Figure 2.
Phosphorylation of S387 stimulates the interaction of procaspase‐8 and Plk1. (A) Bacterially expressed, purified GST‐fused N‐terminal (GST‐NT) and C‐terminal (GST‐CT) subdomains of procaspase‐8 were incubated with lysates of HEK 293T cells that had been transfected with a Flag epitope‐tagged Plk1 or a Flag epitope‐tagged Plk1‐PBD expression construct for pulldown assays. The Flag‐Plk1 or Flag‐Plk1‐PBD protein that associated with GST‐fused subdomains of caspase‐8 was detected by immunoblot with an anti‐Flag antibody. (B) Bacterially expressed, purified GST‐fused Plk1‐PBD was incubated with lysates of HEK 293T cells that had been transfected with a GFP‐tagged caspase‐8 expression construct for pulldown assays. The GFP‐caspase‐8 protein that associated with GST‐fused Plk1 was detected by immunoblot with an anti‐GFP antibody. (C) The putative PBD binding motif in procaspase‐8 is depicted. (D) Bacterially expressed, purified GST‐fused Plk1‐PBD or GST alone were incubated with lysates of HEK 293T cells that had been transfected with expression constructs for Flag‐tagged caspase‐8 or caspase‐8 mutants (S387A, S387E) for pulldown assays. The Flag‐tagged caspase‐8 protein that associated with GST‐fused PBD was detected by immunoblot with an anti‐Flag antibody. (E) Bacterially expressed, purified GST‐fused caspase‐8 or caspase‐8 mutants (S387A, S387E) were first pre‐incubated with Cdk1/cyclin B1 in a cold kinase assay for subsequent incubation with lysates of HEK 293T cells that had been transfected with expression constructs for Flag‐tagged PBD for pulldown assays. The Flag‐tagged Plk1‐PBD that associated with GST‐fused caspase‐8 was detected by immunoblot with an anti‐Flag antibody. (F) Untreated HeLa cells (top) or treated with nocodazole for 12h. Cells were fixed and costained with antibodies for Plk1 and caspase‐8‐pS387. DNA was stained with DAPI (4′,6‐diamidino‐2‐phenylindole). Scale bar, 10μm.