Identification of the in vivo phosphorylation sites for acidic-directed kinases in murine mdr1b P-glycoprotein

Joseph S. Glavy, Susan Band Horwitz, George A. Orr

Research output: Contribution to journalArticle

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Abstract

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 multi-drug 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.

Original languageEnglish (US)
Pages (from-to)5909-5914
Number of pages6
JournalJournal of Biological Chemistry
Volume272
Issue number9
DOIs
StatePublished - Feb 28 1997

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Phosphorylation
P-Glycoprotein
Phosphotransferases
Amino Acids
Casein Kinase II
Phosphopeptides
Peptides
Multiple Drug Resistance
Phosphates
Cells
Acidic Amino Acids
Cell Line
Mutagenesis
Vinblastine
Drug and Narcotic Control
HeLa Cells
Pharmaceutical Preparations
Protein Kinases
Serine
Mass spectrometry

ASJC Scopus subject areas

  • Biochemistry

Cite this

Identification of the in vivo phosphorylation sites for acidic-directed kinases in murine mdr1b P-glycoprotein. / Glavy, Joseph S.; Band Horwitz, Susan; Orr, George A.

In: Journal of Biological Chemistry, Vol. 272, No. 9, 28.02.1997, p. 5909-5914.

Research output: Contribution to journalArticle

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