However, phosphorylation by Cdk5 at Ser396/404 does not cause tau mobility shift to the 68-kDa band (Fig

However, phosphorylation by Cdk5 at Ser396/404 does not cause tau mobility shift to the 68-kDa band (Fig. 3). from a group of neurodegenerative disorders collectively called tauopathies (2, 11). These disorders include frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17), corticobasal degeneration, progressive supranuclear palsy, and Pick disease. Each PHF-tau isolated from autopsied brains of patients suffering from various tauopathies is hyperphosphorylated, displays 60-, 64-, and 68-kDa bands on SDS-gel, and is incapable of binding to microtubules. Upon dephosphorylation, the above referenced PHF-tau migrates as a normal tau on SDS-gel, binds to microtubules, and promotes microtubule assembly (2, 11). These observations suggest that the mechanisms of NFT pathology in various tauopathies may be similar and the phosphorylation-dependent mobility YKL-06-061 shift of tau on SDS-gel may be an indicator of the disease. The tau gene is mutated in familial FTDP-17, and these mutations accelerate NFT pathology in the brain (15C18). Understanding how FTDP-17 mutations promote tau phosphorylation can provide a better understanding of how NFT pathology develops in AD and various tauopathies. However, when expressed in CHO cells, G272V, R406W, V337M, and P301L tau mutations reduce tau phosphorylation (19, 20). In COS cells, although G272V, P301L, and V337M mutations do not show any significant affect, the R406W mutation caused a reduction in tau phosphorylation (21, 22). When expressed in SH-SY5Y cells subsequently differentiated into neurons, the R406W, P301L, and V337M mutations reduce tau phosphorylation (23). In contrast, in hippocampal neurons, R406W increases tau phosphorylation (24). When phosphorylated by recombinant GSK3 DNA polymerase (Stratagene), with a forward primer (5-AAAAAACGCCATATGGCTGAGCCCCGC-3) that contained an NdeI site and a reverse primer (5-AAA AAA GGA TCC TCA CAA ACC CTG CTT GG-3) that contained a BamHI site, and subcloned into bacterial expression vector pET9a (Promega). Various double mutants, each containing the indicated FTDP-17 and S202A mutations, were cloned by PCR using their respective FTDP-17 mutant in pET9a vector as the template and the QuikChange II site-specific mutagenesis kit (Stratagene) following the manufacturer’s instruction manual. Primers used for PCR were 5-CAG CGG CTA CAG CAG CCC CGG CGC CCC AGG CAC TCC CGG CAG CCG C-3 and 5-GCG GCT GCC GGG AGT GCC TGG GGC GCC GGG GCT GCT GTA GCC GCT G-3. All cDNA Rabbit Polyclonal to PMS2 clones and mutations were confirmed by DNA sequencing. overexpressing their respective tau species essentially as described previously (28). Briefly, tau expression was induced by adding isopropyl 1-thio–d-galactopyranoside (0.2 mm) to the overnight bacterial culture. The culture containing isopropyl 1-thio–d-galactopyranoside was allowed to grow for 3 h at 37 C with shaking and then was centrifuged. The pellet was suspended in Pipes buffer (100 mm Pipes (pH 6.8), 1 mm EGTA, 1 mm MgSO4) containing 5 mg/ml benzamidine, 1 g/ml leupeptine, 1 g/ml pepstatin, 1 mm phenylmethylsulfonyl fluoride, and 20 g/ml lysozyme. YKL-06-061 The bacterial suspension was lysed by sonication and then clarified by centrifugation (15,000 rpm, 15 min at 4 C). The supernatant was placed in a boiling water bath for 20 min and subsequently centrifuged. The YKL-06-061 heat-stable proteins in the supernatant were loaded onto a Q-Sepharose Fast Flow column (1 ml; Amersham Biosciences) equilibrated previously in Pipes buffer. The flow-through containing tau was loaded onto an SP-Sepharose Fast Flow column (1.