A Role for p38 Mitogen-activated Protein Kinase-mediated
Threonine 30-dependent Norepinephrine Transporter
Regulation in Cocaine Sensitization and Conditioned Place
Received for publication,October 1, 2014, and in revised form, February 12, 2015 Published, JBC Papers in Press, February 27, 2015, DOI 10.1074/jbc.M114.612192
PadmanabhanMannangatti1, Kamalakkannan NarasimhaNaidu, Mohamad Imad Damaj,
Sammanda Ramamoorthy, and Lankupalle Damodara Jayanthi2
From the Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia 23298
Background: Cocaine-activated p38 MAPK-mediated norepinephrine transporter (NET) threonine 30 phosphorylation up-regulates NET.
Results: p38MAPK inhibition andTAT-NET-Thr30 peptide blocks cocaine-mediatedNETup-regulation and cocaine-induced locomotor sensitization and conditioned place preference (CPP).
Conclusion: Blockade of p38 MAPK-mediated Thr30-dependent NET regulation attenuates cocaine-induced locomotor sensitization, CPP, and CPP reinstatement.
Significance: Regulation of NET plays a mechanistic role in cocaine-mediated behaviors.
The noradrenergic and p38mitogen-activated protein kinase (p38 MAPK) systems are implicated in cocaine-elicited behaviors. Previously, we demonstrated a role for p38 MAPK-mediated norepinephrine transporter (NET) Thr30 phosphorylation in cocaine-induced NET up-regulation (Mannangatti, P.,
Arapulisamy, O., Shippenberg, T. S., Ramamoorthy, S., and Jayanthi, L. D. (2011) J. Biol. Chem. 286, 20239–20250). The present study explored the functional interaction between p38
MAPK-mediated NET regulation and cocaine-induced behaviors. In vitro cocaine treatment of mouse prefrontal cortex synaptosomes resulted in enhanced NET function, surface expression, and phosphorylation. Pretreatment with PD169316, a p38
MAPK inhibitor, completely blocked cocaine-mediated NET up-regulation and phosphorylation. Inmice, in vivo administration of p38 MAPK inhibitor SB203580 completely blocked cocaine-induced NET up-regulation and p38 MAPK activation in the prefrontal cortex and nucleus accumbens. When tested for cocaine-induced locomotor sensitization and conditioned place preference (CPP), mice receiving SB203580 on cocaine challenge day or on postconditioning test day exhibited significantly reduced cocaine sensitization and CPP. A transactivator of transcription (TAT) peptide strategy was utilized to test the involvement of the NET-Thr30 motif. In vitro treatment of synaptosomes with TAT-NET-Thr30 (wild-type peptide) completely blocked cocaine-mediatedNETup-regulation and phosphorylation. In vivo administration of TAT-NET-Thr30 peptide but not TAT-NET-T30A (mutant peptide) completely blocked cocaine-mediated NET up-regulation and phosphorylation. In the cocaine CPP paradigm,mice receiving TAT-NET-Thr30 but notTAT-NET-T30Aonpostconditioning test day exhibited significantly reduced cocaine CPP. Following extinction, TATNET-Thr30 when given prior to cocaine challenge significantly reduced reinstatement of cocaine CPP. These results demonstrate that the direct inhibition of p38 MAPK or the manipulation of NET-Thr30 motif/phosphorylation via a TAT peptide strategy prevents cocaine-induced NET up-regulation, locomotor sensitization, and CPP, suggesting a role for Thr30-linked
NET regulation in cocaine-elicited behaviors.
Efforts with NET3 knock-out (KO) mice provided unequivocal evidence that NET is important for synaptic NE clearance.
More importantly, elevated locomotor activity after psychostimulants and enhanced cocaine reward phenotypes in NET
KOmice underscores the importance of NET in drug addiction (2). The rewarding and powerfully addictive effects of psychostimulants are attributed to dopamine (DA) signaling in the mesolimbic system. However, DA clearance in the prefrontal cortex (PFC) is largely controlled by the NET (3–7), and DArelated behavior is linked toNET function (8). It is possible that cocaine effects on NET and hence on NE/DA signaling may represent one of many neuroadaptations that occur in the development of drug addiction.
NE clearance is a highly orchestrated process involving regulation of NET function via phosphorylation by second messenger-linked signaling pathways downstream of receptor activation (9–11). NET contains multiple consensus sites for several kinases including PKCs andMAPKs. Although PKC is * Thisworkwas supported, inwholeor inpart, byNational Institutes ofHealth
Grants R01-GM081054 (to L. D. J.) and R01-MH083928 (to S. R.). This work was also supported by start-up funds from Virginia Commonwealth University (to L. D. J. and S. R.). 1 Recipient of postdoctoral support from the department of Pharmacology and Toxicology, Virginia Commonwealth University. 2 To whom correspondence should be addressed. Tel.: 804-828-2067; Fax: 804-828-2117; E-mail: firstname.lastname@example.org. 3 The abbreviations used are: NET, norepinephrine transporter; CPP, conditioned place preference; PFC, prefrontal cortex; NAc, nucleus accumbens;
NE, norepinephrine; DA, dopamine; DAT, dopamine transporter; SERT, serotonin transporter; TH, tyrosine hydroxylase; TAT, transactivator of transcription.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 17, pp. 10814–10827, April 24, 2015 © 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. 10814 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290•NUMBER 17•APRIL 24, 2015 implicated in cardiovascular diseases and alcohol addiction (12–14), other PKC isoforms and p38MAPKs are implicated in several brain disorders such as psychostimulant addiction and depression (15, 16). We have documented that phosphorylation of NET may be a common mechanism dictating NET expression and thus NE transport function in maintaining NE homeostasis (1, 17, 18). In particular, our recent in vitro and in vivo studies documented that cocaine increases NET function and surface expression in rodent PFC and NAc via p38MAPKmediated NET phosphorylation (1). Thus, there exists a significant association between cocaine up-regulation of NET and p38 MAPK signaling. This raises the possibility that manipulation of p38MAPK-dependent NET regulation in animals could alter the behavioral response to cocaine.