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J. Biol. Chem., Vol. 281, Issue 33, 23326-23340, August 18, 2006

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Phosphorylation of the Norepinephrine Transporter at Threonine 258 and Serine 259 Is Linked to Protein Kinase C-mediated Transporter Internalization^*

Lankupalle D. Jayanthi¹, Balasubramaniam Annamalai, Devadoss J. Samuvel, Ulrik Gether, and Sammanda Ramamoorthy

From the Department of Neurosciences, Division of Neuroscience Research, Medical University of South Carolina, Charleston, South Carolina 29425 and Molecular Neuropharmacology Group, Department of Pharmacology, Panum Institute, University of Copenhagen, DK-2200 Copenhagen, Denmark

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 (-PMA)-induced phosphorylation of NET occurs on serine and threonine residues. -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 G_q-coupled human neurokinin 1 (hNK-1) receptor coexpressed with the WT-hNET produced effects similar to -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 -PMA or SP. Most interestingly, the plasma membrane insertion of the WT-hNET and hNET double mutant were not affected by -PMA. Although the WT-hNET showed increased endocytosis and redistribution from caveolin-rich plasma membrane domains following -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.

Received for publication, February 7, 2006 , and in revised form, May 15, 2006.

^* This work was supported by National Institutes of Health Grants DA016753 (to L. D. J.) and MH62612 (to S. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Neurosciences, Division of Neuroscience Research, Medical University of South Carolina, Charleston, SC 29425. Tel.: 843-792-8542; Fax: 843-792-4423; E-mail: jayanthi{at}musc.edu.

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