title: Sequential activation of caspases and serine proteases (serpases) during apoptosis.
abstract: Analogous to caspases, serine (Ser) proteases are involved in protein degradation during apoptosis. It is unknown, however, whether Ser proteases are activated concurrently, sequentially, or as an alternative to the activation of caspases. Using fluorescent inhibitors of caspases (FLICA) and Ser proteases (FLISP), novel methods to detect activation of these enzymes in apoptotic cells, we demonstrate that two types of Ser protease sites become accessible to these inhibitors during apoptosis of HL-60 cells. The prior exposure to caspases inhibitor Z-VAD-FMK markedly diminished activation of both Ser protease sites. However, the unlabeled inhibitor of Ser-proteases TPCK had modest suppressive effect- while TICK had no effect- on the activation of caspases. Activation of caspases, thus, appears to be an upstream event and likely a prerequisite for activation of FLISP-reactive sites. Differential labeling with the red fluorescing sulforhodamine-tagged VAD-FMK and the green fluorescing FLISP allowed us to discriminate, within the same cell, between activation of caspases and Ser protease sites. Despite a certain degree of co-localization, the pattern of intracellular caspase- vs FLISP- reactive sites, was different. Also different were relative proportions of activated caspases vs Ser protease sites in individual cells. The observed induction of FLISP-binding sites we interpret as revealing activation of at least two different apoptotic Ser proteases; by analogy to caspases we denote them serpases. Their apparent molecular weight (62-65 kD) suggests that they are novel enzymes.
authors.affiliation: Brander Cancer Research Institute; New York Medical College; Valhalla, New York 10595, USA.
authors.forename: Jerzy
authors.initials: J
authors.lastname: Grabarek
authors.forename: Litong
authors.initials: L
authors.lastname: Du
authors.forename: Gary L
authors.initials: GL
authors.lastname: Johnson
authors.forename: Brian W
authors.initials: BW
authors.lastname: Lee
authors.forename: David J
authors.initials: DJ
authors.lastname: Phelps
authors.forename: Zbigniew
authors.initials: Z
authors.lastname: Darzynkiewicz
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000378:metabolism* / Q000494:pharmacology; D017209:Apoptosis; D001665:Binding Sites; D001667:Binding, Competitive; D002166:Camptothecin / Q000378:metabolism / Q000494:pharmacology; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism / Q000494:pharmacology; D004789:Enzyme Activation; D018922:HL-60 Cells; D006801:Humans; D012697:Serine Endopeptidases / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000378:metabolism* / Q000494:pharmacology; D059004:Topoisomerase I Inhibitors; D014107:Tosyllysine Chloromethyl Ketone / Q000378:metabolism / Q000494:pharmacology; D014108:Tosylphenylalanyl Chloromethyl Ketone / Q000378:metabolism / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: C469935:5(6)-carboxyfluoresceinylleucyl chloromethyl ketone; C469936:5(6)-carboxyfluoresceinylphenylalanyl chloromethyl ketone; D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D015842:Serine Proteinase Inhibitors; D059004:Topoisomerase I Inhibitors; D014409:Tumor Necrosis Factor-alpha; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D014107:Tosyllysine Chloromethyl Ketone; D014108:Tosylphenylalanyl Chloromethyl Ketone; D012697:Serine Endopeptidases; D020169:Caspases; D002166:Camptothecin