Neurofilaments, microtubules and actin filaments are the major cytoskeletal filaments expressed in neuronal cells. Neurofilament proteins are phosphorylated during development and are among the most heavily phosphorylated proteins expressed in brain. However, we know very little about the functional significance of NF protein phosphorylation. An understanding of NF phosphorylation is important since in Alzheimer's Disease (AD) NFs are thought to be hyperphosphorylated and abnormally localized in neurons. We have begun to characterize protein kinases that phosphorylate NF in order to begin to understand the role of NF phosphorylation during normal development and in aging (AD). We have identified by NF-H affinity chromatography a novel NF kinase in mouse brain extracts which we have termed NF kinase 115. NF kinase 115 activity was found in protein lysates of primary mouse neuronal cultures but not in lysates of mouse fibroblast cultures. Since NFs are expressed only in neuronal cells the study of this kinase is likely to be important in understanding the relevance of NF phosphorylation in axonal function. A related kinase has also been found in extracts from human brain tissue. Preliminary results indicate that kinase activity is increased in brain extracts of patients diagnosed as suffering from AD compared to brain extract of a non-AD control. Since AD is associated with altered phosphorylation of many proteins, the study of this novel kinase may also be important in the etiology of AD. In this study we propose to purify the NF kinase 115 from mouse brain extracts in order to characterize the kinase biochemically. We will raise antibodies to the purified kinase in order to further characterize the protein. We will determine the expression of NF kinase 115 during neurite outgrowth of primary neuronal cells and of clonal cells lines that can be induced to extend neurites by NFG treatment. We will determine if increased NF kinase 115 activity correlates with AD. The long term goal of this study is to characterize the novel kinase at a molecular and biochemical level.
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