Phosphorylation from the dopamine transporter (DAT) on N-terminal serines and unidentified
Phosphorylation from the dopamine transporter (DAT) on N-terminal serines and unidentified threonines occurs concomitantly with PKC- and substrate-induced alterations in transporter activity subcellular distribution and dopamine efflux Olaparib but the residues phosphorylated and identities of protein kinases and phosphatases involved are not known. suggests the potential for the enzymes that are active on NDAT to act on DAT and indicates the usefulness of NDAT for guiding future DAT phosphorylation analyses. The dopamine transporter (DAT) is a plasma membrane phosphoprotein expressed in dopaminergic neurons that clears synaptic dopamine (DA) by Na+-Cl? dependent reuptake. This activity controls the availability of extracellular DA for binding to receptors and thus regulates the dynamics of dopaminergic neurotransmission (1). Processes controlled by DA include motor activity emotion and reward and agents Olaparib such as cocaine that inhibit DAT cause elevations in DA levels that lead to motor stimulation and addiction (2). DA levels are also increased by amphetamine (AMPH) and methamphetamine (METH) which are carried by DAT and induce DA efflux by the Olaparib process of reverse transport (3 4 It is thought that dopaminergic disorders such as depression schizophrenia ADHD and Parkinson’s disease may be linked to dysregulation of DAT activity and resulting imbalances in DA clearance (5-9). Various properties of DAT are acutely regulated by protein kinases protein phosphatases and substrate pretreatments (10-12) indicating the ability of DAT to rapidly respond to physiological demands. Regulation of DA transport occurs in response to modulation of protein kinase C (PKC) extracellular-signal regulated protein kinase (ERK) protein kinase B (Akt) and protein phosphatases 1 and 2A (PP1/2A) (13-16) and PKC activity is required for substrate induced transport down-regulation (17-19). Kinase- and substrate-induced DA transport down-regulation are associated with alterations in DAT trafficking and surface levels (16 20 while PKC-induced transport regulation also occurs in part by a trafficking-independent process that is sensitive to cholesterol depletion (25). Basal and AMPH-stimulated DA efflux involve actions of PKC and CaMKII (4 15 26 and activation of PKC stimulates DAT phosphorylation ubiquitylation and degradation (13 27 28 Thus it is clear that several functions of DAT are regulated by multiple phosphorylation pathways. The precise mechanisms by which most of these effects occur remain unknown although DAT N-terminal phosphorylation has been reported to be required for AMPH-induced efflux and AMPH/METH-induced increases in intracellular Ca++ (29) but not for PKC or AMPH-induced DAT endocytosis and down-regulation (18 30 In both rat striatal tissue and heterologous expression systems DAT displays a tonic level of phosphorylation that is increased by PKC activators such as phorbol 12-myristate 13-acetate (PMA) and phosphatase inhibitors such as okadaic acid (OA) (13 18 30 31 implicating the activities of PKC and PP1/2A in DAT phosphate turnover. Reduced DAT phosphorylation has been found after pharmacological inhibition of ERK (32) suggesting a role for this kinase in maintenance of DAT phosphorylation. DAT phosphorylation is also stimulated by and AMPH and METH treatments via a PKC-dependent process (18) and PMA-induced phosphorylation is usually inhibited by Olaparib application of the DA uptake blocker GBR 12909 (19) and at low PMA concentrations by cocaine (33) indicating a role for psychoactive drugs in regulation of transporter phosphorylation and accompanying processes. In addition serine mutagenesis studies have suggested the phosphorylation of DAT by RAB7A CaMKII as a mechanism underlying AMPH-induced DA efflux (15). PKC- and OA-stimulated metabolic phosphorylation of rat striatal DAT has been mapped to a cluster of six closely spaced serines at the distal end of the N-terminal tail (Fig. 1A) (34) and mutagenesis of heterologously expressed rat (r) and human (h) DAT has confirmed utilization of sites in this domain (18 30 The precise residues within this domain that are phosphorylated remain unknown although mutation and deletion studies indicate the use of multiple sites (32 35 This region contains consensus motifs for PKC cAMP-dependent protein kinase (PKA) and CaMKII while a second cluster of.