Characterization of Phosphorylation-defective Mutants of Human P-glycoprotein Expressed in Mammalian Cells (∗)

To assess the role of phosphorylation of the human multidrug resistance MDR1 gene product P-glycoprotein for its drug transport activity, phosphorylation sites within its linker region were subjected to mutational analysis. We constructed a 5A mutant, in which serines at positions 661, 667, 671, 675...

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Bibliographic Details
Published in:The Journal of biological chemistry 1996-01, Vol.271 (3), p.1708-1716
Main Authors: Germann, Ursula A., Chambers, Timothy C., Ambudkar, Suresh V., Licht, Thomas, Cardarelli, Carol O., Pastan, Ira, Gottesman, Michael M.
Format: Article
Language:English
Subjects:
3T3 Cells
Alanine
Amino Acid Sequence
Animals
Aspartic Acid
ATP Binding Cassette Transporter, Subfamily B, Member 1 - biosynthesis
ATP Binding Cassette Transporter, Subfamily B, Member 1 - genetics
ATP Binding Cassette Transporter, Subfamily B, Member 1 - isolation & purification
Base Sequence
Cell Membrane - metabolism
DNA Mutational Analysis
Drug Resistance, Multiple - genetics
Gene Expression
Humans
Mammals
Mice
Molecular Sequence Data
Mutagenesis, Site-Directed
Oligodeoxyribonucleotides
Phosphorylation
Point Mutation
Recombinant Proteins - biosynthesis
Recombinant Proteins - isolation & purification
Restriction Mapping
Serine
Transfection
Vincristine - pharmacology
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Summary:To assess the role of phosphorylation of the human multidrug resistance MDR1 gene product P-glycoprotein for its drug transport activity, phosphorylation sites within its linker region were subjected to mutational analysis. We constructed a 5A mutant, in which serines at positions 661, 667, 671, 675, and 683 were replaced by nonphosphorylatable alanine residues, and a 5D mutant carrying aspartic acid residues at the respective positions to mimic permanently phosphorylated serine residues. Transfection studies revealed that both mutants were targeted properly to the cell surface and conferred multidrug resistance by diminishing drug accumulation. In contrast to wild-type P-glycoprotein, the overexpressed 5A and the 5D mutants exhibited no detectable levels of phosphorylation, either in vivo following metabolic labeling of cells with [32P]orthophosphate or in vitro in phosphorylation assays with protein kinase C, cAMP-dependent protein kinase, or a P-glycoprotein-specific protein kinase purified from multidrug-resistant KB-V1 cells. These results reconfirm that the major P-glycoprotein phosphorylation sites are located within the linker region. Furthermore, the first direct evidence is provided that phosphorylation/dephosphorylation mechanisms do not play an essential role in the establishment of the multidrug resistance phenotype mediated by human P-glycoprotein.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.271.3.1708