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PMID: 23161550 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't

Phosphorylation of dopamine transporter serine 7 modulates cocaine analog binding.

The Journal of biological chemistry ·Vol. 288 ·No. 1 ·2013-01-04 ·Pages 20-32

Moritz AE, Foster JD, Gorentla BK, Mazei-Robison MS, Yang JW, Sitte HH, Blakely RD, Vaughan RA

Abstract

As an approach to elucidating dopamine transporter (DAT) phosphorylation characteristics, we examined in vitro phosphorylation of a recombinant rat DAT N-terminal peptide (NDAT) using purified protein kinases. We found that NDAT becomes phosphorylated at single distinct sites by protein kinase A (Ser-7) and calcium-calmodulin-dependent protein kinase II (Ser-13) and at multiple sites (Ser-4, Ser-7, and Ser-13) by protein kinase C (PKC), implicating these residues as potential sites of DAT phosphorylation by these kinases. Mapping of rat striatal DAT phosphopeptides by two-dimensional thin layer chromatography revealed basal and PKC-stimulated phosphorylation of the same peptide fragments and comigration of PKC-stimulated phosphopeptide fragments with NDAT Ser-7 phosphopeptide markers. We further confirmed by site-directed mutagenesis and mass spectrometry that Ser-7 is a site for PKC-stimulated phosphorylation in heterologously expressed rat and human DATs. Mutation of Ser-7 and nearby residues strongly reduced the affinity of rat DAT for the cocaine analog (-)-2β-carbomethoxy-3β-(4-fluorophenyl) tropane (CFT), whereas in rat striatal tissue, conditions that promote DAT phosphorylation caused increased CFT affinity. Ser-7 mutation also affected zinc modulation of CFT binding, with Ala and Asp substitutions inducing opposing effects. These results identify Ser-7 as a major site for basal and PKC-stimulated phosphorylation of native and expressed DAT and suggest that Ser-7 phosphorylation modulates transporter conformational equilibria, shifting the transporter between high and low affinity cocaine binding states.

MeSH Terms
Animals Binding Sites Chromatography, Thin Layer/methods Cocaine/analogs & derivatives,chemistry Corpus Striatum/drug effects,metabolism Dopamine Plasma Membrane Transport Proteins/chemistry,metabolism Dopamine Uptake Inhibitors/chemistry HEK293 Cells Humans Kinetics Male Mass Spectrometry/methods Mutagenesis, Site-Directed Mutation Phosphorylation Protein Binding Protein Conformation Rats Rats, Sprague-Dawley Serine/chemistry
Chemicals
Dopamine Plasma Membrane Transport Proteins Dopamine Uptake Inhibitors Serine Cocaine
Authors & Affiliations
8 authors, click to expand affiliations / ORCID
Moritz Amy E
Department of Biochemistry and Molecular Biology, University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota 58203-9037, USA.
Foster James D
Gorentla Balachandra K
Mazei-Robison Michelle S
Yang Jae-Won
Sitte Harald H
Blakely Randy D
Vaughan Roxanne A
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Article Info
Journal
The Journal of biological chemistry
Abbr.
J Biol Chem
ISSN
1083-351X
DOI
10.1074/jbc.M112.407874
Published
2013-01-04
Epub
2012-00-16
Pages
20-32
Language
English
Region
United States
NLM ID
2985121R
PMCID
PMC3537014
Subset
IM
Grants
NIDA NIH HHS · R01 DA007390 · United States
NCRR NIH HHS · P20 RR01769 · United States
NIDA NIH HHS · DA07390 · United States
NIDA NIH HHS · R01 DA013147 · United States
NCRR NIH HHS · P20 RR017699 · United States
NIDA NIH HHS · DA13147 · United States
NIMH NIH HHS · F31 MH067472 · United States
NIMH NIH HHS · MH067472 · United States
NIGMS NIH HHS · P20 GM103442 · United States
External Links
PubMed source DOI: 10.1074/jbc.M112.407874 PMC Full Text View journal details
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