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dc.contributor.authorXiao, Jinsong
dc.date.accessioned2012-04-23T19:29:20Z
dc.date.available2012-04-23T19:29:20Z
dc.date.issued1995
dc.identifier.urihttp://hdl.handle.net/10713/1542
dc.descriptionUniversity of Maryland, Baltimore. Molecular and Cell Biology. Ph.D. 1995en_US
dc.description.abstractNeurofilaments (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.en_US
dc.language.isoen_USen_US
dc.subjectBiology, Neuroscienceen_US
dc.subjectBiology, Cellen_US
dc.subject115 kDA polypeptideen_US
dc.subject.meshAlzheimer Disease--metabolismen_US
dc.subject.meshCalcium-Calmodulin-Dependent Protein Kinasesen_US
dc.subject.meshIntermediate Filamentsen_US
dc.titleIdentification and characterization of a novel neurofilament-associated kinase and studies on calcium/calmodulin kinase in Alzheimer's diseaseen_US
dc.typedissertationen_US
dc.contributor.advisorMonteiro, Mervyn J.
dc.identifier.ispublishedYes
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