Our long term objective is to understand the etiology and the pathogenesis of Alzheimer disease (AD) and to find a rational therapeutic treatment of the disease.
The specific aims of this proposal are: (1) to sort the protein kinases into those that do and those that do not make the tau functionally inactive by phosphorylation at one or more of the same abnormal sites as in the AD abnormally phosphorylated tau both proline-directed protein kinases (PDPKs) MAP kinase, GSK-3, and P34 cdc2 and non-PDPKs protein kinase-A1 protein kinase-C, casein kinase (CK)-11 CK-2, CaM kinase-II, and Ser 262 kinase will be employed. Fetal and adult human recombinant taus, normal human tau and AD abnormally phosphorylated tall (AD P-tau)-dephosphorylated with different protein phosphatases will be phosphorylated, Kinetics - of phosphorylation at the abnormal sites determined by Western blots and radioimmunoslot blot assays using antibodies to various abnormal phosphorylation sites of AD P-tau, and the stoichiometry of phosphorylation will be determined. The effect of phosphorylation on the biological activity of will be determined by its binding to tubulin and normal tall, and by assaying its ability to promote assembly of bovine tubulin into microtubules measured by turbidity at 350 nm and by negative stain-electron microscopy (2) Determine the role of protein-protein interaction in the phosphorylation tau by- comparing the phosphorylation of free tau with tau in microtubules, and tau bound to AD P-tau. Both the stoichiometry of phosphorylation and the kinetics of phosphorylation at each site will -be determined a- above. (3) Study the role of site-site interaction in the abnormal phosphorylation of tau. The effect of phosphorylation of tau by one kinase on the kinetics of phosphorylation by a second kinase will be investigated. The site specific phosphorylation at the abnormal sites will be examined by employing antibodies to the phosphor or the non-phosphor state of the epitomes. The tall conformation found to be the best substrate for the abnormal hyperphosphorylation from the Specific Aim #1 will be employed a the substrate for the site-site interaction studies. These studies will reveal the identification of protein kinases that make tall functionally inactive, and whether the abnormal phosphorylation of tau is a result of a specific conformational state of the substrate protein, protein-protein interaction and/or site- sit interaction. Identification of the protein kinase/s responsible far the abnormal phosphorylation in AD brain is critical to devise a rational approach in correcting this defect.
Showing the most recent 10 out of 72 publications
