Cell cycle-dependent phosphorylation and microtubule binding of tau protein stably transfected into Chinese hamster ovary cells

• Published in: Molecular biology of the cell 1995-10, Vol.6 (10), p.1397-1410
• Main Authors: Preuss, U, Döring, F, Illenberger, S, Mandelkow, E M
• Format: Article
• Language: English
• Subjects: Animals; Cell Cycle; CHO Cells; Cricetinae; Humans; Microtubules - chemistry; Microtubules - metabolism; Mitosis; Phosphorylation; Proline - metabolism; Protein Binding; Serine - metabolism; tau Proteins - analysis; tau Proteins - metabolism; Threonine - metabolism; Transfection
• ISSN: 1059-1524; 1939-4586
• DOI: 10.1091/mbc.6.10.1397

Tau protein, a neuronal microtubule-associated protein, is phosphorylated in situ and hyperphosphorylated when aggregated into the paired helical filaments of Alzheimer's disease. To study the phosphorylation of tau protein in vivo, we have stably transfected htau40, the largest human tau isoform, into Chinese hamster ovary cells. The distribution and phosphorylation of tau was monitored by gel shift, autoradiography, immunofluorescence, and immunoblotting, using the antibodies Tau-1, AT8, AT180, and PHF-1, which are sensitive to the phosphorylation of Ser202, Thr205, Thr231, Ser235, Ser396, and Ser404 and are used in the diagnosis of Alzheimer tau. In interphase cells, tau becomes phosphorylated to some extent, partly at these sites; most of the tau is associated with microtubules. In mitosis, the above Ser/Thr-Pro sites become almost completely phosphorylated, causing a pronounced shift in M(r) and an antibody reactivity similar to that of Alzheimer tau. Moreover, a substantial fraction of tau is found in the cytoplasm detached from microtubules. Autoradiographs of metabolically labeled Chinese hamster ovary cells in interphase and mitosis confirmed that tau protein is more highly phosphorylated during mitosis. The understanding of tau phosphorylation under physiological conditions might help elucidate possible mechanisms for the hyperphosphorylation in Alzheimer's disease.