Familial FTDP-17 Missense Mutations Inhibit Microtubule Assembly-promoting Activity of Tau by Increasing Phosphorylation at Ser202 in Vitro

Abstract

In Alzheimer disease (AD), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) and other tauopathies, tau accumulates and forms paired helical filaments (PHFs) in the brain. Tau isolated from PHFs is phosphorylated at a number of sites, migrates as ϳ60-, 64-, and 68-kDa bands on SDS-gel, and does not promote microtubule assembly. Upon dephosphorylation, the PHF-tau migrates as ϳ50 -60-kDa bands on SDS-gels in a manner similar to tau that is isolated from normal brain and promotes microtubule assembly. The site(s) that inhibits microtubule assembly-promoting activity when phosphorylated in the diseased brain is not known. In this study, when tau was phosphorylated by Cdk5 in vitro, its mobility shifted from ϳ60-kDa bands to ϳ64-and 68-kDa bands in a time-dependent manner. This mobility shift correlated with phosphorylation at Ser 202 , and Ser 202 phosphorylation inhibited tau microtubule-assembly promoting activity. When several tau point mutants were analyzed, G272V, P301L, V337M, and R406W mutations associated with FTDP-17, but not nonspecific mutations S214A and S262A, promoted Ser 202 phosphorylation and mobility shift to a ϳ68-kDa band. Furthermore, Ser 202 phosphorylation inhibited the microtubule assembly-promoting activity of FTDP-17 mutants more than of WT. Our data indicate that FTDP-17 missense mutations, by promoting phosphorylation at Ser 202 , inhibit the microtubule assembly-promoting activity of tau in vitro, suggesting that Ser 202 phosphorylation plays a major role in the development of NFT pathology in AD and related tauopathies. Neurofibrillary tangles (NFTs)4 and senile plaques are the two characteristic neuropathological lesions found in the brains of patients suffering from Alzheimer disease (AD).The major fibrous component of NFTs are paired helical filaments (PHFs) (for reviews see Refs. 1-3). Initially, PHFs were found to be composed of a protein component referred to as "A68" (4). Biochemical analysis reveled that A68 is identical to the microtubule-associated protein, tau (4, 5). Some characteristic features of tau isolated from PHFs (PHF-tau) are that it is abnormally hyperphosphorylated (phosphorylated on more sites than the normal brain tau), does not bind to microtubules, and does not promote microtubule assembly in vitro. Upon dephosphorylation, PHF-tau regains its ability to bind to and promote microtubule assembly (6, 7). Tau hyperphosphorylation is suggested to cause microtubule instability and PHF formation, leading to NFT pathology in the brain (1-3).PHF-tau is phosphorylated on at least 21 proline-directed and non-proline-directed sites (8, 9). The individual contribution of these sites in converting tau to PHFs is not entirely clear. However, some sites are only partially phosphorylated in PHFs (8), whereas phosphorylation on specific sites inhibits the microtubule assembly-promoting activity of tau (6, 10). These observations suggest that phosphorylation on a few sites may be responsible and sufficient for causing tau dysfunction in AD.Tau purified ...

Parkinsonian Neurotoxin 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and α-Synuclein Mutations Promote Tau Protein Phosphorylation at Ser262 and Destabilize Microtubule Cytoskeleton in Vitro

“…Protein Concentrations-The concentration of Tau was determined spectrophotometrically by measuring OD at 280 nm as previously described (45). Concentrations of Tau(S214A), phosphorylated Tau(WT), and phosphorylated Tau(S214A) were determined via the Bio-Rad protein assay using Tau(WT) as the standard.…”

“…The pET-3a (A30P) and Myc-pcDNA3.1 (A53T) were described previously (44). FLAG-Tau(WT) and FLAG-Tau(S214A) each in pcDNA3.1 and pET-3a are also described previously (45).…”

“…Proteins and Antibodies-Tau(WT) and Tau(S214A) were purified from their respective bacterial lysates as described previously (45). Purification of ␣-synuclein (WT) and its various mutants A30P, E46K, A53T, and E83P were performed following previously published procedures (46) with some modifications.…”

“…Anti-␣-synuclein monoclonal antibody was purchased from BD Biosciences. Monoclonal antibodies Tau 5, AT8, PHF1, 12E8, anti-Myc, and anti-FLAG are described previously (45). Polyclonal antibodies pS214 and pS262 were purchased from BIOSOURCE Inc.…”

Interaction Between Eye Pigment Genes and Tau-Induced Neurodegeneration inDrosophila melanogaster

“…Tau phosphorylation at S202/T205, as detected by the AT8 antibody , is a common feature in tauopathies and accumulates in fairly late neurofibrillary tangle development (Ikura et al 1998;Wada et al 1998;Augustinack et al 2002;Ferrer et al 2002;Wray et al 2008;Han et al 2009). Thus, increased AT8 signal is predicted to correlate with enhanced toxicity found in white and white 1 brown backgrounds; however, AT8 immunoreactivity was reduced in w 1118 homozygous flies as compared to w 1 /w 1118 heterozygotes ( Figure 4A).…”