Dephosphorylation of human dopamine transporter at threonine 48 by protein phosphatase PP1/2A up-regulates transport velocity

Jae Won Yang, Garret Larson, Lisa Konrad, Madhur Shetty, Marion Holy, Kathrin Jäntsch, Mirja Kastein, Seok Heo, Fatma Asli Erdem, Gert Lubec, Roxanne A. Vaughan, Harald H. Sitte, James D. Foster

Research output: Contribution to journalArticlepeer-review

Abstract

Several protein kinases, including protein kinase C, Ca2/ calmodulin-dependent protein kinase II, and extracellular signal–regulated kinase, play key roles in the regulation of dopamine transporter (DAT) functions. These functions include surface expression, internalization, and forward and reverse transport, with phosphorylation sites for these kinases being linked to distinct regions of the DAT N terminus. Protein phosphatases (PPs) also regulate DAT activity, but the specific residues associated with their activities have not yet been elucidated. In this study, using co-immunoprecipitation followed by MS and immunoblotting analyses, we demonstrate the association of DAT with PP1 and PP2A in the mouse brain and heterologous cell systems. By applying MS in conjunction with a metabolic labeling method, we defined a PP1/2A-sensitive phosphorylation site at Thr-48 in human DAT, a residue that has not been previously reported to be involved in DAT phosphorylation. Site-directed mutagenesis of Thr-48 to Ala (T48A) to prevent phosphorylation enhanced dopamine transport kinetics, supporting a role for this residue in regulating DAT activity. Moreover, T48A-DAT displayed increased palmitoylation, suggesting that phosphorylation/dephosphorylation at this site has an additional regulatory role and reinforcing a previously reported reciprocal relationship between C-terminal palmitoylation and N-terminal phosphorylation.

Original languageEnglish (US)
Pages (from-to)3419-3431
Number of pages13
JournalJournal of Biological Chemistry
Volume294
Issue number10
DOIs
StatePublished - Mar 8 2019
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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