Preservation of caspase-3 subunits from degradation contributes to apoptosis evoked by lactacystin: any single lysine or lysine pair of the small subunit is sufficient for ubiquitination.

Procaspase-3 (p32) is processed by upstream caspases to p12 and p20 subunits, which heterodimerize. Concomitant with formation of the active heterotetramer, p20 is autoprocessed to p17. Treatment of HL-60 cells with lactacystin, a selective inhibitor of the proteasome, exponentially increased caspase-3-like hydrolytic activity and induced apoptosis but had little or no effect on the activity of upstream caspase-8, caspase-9, or granzyme B. Lactacystin treatment decreased the p32 zymogen and evoked the accumulation of the p17 and p12 subunits. Treatment of transfected human retinoblast 911 cells with a proteasome inhibitor evoked the accumulation of epitope-tagged p12, p17, and p20 but had no effect on p32 zymogen. This result suggests that caspase-3 subunits, in contrast to the zymogen, are unstable because of degradation by the ubiquitin-proteasome system. Ubiquitin conjugates of p12 and p17 accumulated in cells that were cotransfected with p12 and a caspase inactive mutant of p17. Substitution of arginine for all eight lysines of p12 almost abolished its ubiquitination. Any single lysine or lysine pair was sufficient for p12 ubiquitination. Lactacystin treatment of HL-60 cells induced proteolytic processing of the X-linked inhibitor of apoptosis (XIAP) and decreased full-length XIAP, which is known to have ubiquitin-protein ligase activity for active caspase-3. These findings indicate that caspase-3 subunits can be degraded by the ubiquitin-proteasome system and suggest that lactacystin induces apoptosis in part by disabling the ubiquitin-protein ligase function of XIAP and by stabilizing active caspase-3 subunits.

[1]  Xiaodong Wang,et al.  Smac, a Mitochondrial Protein that Promotes Cytochrome c–Dependent Caspase Activation by Eliminating IAP Inhibition , 2000, Cell.

[2]  S. Srinivasula,et al.  Molecular Determinants of the Caspase-promoting Activity of Smac/DIABLO and Its Role in the Death Receptor Pathway* , 2000, The Journal of Biological Chemistry.

[3]  M. Hengartner.  The biochemistry of apoptosis , 2000, Nature.

[4]  S. Ōmura,et al.  Ubiquitylation and destruction of endogenous c-mycS by the proteasome: are myc boxes dispensable? , 2000, Archives of biochemistry and biophysics.

[5]  S. Srinivasula,et al.  Generation of Constitutively Active Recombinant Caspases-3 and -6 by Rearrangement of Their Subunits* , 1998, The Journal of Biological Chemistry.

[6]  S H Kaufmann,et al.  Mammalian caspases: structure, activation, substrates, and functions during apoptosis. , 1999, Annual review of biochemistry.

[7]  N. Thornberry,et al.  A Combinatorial Approach Defines Specificities of Members of the Caspase Family and Granzyme B , 1997, The Journal of Biological Chemistry.

[8]  R. Aebersold,et al.  Direct identification of a G protein ubiquitination site by mass spectrometry. , 2002, Biochemistry.

[9]  S. Srinivasula,et al.  Cytochrome c and dATP-Dependent Formation of Apaf-1/Caspase-9 Complex Initiates an Apoptotic Protease Cascade , 1997, Cell.

[10]  L. Staszewski,et al.  Ubiquitin-dependent c-Jun degradation in vivo is mediated by the δ domain , 1994, Cell.

[11]  A. Houweling,et al.  Characterization of 911: a new helper cell line for the titration and propagation of early region 1-deleted adenoviral vectors. , 1996, Human gene therapy.

[12]  R. Takahashi,et al.  Ubiquitin-protein ligase activity of X-linked inhibitor of apoptosis protein promotes proteasomal degradation of caspase-3 and enhances its anti-apoptotic effect in Fas-induced cell death , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[13]  Geng Wu,et al.  Structural basis of IAP recognition by Smac/DIABLO , 2000, Nature.

[14]  D. Green.  Apoptotic Pathways Paper Wraps Stone Blunts Scissors , 2000, Cell.

[15]  M. Weller,et al.  Proteasome Inhibitors Induce p53/p21-Independent Apoptosis in Human Glioma Cells , 1999, Cellular Physiology and Biochemistry.

[16]  F. Chen,et al.  Role of p53 in cell cycle regulation and apoptosis following exposure to proteasome inhibitors. , 2000, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[17]  Emad S. Alnemri,et al.  A conserved XIAP-interaction motif in caspase-9 and Smac/DIABLO regulates caspase activity and apoptosis , 2001, Nature.

[18]  Yili Yang,et al.  Ubiquitin protein ligase activity of IAPs and their degradation in proteasomes in response to apoptotic stimuli. , 2000, Science.

[19]  S. Ōmura,et al.  Structure of lactacystin, a new microbial metabolite which induces differentiation of neuroblastoma cells. , 1991, The Journal of antibiotics.

[20]  H. Drexler.  Activation of the cell death program by inhibition of proteasome function. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[21]  S. Ōmura,et al.  Lactacystin, a specific inhibitor of the proteasome, induces apoptosis in human monoblast U937 cells. , 1995, Biochemical and biophysical research communications.

[22]  J C Reed,et al.  Expression and prognostic significance of IAP-family genes in human cancers and myeloid leukemias. , 2000, Clinical cancer research : an official journal of the American Association for Cancer Research.

[23]  R. Liddington,et al.  Structural Basis for the Inhibition of Caspase-3 by XIAP , 2001, Cell.

[24]  G. Salvesen,et al.  Proteolytic activation of the cell death protease Yama/CPP32 by granzyme B. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[25]  S. Ganesan,et al.  Interaction of the Fanconi anemia proteins and BRCA1 in a common pathway. , 2001, Molecular cell.

[26]  F. Wurm,et al.  Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation. , 1996, Nucleic acids research.

[27]  T. Hunter,et al.  The inhibitor of apoptosis, cIAP2, functions as a ubiquitin-protein ligase and promotes in vitro monoubiquitination of caspases 3 and 7. , 2000, The Journal of biological chemistry.

[28]  D. Vaux,et al.  The anti-apoptotic activity of XIAP is retained upon mutation of both the caspase 3– and caspase 9–interacting sites , 2002, The Journal of cell biology.

[29]  R. Moyer,et al.  Cytotoxic T Lymphocyte-assisted Suicide , 1998, The Journal of Biological Chemistry.

[30]  L. Staszewski,et al.  Ubiquitin-dependent c-Jun degradation in vivo is mediated by the delta domain. , 1994, Cell.

[31]  A. Goldberg,et al.  Proteasome Inhibition Leads to a Heat-shock Response, Induction of Endoplasmic Reticulum Chaperones, and Thermotolerance* , 1997, The Journal of Biological Chemistry.

[32]  R F Standaert,et al.  Inhibition of proteasome activities and subunit-specific amino-terminal threonine modification by lactacystin , 1995, Science.

[33]  Eric A. Hendrickson,et al.  A Sequential Two-Step Mechanism for the Production of the Mature p17:p12 Form of Caspase-3 in Vitro * , 1997, The Journal of Biological Chemistry.

[34]  K. Shinohara,et al.  Apoptosis induction resulting from proteasome inhibition. , 1996, The Biochemical journal.

[35]  Alexander Varshavsky,et al.  The ubiquitin system. , 1998, Annual review of biochemistry.

[36]  J C Reed,et al.  Cleavage of human inhibitor of apoptosis protein XIAP results in fragments with distinct specificities for caspases , 1999, The EMBO journal.

[37]  H. Kizaki,et al.  Peptidyl aldehyde inhibitors of proteasome induce apoptosis rapidly in mouse lymphoma RVC cells. , 1997, Journal of biochemistry.

[38]  C. Pickart,et al.  Mechanisms underlying ubiquitination. , 2001, Annual review of biochemistry.

[39]  S. Ōmura,et al.  Activation of Atypical Protein Kinase C ζ by Caspase Processing and Degradation by the Ubiquitin-Proteasome System* , 2000, The Journal of Biological Chemistry.

[40]  Stephen F. Betz,et al.  Structural basis for binding of Smac/DIABLO to the XIAP BIR3 domain , 2000, Nature.

[41]  J C Reed,et al.  IAP family proteins--suppressors of apoptosis. , 1999, Genes & development.

[42]  J C Reed,et al.  IAPs block apoptotic events induced by caspase‐8 and cytochrome c by direct inhibition of distinct caspases , 1998, The EMBO journal.

[43]  K. Wilkinson.  Ubiquitination and deubiquitination: targeting of proteins for degradation by the proteasome. , 2000, Seminars in cell & developmental biology.

[44]  C. Pickart,et al.  Ubiquitin in chains. , 2000, Trends in biochemical sciences.

[45]  L. Dick,et al.  Mechanistic Studies on the Inactivation of the Proteasome by Lactacystin in Cultured Cells* , 1997, The Journal of Biological Chemistry.

[46]  Robert L Moritz,et al.  Identification of DIABLO, a Mammalian Protein that Promotes Apoptosis by Binding to and Antagonizing IAP Proteins , 2000, Cell.

[47]  A. Zeiher,et al.  Ubiquitin-mediated Degradation of the Proapoptotic Active Form of Bid , 2000, The Journal of Biological Chemistry.

[48]  U. Lopes,et al.  p53-dependent Induction of Apoptosis by Proteasome Inhibitors* , 1997, The Journal of Biological Chemistry.

[49]  Q. Dou,et al.  Bax degradation by the ubiquitin/proteasome-dependent pathway: involvement in tumor survival and progression. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[50]  H. Okayama,et al.  High-efficiency transformation of mammalian cells by plasmid DNA. , 1987, Molecular and cellular biology.

[51]  C. Cepko,et al.  Lineage analysis using retrovirus vectors. , 1993, Methods in enzymology.

[52]  J C Reed,et al.  Pro-caspase-3 Is a Major Physiologic Target of Caspase-8* , 1998, The Journal of Biological Chemistry.

[53]  Arne N. Akbar,et al.  RGD peptides induce apoptosis by direct caspase-3 activation , 1999, Nature.

[54]  L. Hicke.  A New Ticket for Entry into Budding Vesicles—Ubiquitin , 2001, Cell.