Figure 4.

Rodent Par-4 phosphorylation by CK2 prevents caspase-mediated Par-4 cleavage. (a) PC-3 cells transfected with the GFP-tagged constructs wild-type rat Par-4, 124A/D, 223A/D, 124A223A or 124D223D mutants of Par-4 were treated with TRAIL (500 ng/ml, 3 h) in the presence or absence of Z-VAD (15 μM). Par-4 cleavage was determined by western blotting using anti-GFP antibody. Hsp90 was used as a loading control. (b) Wild-type Par-4, 124A/D, 223A/D, 124D223D and 124A223A mutant proteins (GFP tagged) were produced in vitro by a TNT RRL system. Recombinant caspase-8 (upper panel) or caspase-3 and -7 (Casp3, Casp7, lower panel) were incubated with the indicated Par-4 proteins for 3 h at 37 °C, and Par-4 cleavage was assessed by western blotting using GFP antibody. (c) Caspase assay was performed on pre-phosphorylated rat GST-Par-4 (in vitro kinase assay using RecCK2 as described above Figure 2), and cleaved Par-4 was detected by immunoblot. *NS: non-specific bands are probably due to the presence of recombinant Par-4 degraded forms. To note: cleaved GST Par-4 migrates at lower molecular weight than cleaved GFP-Par-4, given GST tag is located in the N-terminus of Par-4 and not on C-terminus as the GFP-tag. (d–f) PC-3 cells transfected with Par-4 wild-type or FLAG-tagged Par-4 (124–332) were treated as above with TRAIL and analyzed for apoptosis by Hoechst staining (d) or FACS analysis of the caspase-3 activity (FLICA) (e) and by immunoblotting analysis of caspase-8 and PARP cleavage (f). Bars represent the mean±S.D. of at least three independent experiments. Flag-Par-4 (124–332) and GFP were revealed in the same membrane using corresponding antibodies (lower panel). Endogenous Par-4 was used as a loading control. *P<0.05, **P<0.01