Abnormal hyperphosphorylation of beta -tubulin and beta -catenin in Alzheimer disease brain.
Item
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Title
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Abnormal hyperphosphorylation of beta -tubulin and beta -catenin in Alzheimer disease brain.
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Identifier
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AAI9986385
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identifier
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9986385
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Creator
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Vijayan, Shrijay.
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Contributor
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Adviser: Khalid Iqbal
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Date
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2000
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Neuroscience
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Abstract
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Microtubule associated protein tau, the major protein subunit of Alzheimer paired helical filaments is abnormally hyperphosphorylated and an imbalance in the protein kinase phosphatase system has been implicated in the abnormal phosphorylation. Furthermore, the activities of phosphoseryl/phosphothreonyl protein phosphatase-1 and -2A, which are expressed in neurons and regulate the phosphorylation of neuronal proteins phosphorylated at serines and threonines, are decreased in Alzheimer disease (AD) brain. We undertook a study to investigate whether proteins other than tau are also hyperphosphorylated in the AD brain. Frontal gray matter from AD and age-matched control cases were homogenized and centrifuged at 100,000 x g for 30 min. While in the 100,000 x g supernatant there were no significant differences in phosphate levels between the two groups, the pellet showed an increase of ∼11 pmoles phosphate per microgram protein in AD over the control cases. Hyperphosphorylation of proteins was examined by employing antibodies to phosphoserine and phosphothreonine on Western blots. Two non-tau phosphoproteins with molecular masses of ∼54 kD and ∼94 kD were found to be hyperphosphorylated in AD. The ∼54 kD non-tau protein was purified by preparative SDS-PAGE and identified as beta-tubulin, by immunolabeling with specific antibody to beta-tubulin, by mass spectrometry and by N-terminal amino acid sequencing. The ∼94 kD protein was identified as beta-catenin, by immunolabeling with antibodies to beta-catenin on Western blots. Hyperphosphorylation of beta-catenin was confirmed by immunoprecipitating beta-catenin followed by Western blotting with P-thr antibodies. We also found that the steady state levels of particulate pool, but not the cytosolic pool, of beta-catenin was ∼39% higher in AD compared with age-matched controls. We investigated the role of APP in the regulation of beta-catenin levels in cultured cos-7 cells. Cos-7 cells were transfected with APP full length (APP FL), DeltacAPP (lacking the C-terminal 44 amino acids), APPswe (M670N,K671L) or as control with vector alone. The APP FL was found to downregulate particulate pool of beta-catenin. This ability was lost when APP was mutated as in APPswe or when 44 amino acids from its cytoplasmic tail were deleted. These studies suggest that normal APP may function by downregulating beta-catenin and when its normal functions are altered as in APPswe it is unable to downregulate beta-catenin levels, resulting in an increase in the particulate pool of beta-catenin. Furthermore, the restoration of normal levels of beta-catenin in the DeltacAPP transfected cells suggest that APP downregulates beta-catenin through its cytoplasmic tail.;Our findings that two non-tau proteins---beta-catenin and probably betabeta-tubulin are hyperphosphorylated support the hypothesis that there is an imbalance in the phosphorylation-dephosphorylation system in the AD brain, resulting in the hyperphosphorylation of several neuronal phospho-proteins, which in turn may result in the alteration of their biological activity, as has been demonstrated for tau.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
