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Serotonin (5‐HT) transporter (SERT) plays a crucial role in serotonergic transmission in the central nervous system, and any aberration causes serious mental illnesses. Nevertheless, the cellular mechanisms that regulate SERT function and trafficking are not entirely understood. Growing evidence suggests that several protein kinases act as modulators. Here, we delineate the molecular mechanisms by which glycogen synthase kinase‐3ß (GSK3ß) regulates SERT. When mouse striatal synaptosomes were treated with the GSK3α/ß inhibitor CHIR99021, we observed a significant increase in SERT function, Vmax, surface expression with a reduction in 5‐HT Km and SERT phosphorylation. To further study how the SERT molecule is affected by GSK3α/ß, we used HEK‐293 cells as a heterologous expression system. As in striatal synaptosomes, CHIR99021 treatment of cells expressing wild‐type hSERT (hSERT‐WT) resulted in a time and dose‐dependent elevation of hSERT function with a concomitant increase in the Vmax and surface transporters because of reduced internalization and enhanced membrane insertion; silencing GSK3α/ß in these cells with siRNA also similarly affected hSERT. Converting putative GSK3α/ß phosphorylation site serine at position 48 to alanine in hSERT (hSERT‐S48A) completely abrogated the effects of both the inhibitor CHIR99021 and GSK3α/ß siRNA. Substantiating these findings, over‐expression of constitutively active GSK3ß with hSERT‐WT, but not with hSERT‐S48A, reduced SERT function, Vmax, surface density, and enhanced transporter phosphorylation. Both hSERT‐WT and hSERT‐S48A were inhibited similarly by PKC activation or by inhibition of Akt, CaMKII, p38 MAPK, or Src kinase. These findings provide new evidence that GSK3ß supports basal SERT function and trafficking via serine‐48 phosphorylation. [ABSTRACT FROM AUTHOR]

The serotonin [5-HT (5-hydroxytryptamine)] transporter (SERT) controls serotonergic neurotransmission in the brain by rapid clearance of 5-HT from the synaptic cleft into presynaptic neurons. SERTs are primary targets for antidepressants for therapeutic intervention of mood disorders. Our previous studies have identified the involvement of several signalling pathways and protein kinases in regulating SERT function, trafficking and phosphorylation. However, whether Akt/PKB (protein kinase) regulates SERT function is not known. In the present study, we made the novel observation that inhibition of Akt resulted in the down-regulation of SERT function through the regulation of SERT trafficking and phosphorylation. Akt inhibitor Akt X {10-(4′-[N-diethylamino)butyl]-2-chlorophenoxazine} reduced the endogenously phosphorylated Akt and significantly decreased 5-HT uptake and 5-HT-uptake capacity. Furthermore, SERT activity is also reduced by siRNA down-regulation of total and phospho-Akt levels. The reduction in SERT activity is paralleled by lower levels of cell-surface SERT protein, reduced SERT exocytosis with no effect on SERT endocytosis and accumulation of SERT in intracellular endocytic compartments with the most prominent localization to late endosomes and lysosomes. Akt2 inhibitor was more effective than Akt1 inhibitor in inhibiting SERT activity. Inhibition of downstream Akt kinase GSK3α/β (glycogen synthase kinase α/β) stimulates SERT function. Akt inhibition leads to a decrease in SERT basal phosphorylation. Our results provide evidence that Akt regulates SERT function and cell-surface expression by regulating the intracellular SERT distribution and plasma membrane availability, which perhaps may be linked to SERT phosphorylation state. Thus any changes in the activation of Akt and/or GSK3α/β could alter SERT-mediated 5-HT clearance and subsequently serotonergic neurotransmission. [ABSTRACT FROM AUTHOR]

Recently, we have demonstrated the phosphorylation- and lipid raft-mediated internalization of the native norepinephrine transporter (NET) following protein kinase C (PKC) activation (Jayanthi, L. D., Samuvel, D. J., and Ramamoorthy, S. (2004) J. Biol. Chem. 279, 19315-19326). Here we tested an hypothesis that PKC-mediated phosphorylation of NET is required for transporter internalization. Phosphoamino acid analysis of 32P-labeled native NETs from rat placental trophoblasts and heterologously expressed wild type human NET (WT-hNET) from human placental trophoblast cells revealed that the phorbol ester (β-PMA)-induced phosphorylation of NET occurs on ser-me and threonine residues. β-PMA treatment inhibited NE transport, reduced plasma membrane hNET levels, and stimulated hNET phosphorylation in human placental trophoblast cells expressing the WT-hNET. Substance P-mediated activation of the Gαq-coupled human neurokinin 1 (hNK-1) receptor coexpressed with the WT-hNET produced effects similar to β-PMA via PKC stimulation. In striking contrast, an hNET double mutant harboring T258A and S259A failed to show NE uptake inhibition and plasma membrane redistribution by β-PMA or SP. Most interestingly, the plasma membrane insertion of the WT-hNET and hNET double mutant were not affected by β-PMA. Although the WT-hNET showed increased endocytosis and redistribution from caveolin-rich plasma membrane domains following β-PMA treatment, the hNET double mutant was completely resistant to these PKC-mediated effects. In addition, the PKC-induced phosphorylation of hNET double mutant was significantly reduced. In the absence of T258A and S259A mutations, alanine substitution of all other potential phosphosites within the hNET did not block PKC-induced phosphorylation and down-regulation. These results suggest that Thr-258 and Ser-259 serve as a PKC-specific phospho-acceptor site and that phosphorylation of this motif is linked to PKC-induced NET internalization. [ABSTRACT FROM AUTHOR]

The serotonin transporter (SERT) is regulated by various signaling mechanisms that may operate to maintain appropriate levels of synaptic serotonin (5-HT). We demonstrate that one of the mitogen-activated protein kinases (MAPKs), p38 MAPK, regulates SERT. Treatment of rat midbrain synaptosomes with p38 MAPK-specific inhibitors, PD169316 [4-(4-fluorophenyl)-2-(4-nitrophenyl)-5-(4-pyridyl)-1H-imidazole] or SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole], reduced 5-HT uptake. An additive SERT inhibition by PD 169316 and β-phorbol 12-myristate 13-acetate (β-PMA) indicated the involvement of a protein kinase C (PKC)-independent MAPK pathway. Kinetic studies indicated a significant decrease in the transport capacity (Vmax) after PD 169316 treatment of synaptosomes. Biotinylation studies showed reduced SERT proteins in the plasma membrane of synaptosomes after p38 MAPK inhibition and PKC activation. Phosphorylation studies using synaptosomes revealed decreased SERT phosphorylation by PD 169316 but increased phosphorylation by β-PMA. D-Amphetamine enhanced SERT basal phosphorylation and PD 169316 blocked this effect. SERT interaction with protein phosphatase 2A catalytic subunit and syntaxin 1A decreased after PD 169316 or β-PMA treatment of synaptosomes. In synaptosomes, PKC activation but not p38 MAPK inhibition resulted in SERT redistribution from cholesterol-rich lipid raft fractions to nonlipid raft fractions. The presence of phospho-p38 MAPK in synaptosomes and human embryonic kidney 293 (HEK-293) cells suggested the presence of constitutively active p38 MAPK in these preparations. Cotransfection of HEK-293 cells with SERT and a constitutively active form of MAP kinase kinase 3b(E) [MKK3b(E)] increased 5-HT transport, and RNA interference targeted to p38 MAPK inhibited 5-HT uptake, confirming the involvement of active p38 MAPK in SERT expression. Although PD 169316 inhibited SERT insertion to the... [ABSTRACT FROM AUTHOR]

Abstract: Presynaptic biogenic amine transporters mediate reuptake of released amines from the synapse, thus regulating serotonin, dopamine and norepinephrine neurotransmission. Medications utilized in the treatment of depression, attention deficit-hyperactivity disorder and other psychiatric disorders possess high affinity for amine transporters. In addition, amine transporters are targets for psychostimulants. Altered expression of biogenic amine transporters has long been implicated in several psychiatric and degenerative disorders. Therefore, appropriate regulation and maintenance of biogenic amine transporter activity is critical for the maintenance of normal amine homoeostasis. Accumulating evidence suggests that cellular protein kinases and phosphatases regulate amine transporter expression, activity, trafficking and degradation. Amine transporters are phosphoproteins that undergo dynamic control under the influence of various kinase and phosphatase activities. This review presents a brief overview of the role of amine transporter phosphorylation in the regulation of amine transport in the normal and diseased brain. Understanding the molecular mechanisms by which phosphorylation events affect amine transporter activity is essential for understanding the contribution of transporter phosphorylation to the regulation of monoamine neurotransmission and for identifying potential new targets for the treatment of various brain diseases. [ABSTRACT FROM AUTHOR]

Kappa opioid receptor (KOR) agonists produce dysphoria and psychotomimesis. While KOR agonists produce pro-depressant-like effects, KOR antagonists produce anti-depressant-like effects in rodent models. The cellular mechanisms and downstream effector(s) by which KOR ligands produce these effects are not clear. KOR agonists modulate serotonin (5-HT) transmission in the brain regions implicated in mood and motivation regulation. Presynaptic serotonin transporter (SERT) activity is critical in the modulation of synaptic 5-HT and, subsequently, in mood disorders. Detailing the molecular events of KOR-linked SERT regulation is important for examining the postulated role of this protein in mood disorders. In this study, we used heterologous expression systems and native tissue preparations to determine the cellular signaling cascades linked to KOR-mediated SERT regulation. KOR agonists U69,593 and U50,488 produced a time and concentration dependent KOR antagonist-reversible decrease in SERT function. KOR-mediated functional down-regulation of SERT is sensitive to CaMKII and Akt inhibition. The U69,593-evoked decrease in SERT activity is associated with a decreased transport V max , reduced SERT cell surface expression, and increased SERT phosphorylation. Furthermore, KOR activation enhanced SERT internalization and decreased SERT delivery to the membrane. These data demonstrate that KOR activation decreases 5-HT uptake by altering SERT trafficking mechanisms and phosphorylation status to reduce the functional availability of surface SERT. [ABSTRACT FROM AUTHOR]

The norepinephrine (NE) transporter (NET) regulates NE signaling by rapidly clearing synaptic NE. Cocaine binds NET and modulates NE transport. These actions contribute to rewarding effects and abuse liability of cocaine. Activation of mitogen-activated protein kinase (MAPK) cascades is implicated in cocaine-induced neuroadaptations. However, the role of MAPK and the mechanisms involved in cocaine modulation of NET are not clear. Acute intra-peritoneal injections of cocaine (20 mg/kg body weight) to rats resulted in increased NE uptake by prefrontal cortex (PFC) synaptosomes with a parallel increase in the surface expression of endogenous NET. Cocaine also enhanced the immunereactivity of phospho-p38 MAPK in the PFC synaptosomes without affecting the total p38 MAPK. In vitro cocaine (30-50 μM) treatment of rat PFC synaptosomes increased native NET function, surface expression, and phosphorylation in a manner sensitive to p38 MAPK inhibition by PD169316. We next examined cocaine-elicited effects on wild-type human NET (hNET) expressed heterologously in human placental trophoblast cells to gain more insights into the mechanisms involved. Cocaine treatment of hNET expressing human placental trophoblast cells up-regulated the function, surface expression, and phosphorylation of hNET in a PD169316-sensitive manner. In addition, cocaine inhibited constitutive endocytosis of hNET. Mutational analysis of serine and threonine residues revealed that substitution of threonine 30, located at the amino terminus of hNET with alanine (T30A-hNET), abolished cocaine-induced up-regulation of NET function, surface expression, and phosphorylation. Furthermore, cocaine did not alter T30A-hNET endocytosis. These studies identify a novel molecular mechanism that cocaine-activated p38 MAPK-mediated phosphorylation of NET-T30 dictates surface NET availability, and hence, NE transport. [ABSTRACT FROM AUTHOR]

Abstract: Cocaine binds with the dopamine transporter (DAT), an effect that has been extensively implicated in its reinforcing effects. However, persisting adaptations in DAT regulation after cocaine self-administration have not been extensively investigated. Here, we determined the changes in molecular mechanisms of DAT regulation in the caudate-putamen (CPu) and nucleus accumbens (NAcc) of rats with a history of cocaine self-administration, followed by 3weeks of withdrawal under extinction conditions (i.e., no cocaine available). DA uptake was significantly higher in the CPu of cocaine-experienced animals as compared to saline-yoked controls. DAT V max was elevated in the CPu without changes in apparent affinity for DA. In spite of elevated CPu DAT activity, total and surface DAT density and DAT-PP2Ac (protein phosphatase 2A catalytic subunit) interaction remained unaltered, although p-Ser- DAT phosphorylation was elevated. In contrast to the CPu, there were no differences between cocaine and saline rats in the levels of DA uptake, DAT V max and K m values, total and surface DAT, p-Ser-DAT phosphorylation, or DAT-PP2Ac interactions in the NAcc. These results show that chronic cocaine self-administration leads to lasting, regionally specific alterations in striatal DA uptake and DAT-Ser phosphorylation. Such changes may be related to habitual patterns of cocaine-seeking observed during relapse. [Copyright &y& Elsevier]

Cellular protein kinases, phosphatases, and other serotonin transporter (SERT) interacting proteins participate in several signaling mechanisms regulating SERT activity. The molecular mechanisms of protein kinase G (PKG)-mediated SERT regulation and the site of transporter phosphorylation were investigated. Treatment of rat midbrain synaptosomes with 8-bromo-cGMP increased SERT activity, and the increase was selectively blocked by PKG inhibitors. The Vmax value for serotonin (5-HT) transport increased following cGMP treatment. However, surface biotinylation studies showed no change in SERT surface abundance following PKG activation. 32P metabolic labeling experiments showed increased SERT phosphorylation in the presence of cGMP that was abolished by selectively inhibiting PKG. Phosphoamino acid analysis revealed that cGMP-stimulated native SERT phosphorylation occurred only on threonine residues. When added to CHO-1 cells expressing SERT, 8-bromo-cGMP stimulated 5-HT transport and SERT phosphorylation. Mutation of SERT threonine 276 to alanine completely abolished cGMP-mediated stimulation of 5-HT transport and SERT phosphorylation. Although the T276A mutation had no significant effect on 5-HT transport or SERT protein expression, mutation to aspartate (T276D) increased the level of 5-HT uptake to that of cGMP-stimulated 5-HT uptake in wild-type SERT-expressing cells and was no longer sensitive to cGMP. These findings provide the first identification of a phosphorylation site in SERT and demonstrate that phosphorylation of Thr-276 is required for cGMP-mediated SERT regulation. They also constitute the first evidence that in the central nervous system PKG activation stimulates endogenous SERT activity by a trafficking-independent mechanism. [ABSTRACT FROM AUTHOR]

• DAT-Ala53 knock-in mice are viable and display no developmental deficits. • DA-transport and the expression of DAT and TH are unaltered in the DAT-Ala53 mice. • DAT-Ala53 mice exhibit hyperactivity in novel environment. • ERK1/2 mediated DAT regulation is blunted in the DAT-Ala53 knock-in mice. • DAT-Ala53 mice exhibit reduced sensitivity to cocaine inhibition and hyperlocomotion. Dopamine (DA) transporter (DAT) is dynamically regulated by several protein kinases and the Thr53 phosphorylation of DAT (pT53-DAT) is documented in heterologous cell models and in rat brain. However, the role of endogenous pT53-DAT in living animals has never been addressed. Here we generated and studied the pT53-lacking DAT mouse model (DAT-Ala53) by CRISPR/Cas9 technology. DAT-Ala53 mice showed normal growth, body weight, body temperature, grip strength, and sucrose preference while pT53-DAT was completely absent. However, DAT-Ala53 mice showed hyperlocomotion, pronounced vertical exploratory behavior, and stereotypy in a novel environment compared to wild-type littermates (WT). DAT-Ala53 mice displayed unaltered levels of monoamines, glutamate, and GABA in the striatum compared to WT. There were also no significant differences between DAT-Ala53 mice and WT in tyrosine hydroxylase (TH) and phospho-TH levels, or in total and surface DAT levels, or in DA-transport kinetic parameters V max and K m. Immunohistochemical and colocalization analyses of TH and DAT in caudate-putamen and nucleus accumbens revealed no significant differences between DAT-Ala53 and WT mice. Interestingly, cocaine's potency to inhibit striatal DA transport and cocaine-induced locomotor activation were significantly reduced in the DAT-Ala53 mice. Also, ERK1/2 inhibitors completely failed to inhibit striatal DA uptake in DAT-Ala53 mice. Collectively, our findings reveal that the mice lacking pT53-DAT display novelty-induced hyperactive phenotype despite having normal transporter protein expression, DA-transport kinetics and DA-linked markers. The results also reveal that the lack of endogenous pT53-DAT renders DAT resistant to ERK1/2 inhibition and also less susceptible to cocaine inhibition and cocaine-evoked locomotor stimulation. [ABSTRACT FROM AUTHOR]