Dephosphorylation of human dopamine transporter at threonine 48 by protein phosphatase PP1/2A up-regulates transport velocity
| Autor: | Seok Heo, James D. Foster, Garret H. Larson, Lisa Konrad, Fatma Asli Erdem, Roxanne A. Vaughan, Marion Holy, Madhur Shetty, Gert Lubec, Harald H. Sitte, Mirja Kastein, Jae-Won Yang, Kathrin Jäntsch |
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| Rok vydání: | 2019 |
| Předmět: |
Threonine
0301 basic medicine Dopamine Lipoylation Biological Transport Active Dopamine transport macromolecular substances Biochemistry Dephosphorylation Mice 03 medical and health sciences Palmitoylation Protein Phosphatase 1 Animals Humans Protein Phosphatase 2 Phosphorylation Molecular Biology Protein kinase C Dopamine transporter Mice Knockout Dopamine Plasma Membrane Transport Proteins 030102 biochemistry & molecular biology biology Chemistry Kinase Brain Cell Biology Protein phosphatase 2 Cell biology 030104 developmental biology nervous system biology.protein |
| Zdroj: | Journal of Biological Chemistry. 294:3419-3431 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.ra118.005251 |
| Popis: | Several protein kinases, including protein kinase C, Ca(2+)/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. |
| Databáze: | OpenAIRE |
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