Figure 6

Caspase-8-Y380 phosphorylation attenuates CD95 DISC activity in cell lysates but not in a lysate-free system. (a) Model of caspase-8 derived from a solution structure of the monomeric unprocessed catalytic domain of the caspase-8 zymogen (PDB: 2K7Z). The active site cysteine 360 has been modelled to replace the original structure’s alanine residue, highlighted in yellow; tyrosine-380 and modelled phosphate are highlighted in red and pink, respectively. (b) (i) Schematics depicting two possible inhibition mechanisms, either an unknown binding partner interacts with the phosphorylated tyrosine, thereby sterically hindering the active site or causing a conformational change, and/or the phosphorylated linker directly interacts with and inhibits the caspase-8 active site. (ii) CD95-IcD pull-downs from Jurkat caspase-8 null cell lysates or the lysate-free fully reconstituted DISC (r-DISC) with CD95-IcD, r-FADD (5 μg), both supplemented with ³⁵S-labelled recombinant procaspase-8b. Beads were analysed for FADD, caspase-8 and active caspase-8 (D384). DISC activity was further bioassayed for r-procaspase-3 (C163A) cleavage.