Cell 150, 429–440 (2012)

Credit: GABRIEL M. SIMON

Caspases are proteases that orchestrate programmed cell death. Substrates for caspases include kinases, and although it is known that cleavage of a kinase can activate or deactivate its enzymatic activity, a systems-wide view of the relationship between caspase-mediated proteolysis in apoptosis and phosphorylation is lacking. Dix et al. now report a proteomic method—combining PROTOMAP, which is used to characterize proteolytic events in cells, with SILAC, which is a quantitative, isotopic in-cell labeling method—called quantitative phospho-PROTOMAP (qP-PROTOMAP) to address this knowledge gap. After validation of the qP-PROTOMAP method, the authors assessed crosstalk between caspase activity and phosphorylation in staurosporine (STS)-induced cell death. The authors compiled all known caspase cleavage sites, aligned by their scissile P1 aspartate, and found that many phosphorylation sites associated with apoptosis clustered within six residues of this cleavage site. An ATP-binding activity–based proteomic analysis revealed that DNA-dependent protein kinase (DNA-PK) had strong activity in STS-treated cells. DNA-PK–selective inhibitors or short hairpin RNA–mediated DNA-PK knockdown blunted phosphorylation at these sites. The authors found that caspase-dependent relocalization of DNA-PK from the nucleus to the cytoplasm was necessary for phosphorylation adjacent to caspase cleavage sites during STS-induced apoptosis. In addition, the authors found that phosphorylation and caspase cleavage are interrelated during apoptosis, with caspase cleavage exposing phosphoryation sites and phosphorylation promoting caspase cleavage. Taken together, these data indicate that large-scale functional crosstalk occurs between phosphorylation and caspase cleavage during apoptosis.