Mechanism Of Hyperphosphorylation Research Articles

Quantitative and combinatory determination of in situ phosphorylation of tau and its FTDP-17 mutants.

Tau is hyperphosphorylated in the brains of patients with tauopathies, such as Alzheimer’s disease and frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). However, neither the mechanism of hyperphosphorylation nor its contribution to pathogenesis is known. We applied Phos-tag SDS-PAGE, a phosphoaffinity electrophoresis, to the analysis of tau phosphorylation in vitro by Cdk5, in cultured cells and in mouse brain. Here, we found that Cdk5-p25 phosphorylated tau in vitro at Ser404, Ser235, Thr205 and Ser202 in this order. In contrast in cultured cells, Ser404 was preferentially phosphorylated by Cdk5-p35, whereas Thr205 was not phosphorylated. Ser202 and Ser235 were phosphorylated by endogenous kinases. Tau exhibited ~12 phosphorylation isotypes in COS-7 cells with different combinations of phosphorylation at Thr181, Ser202, Thr231, Ser235 and Ser404. These phosphorylation sites were similar to tau phosphorylated in mouse brains. FTDP-17 tau with a mutation in the C-terminal region had different banding patterns, indicating a different phosphorylation pattern. In particular, it was clear that the R406W mutation causes loss of Ser404 phosphorylation. These results demonstrate the usefulness of the Phos-tag technique in the quantitative analysis of site-specific in vivo phosphorylation of tau and provide detailed information on in situ combinatory phosphorylation of tau.

Heterogeneous phosphorylation of microtubule-associated protein tau in cultured cells

Event Abstract Back to Event Heterogeneous phosphorylation of microtubule-associated protein tau in cultured cells Taeko Kimura1, Koichi Ishiguro2, Masato Hasegawa3 and Shin-ichi Hisanaga1* 1 Tokyo Metropolitan University, Molecular Neurosicence/Biological Sciences/Science and Engineering, Japan 2 Juntendo University, Neurology, Japan 3 Tokyo Metropolitan Institute of Medical Science, Dementia and Higher Brain Function, Japan Tau, a microtubule-associated protein, is a major component of aggregates seen in brains of tauopathies such as Alzheimer’s disease (AD) and frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). Tau is hyperphosphorylated in the aggregates and therefore the mechanism of hyperphosphorylation has been an intense research subject. Here, we applied the Phos-tag SDS-PAGE technique to characterize tau phosphorylation by cyclin-dependent kinase 5 (Cdk5) in vitro and in cultured cells. Cdk5-p25 phosphorylated tau at Ser404, Ser235, Thr205 and probably Se202 in that order. Among those four major in vitro Cdk5 phosphorylation sites, Ser404 was preferentially phosphorylated by Cdk5 in cultured cells, whereas Thr205 was not. Ser202 and Ser235 were phosphorylated by endogenous kinases. Tau existed in 12 different phosphorylation states in COS-7 cells with Ser202, Thr231 and Ser235 as major phosphorylation sites. p35-Cdk5 and p25-Cdk5 phosphorylated tau to a similar extent, when their expression levels were adjusted. Phosphorylation of FTDP-17 tau mutants was also analyzed by this method. While the mutants in the N-terminal or microtubule-binding region were phosphorylated identically as was wild type tau, the C-terminal mutants showed slightly different banding patterns from wild type tau. In particular, it was clear that R406W mutation resulted in loss of Ser404 phosphorylation. These results not only include novel and interesting information on tau phosphorylation but also indicate usefulness of Phos-tag SDS-PAGE in the analysis of site-specific and cellular phosphorylation pattern of tau. Keywords: Phosphorylation, Alzheimer’s disease, tau, in vitro, protein Conference: 14th Meeting of the Asian-Pacific Society for Neurochemistry, Kuala Lumpur, Malaysia, 27 Aug - 30 Aug, 2016. Presentation Type: Poster Presentation Session Topic: 14th Meeting of the Asian-Pacific Society for Neurochemistry Citation: Kimura T, Ishiguro K, Hasegawa M and Hisanaga S (2016). Heterogeneous phosphorylation of microtubule-associated protein tau in cultured cells. Conference Abstract: 14th Meeting of the Asian-Pacific Society for Neurochemistry. doi: 10.3389/conf.fncel.2016.36.00195 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 04 Aug 2016; Published Online: 11 Aug 2016. * Correspondence: Prof. Shin-ichi Hisanaga, Tokyo Metropolitan University, Molecular Neurosicence/Biological Sciences/Science and Engineering, Hachioji, Tokyo, Japan, hisanaga-shinichi@tmu.ac.jp Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Taeko Kimura Koichi Ishiguro Masato Hasegawa Shin-ichi Hisanaga Google Taeko Kimura Koichi Ishiguro Masato Hasegawa Shin-ichi Hisanaga Google Scholar Taeko Kimura Koichi Ishiguro Masato Hasegawa Shin-ichi Hisanaga PubMed Taeko Kimura Koichi Ishiguro Masato Hasegawa Shin-ichi Hisanaga Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.