okadaic-acid and 3-5-dihydroxyphenylglycine

The regulation of protein phosphorylation requires coordinated interaction between protein kinases and protein phosphatases (PPs). Recent evidence has shown that the Galphaq-protein-coupled metabotropic glutamate receptor (mGluR) 5 up-regulates phosphorylation of MAPK/ERK1/2. However, signaling mechanisms linking mGluR5 to ERK are poorly understood. In this study, roles of a major serine/threonine PP, PP2A, in this event were evaluated in cultured neurons. We found that the PP1/2A inhibitors okadaic acid and calyculin A mimicked the effect of the mGluR5 agonists (RS)-3,5-dihydroxyphenylglycine and (RS)-2-chloro-5-hydroxyphenylglycine in facilitating phosphorylation of ERK1/2 and its upstream kinase, MEK1/2, in a PP2A-dependent but not PP1-dependent manner. Co-administration of either inhibitor with an mGluR5 agonist produced additive phosphorylation of ERK1/2. Enzymatic assays showed a basal level of phosphatase activity of PP2A under normal conditions, and activation of mGluR5 selectively inhibited PP2A, but not PP1, activity. In addition, a physical association of the cytoplasmic C terminus of mGluR5 with PP2A was observed, and ligand activation of mGluR5 reduced mGluR5-PP2A binding. Additional mechanistic studies revealed that mGluR5 activation increased tyrosine (Tyr307) phosphorylation of PP2A, which was dependent on activation of a p60c-Src family tyrosine kinase, but not the epidermal growth factor receptor tyrosine kinase and resulted in dissociation of PP2A from mGluR5 and reduced PP2A activity. Together, we have identified a novel, mGluR5-triggered signaling mechanism involving use- and Src-dependent inactivation of PP2A, which contributes to mGluR5 activation of MEK1/2 and ERK1/2.

Topics: Animals; Blotting, Western; Brain; Cell Survival; Cells, Cultured; Dose-Response Relationship, Drug; Excitatory Amino Acid Agonists; Glycine; Immunoprecipitation; MAP Kinase Kinase 1; Marine Toxins; Microscopy, Confocal; Microscopy, Fluorescence; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neurons; Okadaic Acid; Oxazoles; Peptides; Phenylacetates; Phosphoprotein Phosphatases; Phosphorylation; Protein Phosphatase 2; Protein Structure, Tertiary; Protein-Tyrosine Kinases; Rats; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; Resorcinols; Serine; Signal Transduction; Threonine; Time Factors; Up-Regulation

Activation of group I metabotropic glutamate receptors (mGluRs) up-regulates transcription factor cyclic AMP response element-binding protein (CREB) and Elk-1 phosphorylation via extracellular signal-regulated kinase 1/2 (ERK1/2) in the striatum in vivo. Protein phosphatase 1/2A further regulates immediate early gene expression by inactivating (dephosphorylating) CREB. In this study, using semi-quantitative immunohistochemical and western blot analyses and in situ hybridization histochemistry, we found that intrastriatal infusion of the protein phosphatase 1/2A inhibitor okadaic acid (0.005, 0.05 and 0.5 nmol) increased CREB and Elk-1 phosphorylation and c-Fos immunoreactivity in the injected dorsal striatum in a dose-dependent manner. In addition, okadaic acid (0.05 and 0.5 nM) increased c-fos mRNA expression in the dorsal striatum in a dose-dependent manner. Intrastriatal infusion of the group I agonist 3,5-dihydroxyphenylglycine (DHPG) at 100 and 250 nM also increased CREB and Elk-1 phosphorylation. Pre-treatment of okadaic acid (0.05 nm) did not alter DHPG-induced increases in the phosphorylation of the two transcription factors. These data suggest that protein phosphatase 1/2A in striatal neurons is tonically active in dephosphorylating CREB and Elk-1 and thus suppressing constitutive c-fos mRNA and protein expression. Inhibition of the phosphatase 1/2A may contribute to the group I mGluR-regulated phosphorylation of these transcription factors and c-fos expression.

Topics: Animals; Corpus Striatum; Cyclic AMP Response Element-Binding Protein; DNA-Binding Proteins; Enzyme Inhibitors; ets-Domain Protein Elk-1; Excitatory Amino Acid Agonists; Glycine; Male; Microinjections; Mitogen-Activated Protein Kinases; Neurons; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Protein Phosphatase 1; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-fos; Rats; Rats, Wistar; Resorcinols; RNA, Messenger; Transcription Factors