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J. Biol. Chem., Vol. 282, Issue 16, 11639-11647, April 20, 2007
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1
From the
Department of Neurosciences, Division of Neuroscience Research, Medical University of South Carolina, Charleston, South Carolina 29425 and the Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06519
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.
Received for publication, December 11, 2006 , and in revised form, February 2, 2007.
* This work was supported by National Institutes of Health Grants MH62612 (to S. R.), GM081054 (to L. D. J.) and DA8213 (to G. 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, 173 Ashley Ave., BSB 403, Medical University of South Carolina, Charleston, SC 29425. Tel.: 843-792-3689; Fax: 843-792-4423; E-mail: rama{at}musc.edu.
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