Proline-Directed Phosphorylation of the Dopamine Transporter N-Terminal Domain

Abstract

Phosphorylation of 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, but the residues phosphorylated and identities of protein kinases and phosphatases involved are not known. As one approach to investigating these issues we recombinantly expressed the N-terminal tail of rat DAT (NDAT) and examined its phosphorylation and dephosphorylation properties in vitro. We found that NDAT could be phosphorylated to significant levels by PKCα, PKA, PKG, and CaMKII, which catalyzed serine phosphorylation, and ERK1, JNK, and p38, which catalyzed threonine phosphorylation. We identified Thr53, present in a membrane proximal proline-directed kinase motif as the NDAT site phosphorylated in vitro by ERK1, JNK and p38, and confirmed by peptide mapping and mutagenesis that Thr53 is phosphorylated in vivo. Dephosphorylation studies showed that protein phosphatase 1 catalyzed near-complete in vitro dephosphorylation of PKCα-phosphorylated NDAT, similar to its in vivo and in vitro effects on native DAT. These findings demonstrate the ability of multiple enzymes to directly recognize the DAT N-terminal domain and for kinases to act at multiple distinct sites. The strong correspondence between NDAT and rDAT phosphorylation characteristics suggests the potential for the enzymes that are active on NDAT in vitro to act on DAT in vivo 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 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 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)(14)(15)(16), and PKC activity is required for substrate induced transport down-regulation (17)(18)(19). Kinase-and substrate-induced DA transport down- NIH Public Access Aut...

SLC6 Neurotransmitter Transporters: Structure, Function, and Regulation

“…6 (Granas et al, 2003;Cervinski et al, 2005). Furthermore, in vitro phosphorylation experiments using purified PKC␣ to phosphorylate purified glutathione transferase (GST) fusion proteins containing the DAT N terminus have shown that this domain is a PKC substrate (Fog et al, 2006;Gorentla et al, 2009). However, additional PKC phosphorylation sites outside the N-terminal domain might exist in DAT.…”

Regulation of the Dopamine and Vesicular Monoamine Transporters: Pharmacological Targets and Implications for Disease

“…The DAT N terminus contains several threonine and serine residues that may be phosphorylated by protein kinase C (PKC), protein kinase A (PKA), protein kinase G, and Ca 2+ /calmodulin protein kinase II (Gorentla et al, 2009). Proline-directed phosphorylation of DAT threonine 53 may be involved in maintaining DAT transport (point mutations decrease DAT V max ) and may also be involved in amphetamine (AMPH)-induced reverse transport (Gorentla et al, 2009;Foster et al, 2012).…”

“…The DAT N terminus contains several threonine and serine residues that may be phosphorylated by protein kinase C (PKC), protein kinase A (PKA), protein kinase G, and Ca 2+ /calmodulin protein kinase II (Gorentla et al, 2009). Proline-directed phosphorylation of DAT threonine 53 may be involved in maintaining DAT transport (point mutations decrease DAT V max ) and may also be involved in amphetamine (AMPH)-induced reverse transport (Gorentla et al, 2009;Foster et al, 2012). Acute, in vitro activation of PKC by phorbol esters has been associated with a downregulation of DAT activity, which coincides with DAT phosphorylation and reduced plasma membrane expression (Copeland et al, 1996;Vaughan et al, 1997;Zhu et al, 1997;Daniels and Amara, 1999;Melikian and Buckley, 1999).…”

Cyclin-Dependent Kinase 5 Inhibitors: Inhibition of Dopamine Transporter Activity

“…Cdk5 has been shown to increase phosphorylation of the recombinantly expressed DAT N-terminal tail (Gorentla et al, 2009). To determine whether the pharmacological actions of roscovitine on DAT activity we observed were Cdk5-mediated, we transfected YFP-HA-hDAT-PAE cells with Cdk5-siRNA.…”