LAMIN-B PHOSPHORYLATION BY PROTEIN-KINASE-C-ALPHA AND PROTEOLYSIS DURING APOPTOSIS IN HUMAN LEUKEMIA HL60 CELLS

Protein phosphorylation plays an important role in signal transduction , but its involvement in apoptosis still remains unclear. In this repo rt, the p53-null human leukemia HL60 cells were used to investigate ph osphorylation and degradation of lamin B during apoptosis. We found th at lamin B was phosphorylated within I h after addition of the DNA top oisomerase I inhibitor, camptothecin, and that lamin B phosphorylation preceded lamin B degradation and DNA fragmentation. Using a cell-free system we also found that cytosol from camptothecin-treated cells ind uced lamin B phosphorylation and degradation in isolated nuclei from u ntreated HL60 cells. Lamin B phosphorylation was prevented by the prot ein kinase C (PKC) inhibitor 7-hydroxystaurosporine (UCN-01) but not b y the Cdc2 inhibitor, flavopiridol. Phosphorylation of lamin B was inh ibited by immunodepletion of PKC alpha from activated cytosol and was restored by addition of purified PKC alpha. PKC alpha activity also in creased rapidly as lamin B was phosphorylated after initiation of the apoptotic response in HL60 cells. These data suggest that lamin B is p hosphorylated by PKC alpha and proteolyzed before DNA fragmentation in HL60 cells undergoing apoptosis.