• Login
    View Item 
    •   UMB Digital Archive
    • School, Graduate
    • Theses and Dissertations All Schools
    • View Item
    •   UMB Digital Archive
    • School, Graduate
    • Theses and Dissertations All Schools
    • View Item
    JavaScript is disabled for your browser. Some features of this site may not work without it.

    Browse

    All of UMB Digital ArchiveCommunitiesPublication DateAuthorsTitlesSubjectsThis CollectionPublication DateAuthorsTitlesSubjects

    My Account

    LoginRegister

    Statistics

    Display statistics

    Identification and characterization of a novel neurofilament-associated kinase and studies on calcium/calmodulin kinase in Alzheimer's disease

    • CSV
    • RefMan
    • EndNote
    • BibTex
    • RefWorks
    Find Full text
    Author
    Xiao, Jinsong
    Advisor
    Monteiro, Mervyn J.
    Date
    1995
    Type
    dissertation
    
    Metadata
    Show full item record
    Abstract
    Neurofilaments (NF), the major cytoskeletal component in neuronal cells, are one of the most highly phosphorylated proteins expressed in brain. Apart from the structural role NF play in maintaining neuronal architecture, little else is known of their function. I describe here evidence suggesting that NF may support many other proteins in the neuronal axoplasm including protein kinases. In order to isolate proteins that bind NF, I first expressed the carboxyl-terminal tail domain of the mouse heavy-molecular-weight neurofilament subunit (NF-H) as a fusion protein in bacteria and then used this portion of NF-H as a ligand in affinity chromatography. A number of different proteins were isolated, from mouse brain lysate, that specifically bound to the NF-H column and which did not bind to a control column to which BSA was bound. The proteins eluted from the NF-H column contained kinases able to efficiently phosphorylate NF proteins in vitro. I characterized these kinases further by separating proteins on denaturing polyacrylamide gels and reconstituting kinase activity in situ. Using this assay I identified a number of individual kinases including a 115 kDa polypeptide which showed a significant preference for NF proteins as substrate. Native NF was found to be the best substrate for the 115 kDa kinase, followed by a bacterially expressed NF-H non-fusion protein, and NF-H fusion protein. However, NF-L was a poor substrate. Two different NF monoclonal antibodies, SMI31 and SMI32 (Sternberger Monoclonal Inc.) were used to further demonstrate that the 115 kDa kinase is associated with NF in vivo. The kinase was co-immunoprecipitated along with NF by the two NF monoclonal antibodies but appeared to be preferentially associated with phosphorylated forms of NF. I discuss here some of the novel properties of the 115 kDa NF-associated kinase I have termed NAK115 (for NF-associated kinase with a molecular mass of 115 kDa). Other biochemical properties of NAK115 were also studied. It appears to be a peripheral membrane protein with a pI of 5.4-6.2, and it is expressed at different levels in a variety of mouse tissues. NAK115 exists as a large 570 protein complex in vivo. Purification of NAK115 was also explored. After four steps of purification, NAK115 was enriched around 20 fold.;Alzheimer's disease (AD) is characterized pathologically by two distinguishable deposits in brain, namely senile plaques and neurofibrillary tangles (NFT). Senile plaques are composed of fragments of the amyloid precursor protein, whereas NFT are composed primarily of paired-helical filaments (PHF). The latter are in turn composed principally of the microtubule-associated tau protein. Tau in PHF is highly and unusually phosphorylated. However, neither the mechanism nor the identity of the protein kinases or phosphatases that govern this unusual phosphorylation is known. Using a combination of immunoblotting and kinase assays, I demonstrate that a discreet set of kinases co-purify with PHF. One of these kinases was found by immunoblotting to be {dollar}\alpha{dollar}-calcium-calmodulin dependent kinase II {dollar}(\alpha{dollar}-CaM kinase). Immunogold labeling revealed that {dollar}\alpha{dollar}-CaM kinase was localized to a novel globular structure found at the ends of PHF. Since previous studies have shown {dollar}\alpha{dollar}-CaM kinase to be involved in memory, its association with PHF may have important implications in understanding memory loss in AD. I also discuss the possibility that the association of {dollar}\alpha{dollar}-CaM kinase with PHF may indicate sites where tau protein is converted into PHF.
    Description
    University of Maryland, Baltimore. Molecular and Cell Biology. Ph.D. 1995
    Keyword
    Biology, Neuroscience
    Biology, Cell
    115 kDA polypeptide
    Alzheimer Disease--metabolism
    Calcium-Calmodulin-Dependent Protein Kinases
    Intermediate Filaments
    Identifier to cite or link to this item
    http://hdl.handle.net/10713/1542
    Collections
    Theses and Dissertations All Schools
    Theses and Dissertations School of Medicine

    entitlement

     
    DSpace software (copyright © 2002 - 2022)  DuraSpace
    Quick Guide | Policies | Contact Us | UMB Health Sciences & Human Services Library
    Open Repository is a service operated by 
    Atmire NV
     

    Export search results

    The export option will allow you to export the current search results of the entered query to a file. Different formats are available for download. To export the items, click on the button corresponding with the preferred download format.

    By default, clicking on the export buttons will result in a download of the allowed maximum amount of items.

    To select a subset of the search results, click "Selective Export" button and make a selection of the items you want to export. The amount of items that can be exported at once is similarly restricted as the full export.

    After making a selection, click one of the export format buttons. The amount of items that will be exported is indicated in the bubble next to export format.