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ndltd-OhioLink-oai-etd.ohiolink.edu-osu13118682222021-08-03T06:03:15Z Identification and characterization of a mitosis-associated phosphoepitope up-regulated during neuronal differentiation Feng, Yang <p>Protein components of the neuronal cytoskeleton are fundamentallyimportant for architectural support and organelle transport in axons and dendrites.Among them, microtubules (MTs) and microtubule-associated proteins (MAPs) aremajor components involved in neuronal differentiation and neurodegenerativediseases. Neuronal MTs are relatively stable, yet they are not static structures. Afine-tuned balance in MT stability is required to assure proper plasticity of neuronalprocesses. MAPs are key players in regulating the stability of MTs, and MAPfunction is in turn regulated by phosphorylation. It was found in this study that adramatic increase in MAP1B phosphorylation occurred as PC12 cells developedneurites in response to nerve growth factor. The phosphorylation of MAP1B wasidentified by a monoclonal antibody MPM2, which identifies a mitosis-associatedphosphoepitope. The increase in MPM2 reactivity of MAP1B was also observed insynchronized mitotic PC12 cells, suggesting that related kinase activities areinvolved in pathways inducing cell division and the differentiation of postmitoticneuronal cells. MPM2-reactive MAP1B was localized to neurites of PC12 cells, butnot cell bodies by indirect immunofluorescence and immunogold electronmicroscopy. In order to understand the functional significance of thephosphorylation of MAP1B, the identification of the MPM2 epitope was required.Through proteolytic digestion and peptide microsequencing the epitope was shownto be located in the N-terminal 40 kD of MAP1B. A potential phosphothreonine sitefitting a model of known MPM2 epitopes was identified, and peptides containingthese sequences were synthesized in both phosphorylated and dephosphorylatedforms. The selected phosphopeptide was highly reactive with the MPM2 antibody,while the dephosphopeptide was not. Furthermore, the phosphopeptide had theability to compete for MPM2 antibody binding with both MAP1B and otherphosphoproteins in mitotic cells. These results indicate that the phosphopeptidehad all of the elements required for MPM2 antibody recognition. A phosphorylationstate-specific antibody PMB1 was generated by using the phosphopeptideconjugated to keyhole limpet hemocyanin as immunogen. The purified antibody wasshown to be specific to phosphorylated MAP1B. The phosphorylation site andantibody probe described here are expected to assist in identifying the kinaseresponsible for the modification on MAP1B and understanding the functionalsignificance of this modification in neurogenesis.</p> 1997 English text The Ohio State University / OhioLINK http://rave.ohiolink.edu/etdc/view?acc_num=osu1311868222 http://rave.ohiolink.edu/etdc/view?acc_num=osu1311868222 unrestricted This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws.
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NDLTD
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English
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NDLTD
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author |
Feng, Yang
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spellingShingle |
Feng, Yang
Identification and characterization of a mitosis-associated phosphoepitope up-regulated during neuronal differentiation
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author_facet |
Feng, Yang
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author_sort |
Feng, Yang
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title |
Identification and characterization of a mitosis-associated phosphoepitope up-regulated during neuronal differentiation
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title_short |
Identification and characterization of a mitosis-associated phosphoepitope up-regulated during neuronal differentiation
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title_full |
Identification and characterization of a mitosis-associated phosphoepitope up-regulated during neuronal differentiation
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title_fullStr |
Identification and characterization of a mitosis-associated phosphoepitope up-regulated during neuronal differentiation
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title_full_unstemmed |
Identification and characterization of a mitosis-associated phosphoepitope up-regulated during neuronal differentiation
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title_sort |
identification and characterization of a mitosis-associated phosphoepitope up-regulated during neuronal differentiation
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publisher |
The Ohio State University / OhioLINK
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publishDate |
1997
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url |
http://rave.ohiolink.edu/etdc/view?acc_num=osu1311868222
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work_keys_str_mv |
AT fengyang identificationandcharacterizationofamitosisassociatedphosphoepitopeupregulatedduringneuronaldifferentiation
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_version_ |
1719430130166661120
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