Research reportDopamine transporters are dephosphorylated in striatal homogenates and in vitro by protein phosphatase 1
Introduction
The concentration of dopamine in the synapse is controlled by dopamine transporters (DATs), integral membrane proteins that drive reuptake of the neurotransmitter into the presynaptic cell [14]. DAT belongs to a family of transporters that include those for norepinephrine (NET) and serotonin (SERT), the major sites of action for tricyclic antidepressants and psychostimulants [1], and transporters for other neurotransmitters such as γ-aminobutyric acid (GABA). These proteins have a similar structure of 12 transmembrane spanning domains presumed to form the substrate translocation pathway, and cytoplasmically oriented N- and C-termini that contain numerous potential sites of phosphorylation.
Recent studies have shown that neurotransmitter transporters are phosphoproteins whose activities are controlled by treatments that regulate serine/threonine (S/T) and tyrosine protein kinases [2], [4], [11], [25], [30], [32], [36]. In striatal tissue and cultured cells, activators of PKC and inhibitors of protein phosphatases cause increases in DAT phosphorylation [20], [36]. The same treatments produce reductions in dopamine transport activity that are accompanied by transporter sequestration into internal membrane compartments [7], [9], [28], [39], [40] (reviewed in Refs. [38]). Kinase-stimulated removal of DATs from the cell surface may provide neurons with a mechanism for acute spatial and temporal control of synaptic dopamine levels and subsequent neural signaling. At present, however, a mechanistic relationship between DAT phosphorylation, regulation, and internalization has not been established.
We have recently identified the N-terminal tail of DAT as the major site of basal and stimulated phosphorylation in rat brain tissue [13], but many questions remain with respect to the characteristics of DAT phosphorylation and the relationships of phosphorylation conditions. Phosphopeptide mapping results indicate that both PKC- and OA-stimulated phosphorylation occur in a cluster of six serines present within the first 21 residues of the protein. However, we do not know the number or positions of phosphorylation sites within this cluster, or if OA and PKC-stimulated sites are present on the same, different, or overlapping residues. The kinases and phosphatases that act on DAT to control its overall phosphorylation level are also not known. While activation of PKC leads to stimulated phosphate incorporation into DAT, it is not known if PKC directly phosphorylates DAT or acts via an intermediate kinase, or if basal phosphorylation is mediated by tonic PKC action or involves other kinases.
Results from our laboratory implicate the dephosphorylation of DAT as a crucial aspect of appropriate phosphorylation control [34]. Brief treatment of rat brain tissue with OA produces significant increases in the DAT phosphorylation level [13], [36]. This indicates that in the absence of phosphatase inhibition DAT undergoes robust and rapid tonic dephosphorylation, suggesting a physiological significance to the maintenance of less phosphorylated forms. For example, if PKC-induced phosphorylation of DAT leads to its sequestration [9], [28], then dampening of this process by dephosphorylation would promote retention of DAT at the plasma membrane where it can function to clear dopamine. An alternative scenario is that dephosphorylation of previously endocytosed DATs may be involved in their recycling and return to the cell surface, similar to events that regulate G-protein coupled receptors [24].
Identification of the enzymes that control the DAT phosphorylation level is essential for elucidating the molecular basis of these processes. With respect to dephosphorylation, two previous attempts to identify the phosphatase acting on DAT have yielded inconclusive results. In one study using synaptosomes, DAT dephosphorylation was inhibited by 1–10 μM OA [36], doses that are outside the range of the nanomolar concentrations that inhibit purified PP1 and protein phosphatase 2A (PP2A). The effective concentrations are most consistent with inhibition of protein phosphatase 2B (PP2B, calcineurin), but in the same study the DAT phosphorylation level was unaffected by the PP2B inhibitor cyclosporin. This discrepancy could indicate that the high OA doses required to inhibit DAT dephosphorylation might reflect poor passage of the compound through the plasma membrane, obscuring the true dose–response relationships. A more recent study has shown that SERT, NET, and DAT form co-immunoprecipitating complexes with the catalytic subunit of PP2A [3]. The transporter–phosphatase complexes are disrupted by OA, which would be predicted to result in increased protein phosphorylation levels and suggests a role for PP2A involvement with transporter phosphorylation and regulation. However, the phosphorylation state of DAT and the other transporters was not directly examined in this study, leaving open the issue of the activity of the phosphatase against the proteins. We have further investigated this issue using a variety of approaches to directly examine the DAT phosphorylation state and have obtained strong evidence that DAT is a substrate for endogenous and in vitro dephosphorylation by PP1.
Section snippets
Phosphorylation of DAT in striatal slices
DAT was metabolically labeled with 32PO4 in rat striatal slices using procedures adapted from Halpain et al. [15]. Male Sprague–Dawley rats (175–300 g) were decapitated and the striata were rapidly removed and weighed. The tissue was sliced into 350-μm slices using a McElvain Tissue Chopper, and equivalent amounts of tissue (four to eight slices) were placed into wells of a 12-well culture plate containing oxygenated Krebs-bicarbonate buffer (KBB) consisting of 25 mM NaHCO3, 125 mM NaCl, 5 mM
DAT phosphorylation in striatal homogenates
In previous studies we found that DATs undergo metabolic phosphorylation in rat striatal slices that is rapidly stimulated by PKC activators and protein phosphatase inhibitors [13]. This pattern is similar to that demonstrated previously using striatal synaptosomes and correlates with the regulation of DAT activity induced by these treatments [7], [36]. We have further adapted the striatal slice system to that of a homogenate for the purpose of examining DAT phosphorylation in response to
Discussion
Our results provide strong evidence that DAT is dephosphorylated in vitro and in rat striatal tissue by the serine/threonine phosphatase PP1. Dephosphorylation of DAT in striatal homogenates was strongly inhibited by OA with a dose response compatible with that of in vitro inhibition of purified PP1, while little to no inhibition of dephosphorylation was found at lower doses that inhibit purified PP2A. Similarly, we found significant inhibition of DAT dephosphorylation in striatal homogenates
Acknowledgements
This work was supported by National Institute on Drug Abuse grant DA13147 to R.A.V.
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2024, Advances in PharmacologyDephosphorylation of human dopamine transporter at threonine 48 by protein phosphatase PP1/2A up-regulates transport velocity
2019, Journal of Biological ChemistryCitation Excerpt :This raises the question of the identities of these additional sites. In our previous studies, the OA treatment of rat striatal slices caused increased phosphorylation primarily on distal N-terminal serines (21, 32) and Thr-53 (equivalent to hDAT Ser-53) (16). In addition, our previous MS study pinpointed phosphorylation at hDAT Ser-7 in HEK293 cells upon OA treatment (17).
Model systems for analysis of dopamine transporter function and regulation
2019, Neurochemistry InternationalCitation Excerpt :Signaling pathways are known to be altered in immortalized and cancer cell lines (Hanahan and Weinberg, 2011), and model systems may lack the repertoire of enzymes and signals that perform these functions in neurons. We have previously noted differences in DAT glycosylation, phosphorylation, and palmitoylation characteristics between different brain regions and between brain and cell systems (Foster et al., 2003; Foster and Vaughan, 2011; Lew et al., 1992), supporting this idea. Membrane cholesterol is also key to DAT function, with transport activity being reduced or enhanced, respectively, by depletion or supplementation of cholesterol (Adkins et al., 2007; Foster et al., 2008; Hong and Amara, 2010; Jones et al., 2012).
Phosphorylation of the Amino Terminus of the Dopamine Transporter: Regulatory Mechanisms and Implications for Amphetamine Action
2018, Advances in PharmacologyCitation Excerpt :These studies showed that DAT phosphorylation is rapidly elevated by activation of protein kinase C (PKC) with phorbol 12-myristate, 13-acetate (PMA) (Huff et al., 1997; Karam et al., 2017; Vaughan et al., 1997), diacylglycerol analogs (Vaughan et al., 1997), or Gq activation by G protein-coupled receptor agonists (Granas et al., 2003). DAT phosphorylation was also shown to be strongly enhanced by treating cells with protein phosphatase inhibitors such as okadaic acid (OA), indicating that the transporter is subject to ongoing kinase activity even in the absence of exogenous kinase activators (Foster, Pananusorn, Cervinski, Holden, & Vaughan, 2003; Foster et al., 2002; Huff et al., 1997; Karam et al., 2017; Vaughan et al., 1997). Critically, treatment with psychostimulants such as AMPH and methamphetamine (METH) stimulates an increase in DAT phosphorylation in both heterologously expressing cells and in rat brain tissue (Cervinski, Foster, & Vaughan, 2005; Karam et al., 2017).
Phosphorylation mechanisms in dopamine transporter regulation
2017, Journal of Chemical NeuroanatomyDopamine transporter phosphorylation site threonine 53 regulates substrate reuptake and amphetamine-stimulated efflux
2012, Journal of Biological ChemistryCitation Excerpt :In both cases, Thr(P)53 Ab-mediated precipitation was blocked with Thr(P)53 peptide but not with the dephosphopeptide (Fig. 3C, top), demonstrating specificity for Thr53 phosphorylation. DAT phosphorylation has been studied primarily by metabolic labeling with 32PO4 and has been demonstrated to be modulated by PKC, AMPH, and protein phosphatases (17, 21, 22, 45–47). However, the vast majority of basal and stimulated 32PO4 labeling occurs on distal N-terminal serines (21), making this method unfeasible for characterization of Thr53 phosphorylation responses.
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Present address: Department of Molecular Pharmacology and Toxicology, School of Pharmacy, University of Southern California, Los Angeles, CA 90089, USA.