Regulation of Amphetamine-stimulated Dopamine Efflux by Protein Kinase C

Department of Pharmacology, University of Michigan, Ann Arbor, Michigan, United States
Journal of Biological Chemistry (Impact Factor: 4.57). 04/2005; 280(12):10914-9. DOI: 10.1074/jbc.M413887200
Source: PubMed


Evidence suggests that protein kinase C (PKC) and intracellular calcium are important for amphetamine-stimulated outward transport of dopamine in rat striatum. In this study, we examined the effect of select PKC isoforms on amphetamine-stimulated dopamine efflux, focusing on Ca(2+)-dependent forms of PKC. Efflux of endogenous dopamine was measured in superfused rat striatal slices; dopamine was measured by high performance liquid chromatography. The non-selective classical PKC inhibitor Gö6976 inhibited amphetamine-stimulated dopamine efflux, whereas rottlerin, a specific inhibitor of PKC delta, had no effect. A highly specific PKC beta inhibitor, LY379196, blocked dopamine efflux that was stimulated by either amphetamine or the PKC activator, 12-O-tetradecanoylphorbol-13-acetate. None of the PKC inhibitors significantly altered [3H]dopamine uptake. PKC beta(I) and PKC beta(II), but not PKC alpha or PKC gamma, were co-immunoprecipitated from rat striatal membranes with the dopamine transporter (DAT). Conversely, antisera to PKC beta(I) and PKC beta(II) but not PKC alpha or PKCg amma were able to co-immunoprecipitate DAT. Amphetamine-stimulated dopamine efflux was significantly enhanced in hDAT-HEK 293 cells transfected with PKC beta(II) as compared with hDAT-HEK 293 cells alone, or hDAT-HEK 293 cells transfected with PKCa lpha or PKC beta(I). These results suggest that classical PKC beta(II) is physically associated with DAT and is important in maintaining the amphetamine-stimulated outward transport of dopamine in rat striatum.

Download full-text


Available from: Margaret E Gnegy, Jan 20, 2015
  • Source
    • "In vivo, they can behave as unique isoenzymes with distinct properties. Although both PKCβI and PKCβII couple to DAT (Johnson et al. 2005b, Hadlock et al. 2011), the PKCβII isozyme enhanced AMPHstimulated reverse transport of DAT (Johnson et al. 2005b). In purified striatal synaptosomes from PKCβ +/+ mice, the selective activation of PKCβII but not PKCβI by quinpirole may be due to differing cellular localization of the proteins. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The strength and duration of extracellular dopamine concentrations are regulated by the presynaptic dopamine transporter (DAT) and dopamine D2 autoreceptors (D2autoRs). There is a functional interaction between these two proteins. Activation of D2autoRs increases DAT trafficking to the surface whereas disruption of this interaction compromises activities of both proteins and alters dopaminergic transmission. Previously we reported that DAT expression and activity are subject to modulation by protein kinase Cβ (PKCβ). Here, we further demonstrate that PKCβ is integral for the interaction between DAT and D2autoR. Inhibition or absence of PKCβ abolished the communication between DAT and D2autoR. In mouse striatal synaptosomes and transfected N2A cells, the D2autoR-stimulated membrane insertion of DAT was abolished by PKCβ inhibition. Moreover, D2autoR-stimulated DAT trafficking is mediated by a PKCβ-ERK (extracellular signal-regulated kinase) signaling cascade where PKCβ is upstream of ERK. The increased surface DAT expression upon D2autoR activation resulted from enhanced DAT recycling as opposed to reduced internalization. Further, PKCβ promoted accelerated DAT recycling. Our study demonstrates that PKCβ critically regulates D2autoR-activated DAT trafficking and dopaminergic signaling. PKCβ is a potential drug target for correcting abnormal extracellular dopamine levels in diseases such as drug addiction and schizophrenia. © 2013 The Authors Journal of Neurochemistry © 2013 International Society for Neurochemistry.
    Full-text · Article · Mar 2013 · Journal of Neurochemistry
  • Source
    • "Ser and Thr that are phosphorylation targets for multiple kinases such as PKC [70] [71] [72] [73] [74] [75] [76] [77] and CaMKIIα [79]. N-terminal and/or the C-terminal phosphorylation on DAT structure have been shown to regulate DAT biology [100] [101] [102]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The development of effective therapeutic interventions for neurodegeneration requires a better understanding of the early events that precede neuronal loss. Recent work in various disease models has begun to emphasize the significance of presynaptic dysfunction as an early event that occurs before manifestation of neurological disorders. Dysregulation of dopamine (DA) homeostasis is implicated in neurodegenerative diseases, drug addiction, and neuropsychiatric disorders. The neuronal plasma membrane dopamine transporter (DAT) is essential for the maintenance of DA homeostasis in the brain. α-synuclein is a 140-amino acid protein that forms a stable complex with DAT and is linked to the pathogenesis of neurodegenerative disease. In this review we will examine the prevailing hypotheses for α-synuclein-regulation of DAT biology.
    Full-text · Article · Sep 2012
  • Source
    • "Therefore, it is possible that enhanced ubiquitination and endocytosis are dependent on activation of PKCα and PKCβ whereas transporter phosphorylation is an independent process that is triggered by PKCβ activation. Several published data support this hypothesis (Johnson et al., 2005; Chen et al., 2009; Robertson et al., 2009). In recent years, many membrane proteins such as receptors, channels and transporters are described to be phosphorylated and ubiquitinated, these modifications may represent regulatory mechanisms for controlling one or multiple properties of the protein. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The extracellular levels of the neurotransmitter glycine in the brain are tightly regulated by the glycine transporter 1 (GlyT1) and the clearance rate for glycine depends on its rate of transport and the levels of cell surface GlyT1. Over the years, it has been shown that PKC tightly regulates the activity of several neurotransmitter transporters. In the present work, by stably expressing three N-terminus GlyT1 isoforms in porcine aortic endothelial cells and assaying for [(32)P]-orthophosphate metabolic labeling, we demonstrated that the isoforms GlyT1a, GlyT1b, and GlyT1c were constitutively phosphorylated, and that phosphorylation was dramatically enhanced, in a time dependent fashion, after PKC activation by phorbol ester. The phosphorylation was PKC-dependent, since pre-incubation of the cells with bisindolylmaleimide I, a selective PKC inhibitor, abolished the phorbol ester-induced phosphorylation. Blotting with specific anti-phospho-tyrosine antibodies did not yield any signal that could correspond to GlyT1 tyrosine phosphorylation, suggesting that the phosphorylation occurs at serine and/or threonine residues. In addition, a 23-40%-inhibition on V(max) was obtained by incubation with phorbol ester without a significant change on the apparent Km value. Furthermore, pre-incubation of the cells with the selective PKCα/β inhibitor Gö6976 abolished the downregulation effect of phorbol ester on uptake and phosphorylation, whereas the selective PKCβ inhibitors (PKCβ inhibitor or LY333531) prevented the phosphorylation without affecting glycine uptake, defining a specific role of classical PKC on GlyT1 uptake and phosphorylation. Taken together, these data suggest that conventional PKCα/β regulates the uptake of glycine, whereas PKCβ is responsible for GlyT1 phosphorylation.
    Full-text · Article · Aug 2011 · Neurochemistry International
Show more