Regulation of a site-specific phosphorylation of the microtubule-associated protein 2 during the development of cultured neurons.
The phosphorylation state of cytoskeletal proteins, including certain microtubule-associated proteins, may influence the development and plasticity of axons and dendrites in mammalian neuron in response to appropriate extracellular stimuli. In particular, high molecular weight microtubule-associated protein 2, has been implicated in dendrite growth and synaptic plasticity and is thought to be modulated by phosphorylation and dephosphorylation. We have previously determined that threonines 1620/1623 on the microtubule-associated protein 2 molecule become phosphorylated in vivo and are targets for proline-directed protein kinases in vitro. Using the phosphorylated site-specific antibody 305, we now report the decreased phosphorylation state of high molecular weight microtubule-associated protein 2 during the development of cultured cerebellar granule neurons. Phosphorylation of high molecular weight microtubule-associated protein 2 at this site is significantly inhibited by lithium in short-term cultured neurons, which suggests the implication of glycogen synthase kinase-3. In long-term cultured neurons, it is also partially inhibited by PD098059, an inhibitor of extracellular signal-regulated protein kinase activation, which indicates an additional contribution of this kinase to high molecular weight microtubule-associated protein 2 phosphorylation at this stage. Both in short-term and long-term cultured neurons, okadaic acid augments high molecular weight microtubule-associated protein 2 phosphorylation at this site through the inhibition of protein phosphatases 1 and/or 2A. Finally, glutamate receptor activation leads to a dephosphorylation of high molecular weight microtubule-associated protein 2 at this site which can also be effectively prevented by okadaic acid. These results are consistent with the participation of glycogen synthase kinase-3, extracellular signal-regulated protein kinases and protein phosphatases 1 and 2A, in the regulation of microtubule-associated protein 2 phosphorylation within living neurons, which may be modulated by extracellular signals like the neurotransmitter glutamate.
Centro de Biología Molecular Severo Ochoa, Facultad de Ciencias, Universidad Autónoma de Madrid, Cantoblanco, Spain.
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Sánchez Martin
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Díaz-Nido
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Avila
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D038362:Glycogen Synthase Kinase 3; D038341:Glycogen Synthase Kinases; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D019950:Mitogen-Activated Protein Kinase 1; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D016501:Neurites / Q000502:physiology; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D019319:Okadaic Acid / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D010769:Phosphothreonine / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar
D004791:Enzyme Inhibitors; D005419:Flavonoids; D008869:Microtubule-Associated Proteins; D009419:Nerve Tissue Proteins; D010769:Phosphothreonine; D019319:Okadaic Acid; D011494:Protein Kinases; D038341:Glycogen Synthase Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D038362:Glycogen Synthase Kinase 3; D010749:Phosphoprotein Phosphatases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one