Dopamine transporters are dephosphorylated in striatal homogenates and in vitro by protein phosphatase 1.
Dopamine transporters (DATs) undergo increased phosphorylation upon treatment of striatal tissue or cultured cells with protein kinase C activators and protein phosphatase inhibitors. Phosphorylation conditions also lead to reductions in dopamine transport activity, which may function to regulate synaptic dopamine levels and control the extent and duration of dopaminergic signaling. Treatment of rat striatal tissue with okadaic acid (OA), a broad-spectrum protein phosphatase inhibitor, produces apparent maximal increases in DAT phosphorylation, suggesting that dephosphorylation is a crucial regulator of the DAT phosphorylation state. We used a combination of endogenous and in vitro approaches to identify the phosphatase(s) responsible for this activity. In homogenates prepared from (32)PO(4)-labeled rat striatal slices, OA inhibited DAT dephosphorylation with an IC(50) of 40 nM, a dose most compatible with inhibition of protein phosphatase 1 (PP1). Dephosphorylation of DAT in striatal homogenates was also inhibited by PP1 inhibitor 2, while little effect was produced by protein phosphatase 2A inhibitor 1. In vitro dephosphorylation assays showed substantial removal of (32)PO(4) from DATs by PP1 but not by protein phosphatase 2A, protein phosphatase 2B, or protein tyrosine phosphatase, and this effect was blocked by OA, verifying that the (32)PO(4) loss from DAT was due to dephosphorylation. These results demonstrate that DAT is a direct substrate for PP1 in vitro and suggest that PP1 is a major DAT phosphatase in rat striatum.
Foster
James D
JD
Department of Biochemistry and Molecular Biology, University of North Dakota School of Medicine and Health Sciences, 501 N. Columbia Road, Grand Forks, ND 58202, USA.
Pananusorn
Benchaporn
B
Cervinski
Mark A
MA
Holden
Heather E
HE
Vaughan
Roxanne A
RA
D000818:Animals; D003342:Corpus Striatum / Q000378:metabolism*; D050483:Dopamine Plasma Membrane Transport Proteins; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008297:Male; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins / Q000378:metabolism*; D009419:Nerve Tissue Proteins; D019319:Okadaic Acid / Q000494:pharmacology; D009924:Organ Culture Techniques; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2; D011506:Proteins / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D000097847:Protein Phosphatase Inhibitory Proteins
D050483:Dopamine Plasma Membrane Transport Proteins; D004791:Enzyme Inhibitors; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases; D011493:Protein Kinase C; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2; D011506:Proteins; C493216:Slc6a3 protein, rat; C024833:protein phosphatase inhibitor-2; D000097847:Protein Phosphatase Inhibitory Proteins