PKCβ-dependent phosphorylation of the glycine transporter 1

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 [32P]-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 Vmax 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 phosphorylation that conventional PKCα/β regulates the uptake of glycine, whereas PKCβ is responsible for GlyT1 phosphorylation.

PKC-Dependent GlyT1 Ubiquitination Occurs Independent of Phosphorylation: Inespecificity in Lysine Selection for Ubiquitination

“…Several studies have demonstrated the role of PKC on endocytosis and posttranslational modifications of most transporters of the SLC6 family including GlyT1 [ 10 , 24 , 25 ]. For the dopamine transporter, we previously demonstrated that accelerated endocytosis triggered by PKC activation was a result of enhanced DAT ubiquitination [ 12 , 25 ], events further confirmed by other groups [ 26 ].…”

“…The cells were incubated for 2 h with cycloheximide (50 μg/ml) to stop the delivery and synthesis of new proteins followed by treatment with DMSO (6 h) or 1 μM PMA for 6, 4, or 2 h. After incubations, the cells were washed and lysed as previously described. The cleared lysate was quickly mixed with loading dye and the proteins denatured by heating as previously described [ 10 , 11 , 21 ]. It is worth mentioning that glycine transporters expressed in cultured cells is not aggregated by heating, unlike the endogenous from tissue that aggregates.…”

“…For most members of the SLC6 family, both of these properties are tightly-connected to a signaling pathway that involves P rotein K inase C (PKC). For several SLC6 family members, including GlyT1, PKC activation by phorbol ester (PMA) diminishes neurotransmitter maximal transport capacity and increases both transporter phosphorylation and interaction with the SNARE protein syntaxin 1A [ 8 , 9 , 10 ]. These properties, which result in inhibition of transporter activity, have been attributed to increased endocytosis.…”

Kinase-dependent Regulation of Monoamine Neurotransmitter Transporters

“…Studies by Vargas-Medrano et al (2011) showed that inhibition of PKCb abolished b-PMA-induced increases in DAT phosphorylation in PAE cells, suggesting that 904 this isoform is required for DAT phosphorylation upon PKC activation. Considering work by Chen et al (2013) discussed above seems to indicate that PKCb may act to elevate DAT surface levels, it is possible that phosphorylation of DAT by PKC activation actually supports DAT surface expression, despite the net decrease in surface levels upon nonspecific PKC activation by b-PMA.…”

Glycine transporters type 1 inhibitor promotes brain preconditioning against NMDA-induced excitotoxicity

“…GlyT1 protein is associated to glutamatergic synapses where its main function is to control the level of glycine (Zafra et al, 1995a;Zafra et al, 1995b;Cubelos et al, 2005;Jim enez et al, 2011;Vargas-Medrano et al, 2011). The inhibition of GlyT1 promotes an increase on glycine levels in synaptic terminals, which potentiates the NMDAR activation (Bergeron et al, 1998;Chen et al, 2003;Kinney et al, 2003;Lim et al, 2004;Zhang et al, 2009).…”