Abstract
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.
Highlights
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)
Recombinant tau was incubated with Cyclin-dependent kinase 5 (Cdk5)-p25 in the presence of ATP, and phosphorylation was assessed by upward shift on an immunoblot generated using Phos-tag SDS-PAGE and the phosphorylation-independent tau antibody Tau[5]
We characterized the phosphorylation of tau by Cdk[5] in vitro, in cultured cells and in brain tissue using the Phos-tag SDS-PAGE technique
Summary
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). Tau exhibited ~12 phosphorylation isotypes in COS-7 cells with different combinations of phosphorylation at Thr[181], Ser[202], Thr[231], Ser[235] and Ser[404] These phosphorylation sites were similar to tau phosphorylated in mouse brains. Cdk[5] phosphorylates mainly Ser[202], Thr[205], Ser[235] and Ser[404] in vitro[17,18,19], up to 13 sites have been reported to date[7] While these major Cdk[5] sites are phosphorylated in tau from healthy neurons, they are abnormally phosphorylated in tau from www.nature.com/scientificreports/. A basic understanding of tau phosphorylation is required, such as which site is phosphorylated to what extent under which conditions It is not clear yet whether the major Cdk[5] phosphorylation sites are phosphorylated in vitro and in cells. We identified a number of novel and interesting aspects of the phosphorylation of tau, including FTDP-17 mutant tau, by Cdk[5]
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