Identification of phosphorylation sites on caspase-8 and caspase-3. (A) Active phosphorylated p38-MAPK immunoprecipitates from freshly isolated neutrophils were incubated with [γ-32P]ATP and recombinant procaspase-8 or procaspase-3. The proteins were separated by SDS–gel electrophoresis, and the separated proteins were digested in situ with trypsin. The obtained phosphopeptides were separated on cellulose TLC glass plates (elect.), followed by ascending chromatography (chrom.). The indicated electrophoresis direction is from the anode to the cathode. The plates were analyzed in a PhosphorImager as well as exposed to an X-ray film. (B) The phosphopeptides from caspase-8 or caspase-3 were eluted from the TLC plates and subjected to two-dimensional phosphoamino acid analysis. The locations of the phosphoamino acids (top) were compared with that of phosphoamino acid markers (bottom) as follows: serine (S), threonine (T), and tyrosine (Y). The phosphopeptides obtained from A were subjected to amino acid sequencing (C), and the radioactivity released in each cycle was measured by spotting onto TLC plates and exposure on a Fuji image analyzer. The phosphorylated serine residues, 364 for caspase-8 (C) and 150 for caspase-3 (C), are indicated in the sequence of the putative fragment from caspase-8 and caspase-3, respectively. The illustrated phosphomapping is representative of three experiments.
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