Contributions of protein phosphatases PP1, PP2A, PP2B and PP5 to the regulation of tau phosphorylation Fei Liu, Inge Grundke-Iqbal, Khalid Iqbal and Cheng-Xin Gong Department of Neurochemistry, New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, New York 10314, USA Keywords: Alzheimer’s disease, dephosphorylation, human brain, hyperphosphorylation, protein phosphatase 2A Abstract Abnormal hyperphosphorylation of tau is believed to lead to neurofibrillary degeneration in Alzheimer’s disease (AD) and other tauopathies. Recent studies have shown that protein phosphatases (PPs) PP1, PP2A, PP2B and PP5 dephosphorylate tau in vitro, but the exact role of each of these phosphatases in the regulation of site-specific phosphorylation of tau in the human brain was unknown. Hence, we investigated the contributions of these PPs to the regulation of tau phosphorylation quantitatively. We found that these four phosphatases all dephosphorylated tau at Ser199, Ser202, Thr205, Thr212, Ser214, Ser235, Ser262, Ser396, Ser404 and Ser409, but with different efficiencies toward different sites. The K m values of tau dephosphorylation catalysed by PP1, PP2A and PP5 were 8–12 lm, similar to the intraneuronal tau concentration of human brain, whereas the K m of PP2B was fivefold higher. PP2A, PP1, PP5 and PP2B accounted for  71%,  11%,  10% and  7%, respectively, of the total tau phosphatase activity of human brain. The total phosphatase activity and the activities of PP2A and PP5 toward tau were significantly decreased, whereas that of PP2B was increased in AD brain. PP2A activity negatively correlated to the level of tau phosphorylation at the most phosphorylation sites in human brains. Our findings indicate that PP2A is the major tau phosphatase that regulates its phosphorylation at multiple sites in human brain. The abnormal hyperphosphorylation of tau is partially due to a downregulation of PP2A activity in AD brain. Introduction Alzheimer’s disease (AD) is characterized by two hallmark brain lesions ) extracellular deposits of b-amyloid and intracellular neuro- fibrillary tangles (NFTs) ) the latter of which directly correlates to the severity of dementia symptoms (Alafuzoff et al., 1987; Arriagada et al., 1992; Riley et al., 2002). NFTs are composed of bundles of paired helical filaments (PHFs) and straight filaments, the major protein component of which is the abnormally hyperphosphorylated tau (Grundke-Iqbal et al., 1986a,b; Lee et al., 1991). Tau is a phosphoprotein, normally containing two–three phosphate groups per molecule. In AD brain, tau is abnormally hyperphosphorylated with 9–10 mol of phosphate per mole of tau (Ksiezak-Reding et al., 1992; Ko ¨pke et al., 1993). More than 30 phosphorylation sites have been identified in the hyperphosphorylated tau isolated from AD brain (for review, see Gong et al., 2005). The abnormal hyperphosphorylation of tau is believed to be responsible for its loss of biological activity, for its gain of toxic activity and for its aggregation into PHFs (Iqbal et al., 1986; Alonso et al., 1994; 2001a,b; Lucas et al., 2001; Fath et al., 2002; Jackson et al., 2002; Perez et al., 2002). Hence, the abnormal hyperphosphorylation of tau appears to be critical to the pathogenesis of AD. The molecular mechanism leading to the abnormal hyperphosph- orylation of tau in AD is not well understood. Tau phosphorylation is catalysed by tau protein kinases and reversed by tau protein phosphatases (PPs). More than a dozen protein kinases have been shown to phosphorylate tau in vitro (for review, see Gong et al., 2005). There are five phosphoserine ⁄ phosphothreonine PPs, i.e. PP1, PP2A, PP2B, PP2C and PP5, which are highly expressed in mammalian brains. All of these phosphatases, except PP2C, dephosphorylate tau in vitro and possibly in vivo as well (for review, see Gong et al., 2005). It has been reported that the expressions and activities of some phosphatases are decreased in the affected areas of AD brain (Gong et al., 1993, 1995; Lian et al., 2001; Loring et al., 2001; Vogelsberg- Ragaglia et al., 2001; Sontag et al., 2004; Liu et al., 2005). These observations suggest that a downregulation of tau phosphatases in AD brain might underlie the abnormal hyperphosphorylation of tau and other neuronal proteins. However, the exact contribution of each of these phosphatases in regulating tau phosphorylation is not known. In addition, each phosphatase catalyses dephosphorylation of tau at different phosphorylation sites with different efficiencies. Because the impact of tau phosphorylation on its biological activity, on its gain of toxic function and on its polymerization into PHFs is site-specific, understanding the role of each phosphatase in the regulation of tau phosphorylation at individual phosphorylation sites is critical. In the present study, we carried out quantitative and kinetic analyses, and elucidated for the first time the relative contributions of PP1, PP2A, PP2B and PP5 in the regulation of tau phosphorylation and in the abnormal hyperphosphorylation of tau in AD brain. Materials and methods Materials The longest isoform of human brain tau (tau 441 ), cyclin-dependent kinase 5 and its activator p25 (cdk5 ⁄ p25), and rat PP5 were cloned, expressed and purified from Escherichia coli, as described previously Correspondence: Dr C.-X. Gong or Dr F. Liu, as above. E-mail: cxgong@ultinet.net; feiliu63@hotmail.com Received 7 July 2005, revised 10 August 2005, accepted 18 August 2005 European Journal of Neuroscience, Vol. 22, pp. 1942–1950, 2005 ª Federation of European Neuroscience Societies doi:10.1111/j.1460-9568.2005.04391.x