The regulation of glycine transporter GLYT1 is mainly mediated by protein kinase Cα in C6 glioma cells
ABSTRACT Glycine has been shown to possess important functions as a bidirectional neurotransmitter. At synaptic clefts, the concentration of glycine is tightly regulated by the uptake of glycine released from nerve terminals into glial cells by the transporter GLYT1. It has been recently demonstrated that protein kinase C (PKC) mediates the downregulation of GLYT1 activity in several cell systems. However, it remains to be elucidated which subtypes of PKC might be important in the regulation of GLYT1 activity. In this study, we attempted to make clear the mechanism of the phorbol 12-myristate 13-acetate (PMA)-suppressed uptake of glycine in C6 glioma cells which have the native expression of GLYT1. In C6 cells, the expression of PKCalpha, PKCdelta, and PKCvarepsilon of the PMA-activated subtypes was detected. The PMA-suppressed action was fully reversed by the removal of both extracellular and intracellular Ca(2+). Furthermore, the inhibitory effects of PMA or thymeleatoxin (THX), which is a selective activator of conventional PKC (cPKC), were blocked by the downregulation of all PKCs expressed in C6 cells by long-term incubation with THX, or pretreatment with GF109203X or Gö6983, which are broad inhibitors of PKC, or Gö6976, a selective inhibitor of cPKC. On the other hand, treatment of C6 cells with ingenol, a selective activator of novel PKCs, especially PKCdelta and PKCvarepsilon, did not affect the transport of glycine. Silencing of PKCdelta expression by using RNA interference or pretreatment with the inhibitor peptide for PKCvarepsilon had no effect on the PMA-suppressed uptake of glycine. Together, these results suggest PKCalpha to be a crucial factor in the regulation of glycine transport in C6 cells.
- SourceAvailable from: Lise Sofie Haug Nissen-Meyer
[Show abstract] [Hide abstract]
- "For EAAC1, PKC seems to increase the surface expression together with PI3-kinase, whereas PP2A dephosphorylation elicits the internalization. Also the glycine transporter is being regulated by PKC (49–51). As PKC is ubiquitously expressed but strictly compartmentalized, its subcellular activation is differentially executed through a myriad of signal pathways, accounting for a large and diverse part of total phosphorylation phenomena in all cell types. "
ABSTRACT: The system N transporter SN1 (also known as SNAT3) is enriched on perisynaptic astroglial cell membranes. SN1 mediates electroneutral and bidirectional glutamine transport, and regulates the intracellular as well as the extracellular concentrations of glutamine. We hypothesize that SN1 participates in the glutamate/γ-aminobutyric acid (GABA)-glutamine cycle and regulates the amount of glutamine supplied to the neurons for replenishment of the neurotransmitter pools of glutamate and GABA. We also hypothesize that its activity on the plasma membrane is regulated by protein kinase C (PKC)-mediated phosphorylation and that SN1 activity has an impact on synaptic plasticity. This review discusses reports on the regulation of SN1 by PKC and presents a consolidated model for regulation and degradation of SN1 and the subsequent functional implications. As SN1 function is likely also regulated by PKC-mediated phosphorylation in peripheral organs, the same mechanisms may, thus, have impact on e.g., pH regulation in the kidney, urea formation in the liver, and insulin secretion in the pancreas.Frontiers in Endocrinology 10/2013; 4:138. DOI:10.3389/fendo.2013.00138
[Show abstract] [Hide abstract]
- "Different quantities of total lysate from PAE and C6 glioma cells were subjected to Western blotting with anti-PKC antibodies from different sources. As mentioned above, PKCα was detected in control and PAE cell lysates with the anti-PKCα antibody, consistent with earlier observations in C6 glioma cells by Morioka et al. (Morioka et al., 2008). By contrast, blotting with monoclonal (E3) or a polyclonal (C-16, not shown) anti PKCβ1 antibodies readily detected a ∼75 kDa band in controls and C6 glioma cells corresponding to PKCβ1, showing the expression of this isozyme and to similar levels in both C6 and PAE cells. "
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.Neurochemistry International 08/2011; 59(8):1123-32. DOI:10.1016/j.neuint.2011.08.006 · 3.09 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: We studied the amino acid and lipid dynamics during embryogenesis of Homarus gammarus. Major essential amino acids (EAA) in the last stage of embryonic development were arginine, lysine and leucine; major nonessential amino acids (NEAA) were glutamic acid, aspartic acid, valine and glycine. The highest percent of utilization occurred in respect to EAA (27.8%), mainly due to a significant decrease (p<0.05) of methionine (38.3%) and threonine (36.0%). NEAA also decreased significantly (p<0.05, 11.4%), namely serine (38.1%), tyrosine (26.4%) and glutamic acid (25.7%). In contrast, the free amino acid content increased significantly (p<0.05) during embryonic development, especially the free nonessential amino acids (FNEAA). In the last stage, the most abundant FNEAA were glycine, proline, alanine and taurine, and the major free essential amino acids (FEAA) were arginine, lysine and leucine. Lipid content decreased significantly (p<0.05) during embryonic development. A substantial decrease in all neutral lipid classes was observed (>80% of utilization). Major fatty acids were 16:0, 18:0, 18:1n-9, 18:2n-6, 18:3n-3, 20:5n-3 and 22:6n-3. Unsaturated (UFA) and saturated fatty acids (SFA) were used up at similar rates (76.5% and 76.3%, respectively). Within UFA, monounsaturates (MUFA) were consumed more than polyunsaturates (PUFA) (82.9% and 67.5%, respectively).Comparative Biochemistry and Physiology Part B Biochemistry and Molecular Biology 02/2005; 140(2):241-9. DOI:10.1016/j.cbpc.2004.10.009 · 1.55 Impact Factor