CELL CYCLE-DEPENDENT PHOSPHORYLATION AND MICROTUBULE-BINDING OF TAU-PROTEIN STABLY TRANSFECTED INTO CHINESE-HAMSTER OVARY CELLS

Tau protein, a neuronal microtubule-associated protein, is phosphoryla ted in situ and hyperphosphorylated when aggregated into the paired he lical filaments of Alzheimer's disease. To study the phosphorylation o f 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-l, AT8, AT180, and PHF-1, which are sensitive to the phosphorylation of S er202, Thr205, Thr231, Ser235, Ser396, and Ser404 and are used in the diagnosis of Alzheimer tau. In interphase cells, tau becomes phosphory lated to some extent partly at these sites; most of the tau is associa ted with microtubules. In mitosis, the above Ser/Thr-Pro sites become almost completely phosphorylated, causing a pronounced shift in M(r) a nd 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 m ore highly phosphorylated during mitosis. The understanding of tau pho sphorylation under physiological conditions might help elucidate possi ble mechanisms for the hyperphosphorylation in Alzheimer's disease.