Phosphatidylinositol 3-Kinase, Protein Kinase C, and MEK1/2 Kinase Regulation of Dopamine Transporters (DAT) Require N-terminal DAT Phosphoacceptor Sites

Abstract

The dopamine transporter (DAT) modulates dopamine neurotransmission and is a primary target for psychostimulant influences on locomotion and reward. Selective DAT expression by dopaminergic neurons has led to use of cocaine analog DAT radioligands to assess rates of progression of dopamine neuronal degeneration in Parkinson's disease. We have documented that DAT is a phosphoprotein that is regulated by phosphorylation through pathways that include protein kinase C cascades. We now extend this work using drugs selective for phosphatidylinositol 3-kinase (PI3K), protein kinase C, MEK1/2, p38 kinase, and Ca 2؉ /calmodulin kinase II. We compare the drug effects on wild type DAT to the effects on 20 DAT mutants and a DAT deletion. PI3K and MEK1/2 modulators exert strong effects on DAT expression patterns and dopamine uptake V max . PKC principally modulates V max . Neither p38 nor Ca 2؉ /calmodulin kinase II agents exert significant influences on wild type DAT. Several mutants and a DAT with an N-terminal deletion display alterations that interact with the effects of kinase modulators, especially S7A for PKC effects; T62A, S581A, and T612A for PI3K effects; and S12A and T595A mutants for MEK1/2 effects. 32 P-Labeling studies confirm several of these effects of kinase pathway modulators on DAT phosphorylation. DAT expression and activities can be regulated by kinase cascades that require phosphoacceptor sites most concentrated in its N terminus. These results have a number of implications for DAT regulation and mandate caution in using DAT radioligand binding to infer changes in dopaminergic neuronal integrity after treatments that alter activities of these kinase pathways.The plasma membrane dopamine transporter (DAT) 1 functions to terminate dopaminergic neurotransmission by re-uptake of synaptic dopamine molecules into presynaptic neurons. Modulators of DAT function thus regulate the intensity and duration of dopaminergic neurotransmission (1, 2). DAT blockade by psychostimulants plays a key role in their rewarding and locomotor-stimulating properties (1, 3, 4). DAT uptake of dopamine-selective neurotoxins such as 6-hydroxydopamine and 1-methyl-4-phenylpyridinium is central to the cell type specificity of these model toxins for parkinsonian neurodegeneration.DAT is the most selective current marker for selected populations of dopaminergic neurons. It is expressed only in dopaminergic cells, with relatively high levels of expression in substantia nigra pars compacta neurons, intermediate levels in ventral tegmental area dopaminergic neurons, and low levels in arcuate nucleus neurons. These features, and the high affinity with which DAT can recognize cocaine analogs, have led to use of in vitro and in vivo assessments of DAT binding to seek evidence for degeneration of dopaminergic systems (5). DAT functions including its velocity of dopamine transport and its expression on plasma membranes can be regulated by several kinase or phosphatase pathways. DAT activity can be altered by activation of protein kinase C (PKC),...

n-3 Polyunsaturated fatty acid deprivation in rats decreases frontal cortex BDNF via a p38 MAPK-dependent mechanism

“…15 Fluoxetine also activates the p38 MAPK cascade, 80 suggesting that the depressive symptoms observed in n-3 PUFA deprived rats are related to decreased p38 MAPK activity. Constitutive p38 MAPK activity maintains the expression of rat brain dopamine 81,82 and serotonin transporters 83 and n-3 PUFA deprivation upregulates D1 and D2 receptors 84 and decreases stimulated serotonin release, 85 all of which are believed to be involved in depression.…”

Kappa Opioid Receptor-Induced Aversion Requires p38 MAPK Activation in VTA Dopamine Neurons

“…In serotonergic neurons, KOR activation of p38␣ MAPK caused translocation of the serotonin transporter (SERT, SLC6A4 ) from an intracellular, endosomal compartment to the cell surface to increase the rate of serotonin reuptake (Bruchas et al, 2011;Schindler et al, 2012). KOR activation of p38␣ MAPK could potentially have affected the DAT (SLC6A3) because in vivo administration of the agonist U69,593 increased dopamine uptake (Thompson et al, 2000); however, p38 MAPK did not affect dopamine uptake in DATexpressing COS cells (Lin et al, 2003), and we did not observe an effect of p38 activation on dopamine uptake in the present study.…”

“…Uncontrolled, sustained stressful experiences increase depressive and anxiety disorders in humans and in animal models of these behaviors (Neumann et al, 2011;Kormos and Gaszner, 2013). Stress expo-sure also increases the rewarding valence of drugs of abuse, promotes escalation of addictive drug consumption, and stimulates relapse in abstinent individuals (Brady and Sinha, 2005;Mantsch et al, 2015). Developing proresilience medications based on an understanding of how stress controls these behaviors has the potential for a novel therapeutic advance.…”

“…Key to this understanding is the analysis of how stress-evoked release of neurotransmitters affects neural circuit physiology and decision-making processes. A series of recent studies indicate that stress exposure produces its dysphoric, anxiogenic, and proaddictive effects through the activation of extrahypothalamic cortocotrophin releasing factor, which releases the endogenous dynorphin opioid neuropeptides in brain (Land et al, 2008;Mantsch et al, 2015). Dynorphin selectively activates opioid receptors (KOR), and rodents treated with receptor antagonists or genetically lacking either prodynorphin or KOR have reduced immobility in forced swim assays (typical of antidepressant drug actions), reduced social avoidance behaviors following repeated social defeat, reduced escalation of psychostimulant self-administration, and reduced reinstatement of extinguished ethanol or cocaine selfadministration (Bruchas et al, 2010;Van't Veer and Carlezon, 2013;Lalanne et al, 2014).…”

Enhanced Ubiquitylation and Accelerated Degradation of the Dopamine Transporter Mediated by Protein Kinase C

“…Phosphorylation of the amino-terminal tail of DAT by PKC has been observed in rat striatal synaptosomal preparations and transfected mammalian cell lines (10,19,20). However, truncations or point mutations of the phosphorylation sites in DAT did not inhibit PKC-induced down-regulation of DAT, suggesting that this PKC effect is not mediated by DAT phosphorylation (17,21,22). On the other hand, amino-terminal phosphorylation has been shown to be important for reversed dopamine transport by DAT (23).…”