Figure 3.
Mapping of Plk1 phosphorylation sites in procaspase‐8. (A) Scheme of procaspase‐8 and its subdomains (left panel). Bacterially expressed, purified GST‐fused N‐terminal (GST‐NT) and C‐terminal (GST‐CT) subdomains of procaspase‐8 were incubated with Plk1 and [γ‐32P]ATP, analyzed by SDS‐PAGE, and visualized by autoradiography (left panel). This was followed by phospho amino acid analysis (right panel). Recombinant GST‐fused subdomains of procaspase‐8 were analyzed by SDS‐PAGE (lower left panel). Relative radioactive signal intensity of GST‐fused N‐terminal (GST‐NT) and C‐terminal (GST‐CT) subdomains of caspase‐8 phosphorylated by Plk1 is depicted as bar graph (lower right panel). (B) Bacterially expressed, purified GST‐fused N‐terminal (GST‐NT) and C‐terminal (GST‐CT) subdomains of procaspase‐8 were incubated with Plk1 or Cdk1/cyclin B1 and [γ‐32P]ATP, analyzed by SDS‐PAGE, and visualized by autoradiography. (C) Wild‐type (WT) and mutated (S217A, S219A, S230A, S305A, S317A, S347A) forms of the p18 subdomain of caspase‐8 were subjected to kinase assays with Plk1 including [γ‐32P]ATP, analyzed by SDS‐PAGE, and autoradiography. (D) Comparative phosphorylation of the mutated C‐terminal domain (CT) of procaspase‐8 including p18 and p10 (S305A, S387A) in kinase assays including [γ‐32P]ATP using Plk1 or Cdk1/cyclin B1, analyzed by SDS‐PAGE, and autoradiography. (E) Wild‐type (WT) and mutated (S387A, S305A) recombinant full‐length GST‐caspase‐8 proteins were subjected to kinase assays using Plk1 including [γ‐32P]ATP, analyzed by SDS‐PAGE, and autoradiography (F) Caspase‐8‐depleted HeLa cells were transfected with expression vectors for wild‐type caspase‐8 or caspase‐8 (S305A), fixed and costained with antibodies for caspase‐8 (pS305). DNA was stained with DAPI (4′,6‐diamidino‐2‐phenylindole). Scale bar, 10μm. (G) Cells were transfected with Flag‐Casp8 WT or Flag‐Casp8 (S305A) and treated with or without nocodazole. Lysates were prepared and subjected to anti‐Flag antibody IP, followed by western blot analysis with an antiphospho‐serine antibody. The same membrane was stripped and probed with an anti‐Flag antibody (left panel). Cells were transfected with siRNActrl or siRNA‐Plk1, 24h later followed by a transfection with Flag‐Casp8 WT or Flag‐Casp8 (S305A) and treated with nocodazole. Lysates were prepared and subjected to anti‐Flag antibody IP, followed by western blot analysis with an antiphospho‐serine antibody. The same membrane was stripped and probed with anti‐Flag and anti‐Plk1‐antibodies (right panel).