Y465 phosphomimetic mutation inhibits caspase-8A cleavage.

<p>A) Upon activation, caspase-8 is autocatalytically cleaved first after D391, and then after D233. Using an anti-GFP antibody that binds to the N-terminus of the molecule, a 68-kDa fragment is visualized when GFP-caspase-8A is cleaved after D391, whereas a 50-kDa fragment is visualized when GFP-caspase-8A is cleaved after D233. B) Using an anti-caspase-8 antibody that binds to the C-terminus of the molecule, a 30-kDa fragment is visualized when GFP-caspase-8A is cleaved after D233, whereas a 12-kDa fragment is visualized when GFP-caspase-8A is cleaved after D391. A 55-kDa band represents endogenous caspase-8. C) HEK293 cells were transfected with GFP-caspase-8A WT or mutants for 24 hours. Whole cell lysates were subjected to Western blot analysis with anti-GFP antibody that recognizes the N-terminal end of GFP-caspase-8A. D) Whole cell lysates were subjected to Western blot analysis with anti-caspase-8 antibody that recognizes the C-terminal end of GFP-caspase-8A. E) Both the inactive C377S and Y465E GFP-caspase-8A mutants failed to undergo cleavage at D391, the essential step of the activation of caspase-8. F-H) HEK293 cells were transfected with GFP-caspase-8B (F), Myc-caspase-8B (G), GFP-caspase-8A (H) WT or mutants for 24 hours. Whole cell lysates were subjected to Western blot analysis with anti-GFP antibody that recognizes the N-terminal end of GFP-caspase-8 and anti-Myc antibody that recognizes the C-terminal end of GFP-caspase-8 (active caspase-8).