Palmitoylation Controls Dopamine Transporter Kinetics, Degradation, and Protein Kinase C-dependent Regulation

Abstract

Palmitoylation is a lipid modification that confers diverse functions to target proteins and is a contributing factor for many neuronal diseases. In this study, we demonstrate using [ 3 H]palmitic acid labeling and acyl-biotinyl exchange that native and expressed dopamine transporters (DATs) are palmitoylated, and using the palmitoyl acyltransferase inhibitor 2-bromopalmitate (2BP), we identify several associated functions. Treatment of rat striatal synaptosomes with 2BP using lower doses or shorter times caused robust inhibition of transport V max that occurred with no losses of DAT protein or changes in DAT surface levels, indicating that acute loss of palmitoylation leads to reduction of transport kinetics. Treatment of synaptosomes or cells with 2BP using higher doses or longer times resulted in DAT protein losses and production of transporter fragments, implicating palmitoylation in regulation of transporter degradation. Site-directed mutagenesis indicated that palmitoylation of rat DAT occurs at Cys-580 at the intracellular end of transmembrane domain 12 and at one or more additional unidentified site(s). Cys-580 mutation also led to production of transporter degradation fragments and to increased phorbol ester-induced down-regulation, further supporting palmitoylation in opposing DAT turnover and in opposing protein kinase C-mediated regulation. These results identify S-palmitoylation as a major regulator of DAT properties that could significantly impact acute and long term dopamine transport capacity.In the central nervous system, dopamine (DA) 2 controls numerous processes, including motor activity, emotion, and reward; and many diseases, including Parkinson disease, depression, attention deficit hyperactivity disorder, and schizophrenia, are related to abnormalities in dopaminergic function (1). The plasmalemmal dopamine transporter (DAT) actively transports DA from the extracellular space into the presynaptic neuron, and it is the primary mechanism controlling the concentration and duration of DA in the synapse (2). This activity is crucial for proper dopaminergic neurotransmission, and transport dysregulation is hypothesized to contribute to dopaminergic disorders (3). DAT is a major target for many abused and therapeutic drugs that inhibit transport or stimulate DA efflux (4, 5), leading to increased extracellular DA levels that induce psychomotor stimulation and drug addiction, or therapeutically modulate DA levels in mood and psychiatric disorders.DAT is subject to extensive acute and chronic regulatory processes that modulate DA neurotransmission during momentary physiological demands and long term disease and drug addiction states (6, 7), but the mechanisms remain poorly understood. In model cell systems, regulation of transporter surface levels has been established as an element of acute control, as DAT surface levels are modulated by constitutive and regulated DAT endocytosis and membrane recycling (8 -11). Activation of protein kinase C (PKC) stimulates endocytosis of transporters through ear...

Kinase-dependent Regulation of Monoamine Neurotransmitter Transporters

“…The Vaughan laboratory more recently provided evidence that the trafficking-independent effects of PKC may involve regulation of palmitoylation of DAT. Inhibition of DAT palmitoylation by the palmitoyl acyltransferase inhibitor 2-bromopalmitate in rat striatal synaptosomes led to decreases in the V max of DA uptake independent of changes in DAT surface levels, supporting a role for this modification in trafficking-independent regulation of DAT activity (Foster and Vaughan, 2011). Importantly, activation and inhibition of PKC by PMA/BIM-1 decreased and increased palmitoylation of DAT, respectively, and mutation of the PKC target Ser7 to alanine abolished these effects on palmitoylation (Moritz et al, 2015).…”

“…This is a critical issue as these regions support the binding of a growing class of interacting proteins that dictate transporter localization, stability, and activity. Cytoplasmic domains also contain sites of posttranslational modifications, including lipidation (Foster and Vaughan, 2011) and phosphorylation . Together, these modes of posttranslational regulation amply demonstrate that the MA reuptake process itself is under the control of signaling proteins whose actions provide additional points of control for MA signaling.…”

Reciprocal Phosphorylation and Palmitoylation Control Dopamine Transporter Kinetics

“…These resi-dues are not necessary for PKC-stimulated endocytosis of the transporter (21,22), but their role in kinetic regulation of uptake has not yet been investigated. DAT is also palmitoylated on Cys-580 at the membrane-cytoplasm interface of the most C-terminal transmembrane helix (17). Acute inhibition of transporter palmitoylation enhances PKC-stimulated downregulation and induces significant trafficking-independent decreases in transport V max (17), also suggesting a role for this modification in catalytic regulation of transport.…”

“…Most studies to date have focused on membrane trafficking as the mechanism underlying PKC-induced transport reductions (10 -14), but recent findings have shown that significant levels of downregulation are retained when transporter endocytosis is blocked or impaired (15)(16)(17), indicating the presence of a kinetic regulatory mechanism that operates in concert with internalization.…”

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

“…Palmitoylation regulates DAT function and structure by maintaining V max and reducing PKC-mediated plasma membrane turnover (Foster and Vaughan, 2011). Although much of this process requires elucidation, palmitoylation is important in activity-driven changes in synapse morphology and function (Kang et al, 2008) and may serve as a future target for pharmacological manipulation.…”