Casado, M. et al. Phosphorylation and modulation of brain glutamate transporters by protein kinase C. J. Biol. Chem. 268, 27313−27317

Centro de Biología Molecular, Facultad de Ciencias, Universidad Autónoma de Madrid, Spain.
Journal of Biological Chemistry (Impact Factor: 4.57). 01/1994; 268(36):27313-7.
Source: PubMed


High affinity sodium- and potassium-coupled L-glutamate transport into presynaptic nerve terminals and fine glial processes removes the neurotransmitter from the synaptic cleft, thereby terminating glutamergic transmission. This report describes that the purified L-glutamate transporter from pig brain is phosphorylated by protein kinase C, predominantly at serine residues. Upon exposure of C6 cells, a cell line of glial origin, to 12-O-tetradecanoylphorbol-13-acetate, about a 2-fold stimulation of L-glutamate transport is observed within 30 min. Concomitantly, the level of phosphorylation increases with similar kinetics. The phorbol ester also stimulates L-glutamate transport in HeLa cells infected with a recombinant vaccinia virus expressing T7 RNA polymerase and transfected with pT7-GLT-1. The latter is a recently cloned rat brain glutamate transporter of glial origin. Mutation of serine 113 to asparagine does not affect the levels of expressed transport but abolishes its stimulation by the phorbol ester. To our knowledge, this is the first direct demonstration of the regulation of a neurotransmitter transporter by phosphorylation.

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    • "No evident triple colocalization EAAC1–adducin–actin was observed. Fig. 2 shows the EAAC1 distribution in C6 cells treated with Phorbol 12,13-dibutyrate (PDBu) for 15 min, the experimental time associated with the maximal PKC-dependent increase in the transporter activity (Casado et al., 1993). "
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    ABSTRACT: Excitatory amino acid carrier 1 (EAAC1) belongs to the family of the Na(+)-dependent glutamate carriers. Although the association between defective EAAC1 function and neurologic disease has been repeatedly studied, EAAC1 regulation is not yet fully understood. We have reported that in C6 glioma cells both the activity and membrane targeting of EAAC1 require the integrity of actin cytoskeleton. Here we show that, in the same model, EAAC1 partially co-localizes with actin filaments at the level of cell processes. Moreover, perinuclear spots in which EAAC1 co-localizes with the actin binding protein alpha-adducin are observed in some cells and, consistently, faint co-immunoprecipitation bands between EAAC1 and alpha-adducin are detected. Co-localization and partial co-immunoprecipitation of EAAC1 and adducin are still detectable after cell treatment with phorbol esters, a condition that leads to a protein kinase C (PKC)-dependent increase of EAAC1 expression on the membrane and to the phosphorylation of adducin. A co-immunoprecipitation band was also detected in protein extracts of rat hippocampus. The amount of adducin co-immunoprecipitated with EAAC1 increases after the treatment of C6 cells with retinoic acid, a differentiating agent that induces EAAC1 overexpression in this cell model. Moreover, in clones of C6 cells transfected with a hemagglutinin (HA)-tagged adducin, the bands of EAAC1 immunoprecipitated by an anti-HA antiserum were proportional to EAAC1 expression. These results suggest the existence of a pool of EAAC1 transporters associated with the actin binding protein alpha-adducin in a PKC-insensitive manner.
    Full-text · Article · Aug 2010 · Neuroscience
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    • "This is mainly because the effect of a particular PK does not only depend on the type of the transporter but also on the cell system used. Several groups studied the effects of PKC on GLT1 activity and obtained contradictory results showing that activation of PKC increases, decreases or has no effect on GLT1 activity using either HELA-, MDCK-, MCB or L-M(TK-) cells (Casado et al., 1991, 1993; Carrick and Dunlop, 1999; Tan et al., 1999). Similarly, contradictory results were obtained when the effects of PKC activation on GLAST activity were analysed (Casado et al., 1991; Gonzalez and Ortega, 1997; Daniels and Vickroy, 1999). "
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    ABSTRACT: Glutamate is the main excitatory neurotransmitter in the vertebrate CNS. Removal of the transmitter from the synaptic cleft by glial and neuronal glutamate transporters (GLTs) has an important function in terminating glutamatergic neurotransmission and neurological disorders. Five distinct excitatory amino-acid transporters have been characterized, among which the glial transporters excitatory amino-acid transporter 1 (EAAT1) (glutamate aspartate transporter) and EAAT2 (GLT1) are most important for the removal of extracellular glutamate. The purpose of this study was to describe the effect of the commonly used anaesthetic etomidate on glutamate uptake in cultures of glial cells. The activity of the transporters was determined electrophysiologically using the whole cell configuration of the patch-clamp recording technique. Glutamate uptake was suppressed by etomidate (3-100 microM) in a time- and concentration-dependent manner with a half-maximum effect occurring at 2.4+/-0.6 microM. Maximum inhibition was approximately 50% with respect to the control. Etomidate led to a significant decrease of V(max) whereas the K(m) of the transporter was unaffected. In all cases, suppression of glutamate uptake was reversible within a few minutes upon washout. Furthermore, both GF 109203X, a nonselective inhibitor of PKs, and H89, a selective blocker of PKA, completely abolished the inhibitory effect of etomidate. Inhibition of glutamate uptake by etomidate at clinically relevant concentrations may affect glutamatergic neurotransmission by increasing the glutamate concentration in the synaptic cleft and may compromise patients suffering from acute or chronic neurological disorders such as CNS trauma or epilepsy.
    Full-text · Article · Dec 2008 · British Journal of Pharmacology
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    • "The activity and cell surface expression of GLT-1 is regulated by various signaling molecules including PKC and scaffolding proteins (for reviews, see Danbolt, 2001; González and Robinson, 2004; Beart and O'Shea, 2007). Although Casado and colleagues originally suggested that activation of PKC increases activity in GLT-1-transfected HeLA cells (Casado et al., 1993), we were unable to replicate this result (Tan et al., 1999). In primary cultures derived from rat brain and Y-79 human retinoblastoma cells that endogenously express GLT-1, activation of PKC rapidly (within min) decreases GLT-1-mediated transport activity and reduces the amount of GLT-1 that is observed at the plasma membrane (Ganel and Crosson, 1998 Kalandadze et al., 2002; Zhou and Sutherland, 2004; Guillet et al., 2005). "
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    ABSTRACT: Activation of protein kinase C (PKC) decreases the activity and cell surface expression of the predominant forebrain glutamate transporter, GLT-1. In the present study, C6 glioma were used as a model system to define the mechanisms that contribute to this decrease in cell surface expression and to determine the fate of internalized transporter. As was previously observed, phorbol 12-myristate 13-acetate (PMA) caused a decrease in biotinylated GLT-1. This effect was blocked by sucrose or by co-expression with a dominant-negative variant of dynamin 1, and it was attenuated by co-expression with a dominant-negative variant of the clathrin heavy chain. Depletion of cholesterol with methyl-beta-cyclodextrin, co-expression with a dominant-negative caveolin-1 mutant (Cav1/S80E), co-expression with dominant-negative variants of Eps15 (epidermal-growth-factor receptor pathway substrate clone 15), or co-expression with dominant-negative Arf6 (T27N) had no effect on the PMA-induced loss of biotinylated GLT-1. Long-term treatment with PMA caused a time-dependent loss of biotinylated GLT-1 and decreased the levels of GLT-1 protein. Inhibitors of lysosomal degradation (chloroquine or ammonium chloride) or co-expression with a dominant-negative variant of a small GTPase implicated in trafficking to lysosomes (Rab7) prevented the PMA-induced decrease in protein and caused an intracellular accumulation of GLT-1. These results suggest that the PKC-induced redistribution of GLT-1 is dependent upon clathrin-mediated endocytosis. These studies identify a novel mechanism by which the levels of GLT-1 could be rapidly down-regulated via lysosomal degradation. The possibility that this mechanism may contribute to the loss of GLT-1 observed after acute insults to the CNS is discussed.
    Full-text · Article · Mar 2008 · Neurochemistry International
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