Directed Mutagenesis
Labels
Digestion
Amino Acid Sequence
Exons

ASJC Scopus subject areas

  • Biochemistry

Cite this

Identification of the major phosphorylation domain of murine mdr1b P-glycoprotein : Analysis of the protein kinase A and protein kinase C phosphorylation sites. / Orr, George A.; Han, Edward K H; Browne, Paul C.; Nieves, Edward; O'Connort, Brigid M.; Yang, Chia-Ping H.; Band Horwitz, Susan.

In: Journal of Biological Chemistry, Vol. 268, No. 33, 25.11.1993, p. 25054-25062.

Research output: Contribution to journalArticle

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abstract = "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 mdrlb P-glycoprotein. After labeling of J7.V1-1 cells with [32P]Pi, or labeling membranes with [γ-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.",
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AU - Han, Edward K H

AU - Browne, Paul C.

AU - Nieves, Edward

AU - O'Connort, Brigid M.

AU - Yang, Chia-Ping H.

AU - Band Horwitz, Susan

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