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Blood, Vol. 92 No. 9 (November 1), 1998:
pp. 3042-3049
RAPID COMMUNICATION
Phosphorylated Forms of Activated Caspases Are Present in Cytosol From
HL-60 Cells During Etoposide-Induced Apoptosis
Luis M. Martins,
Timothy J. Kottke,
Scott H. Kaufmann, and
William C. Earnshaw
From the Institute of Cell & Molecular Biology, University of
Edinburgh, Edinburgh, UK; and the Division of Oncology Research, Mayo
Clinic, Rochester, MN.
Treatment of HL-60 human leukemia cells with etoposide induces
apoptotic cell death and activation of at least 18 electrophoretically distinct 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 phosphorylated. Phosphatase treatment of cytosolic extracts containing active caspases followed by affinity labeling with
N-(N -benzyloxycarbonylglutamyl-N -biotinyllysyl)aspartic
acid [(2,6-dimethylbenzoyl)oxy] methyl ketone (Z-EK(bio)D-aomk)
showed a mobility shift in several of the labeled species, suggesting
that phosphorylated forms of these enzymes are present in the extracts.
Metabolic labeling with 32P followed by etoposide treatment
and subsequent affinity purification of affinity-labeled caspases
confirmed that at least three caspase species were
phosphorylated. To detect effects of the phosphorylation on enzymatic
activity, caspase-mediated cleavage of
aspartylglutamylvalinylaspartyl-7-amino-4-trifluoromethylcoumarin (DEVD-AFC) and poly(ADP-ribose) polymerase (PARP) by phosphorylated and
dephosphorylated extracts was measured. No significant changes in
Km or vmax were detected using DEVD-AFC. In
contrast, a slight, but significant enhancement of PARP cleavage was
observed in dephosphorylated extracts, suggesting that phosphorylation
of active caspases could have an inhibitory effect on enzyme activity.
These observations, which provide the first evidence that caspases are
phosphoproteins, suggest that caspases may be targets for some of the
growing list of protein kinases that are involved in apoptotic events.
© 1998 by The American Society of Hematology.
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