Phosphorylation of Dopamine Transporter Serine 7 Modulates Cocaine Analog Binding |
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Authors: | Amy E. Moritz James D. Foster Balachandra K. Gorentla Michelle S. Mazei-Robison Jae-Won Yang Harald H. Sitte Randy D. Blakely Roxanne A. Vaughan |
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Affiliation: | From the ‡Department of Biochemistry and Molecular Biology, University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota 58203-9037.;the §Departments of Pharmacology and Psychiatry, Vanderbilt University, Nashville, Tennessee 37232-8548, and ;the ¶Center of Physiology and Pharmacology, Institute of Pharmacology, Medical University of Vienna, Waehringerstrasse 13a, A-1090 Vienna, Austria |
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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. |
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Keywords: | Calcium-Calmodulin-dependent Protein Kinase (CaMK) Mass Spectrometry (MS) Protein Conformation Protein Kinase A (PKA) Protein Kinase C (PKC) Peptide Mapping Zinc Binding |
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