PHOSPHORYLATED FORMS OF ACTIVATED CASPASES ARE PRESENT IN CYTOSOL FROM HL-60 CELLS DURING ETOPOSIDE-INDUCED APOPTOSIS

Treatment of HL-60 human leukemia cells with etoposide induces apoptot ic cell death and activation of at least 18 electrophoretically distin ct cysteine-dependent aspartate-directed protease (caspase) isoforms, several of which differ only in their isoelectric points. The purpose of the present study was to determine whether activated caspases are p hosphorylated, Phosphatase treatment of cytosolic extracts containing active caspases followed by affinity labeling with ycarbonylglutamyl-N -epsilon-biotinyllysyl)aspartic acid [(2,6-dimethylbenzoyl)oxy] methyl ketone (Z-EK(bio)D-aomk) showed a mobility shift in several of the la beled species, suggesting that phosphorylated forms of these enzymes a re present in the extracts. Metabolic labeling with P-32 followed by e toposide treatment and subsequent affinity purification of affinity-la beled caspases confirmed that at least three caspase species were phos phorylated. To detect effects of the phosphorylation on enzymatic acti vity, caspase-mediated cleavage of lvalinylaspartyl-7-amino-4-trifluor omethylcoumarin (DEVD-AFC) and poly(ADP-ribose) polymerase (PARP) by p hosphorylated and dephosphorylated extracts was measured. No significa nt changes in K-m or v(max) were detected using DEVD-AFC. In contrast, a slight, but significant enhancement of PARP cleavage was observed i n dephosphorylated extracts, suggesting that phosphorylation of active caspases could have an inhibitory effect on enzyme activity. These ob servations, which provide the first evidence that caspases are phospho proteins, suggest that caspases may be targets for some of the growing list of protein kinases that are involved in apoptotic events. (C) 19 98 by The American Society of Hematology.