To establish whether protein kinase C was involved in the nuclear events underlying cell differentiation and proliferation, rat pheochromocytoma PC12 cells, serum-starved for 24 h, were treated with either differentiating doses of nerve growth factor or high serum concentrations, which represented a powerful mitogenic stimulus. Western blot analysis with isoform-specific antibodies, performed on whole cell homogenates, cytoplasms, and purified nuclei, showed that PKC isotypes α, βI, βII, δ, ε, η, and ζ were expressed in PC12 cells and that all of them, except for βI, were found at the nuclear level, variably modulated depending on the cell treatment. Compared to serum-stimulated cells, in which an early (1 day) and marked rise of protein kinase C activity was followed by a plateau, nerve growth factor- treated cells showed a progressive increase of protein kinase C activity coincident with the onset and maintenance of the differentiated phenotype. Western blot analysis of nuclei isolated from fully differentiated cells demonstrated an increase of protein kinase C α, paralleled by enhanced phosphotransferase activity along with the nerve growth factor treatment, and complete loss of the δ isotype. In contrast, in nuclei of proliferating PC12 cells, after an early but modest increase at 1 day of mitogenic stimulation, protein kinase C activity reached a plateau. Isotype-specific analysis indicated a concomitant increase of protein kinase C βII, δ, and ζ and the appearance of protein kinase C ε and η at the nuclear level. Considering the relative intensity of the cytoplasmic and nuclear immunoreactive bands under the three conditions examined, clear-cut translocation to the nucleus occurred for PKC ε and η in serum-stimulated cells. Additional nuclear accumulation of PKC by translocation from the cytoplasm was prominently induced for the ζ isoform after mitogenic stimulation and for PKC α during prolonged NGF treatment. Our data suggest that nuclear translocation and selective activation of distinct protein kinase C isoforms play a relevant role in the control of proliferation and differentiation of the same cell type and that nuclear protein kinase C is crucial to the induction and persistence of the differentiated neuronal phenotype of PC12 cells.
ASJC Scopus subject areas
- Cell Biology