title: Phosphatidylinositol 3-kinase, protein kinase C, and MEK1/2 kinase regulation of dopamine transporters (DAT) require N-terminal DAT phosphoacceptor sites.
abstract: 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 Vmax. PKC principally modulates Vmax. 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.
authors.affiliation: Molecular Neurobiology Branch, NIDA-IRP, National Institutes of Health, Baltimore, Maryland 21224, USA.
authors.forename: Zhicheng
authors.initials: Z
authors.lastname: Lin
authors.forename: Ping-Wu
authors.initials: PW
authors.lastname: Zhang
authors.forename: Xuguang
authors.initials: X
authors.lastname: Zhu
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Melgari
authors.forename: Robin
authors.initials: R
authors.lastname: Huff
authors.forename: Rachel L
authors.initials: RL
authors.lastname: Spieldoch
authors.forename: George R
authors.initials: GR
authors.lastname: Uhl
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D004298:Dopamine / Q000378:metabolism; D050483:Dopamine Plasma Membrane Transport Proteins; D007150:Immunohistochemistry; D018374:LLC-PK1 Cells; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins / Q000378:metabolism*; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats
chemical_list: D017931:DNA Primers; D004298:Dopamine; D050483:Dopamine Plasma Membrane Transport Proteins; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D020929:Mitogen-Activated Protein Kinase Kinases; D009419:Nerve Tissue Proteins; D019869:Phosphatidylinositol 3-Kinases; D011493:Protein Kinase C; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases; C493216:Slc6a3 protein, rat