Nucleolin is a protein kinase C-ζ substrate -: Connection between cell surface signaling and nucleus in PC12 cells

We have previously shown that protein kinase C (PKC)-zeta is activated and required for nerve growth factor (NGF)-induced differentiation of rat pheochromocytoma PC12 cells (Wooten, M, W,, Zhou, G., Seibenhener M. L,, and Coleman, E. S, (1994) Cell Growth & Diff. 5, 395-403; Coleman, E, S,, and Wooten, M, W, (1994) J, Mol, Neurosci, 5, 39-57), Here we report the characterization and identification of a 106-kDa nuclear protein as a specific substrate of PKC-zeta, NGF treatment of PC12 cells resulted in translocation of PKC-zeta and coincident phosphorylation of a protein that was localized within the nucleoplasm of nuclei isolated from PC12 cells, Addition of PKC-zeta pseudosubstrate peptide in vitro or myristoylated peptide in vivo diminished phosphorylation of pp106 in a dose-dependent fashion, Likewise, addition of purified PKC-zeta, but neither PKC-alpha nor delta, to nuclear extracts resulted in an incremental increase in the phosphorylation of pp106, Expression of dominant-negative PKC-zeta inhibited NGF-induced phosphorylation of pp106, by comparison overexpression of PKC-zeta enhanced basal phosphorylation without a noticeable effect upon NGF-induced effects, Amino acid sequence analysis of four peptides derived from purified pp106 revealed that this protein was homologous to nucleolin, Using an in vitro reconstitution system, purified nucleolin was likewise shown to be phosphorylated by purified PKC-zeta. The staining intensity of both enzyme and substrate in the nucleus increased upon treatment with NGF, In vivo labeling with P-32(i) and stimulation of PC12 cells with NGF followed by immunoprecipitation with anti-nucleolin antibody corroborated the in vitro approach documenting enhanced phosphorylation of nucleolin by NGF treatment, Taken together, the findings presented herein document that nucleolin is a target of PKC-zeta that serves to relay NGF signals from cell surface to nucleus in PC12 cells.