PRESENCE OF A BETA(II) PROTEIN-KINASE C-SELECTIVE NUCLEAR-MEMBRANE ACTIVATION FACTOR IN HUMAN LEUKEMIA-CELLS

In human promyelocytic (HL60) leukemia cells beta(II) protein kinase C (PKC) is selectively translocated to the nucleus in response to proli ferative stimuli. At the nucleus, beta(II) PKC directly phosphorylates the nuclear envelope polypeptide lamin B at two consensus PKC phospho rylation sites, Ser(395) and Ser(405). Phosphorylation of these sites by beta(II) PKC leads to solubilization of lamin B indicative of mitot ic nuclear envelope breakdown in vitro (Hocevar, B. A., Burns, D. J., and Fields, A. P. (1993) J. Biol. Chem. 268, 7545-7552). We have now i nvestigated the molecular basis for beta(II) PKC-selective nuclear tra nslocation and lamin B phosphorylation using an in vitro reconstitutio n system. We find that beta(II) PKC phosphorylates nuclear envelope la min B at 10-20 times the rate of alpha PKC, whereas both kinases phosp horylate soluble lamin B at similar rates. Comparative tryptic phospho peptide analysis demonstrates that alpha PKC and beta(II) PKC phosphor ylate identical sites, Ser(395) and Ser(405), on soluble lamin B. Thes e data suggest that a component(s) of the nuclear envelope confers bet a(II) PKC-selective nuclear activation and lamin B phosphorylation. Ex traction of nuclear envelopes with either non-ionic detergent (2% n-oc tyl glucoside) or organic solvent (CHCl3/ CH3OH/H2O; 10:10:3) abolishe s beta(II) PKC-selective phosphorylation of nuclear lamin B. Nuclear m embrane extracts reconstitute beta(II) PKC-selective phosphorylation, indicating the presence of a beta(II) PKC-selective nuclear membrane a ctivation factor (NMAF). NMAF selectively activates beta(II) PKC histo ne H1 kinase activity 3-4-fold above the level achieved with optimal c oncentrations of Ca2+, diacylglycerol, and phosphatidylserine. Finally , NMAF activity is not affected by exhaustive protease treatment, sugg esting that it is a nuclear membrane lipid(s) or lipid metabolite. The se data suggest that NMAF plays a physiologic role in the nuclear acti vation of beta(II) PKC.