Effect of FTDP-17 mutations on phosphorylation and dephosphorylation of tau protein in vitro

Microtubule-associated protein tau binds to microtubules and stabilizes microtubule structure. By stabilizing the microtubule cytoskeleton, tau plays an important role in the maintenance of neuronal survival. Tau is abnormally hyperphosphorylated and forms neurofibrillary tangles (NFTs) in the brains of the patients suffering from a number of neurodegenerative diseases termed tauopathies, which include Alzheimer's disease (AD) and frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17). Missense tau mutations have been discovered to cosegregate with FTDP-17. The mechanism by which these mutations promote tau hyperphosphorylation and NFT pathology remains unknown. The goal of this study was to determine the mechanism by which FTDP-17 mutations enhance tau hyperphosphorylation and promote tau pathology.In vitro phosphorylation of wild type (WT) tau and FTDP-17 mutant tau G272V, P301L, V337M and R406W by Cyclin-dependent-kinase 5 (Cdk5) (implicated in tau phosphorylation in AD and various tauopathies) was carried out. Tau (WT), which migrates as a single band of ∼60 kDa in size, migrated as three bands with sizes of 60, 64 and ∼68 kDa on an SDS gel upon phosphorylation by CdkS. Since tau isolated from NFTs migrates as three bands of sizes ∼60, ∼64 and ∼68 kDa, these observations suggest that phosphorylation of tau by Cdk5 converts normal tau to diseased tau. Importantly, each FTDP-17 mutation promoted Cdk5 phosphorylation-induced conversion of normal tau to diseased tau. In addition, the FTDP-17 mutations also significantly reduced the ability of phosphorylated tau to bind and promote microtubule assembly in vitro.Protein phosphatase 1 (PP1), protein phosphatase 2A (PP2A) and protein phosphatase 2B (PP2B) have been implicated in the dephosphorylation of tau in the brain. Since the phosphorylation state of a protein is regulated by the activities of kinases and phosphatases acting on that protein, the effect of various FTDP-17 mutations on tau dephosphorylation by PP1, PP2A and PP2B was analyzed. When expressed in HEK293 cells, tau (WT) was phosphorylated by the endogenous kinases of the cell. When transfected cells were lysed and incubated in vitro, endogenously phosphorylated tau was dephosphorylated by cellular PP1, PP2A and PP2B present in the lysate. Using specific inhibitors of PP1, PP2A and PP2B, dephosphorylation of WT and FTDP-17 mutant tau G272V, P301L, V337M and R406W by the above phosphatases was examined. It was observed that each FTDP-17 mutation selectively inhibited tau dephosphorylation by PP1, PP2A and PP2B in a site-specific manner.Taken together, the results of this study indicate that FTDP-17 mutations not only promote tau phosphorylation but also inhibit tau dephosphorylation. Furthermore, FTDP-17 tau mutations and tau phosphorylation synergistically act to promote tau dysfunction associated with NFT pathology