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, William C. Earnshaw

Research output: Contribution to journalArticle

47 Scopus citations

Abstract

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-(Nα-benzyloxycarbonylglutamyl-N(ε)- 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 K(m) or V(max) 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.

Original languageEnglish (US)
Pages (from-to)3042-3049
Number of pages8
JournalBlood
Volume92
Issue number9
StatePublished - Nov 1 1998

ASJC Scopus subject areas

  • Biochemistry
  • Immunology
  • Hematology
  • Cell Biology

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