THE CELL CYCLE-DEPENDENT NUCLEAR IMPORT OF V-JUN IS REGULATED BY PHOSPHORYLATION OF A SERINE ADJACENT TO THE NUCLEAR-LOCALIZATION SIGNAL

Cell cycle-dependent phosphorylation and nuclear import of the tumorig enic transcription factor viral Jun (v-Jun) were investigated in chick en embryo fibroblasts. Nuclear accumulation of v-Jun but not of cellul ar Jun (c-Jun) is cell cycle dependent, decreasing in G1 and increasin g in G2. The cell cycle-dependent regulation of v-Jun was mapped to a single serine residue at position 248 (Ser(248)), adjacent to the nucl ear localization signal (NLS). Ser(248) Of v-Jun represents an amino a cid substitution, replacing cysteine of c-Jun. It was shown by peptida se digestion and immunoprecipitation with antibody to the NLS that v-J un is phosphorylated at Ser(248) in the cytoplasm but not in the nucle us. This phosphorylation is high in G1 and low in G2. Nuclear accumula tion of v-Jun is correlated with underphosphorylation at Ser(248). The regulation of nuclear import by phosphorylation was also examined usi ng NLS peptides with Ser(248) of v-Jun. Phosphorylation of the serine inhibited nuclear import mediated by the NLS peptide in vivo and in vi tro. The protein kinase inhibitors staurosporine and H7 stimulated but the phosphatase inhibitor okadaic acid inhibited nuclear import media ted by the NLS peptide. The cytosolic activity of protein kinases phos phorylating Ser(248) increased in G0 and decreased during cell cycle p rogression, reaching a minimum in G2, whereas phosphatase activity dep hosphorylating Ser(248) was not changed. These results show that nucle ar import of v-Jun is negatively regulated by phosphorylation at Ser(2 48) in the cytoplasm in a cell cycle-dependent manner.