Ubiquitin and ubiqutinated proteins in PC12 cells induced by NGF

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Changes in ubiquitin-immunoreactivity after nerve growth factor (NGF) treatment were investigated in PC12h cells. Ubiquitin-immunoreactivity was increased in the nucleus of NGF-treated cells. The quantitiy analysis revealed that, after 7days of NGF treatment, almost 20% of cells had ubiquitin-immunoeractive nuclei and the frequency was increased thereafter. Levels of free ubiquitin and multi-ubiquitin chains were measured by radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), respectively. Measurements were carried out for four subcellular fractions : urea-and water-soluble extracts of nuclei and cytoplasm. Decrease in free ubiquitin was observed in water-soluble cytoplasmic extracts of NGF-treated cells, though increase in multi-ubiquitin chains in the same fraction was not observed. As for nuclei, increase in multi-ubiquitin chains and concomitant decrease in free ubiquitin were found in the water-soluble extracts after NGF treatment. Lvels of multi-ubiquitin chains did not change in urea-soluble cytoplasmic extracts as well nuclear urea-soluble ones afer NGF treatment. These results indicated that multi-ubiquitination of nuclear proteins is increased during NGF-induced neuronal differentiation of PC12h cells.