Phosphorylation site mutations in the human multidrug transporter modulate its drug-stimulated ATPase activity

Katalin Szabó, E. Bakos, E. Welker, Marianna Müllerf, Hugh R. Goodfellow, Christopher F. Higgins, A. Váradi, B. Sarkadi

Research output: Contribution to journalArticle

48 Citations (Scopus)

Abstract

In the human multidrug transporter (MDR1), three serine residues located in the 'linker' region of the protein are targets of in viva phosphorylation. These three serines, or all eight serines and threonines in the linker, were substituted by alanines (mutants 3A and 8A) or with glutamic acids (mutants 3E and 8E). The wild-type and mutant proteins were expressed in baculovirus- infected Spodoptera frugiperda (Sf9) ovarian insect cells, and the vanadate- sensitive, drug-stimulated ATPase activity was measured in isolated membrane preparations. The maximum drug-stimulated MDR1-ATPase activity was similar for the wild-type and the mutant proteins. However, wild-type MDR1, which is known to be phosphorylated in Sf9 membranes, and the 3E and 8E mutants, which mimic the charge of phosphorylation, achieved half-maximum activation of MDR1-ATPase activity at lower verapamil, vinblastine, or rhodamine 123 concentrations than the nonphosphorylatable 3A and 8A variants. For some other drugs (e.g. valinomycin or calcein acetoxymethylester) activation of the MDR1-ATPase for any of the mutants was indistinguishable from that of the wild-type protein. Kinetic analysis of the data obtained for the 3A and 8A MDR1 variants indicated the presence of more than one drug interaction site, exhibiting an apparent negative cooperativity. This phenomenon was not observed for the wild-type or the 3E and 8E MDR1 proteins. The dependence of the MDR1-ATPase activity on ATP concentration was identical in the wild-type and the mutant proteins, and Hill plots indicated the presence of more than one functional ATP-binding site. These results suggest that phosphorylation of the linker region modulates the interaction of certain drugs with MDR1, especially at low concentrations, although phosphorylation does not alter the maximum level of MDR1-ATPase activity or its dependence on ATP concentration.

Original languageEnglish
Pages (from-to)23165-23171
Number of pages7
JournalJournal of Biological Chemistry
Volume272
Issue number37
DOIs
Publication statusPublished - 1997

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Phosphorylation
Adenosine Triphosphatases
Mutation
Mutant Proteins
Pharmaceutical Preparations
Serine
Adenosine Triphosphate
Drug Interactions
Chemical activation
Drug interactions
Glutamates
Membranes
Rhodamine 123
Valinomycin
Spodoptera
Vanadates
Vinblastine
Baculoviridae
P-Glycoprotein
Threonine

ASJC Scopus subject areas

  • Biochemistry

Cite this

Phosphorylation site mutations in the human multidrug transporter modulate its drug-stimulated ATPase activity. / Szabó, Katalin; Bakos, E.; Welker, E.; Müllerf, Marianna; Goodfellow, Hugh R.; Higgins, Christopher F.; Váradi, A.; Sarkadi, B.

In: Journal of Biological Chemistry, Vol. 272, No. 37, 1997, p. 23165-23171.

Research output: Contribution to journalArticle

Szabó, Katalin ; Bakos, E. ; Welker, E. ; Müllerf, Marianna ; Goodfellow, Hugh R. ; Higgins, Christopher F. ; Váradi, A. ; Sarkadi, B. / Phosphorylation site mutations in the human multidrug transporter modulate its drug-stimulated ATPase activity. In: Journal of Biological Chemistry. 1997 ; Vol. 272, No. 37. pp. 23165-23171.
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AU - Szabó, Katalin

AU - Bakos, E.

AU - Welker, E.

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AU - Goodfellow, Hugh R.

AU - Higgins, Christopher F.

AU - Váradi, A.

AU - Sarkadi, B.

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