Dopamine transporter phosphorylation site threonine 53 regulates substrate reuptake and amphetamine-stimulated efflux.
In the central nervous system, levels of extraneuronal dopamine are controlled primarily by the action of the dopamine transporter (DAT). Multiple signaling pathways regulate transport activity, substrate efflux, and other DAT functions through currently unknown mechanisms. DAT is phosphorylated by protein kinase C within a serine cluster at the distal end of the cytoplasmic N terminus, whereas recent work in model cells revealed proline-directed phosphorylation of rat DAT at membrane-proximal residue Thr(53). In this report, we use mass spectrometry and a newly developed phospho-specific antibody to positively identify DAT phosphorylation at Thr(53) in rodent striatal tissue and heterologous expression systems. Basal phosphorylation of Thr(53) occurred with a stoichiometry of ~50% and was strongly increased by phorbol esters and protein phosphatase inhibitors, demonstrating modulation of the site by signaling pathways that impact DAT activity. Mutations of Thr(53) to prevent phosphorylation led to reduced dopamine transport V(max) and total apparent loss of amphetamine-stimulated substrate efflux, supporting a major role for this residue in the transport kinetic mechanism.
Department of Biochemistry and Molecular Biology, University of North Dakota School of Medicine and Health Sciences, Grand Forks, ND 58202-9037, USA.
James D
JD
Foster
Jae-Won
JW
Yang
Amy E
AE
Moritz
Sathyavathi
S
Challasivakanaka
Margaret A
MA
Smith
Marion
M
Holy
Kyle
K
Wilebski
Harald H
HH
Sitte
Roxanne A
RA
Vaughan
D019943:Amino Acid Substitution; D000661:Amphetamine / Q000494:pharmacology*; D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects / Q000235:genetics; D045744:Cell Line, Tumor; D015259:Dopamine Agents / Q000494:pharmacology*; D050483:Dopamine Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D010766:Phosphorylation / Q000187:drug effects / Q000235:genetics; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects* / Q000235:genetics; D013552:Swine; D013912:Threonine / Q000235:genetics / Q000378:metabolism
D015259:Dopamine Agents; D050483:Dopamine Plasma Membrane Transport Proteins; D013912:Threonine; D000661:Amphetamine