LOCALIZATION AND IN-SITU PHOSPHORYLATION STATE OF NUCLEAR-TAU

The localization and phosphorylation state of tau in LA-N-5 neuroblast oma cells was examined Our results demonstrate that there are two popu lations of tau in LA-N-5 cells: cytosolic tau and nuclear tau Indirect immunofluorescent microscopy revealed that nuclear tau is specificall y localized to the nucleolus while cytosolic tau is diffusely distribu ted, To localize and quantitate tau in LA-N-5 cells by subcellular fra ctionation, a method was developed to extract tau from the nucleus whi le preserving the endogenous state of the protein. These studies revea led that 16% of the total tau protein in LA-N-5 cells is located in th e nucleus and more specifically was found predominantly in the chromat in fraction containing DNA, chromatin, and associated proteins. The ph osphorylation state of nuclear and cytosolic tau was examined by label ing LA-N-5 cells with P-32(i) and immunoprecipitating tau from the dif ferent fractions. These data demonstrated that nuclear tau and cytosol ic tau are phosphorylated approximately to the same extent. To determi ne if the phosphorylation of nuclear tau occurs in the nucleus, LA-N-B nuclei were isolated, incubated with [gamma-P-32]ATP, extracted, and tau was immunoprecipitated. Although numerous nuclear proteins were P- 32-labeled, tau was not phosphorylated. These results suggest that nuc lear tau is not phosphorylated in the nucleus but rather in the cytoso l prior to transport into the nucleus. The specific localization of nu clear tau strongly suggests that it has a functional role in the nucle us. However, further studies are necessary to determine the function o f nuclear tau and how it may be regulated by phosphorylation. (C) 1995 Academic Press, Inc.