| Popis: |
In human promyelocytic (HL60) leukemia cells beta II protein kinase C (PKC) is selectively translocated to the nucleus in response to proliferative stimuli. At the nucleus, beta II PKC directly phosphorylates the nuclear envelope polypeptide lamin B at two consensus PKC phosphorylation sites, Ser395 and Ser405. Phosphorylation of these sites by beta II PKC leads to solubilization of lamin B indicative of mitotic 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 investigated the molecular basis for beta II PKC-selective nuclear translocation and lamin B phosphorylation using an in vitro reconstitution system. We find that beta II PKC phosphorylates nuclear envelope lamin B at 10-20 times the rate of alpha PKC, whereas both kinases phosphorylate soluble lamin B at similar rates. Comparative tryptic phosphopeptide analysis demonstrates that alpha PKC and beta II PKC phosphorylate identical sites, Ser395 and Ser405, on soluble lamin B. These data suggest that a component(s) of the nuclear envelope confers beta II PKC-selective nuclear activation and lamin B phosphorylation. Extraction of nuclear envelopes with either non-ionic detergent (2% n-octyl glucoside) or organic solvent (CHCl3/CH3OH/H2O; 10:10:3) abolishes beta II PKC-selective phosphorylation of nuclear lamin B. Nuclear membrane extracts reconstitute beta II PKC-selective phosphorylation, indicating the presence of a beta II PKC-selective nuclear membrane activation factor (NMAF). NMAF selectively activates beta II PKC histone H1 kinase activity 3-4-fold above the level achieved with optimal concentrations of Ca2+, diacylglycerol, and phosphatidylserine. Finally, NMAF activity is not affected by exhaustive protease treatment, suggesting that it is a nuclear membrane lipid(s) or lipid metabolite. These data suggest that NMAF plays a physiologic role in the nuclear activation of beta II PKC. |
