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 Thr53. In this report, we use mass spectrometry and a newly developed phospho-specific antibody to positively identify DAT phosphorylation at Thr53 in rodent striatal tissue and heterologous expression systems. Basal phosphorylation of Thr53 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 Thr53 to prevent phosphorylation led to reduced dopamine transport Vmax and total apparent loss of amphetamine-stimulated substrate efflux, supporting a major role for this residue in the transport kinetic mechanism. Background: DAT activity is regulated by protein kinases. Results: We identify Thr53 as a DAT phosphorylation site in rat striatum by mass spectrometry and a phospho-specific antibody; Thr53 mutation reduced dopamine influx and ablated transporter-mediated efflux. Conclusion: Phosphorylation of DAT Thr53 is involved in transport activity. Significance: These results identify Thr53 phosphorylation of DAT in vivo and elucidate associated functional properties.