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J. Biol. Chem., Vol. 278, Issue 22, 20162-20170, May 30, 2003

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

Zhicheng Lin, Ping-Wu Zhang , Xuguang Zhu , Jean-Marc Melgari , Robin Huff, Rachel L. Spieldoch and George R. Uhl 

From the Molecular Neurobiology Branch, NIDA-IRP, National Institutes of Health, Baltimore, Maryland 21224

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 Ca²⁺/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 Ca²⁺/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. ³²P-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.

Received for publication, September 18, 2002 , and in revised form, March 25, 2003.

^* This work was supported by NIDA/IRP of the National Institutes of Health. 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.

These authors contributed equally to this work.

To whom correspondence should be addressed: Molecular Neurobiology Branch, NIDA-IRP, National Institutes of Health, 5500 Nathan Shock Dr., Baltimore, MD 21224. Tel.: 410-550-2843 (ext. 146); Fax: 410-550-2846; E-mail: guhl{at}intra.nida.nih.gov.

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