okadaic-acid and 3-4-dihydroxyphenylglycol

Fragile X syndrome is caused by the loss of fragile X mental retardation protein (FMRP), which represses and reversibly regulates the translation of a subset of mRNAs in dendrites. Protein synthesis can be rapidly stimulated by mGluR-induced and protein phosphatase 2a (PP2A)-mediated dephosphorylation of FMRP, which is coupled to the dissociation of FMRP and target mRNAs from miRNA-induced silencing complexes. Here, we report the rapid ubiquitination and ubiquitin proteasome system (UPS)-mediated degradation of FMRP in dendrites upon DHPG (3,5-dihydroxyphenylglycine) stimulation in cultured rat neurons. Using inhibitors to PP2A and FMRP phosphomutants, degradation of FMRP was observed to depend on its prior dephosphorylation. Translational induction of an FMRP target, postsynaptic density-95 mRNA, required both PP2A and UPS. Thus, control of FMRP levels at the synapse by dephosphorylation-induced and UPS-mediated degradation provides a mode to regulate protein synthesis.

Topics: Analysis of Variance; Animals; Boronic Acids; Bortezomib; Cells, Cultured; Dendrites; Disks Large Homolog 4 Protein; Drosophila Proteins; Embryo, Mammalian; Enzyme Inhibitors; Female; Fragile X Mental Retardation Protein; Gene Expression Regulation; Green Fluorescent Proteins; Hippocampus; Immunoprecipitation; Intracellular Signaling Peptides and Proteins; Leupeptins; Male; Membrane Proteins; Methoxyhydroxyphenylglycol; Mutation; Neurons; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Protein Biosynthesis; Pyrazines; Rats; Rats, Sprague-Dawley; Receptors, Metabotropic Glutamate; RNA, Messenger; Serine; Signal Transduction; Synapses; Transfection; Ubiquitination

Group 1 metabotropic glutamate receptor (mGluR)-stimulated protein synthesis and long-term synaptic depression (mGluR-LTD) are altered in the mouse model of fragile X syndrome, Fmr1 knock-out (KO) mice. Fmr1 encodes fragile X mental retardation protein (FMRP), a dendritic RNA binding protein that functions, in part, as a translational suppressor. It is unknown whether and how FMRP acutely regulates LTD and/or the rapid synthesis of new proteins required for LTD, such as the activity-regulated cytoskeletal-associated protein (Arc). The protein phosphatase PP2A dephosphorylates FMRP, which contributes to translational activation of some target mRNAs. Here, we report that PP2A and dephosphorylation of FMRP at S500 are required for an mGluR-induced, rapid (5 min) increase in dendritic Arc protein and LTD in rat and mouse hippocampal neurons. In Fmr1 KO neurons, basal, dendritic Arc protein levels and mGluR-LTD are enhanced, but mGluR-triggered Arc synthesis is absent. Lentiviral-mediated expression of wild-type FMRP in Fmr1 KO neurons suppresses basal dendritic Arc levels and mGluR-LTD, and restores rapid mGluR-triggered Arc synthesis. A phosphomimic of FMRP (S500D) suppresses steady-state dendritic Arc levels but does not rescue mGluR-induced Arc synthesis. A dephosphomimic of FMRP (S500A) neither suppresses dendritic Arc nor supports mGluR-induced Arc synthesis. Accordingly, S500D-FMRP expression in Fmr1 KO neurons suppresses mGluR-LTD, whereas S500A-FMRP has no effect. These data support a model in which phosphorylated FMRP functions to suppress steady-state translation of Arc and LTD. Upon mGluR activation of PP2A, FMRP is rapidly dephosphorylated, which contributes to rapid new synthesis of Arc and mGluR-LTD.

Topics: Animals; Animals, Newborn; Cells, Cultured; Cytoskeletal Proteins; Dendrites; Electric Stimulation; Enzyme Inhibitors; Excitatory Postsynaptic Potentials; Fragile X Mental Retardation Protein; Gene Expression Regulation; Green Fluorescent Proteins; Hippocampus; Humans; In Vitro Techniques; Long-Term Synaptic Depression; Methoxyhydroxyphenylglycol; Mice; Mice, Inbred C57BL; Mice, Knockout; Models, Biological; Nerve Tissue Proteins; Neurons; Okadaic Acid; Patch-Clamp Techniques; Phosphorylation; Protein Biosynthesis; Protein Phosphatase 2; Rats; Rats, Long-Evans; Receptors, Metabotropic Glutamate; Serine; Sodium Channel Blockers; Tetrodotoxin; Time Factors; Transfection

Previous reports have shown that activation of N-methyl-D-aspartate (NMDA) receptors potentiates responses to activation of the group I metabotropic glutamate receptor mGluR5 by reversing PKC-mediated desensitization of this receptor. NMDA-induced reversal of mGluR5 desensitization is dependent on activation of protein phosphatases. However, the specific protein phosphatase involved and the precise mechanism by which NMDA receptor activation reduces mGluR desensitization are not known. We have performed a series of molecular, biochemical, and genetic studies to show that NMDA-induced regulation of mGluR5 is dependent on activation of calcium-dependent protein phosphatase 2B/calcineurin (PP2B/CaN). Furthermore, we report that purified calcineurin directly dephosphorylates the C-terminal tail of mGluR5 at sites that are phosphorylated by PKC. Finally, immunoprecipitation and GST fusion protein pull-down experiments reveal that calcineurin interacts with mGluR5, suggesting that these proteins could be colocalized in a signaling complex. Taken together with previous studies, these data suggest that activation of NMDA receptors leads to activation of calcineurin and that calcineurin modulates mGluR5 function by directly dephosphorylating mGluR5 at PKC sites that are involved in desensitization of this receptor.

Topics: Animals; Autoradiography; Brain; Calcineurin; CHO Cells; Cricetinae; Cricetulus; Dose-Response Relationship, Drug; Drug Synergism; Electric Stimulation; Enzyme Activation; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Glutamic Acid; Glutathione Transferase; Hydroxylation; Immunoblotting; Immunoprecipitation; Membrane Potentials; Methoxyhydroxyphenylglycol; Mutagenesis; N-Methylaspartate; Okadaic Acid; Oocytes; Patch-Clamp Techniques; Phosphatidylinositols; Protein Kinase C; Rats; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; Transfection; Xenopus

Casein kinase 1 (CK1) is a highly conserved serine/threonine kinase, present in virtually all cell types, in which it phosphorylates a wide variety of substrates. So far, no role has been found for this ubiquitous protein kinase in the physiology of nerve cells. In the present study, we show that CK1 regulates fast synaptic transmission mediated by glutamate, the major excitatory neurotransmitter in the brain. Through the use of CK1 inhibitors, we present evidence that activation of CK1 decreases NMDA receptor activity in the striatum via a mechanism that involves activation by this kinase of protein phosphatase 1 and/or 2A and resultant increased dephosphorylation of NMDA receptors. Indeed, inhibition of CK1 increases NMDA-mediated EPSCs in medium spiny striatal neurons. This effect is associated with an increased phosphorylation of the NR1 and NR2B subunits of the NMDA receptor and is occluded by the phosphatase inhibitor okadaic acid. The mGluR1, but not mGluR5, subclass of metabotropic glutamate receptors uses CK1 to inhibit NMDA-mediated synaptic currents. These results provide the first evidence for a role of CK1 in the regulation of synaptic transmission in the brain.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Bicuculline; Casein Kinase 1 epsilon; Casein Kinase I; Casein Kinase Ialpha; Casein Kinase Idelta; Corpus Striatum; Egtazic Acid; Evoked Potentials; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Glutamic Acid; Kainic Acid; Methoxyhydroxyphenylglycol; Mice; Mice, Inbred C57BL; N-Methylaspartate; Neocortex; Nerve Tissue Proteins; Okadaic Acid; Phosphoprotein Phosphatases; Protein Phosphatase 1; Receptors, Metabotropic Glutamate; Receptors, N-Methyl-D-Aspartate; Synaptic Transmission; Tetrodotoxin

We have shown earlier that activation of metabotropic glutamate (mGlu) receptors using a group I-specific mGlu receptor agonist, (RS)-3,5-dihydroxyphenylglycine (DHPG), can induce long-term depression (LTD) in the CA1 region of the hippocampus. In an attempt to determine the signal transduction mechanisms involved in this form of synaptic plasticity, we have tested the effects of a range of inhibitors on DHPG-induced LTD. In vitro grease-gap electrophysiological recordings were performed in the rat hippocampal CA1 region. We have found that DHPG-induced LTD is resistant to the two potent protein kinase C (PKC) inhibitors, Gö 6976 (10 microM) and Gö 6983 (10 microM), the potent and selective protein kinase A (PKA) inhibitor, KT 5720 (10 microM), and the potent broad spectrum kinase inhibitor, staurosporine (10 microM). In contrast, non-selective inhibitors of protein phosphatases (PP1 and PP2A), okadaic acid (1 microM) or calyculin A (1 microM), facilitated DHPG-induced LTD. However, an inhibitor of protein phosphatase 2B, FK 506 (1 microM), did not influence this process. The PP1/PP2A protein phosphatase inhibitors, but none of the other agents tested, also inhibited (S)-alpha-methyl-4-carboxyphenylglycine (MCPG)-induced reversal of DHPG-induced LTD. These data suggest that activation of neither PKC nor PKA is involved in DHPG-induced LTD. They do, however, suggest that the process is under regulation by protein phosphorylation and dephosphorylation.

Topics: Animals; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Female; Hippocampus; Methoxyhydroxyphenylglycol; Neuronal Plasticity; Okadaic Acid; Phosphoprotein Phosphatases; Protein Kinase C; Rats; Rats, Wistar