Okadaic acid-induced, naringin-sensitive phosphorylation of glycine N-methyltransferase in isolated rat hepatocytes

Abstract

Glycine N-methyltransferase (GNMT) is an abundant cytosolic enzyme that catalyses the methylation of glycine into sarcosine, coupled with conversion of the methyl donor, S -adenosylmethionine (AdoMet), into S -adenosylhomocysteine (AdoHcy). GNMT is believed to play a role in monitoring the AdoMet/AdoHcy ratio, and hence the cellular methylation capacity, but regulation of the enzyme itself is not well understood. In the present study, treatment of isolated rat hepatocytes with the protein phosphatase inhibitor okadaic acid, was found to induce an overphosphorylation of GNMT, as shown by proteomic analysis. The analysis comprised two-dimensional gel electrophoretic separation of (32)P-labelled phosphoproteins and identification of individual protein spots by matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry. The identity of GNMT was verified by N-terminal Edman sequencing of tryptic peptides. Chromatographic separation of proteolytic peptides and (32)P-labelled amino acids suggested that GNMT was phosphorylated within a limited region, and only at serine residues. GNMT phosphorylation could be suppressed by naringin, an okadaic acid-antagonistic flavonoid. To assess the possible functional role of GNMT phosphorylation, the effect of okadaic acid on hepatocytic AdoMet and AdoHcy levels was examined, using HPLC separation for metabolite analysis. Surprisingly, okadaic acid was found to have no effect on the basal levels of AdoMet or AdoHcy. An accelerated AdoMet-AdoHcy flux, induced by the addition of methionine (1 mM), was likewise unaffected by okadaic acid. 5-Aminoimidazole-4-carboxamide riboside, an activator of the hepatocytic AMP-activated protein kinase, similarly induced GNMT phosphorylation without affecting AdoMet and AdoHcy levels. Activation of cAMP-dependent protein kinase by dibutyryl-cAMP, reported to cause GNMT phosphorylation under cell-free conditions, also had little effect on hepatocytic AdoMet and AdoHcy levels. Phosphorylation of GNMT would thus seem to play no role in regulation of the intracellular AdoMet/AdoHcy ratio, but could be involved in other GNMT functions, such as the binding of folates or aromatic hydrocarbons.

CaM-kinaseII-dependent commitment to microcystin-induced apoptosis is coupled to cell budding, but not to shrinkage or chromatin hypercondensation

“…In line with this, naringin was found to only marginally affect the protein phosphorylation in okadaic acidstimulated hepatocytes. 30 Furthermore, hepatocytes stimulated by ROS generators like azide fail to reproduce the Figure 4 Determination of the point when CaMKII activity is no more required for the progression of various MC-induced cell death-associated parameters. Hepatocytes in suspension culture were incubated with 0.5 (K) or 2.0 mM (J) of MC.…”

Preclinical Evidence for the Pharmacological Actions of Naringin: A Review

“…Likewise, in another study by Berven and colleagues, naringin (100 µM) has exerted protective effects in isolated rat hepatocytes against okadaic acid-induced apoptotic cell death and disruption of the keratin intermediate filament network and canalicular sheaths, though these effects could not be replicated in vivo [149]. In other studies, it significantly prevented the okadaic acid-induced inhibition of hepatocyte autophagy and endocytosis at a dose range of 5-100 µM [150] and phosphorylation of intracellular proteins in rat hepatocytes such as glycine N-methyltransferase at a dose of 100 µM [151] and plectin at dose of 100 µM [152].…”

Regulation of human methylenetetrahydrofolate reductase by phosphorylation

“…To test our hypothesis, adenosine was added to the medium. Addition of adenosine decreases the intracellular AdoMet͞AdoHcy ratio (18). MTHFR exhibited a mobility characteristic of unmodified enzyme when adenosine was added to the medium, even when methionine was present (Fig.…”