Figure 5.

Human Par-4 is phosphorylated both in vitro and in vivo on S231 (ortholog of rodent S223) by CK2 and this phosphorylation impairs apoptosis. (a) Conservation of the CK2 recognition motif (including the serine 231 of human Par-4) was evaluated in different species. (b) Recombinant Myc-tagged human Par-4 (hPar-4) or 231D mutant proteins were produced with TNT RRL system. An in vitro kinase assay was performed with the immunoprecipitates as substrates, in the presence of recombinant CK2 and [γ−32P]ATP. Phosphorylated Par-4 was detected by autoradiography. Production of recombinant Myc tagged proteins were checked by western blotting (right panel). (c) Phosphorylation of endogenous Par-4 was detected by western blotting (left panel), using the human anti-phosphoserine231-Par-4 antibody (Ph231 hPar-4), in PC-3 cells treated (+) or not (−) with TRAIL for 9 h, and densitometry analysis was done. The blocking peptide was used in order to test the specificity of the phospho-antibody. In parallel, the percentage of apoptosis induced by TRAIL (9 h) was assessed by Hoetsch staining. Bars represent the mean±S.D. of at least three independent experiments (right panel). (d–f) HCT116 cells were transfected with empty Myc vector control (Mock), human Myc-tagged Par-4, 231A or 231D mutant Par-4 (mimicking unphosphorylated and phosphorylated hPar-4, respectively) and then treated with recombinant TRAIL (150 ng/ml, 3 h). Expression of the different constructs was assessed by western blotting using Myc antibody, and apoptosis was monitored by immunoblot analysis of caspase-8 and PARP cleavage (d) and by DAPI staining (e and f). Bar graph shows semi-quantified densitometry from PARP and Caspase-8 western blotting analysis. Bars represent the mean±S.D. of at least four independent experiments. Hsp90 was used as a loading control. Bar=10 μm, magnification × 63; *P<0.05. a.u, arbitrary unit