NUCLEOLIN IS A PROTEIN-KINASE C-ZETA 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; Cole man, E, S,, and Wooten, M, W, (1994) J, Mol, Neurosci, 5, 39-57), Here we report the characterization and identification of a 106-kDa nuclea r protein as a specific substrate of PKC-zeta, NGF treatment of PC12 c ells resulted in translocation of PKC-zeta and coincident phosphorylat ion of a protein that was localized within the nucleoplasm of nuclei i solated from PC12 cells, Addition of PKC-zeta pseudosubstrate peptide in vitro or myristoylated peptide in vivo diminished phosphorylation o f pp106 in a dose-dependent fashion, Likewise, addition of purified PK C-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 o f pp106, by comparison overexpression of PKC-zeta enhanced basal phosp horylation without a noticeable effect upon NGF-induced effects, Amino acid sequence analysis of four peptides derived from purified pp106 r evealed that this protein was homologous to nucleolin, Using an in vit ro reconstitution system, purified nucleolin was likewise shown to be phosphorylated by purified PKC-zeta. The staining intensity of both en zyme and substrate in the nucleus increased upon treatment with NGF, I n vivo labeling with P-32(i) and stimulation of PC12 cells with NGF fo llowed by immunoprecipitation with anti-nucleolin antibody corroborate d the in vitro approach documenting enhanced phosphorylation of nucleo lin by NGF treatment, Taken together, the findings presented herein do cument that nucleolin is a target of PKC-zeta that serves to relay NGF signals from cell surface to nucleus in PC12 cells.