title: Phosphorylation of dopamine transporter serine 7 modulates cocaine analog binding.
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.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota 58203-9037, USA.
authors.forename: Amy E
authors.initials: AE
authors.lastname: Moritz
authors.forename: James D
authors.initials: JD
authors.lastname: Foster
authors.forename: Balachandra K
authors.initials: BK
authors.lastname: Gorentla
authors.forename: Michelle S
authors.initials: MS
authors.lastname: Mazei-Robison
authors.forename: Jae-Won
authors.initials: JW
authors.lastname: Yang
authors.forename: Harald H
authors.initials: HH
authors.lastname: Sitte
authors.forename: Randy D
authors.initials: RD
authors.lastname: Blakely
authors.forename: Roxanne A
authors.initials: RA
authors.lastname: Vaughan
mesh_terms: D000818:Animals; D001665:Binding Sites; D002855:Chromatography, Thin Layer / Q000379:methods; D003042:Cocaine / Q000031:analogs & derivatives / Q000737:chemistry*; D003342:Corpus Striatum / Q000187:drug effects / Q000378:metabolism; D050483:Dopamine Plasma Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism; D018765:Dopamine Uptake Inhibitors / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008297:Male; D013058:Mass Spectrometry / Q000379:methods; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D017207:Rats, Sprague-Dawley; D012694:Serine / Q000737:chemistry*
chemical_list: D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors; D012694:Serine; D003042:Cocaine