FASEB J.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by YANG, Y.
Right arrow Articles by YU, X.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by YANG, Y.
Right arrow Articles by YU, X.
(The FASEB Journal. 2003;17:790-799.)
© 2003 FASEB

Regulation of apoptosis: the ubiquitous way

YILI YANG1 and XIAODAN YU*

Regulation of Cell Growth Laboratory, Center for Cancer Research, National Cancer Institute at Frederick, National Institutes of Health, Frederick, Maryland, USA; and
* Department of Cell Biology, Institute of Basic Medical Sciences, Beijing 100850, China

1Correspondence: NCI-Frederick, 1050 Boyles St., Building 560, Room 22-45, Frederick, MD 21702, USA. E-mail: yangyili{at}ncifcrf.gov


   ABSTRACT
TOP
 ABSTRACT
THE UBIQUITINATION SYSTEM
 UBIQUITINATION IS ASSOCIATED...
 THE RING-CONTAINING IAPS -...
 CONCLUSIONS
 REFERENCES
 
Ubiquitin is a ubiquitously expressed 76 amino acid protein that can be covalently attached to target proteins, leading to their ubiquitination. Many ubiquitinated proteins are degraded by the proteasome, a 2000 kDa ATP-dependent proteolytic complex. Numerous studies have demonstrated that the ubiquitination and proteasome system plays an important role in controlling the levels of various cellular proteins and therefore regulates basic cellular processes such as cell cycle progression, signal transduction, and cell transformation. Ubiquitination also directly affects the function and location of target proteins. Recent studies found that ubiquitination-mediated degradation and change in activity regulate many molecules of the cell death machinery, such as p53, caspases, and Bcl-2 family members. Ring finger-containing members of the IAP (inhibitor of apoptosis) family proteins themselves can function as ubiquitin protein ligases to ubiquitinate their target proteins or promote autoubiquitination. It has been demonstrated that degradation of the IAP proteins is required for apoptosis to occur in some systems, indicating apoptosis proceeds by activating death pathways as well as eliminating "roadblocks" through ubiquitination. These new findings also suggest that ubiquitination is one of the major mechanisms that regulate apoptotic cell death and could be a unique target for therapeutic intervention.—Yang, Y., Yu, X. Regulation of apoptosis: the ubiquitous way.


Key Words: ubiquitination • caspase • IAP • ring finger protein • ubiquitin protein ligase (E3)


   THE UBIQUITINATION SYSTEM
TOP
 ABSTRACT
 THE UBIQUITINATION SYSTEM
UBIQUITINATION IS ASSOCIATED...
 THE RING-CONTAINING IAPS -...
 CONCLUSIONS
 REFERENCES
 
UBIQUITIN is a highly conserved 76 amino acid protein that is expressed ubiquitously in all eukaryotic cells. Under the sequential action of E1 (ubiquitin-activating enzyme), E2 (ubiquitin conjugating enzyme or UBC), and E3 (ubiquitin protein ligase), ubiquitin is covalently conjugated to cellular proteins through the isopeptide bond formed between its carboxyl-terminal Gly (G76) and the {varepsilon}-amino group of a lysine residue in the substrate. The same set of enzymes also catalyzes the formation of the isopeptide bond between G76 and the lysine residue (usually the K48) of previously conjugated ubiquitin, leading to formation of polyubiquitin chain (1) . Other factors may participate in elongation of the polyubiquitin chains under certain circumstances. For example, yeast protein Ufd2 is required for efficient polyubiquitination of substrate catalyzed by Ufd4 (E3) and Ubc4 (E2) (2) . Proteins attached with polyubiquitin chains usually are degraded by the proteasome, a large multri-subunit complex consisting of 20S catalytic core and two 19S subunits that recognize ubiquitinated proteins and remove attached ubiquitins (3) . The catalytic core has many different types of proteolytic activities to digest substrate proteins. Its activities can be modified by incorporation of different catalytic subunits and by association with alternative accessory factors to perform specific functions such as antigen processing. An interesting issue is how the polyubiquitinated proteins encounter and recognize proteasomes. Recent studies have indicated that proteins like Cdc48/VCP may act as chaperones to facilitate the degradation of ubiquitinated proteins at proteasomes (4) . Tagging proteins with ubiquitins is involved in many other cellular events such as activation of kinases and transcription factors, DNA repair, endocytosis, and intracellular trafficking of proteins (5) . Therefore, like phosphorylation, ubiquitination is a mechanism of signal transduction in the cells. It is not a surprise that cells have a large number of deubiquitination enzymes (DUBs) to facilitate the degradation of ubiquitinated proteins and regulate the functions of various proteins (Fig. 1 ) (6 , 7) .



View larger version (24K):
[in this window]
[in a new window]
  		Figure 1. Ubiquitination leads to protein degradation and alteration of protein functions.

The E1 enzyme initiates the ubiquitination process by binding with ubiquitin in the presence of ATP and forming a thiol ester bond between Cys residue in its active site and G76 of ubiquitin. There is only a single E1 gene in most organisms. Studies in cells harboring temperature-sensitive mutation of E1 gave the first definitive evidence that ubiquitination is essential for the cell cycle (1) . E2 accepts activated ubiquitin from E1 with its own Cys residue. However, there are multiple E2s in eukaryotic cells due to presence of isoforms and the emergence of new genes during evolution. There are at least 11 E2s in Saccharomyces cerevisiae and >25 E2s in mammalian cells. All E2s share a characteristic UBC domain consisting of ~150 amino acid residues that contains the active site and interacts with ubiquitin, E1, and E3. In vitro and in vivo experiments demonstrated that although E2s are interchangeable under certain circumstances, different E2s are often required for the ubiquitination of different substrates. For example, Ubc3 is essential for degradation of cell cycle-related proteins such as G1 cyclins whereas Ubc7 is mainly involved in ER-associated protein degradation (8) .

E3 is the enzyme that recognizes target proteins and transfers activated ubiquitin to them. Two types of E3s have been identified so far. The HECT domain E3 was initially discovered when studying the oncogenic human papillomaviruses (HPV) (9) . E6 protein encoded by HPV genome bound to a cellular protein named E6-AP (E6-associated protein) that functioned as E3 to catalyze the ubiquitination and degradation of tumor suppressor p53 in the host cells. The carboxyl terminal of E6-AP is required for its E3 activity and contains the Cys that participates in the transfer of ubiquitin to target proteins. Subsequent studies found that other E3s had a region of ~350 amino acid residues homologous to the carboxyl-terminal of E6-AP, and the name HECT (homologous to E6-AP carboxyl terminus) domain E3 was coined (10) . It has been estimated there are probably >30 HECT domain E3s in mammalian cells. The amino terminal of many HECT domain E3s have substrate binding region such as the WW domain, characterized by a pair of tryptophan (W) residues and conserved prolines, which recognizes phosphorylated proline-rich regions in the target proteins (11) . Besides regulating p53, E6-AP ubiquitinates some other proteins, and its mutation leads to the severe neurological disease Angelman syndrome (12) . Another extensively studied HECT domain E3 Nedd4 regulates the level of epithelial Na+ channel (ENaC) through ubiquitination. Mutation of Nedd4 leads to Liddle’s syndrome (hypertension and hypokalemic alkalosis) due to increased expression and activity of ENaC on the cell surface (13) .

Ring finger consists of eight conserved Cys and His residues forming a cross-brace structure to chelate two Zinc ions. Numerous studies during the last 3 years demonstrated that some ring finger-containing proteins are E3s that catalyze autoubiquitination and/or the ubiquitination of their substrates. For example, oncogenic protein Mdm2 promotes the ubiquitination and degradation of p53, a major mechanism regulating intracellular level of p53 (14 , 15) . The carboxyl-terminal ring finger of Mdm2 is required for ubiquitination of p53 in the presence of E1 and E2, whereas the amino-terminal of Mdm2 is the major p53 binding site. Ring finger-containing protein c-Cbl ubiquitinates some growth factor receptors, leading to their down-regulation (16) . Mutational analysis and crystal structure revealed that ring finger is responsible for binding with E2 (17) and therefore brings E2 and substrate together. Although recombinant ring finger fusion proteins can promote their autoubiquitination in vitro, it appears that ring finger does not directly participate in the transfer of ubiquitin from E2 to substrates, and it is not known yet whether the ring finger activates E2s. Small ring finger proteins Rbx1 and Apc11 are essential components of multri-subunit E3s SCF (Skp1/Cullin/F-box protein), APC (anaphase-promoting complex), and VHL-CBC (von Hippel-Lindau/Cul2/elongin B/elongin C) (16) . The cullin protein in these E3s forms a scaffold that associates with the ring finger protein at one end of the molecule and the substrate-recognizing protein at the other (18) . The ring finger-containing proteins bind with E2 and function similarly to the ring fingers in the single polypeptide E3s, such as Mdm2 and c-Cbl. These complex E3s can bind many different substrate-recognizing subunits (such as different F-box proteins in SCF E3s) and therefore ubiquitinate a variety of target proteins. There are an estimated 200 ring finger proteins in mammalian cells, the majority of which have not been characterized. Based on the structure of ring finger and its interaction with E2s, it is conceivable that we could learn to predict whether a particular ring finger confers E3 activity. Two alternatives of the ring finger domain, the U-box and PHD domains, can also bring E3 activity to proteins (19 20 21) . It is likely that both U-box and PHD domains use mechanisms similar to that of ring finger to facilitate ubiquitination, as the structure of the PHD domain is similar to that of the ring finger; the U-box shares significant sequence homology with ring finger and, like ring finger, can directly bind to E2 (22 23 24) .

Two types of ubiquitin-related proteins have been identified in cells. The ubiquitin-like molecules (UBLs) are small proteins structurally similar to ubiquitin; like ubiquitin, they can be conjugated to various target proteins. Modification with UBLs does not target proteins for degradation; rather, it affects their functions in many different ways. For example, sumoylation, modifying proteins with the small ubiquitin-related modifier (SUMO), affects protein and protein interaction, translocation, activities of transcription factors, and may save the target proteins from ubiquitination and degradation (25) . Sumoylation uses enzymes similar to those participating in ubiquitination to activate and ligate SUMO to target proteins, suggesting these two processes are evolutionally closely related. There are growing lists of proteins that contain ubiquitin domain (termed UDPs) and not known to be conjugated to other proteins. It remains to be determined whether the ubiquitin domain plays a unique role in the function of various UDPs (26 , 27) .


   UBIQUITINATION IS ASSOCIATED WITH APOPTOSIS
TOP
 ABSTRACT
 THE UBIQUITINATION SYSTEM
 UBIQUITINATION IS ASSOCIATED...
THE RING-CONTAINING IAPS -...
 CONCLUSIONS
 REFERENCES
 
Observations in the early 1990s began to reveal a link between ubiquitination and apoptosis or programmed cell death. Studies using the hawk moth (Manduca sexta) found ubiquitin mRNA and ubiquitinated proteins were dramatically increased in the intersegmental muscles cells undergoing programmed cell death (28) . In these dying cells, there were increased ubiquitinating enzymes activities as well as increased proteasome components and their proteolytic activities. Despite many negative results, a correlation between apoptosis and increased ubiquitin or ubiquitinated proteins was further shown in several other model systems, such as muscles from dystrophin-deficient mice, thyroxin- or serum deprivation-induced cell death, and apoptosis triggered by DNA-damaging agents (29) . Antisense oligonucleotides of ubiquitin can inhibit protein ubiquitination and apoptosis induced by {gamma}-radiation in lymphocytes (30) , indicating protein ubiquitination is essential for lymphocyte apoptosis. Consistent with these results, proteasome inhibitors effectively blocked DNA-damaging agents and glucocorticoid-induced thymocyte death (31) and nerve growth factor withdrawal-induced sympathetic neuron death (32) , suggesting that proteasome degradation is required for mitochondria-dependent apoptosis. Subsequent studies indicated that glucocorticoids increased proteasomal proteolytic activity at the premitochondria step of thymocyte apoptosis (33) . It is not clear what the target molecules of the increased ubiquitination and proteasome activity are or how the ubiquitination and proteasome system interacts with the apoptosis machinery.

The tremendous progress in understanding the ubiquitination process over the last couple of years made it possible to identify ubiquitination substrates and study the role of ubiquitination in regulating the levels and functions of many apoptosis-related proteins. The tumor suppressor p53 plays a critical role in maintaining the integrity of genome and preventing tumor development. When cells are subjected to DNA-damaging agents, oncogene activation, hypoxia, and lack of growth factors, the intracellular level of p53 increases rapidly to induce growth arrest and/or apoptosis (34) . It has been demonstrated that the level of p53 is mainly regulated at the post-transcriptional stage through ubiquitination-dependent degradation (14 , 15) . The ring-containing protein Mdm2 is the E3 that ubiquitinates p53 and keeps it at a low level in unstimulated cells. Ubiquitination of p53 by Mdm2 also facilitates the export of p53 from nucleus to cytoplasm, where it could not activate transcription of target genes (35) . Upon stimulation of cells with chemotherapeutic agents or radiation, phosphorylation of p53 prevents its interaction with Mdm2 and leads to inhibition of ubiquitination and degradation. Under certain circumstances, such as oncogene activation, the expression of an Mdm2 binding protein, ARF, is increased, which directly inhibits Mdm2’s E3 activity and therefore increases intracellular p53 level (36) . Deubiquitination enzyme herpes virus-associated ubiquitin-specific protease (HAUSP) can specifically remove ubiquitin from p53 and stabilize it, even in the presence of Mdm2 (7) . Overexpression of HAUSP suppressed colony formation in a p53-dependent manner, presumably through p53-induced growth arrest and/or apoptosis. It is not known yet whether or how HAUSP is regulated by p53-inducing stimuli such as DNA damage and oncogene activation. On the other hand, increased p53 up-regulates Mdm2 gene transcription, forming a negative feedback to prevent the unwanted high level of p53 in the cells. The importance of regulating p53 level by Mdm2 was illustrated by the observation that the early embryonic lethality of Mdm2-deficient mice was completely rescued by the simultaneous deletion of p53 (35) . Surprisingly, deletion of Mdm2 homologue MdmX also led to embryonic death, which can be rescued by loss of p53 (37 38 39) . Although MdmX has a ring finger and can bind to p53, it does not possess E3 activity toward p53. Recent studies indicated that MdmX formed a heterodimer with Mdm2 that led to its stabilization (40) . This is likely because Mdm2 promotes its own ubiquitination and degradation in the absence of MdmX. However, overexpressed MdmX might compete with Mdm2 in binding with p53 and therefore inhibit Mdm2-mediated p53 ubiquitination and degradation, as earlier studies reported. Like mutation of p53 itself, abnormalities in these regulating mechanisms such as enhanced p53 degradation due to expression of E6 after infection of oncogenic HPV, loss of ARF and amplification of Mdm2 or MdmX gene have been demonstrated to play critical roles in the development of certain tumors (41 , 42) . It is conceivable that dysfunction of p53 pathway may contribute to the development of most, if not all, human cancers.

Bcl-2 family proteins are critical regulators of apoptosis (43) . The anti-apoptotic members of the family such as Bcl-2, Bcl-xL, and Bcl-w all contain the conserved Bcl-2 homology (BH) regions BH1, BH2, and BH3, which form a hydrophobic groove that binds to BH3 domain of other family members (44) . The Bcl-2 family has two types of proapoptotic members: the Bax and Bak subfamily and the BH3-only proapoptotic members such as Bid, Bad, Bim/Bod, and PUMA (45) . Studies with gene-targeted cells indicated that the presence of Bax or Bak is required for many forms of apoptosis (46 , 47) , and each type of cell needs at least one of the anti-apoptotic Bcl-2 family member s to survive (43 , 48) . The BH3-only proteins are activated in response to various apoptotic stimuli and are able to induce apoptosis through interacting directly with Bax and Bak (48) or binding to the hydrophobic groove of anti-apoptotic members such as Bcl-2 or Bcl-xL and removing the inhibition of these anti-apoptotic molecules on Bax and Bak. Besides being controlled through transcription, phosphorylation, and proteolytic cleavage, it is becoming evident that Bcl-2 family members are regulated by ubiquitination and proteasome degradation systems. Bcl-2 was ubiquitinated and degraded upon stimulation of human umbilical vein endothelial cells with TNF-{alpha} (49) . Expression of Bcl-2 that had all four lysine residues mutated conferred significantly more resistance to apoptosis than that of wild-type Bcl-2, suggesting down-regulation of Bcl-2 is required for TNF-{alpha} to induce apoptosis in the cells. It appeared that the increased ubiquitination resulted from dephosphorylation of Bcl-2 induced by TNF-{alpha} (50) . MAP kinase (ERK1/2) can phosphorylate Bcl-2 in vitro and in transfected cells, and its inhibition by a specific inhibitor or TNF-{alpha} correlated with Bcl-2 dephosphorylation and degradation. Mutated Bcl-2 with threonine/serine of all three potential MAP kinase sites changed into aspartic acid residues (phospho-mimetic mutation) was resistant to TNF-{alpha}-induced degradation, whereas changes of PKC and cAMP-dependent protein kinase sites did not affect the stability of Bcl-2. MAP kinase-specific phosphatase-3 is likely to be the enzyme responsible for removal of phosphorus from Bcl-2. Proapoptotic Bc-2 family members Bax and Bik are degraded by the ubiquitination and proteasome systems (51 , 52) . Their accumulation in the cells might be at least partially responsible for the apoptosis induced by proteasome inhibitors. Decreased intracellular level of Bax was accompanied by increased Bax-degrading activity in aggressive human prostate adenocarcinomas (51) , indicating degradation of proapoptotic Bax might be the survival mechanism of some tumor cells. Bid is a BH3 region-only Bcl-2 family member located primarily in the cytoplasm. After activation of caspase 8, the carboxyl terminal of Bid is cleavaged to generate the truncated Bid (tBid), which then translocates to mitochondria and leads to the release of apoptosis-promoting molecules. Like Bax and Bik, tBid is degraded through ubiquitination and the proteasome system (53) . Expression of the mutated tBid that could not be ubiquitinated enhanced cytochrome c release and cell death. These studies demonstrated that the ubiquitination and proteasome systems could change the ratio of Bcl-2 family members and therefore alter the susceptibility of cells to various apoptotic stimuli. It is hoped that we will identify in the not-so-distant future the enzymes responsible for ubiquitination of Bcl-2 family members, which could provide valuable targets for modulating apoptosis of normal and tumor cells.

Many molecules of the extrinsic apoptosis pathway are closely related to the ubiquitination and proteasome system. For example, the intracellular domains of Fas and type I TNF receptor bind with SUMO-1 and Ubc9 (54 , 55) , suggesting that they are targets of SUMO modification or may function as a scaffold to promote sumoylation. The death receptor-associated protein Daxx can also bind with SUMO-1 and Ubc9 (56) and was conjugated with SUMO-1 (57) . Since the SUMO-1 and Ubc9 binding site is overlapped with the Fas binding region of Daxx, it is conceivable that SUMO association or sumoylation may affect signal transduction of death receptor. Since we now know that sumoylation modulates many proteins and cellular processes, the mechanisms and physiological significance of the earlier transfection experiments indicating that overexpression of SUMO-1 altered the susceptibility to apoptosis remain to be determined. Another Fas-associated protein, FAF1, contains an ubiquitin-like domain required for the apoptosis induced by its overexpression (27) . cFLIP contains two death effector domains and acts as a dominant negative inhibitor of death receptor-mediated activation of caspase 8 (58) . Lack of this protein made cells become highly sensitive to FasL or TNF (59) . The resistance of certain melanoma cells to the death receptor ligand TRAIL has been attributed to the expression of cFLIP (60) . It was shown recently that agents that bind to the peroxisome proliferation-activated receptor {gamma} sensitize tumor, but not untransformed, cells to TRAIL (61) . The increased sensitivity was closely correlated with their ability to reduce the level of c-FLIP and could be overcome by enforced expression of c-FLIP. Further studies indicated that the down-regulation of c-FLIP was due to its ubiquitination and subsequent proteasomal degradation. However, it has not been addressed yet how these compounds selectively induced the ubiquitination and degradation of c-FLIP and which E3 is responsible for its ubiquitination. Since other chemotherapeutic agents synergize with TRAIL in killing tumor cells (62) , it would be interesting to explore whether they also cause ubiquitination and degradation of c-FLIP.

Ubiquitination is a critical process in regulating many signal transduction pathways, transcription factors, and adaptor molecules that have profound effects on the growth and death of cells. For example, suppressors of cytokine signaling (SOCS) are a family of intracellular proteins that contain a center SH2 domain and characteristic carboxyl-terminal SOCS box (63) . They are often induced by cytokines and in turn suppress the Jak-Stat pathways that mediate the signal of cytokines, forming a negative feedback to limit cytokines’ biological effects. Some of the SOCS family members directly bind to Jaks and inhibit their kinase activities; others occupy the cytokine receptors and block the recruitment of Stats. The SOCS domain can bind to elongins B and C, which are components of complex E3 VHL-CBC (64 , 65) . It has been shown that SOCS1 can act as an adaptor to facilitate ubiquitination and degradation of proteins bound to its SH2 domain, such as oncogenic fusion protein Tel-JAK2 (66 , 67) and guanine nucleotide exchange factor Vav (68) . It is now believed that SOCS-induced ubiquitination and degradation is an important mechanism to down-regulate cytokine’s effects. Ubiquitination also affects signal transduction in a nondegradating manner. For example, TRAF6 is an important signaling mediator for Toll-like and IL-1 receptors as well as the TNF receptor family. The ring finger-dependent E3 activity of TRAF6, which catalyzes the formation of K-63 polyubiquitin chain, is required for activating TAK1, which in turn activates I{kappa}B kinase (IKK) and kinase for JNK (69 , 70) . Activated IKK phosphorylates I{kappa}B and leads to its ubiquitination and degradation, which enable the activation of NF{kappa}B, a critical apoptosis-regulating transcription factor (71) .


   THE RING-CONTAINING IAPS – E3S AS APOPTOSIS INHIBITORS AND BEYOND
TOP
 ABSTRACT
 THE UBIQUITINATION SYSTEM
 UBIQUITINATION IS ASSOCIATED...
 THE RING-CONTAINING IAPS -...
CONCLUSIONS
 REFERENCES
 
The IAP (inhibitor of apoptosis) family proteins are characterized by containing one to three BIR (baculovirus IAP repeats) domains, each consisting of ~70 amino acid residues (72) . Since the founding member was discovered in baculovirus almost a decade ago, cellular IAPs have been identified in species from yeast to human (73) . Among the eight mammalian IAPs, cIAP1, cIAP2, XIAP, and NAIP have three BIR domains, whereas survivin, BRUCE, ILP-2, and ML-IAP only have one (73 74 75) . Based mainly on transfection experiments, many IAPs have been shown to be bona fide apoptosis inhibitors. XIAP appeared to be the most potent one and blocked cell death through inhibiting caspases 3, 7, and 9. Whereas the BIR3 of XIAP directly bound to the small subunit of caspase 9, it was the linker region between BIR1 and BIR2, which contacted with caspase 3 or 7, and prevented their active sites from binding with substrates (76) . Perhaps to avoid the inhibition of extrinsic and intrinsic death pathways at the same time, testis expresses ILP-2 (that contains one BIR3-like BIR), which inhibits caspase 9 but not caspase 3, and therefore prevents only mitochondria-dependent apoptosis (74) . Besides acting as endogenous caspases inhibitors, IAP family proteins such as XIAP, cIAP1, and cIAP2 participate in receptor-mediated signal transduction (77) . A subgroup of IAPs, including survivin in mammalian cells and BIR-1 and BIR-2 in Caenorhabditis elegans, appears to function not mainly as caspases inhibitors, but to regulate mitotic spindle formation and cytokinesis (78 79 80) . This unique feature has led to the suggestion that these proteins should simply be called BIR domain-containing proteins (81) .

The majority of the caspase-inhibiting IAPs possess a carboxyl-terminal ring finger motif. In an effort to understand why proteasome inhibitors could effectively block thymocyte apoptosis, we found that XIAP, cIAP1, and cIAP2 were degraded in a proteasome-dependent manner when the thymocytes were stimulated with apoptotic stimuli such as glucocorticoids or etoposide (82) . It was found that these IAPs have ring finger-dependent E3 activities that catalyze autoubiquitination in vitro and in cells. Overexpression cIAP1 caused its autoubiquitination and degradation. This may explain why transfection of cIAP1 or cIAP2 failed to protect cells from Bax, TNF-{alpha}, or Sindbis virus-induced apoptosis whereas expression of ring domain-deleted cIAP1 or cIAP2 did (83) . In contrast to wild-type XIAP, mutated XIAP without E3 activity was not down-regulated by apoptotic stimuli in T cell hybridomas and provided better protection against apoptosis (82) , indicating that autoubiquitination and degradation of XIAP are required for these cells to undergo apoptosis. The involvement of IAP-associated E3 activity in inhibiting apoptosis is further supported by the finding that cIAP2 can promote mono-ubiquitination of caspase 3 and 7 (84) and that XIAP catalyzes ubiquitination and degradation of caspase 3 (85) . However, mice deficient in XIAP developed normally and cells from XIAP-/- mice are not more sensitive to apoptotic stimuli (86) , suggesting there might be compensation mechanisms during development.

The importance of eliminating IAPs during apoptosis is clearly illustrated by studies using Drosophila, which has two ring finger-containing IAPs: DIAP1 and DIAP2. Pioneer researches found that induction of apoptosis in Drosophila requires Reaper, Hid, and Grim; their overexpression resulted in excess cell death that could be suppressed by expression of DIAP1 (87 , 88) . Further studies demonstrated that DAP1 is essential for the survival of Drosophila embryonic cells. Reaper, Him, and Grim all have the so-called RHG motif at their amino termini, which could interact with DIAP1. They induce apoptosis through direct interaction with DIAP1 and suppressing its caspase-inhibiting activity (89 , 90) . Recently, a series of studies using different in vivo and in vitro systems demonstrated that Reaper, Hid, and Grim not only bound with DIAP1, but also down-regulated DIAP1 (91) . Intriguingly, in some systems, Reaper and Grim but not Hid induced the ubiquitination and degradation of DIAP1 (92 93 94) ; in other systems, it was Hid but not Reaper and Grim that caused DIAP1 ubiquitination and degradation in a ring-dependent manner (95) . In either situation, DIAP1 degradation is required for apoptosis to occur. Reaper and Grim also caused general suppression of protein translation, which contributed to the reduction of short-lived DIAP1 (94 , 95) . Genetic modifier screen found that certain mutations of DIAP ring finger enhanced Reaper-induced cell death but inhibited Hid-induced apoptosis (96) , suggesting that additional factors are involved in their interactions with DIAP1. At least two E2s—Morgue and UBCD1—can bind DIAP1 and promote its autoubiquitination; the reduction of Morgue or UBCD1 suppresses Reaper and Grim-induced DIAP1 down-regulation and apoptosis (92 , 93 , 97) . Morgue has an F-box, suggesting it might be a substrate-recognizing subunit of a SCF E3. If so, it would be interesting to know whether this putative E3 participates in the ubiquitination and degradation of DIAP1. Finally, like mammalian IAPs, DIAP1 binds with Drosophila caspase Dronc and induces its ubiquitination in a ring-dependent manner (96) . This interaction is likely important as DIAP1 binding protein Jafrac2 competes with Dronc for the binding of DIAP1 and promotes cell death (98) .

SMAC (second mitochondria-derived activator of caspase, also known as Diablo) is synthesized as a 239 amino acid precursor protein (99 , 100) . The first 55 amino acids of the molecule are required for its import into mitochondria, where the sequence is cleaved. Upon apoptotic stimuli such as DNA damage and serum deprivation, matured SMAC is released from the mitochondria and removes the inhibition of XIAP on caspase 9 (101 102 103) . Apparently this is because the amino terminal of SMAC has a motif similar to that of the small subunit of caspase 9, which is responsible for binding of caspase 9 to the groove on the surface of XIAP BIR3 domain (104) . This four amino acid motif is similar to the RHG motif of Drosophila proteins Reaper, Hid, and Grim, raising the possibility that SMAC may be able to promote the ubiquitination and degradation of XIAP. Though this remains to be proved or disproved, it has been shown that XIAP is able to promote the ubiquitination and degradation of SMAC (105) . Although XIAP-deficient mice developed normally and cells from these mice are not more sensitive to apoptotic stimuli (86) , three different laboratories have demonstrated recently that the SMAC and XIAP interaction does play an important role in apoptosis. It was found that TRAIL (TNF-related apoptosis-inducing ligand) could not induce apoptosis effectively in Bax-deficient cells, apparently due to formation of p20/p12 caspase 3 but not the fully active p17/p12 dimer. This incompletely processed p20/p12 caspase 3 was associated with XIAP due to the lack of release of SMAC from mitochondria in the absence of Bax (106) . When SMAC was expressed in the cytoplasm of these Bax -/- cells, TRAIL could induce the generation of p17/p12 caspase 3 as well as apoptosis, indicating that interaction of SMAC with XIAP is required for full activation of caspase and a cell death program. Consistent with these results, Fas engagement could not induce full processing of caspase 3 in Jurkat cells that overexpress Bcl-2 or Bcl-xL (107) , which prevented the release of SMAC from mitochondria. In this report, the ability of SMAC to remove the inhibition of XIAP on the full activation of caspase 3 was directly demonstrated through reconstitution of all the components in vitro. Similar results were observed in hepatocytes from Bid-deficient mice (108) , since Bid-mediated activation of the mitochondria death pathway is required for anti-Fas antibody to induce apoptosis in hepatocytes. Serine protease Omi/HtrA2 also contains the four amino acid motif that interacts with XIAP and blocks its ability to inhibit caspase (109 110 111 112) . Like SMAC, Omi/HtrA2 is released from mitochondria upon apoptotic stimuli. Enforced expression of Omi/HtrA2-sensitized cells for apoptosis, and decreased Omi/HtrA2 made cells more resistant to death stimuli. The physiological role of Omi/HtrA2 and its relationship with SMAC remain to be determined.

The E3 activity of IAPs may also regulate apoptosis indirectly. The type I TNF receptor (TNF-RI) has an intracellular death domain and is able to activate caspase 8 to induce apoptosis. Through adaptor molecules TRADD and TRAF2, it can activate Jun kinase and induce NF{kappa}B, which are anti-apoptotic under many circumstances. The importance of TRAF2 in protecting cells from apoptosis is clearly illustrated by the observation that cells from TRAF2-deficient mice are significantly more sensitive to TNF-{alpha}-induced apoptosis (113) . Type II TNF receptor (TNF-RII) does not have the death domain and associates with the complex of TRAF1/TRAF2/cIAP1/cIAP2 (114) . Paradoxically, although engagement of TNF-RII leads to activation of NF{kappa}B, it enhances TNF-RI induced apoptosis in both normal and transformed cells (115 , 116) . It has been shown that in the presence of TNF-RII and TNF-RI, TNF-{alpha} caused ubiquitination and proteasomal degradation of TRAF2, which was accompanied by quicker reduction of Jun kinase activity than that in cells expressing only TNF-RI (117) . Further studies demonstrated that cIAP1 ubiquitinated TRAF2 in vitro and in vivo in a ring finger-dependent manner. Overexpression of cIAP1 that does not possess E3 activity suppressed TNF-{alpha}-induced down-regulation of TRAF2 and cell death, indicating that cIAP1-mediated ubiquitination and degradation of TRAF2 are responsible for the enhanced apoptosis. Therefore, cIAP1 acts as a proapoptotic molecule rather than an inhibitor of apoptosis under this scenario. It has been shown that cIAP1 can be cleaved by caspases to generate a carboxyl-terminal fragment that includes the ring domain. In transfection experiments, this fragment suppressed the anti-apoptotic activity of the cIAP1 BIR domain (83) . Although the physiological significance of this observation remains to be established, it shows again that cIAP1 can have proapoptotic effect during apoptotic cell death, presumably due to its E3 activity.


   CONCLUSIONS
TOP
 ABSTRACT
 THE UBIQUITINATION SYSTEM
 UBIQUITINATION IS ASSOCIATED...
 THE RING-CONTAINING IAPS -...
 CONCLUSIONS
REFERENCES
 
Ubiquitination is a particular mechanism of signal transduction in the cells. In addition to modulating the functions and interactions of various proteins, it controls the level of proteins by targeting them for proteasomal degradation. Identification of ring finger-containing proteins such as E3s or E3 components has made it possible to characterize ubiquitination enzymes and substrates under many circumstances. Many components of the cellular apoptosis machinery are targets of ubiquitination, and the ring finger-containing IAPs are themselves E3s. This E3 activity appears to serve as a self-destruction mechanism to eliminate the "roadblockers" of apoptosis pathway and may play an important role in receptor-mediated signal transduction (Fig. 2 ). These findings not only advanced our understanding of apoptosis regulation, but provided a unique opportunity for therapeutic intervention. For example, targeting the ring finger of Mdm2 could increase the intracellular level of p53, which in turn induces apoptosis in transformed cells. Due to the specificity of the ubiquitination reaction, especially the interaction between E3 and substrate and the critical role of apoptosis in various pathological conditions, it is conceivable that targeting the ubiquitination process could become an effective therapeutics in the not-so-distant future.



View larger version (31K):
[in this window]
[in a new window]
  		Figure 2. Ubiquitination is an important mechanism in regulating cell death pathways.


   ACKNOWLEDGMENTS
 
We are grateful to Drs. J. Ashwell, K. Vousden, and A. M. Weissman for many invaluable discussions. We apologize to colleagues whose important contributions have been cited only indirectly due to space limitations.

Received for publication October 14, 2002. Accepted for publication January 9, 2003.


   REFERENCES
TOP
 ABSTRACT
 THE UBIQUITINATION SYSTEM
 UBIQUITINATION IS ASSOCIATED...
 THE RING-CONTAINING IAPS -...
 CONCLUSIONS
 REFERENCES
 

  1. Hershko, A., Ciechanover, A. (1998) The ubiquitin system. Annu. Rev. Biochem. 67,425-479[CrossRef][Medline]
  2. Koegl, M., Hoppe, T., Schlenker, S., Ulrich, H. D., Mayer, T. U., Jentsch, S. (1999) A novel ubiquitination factor, E4, is involved in multiubiquitin chain assembly. Cell 96,635-644[CrossRef][Medline]
  3. Almond, J. B., Cohen, G. M. (2002) The proteasome: a novel target for cancer chemotherapy. Leukemia 16,433-443[CrossRef][Medline]
  4. Dai, R. M., Li, C. C. (2001) Valosin-containing protein is a multi-ubiquitin chain-targeting factor required in ubiquitin-proteasome degradation. Nat. Cell Biol. 3,740-744[CrossRef][Medline]
  5. Weissman, A. M. (2001) Themes and variations on ubiquitylation. Nat. Rev. Mol. Cell Biol. 2,169-178[CrossRef][Medline]
  6. Yao, T., Cohen, R. E. (2002) A cryptic protease couples deubiquitination and degradation by the proteasome. Nature (London) 419,403-407[CrossRef][Medline]
  7. Li, M., Chen, D., Shiloh, A., Luo, J., Nikolaev, A. Y., Qin, J., Gu, W. (2002) Deubiquitination of p53 by HAUSP is an important pathway for p53 stabilization. Nature (London) 416,648-653[CrossRef][Medline]
  8. Pickart, C. M. (2001) Mechanisms underlying ubiquitination. Annu. Rev. Biochem. 70,503-533[CrossRef][Medline]
  9. Scheffner, M., Huibregtse, J. M., Vierstra, R. D., Howley, P. M. (1993) The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53. Cell 75,495-505[CrossRef][Medline]
  10. Huibregtse, J. M., Scheffner, M., Beaudenon, S., Howley, P. M. (1995) A family of proteins structurally and functionally related to the E6-AP ubiquitin-protein ligase. Proc. Natl. Acad. Sci. USA 92,2563-2567[Abstract/Free Full Text]
  11. Lu, P. J., Zhou, X. Z., Shen, M., Lu, K. P. (1999) Function of WW domains as phosphoserine- or phosphothreonine-binding modules. Science 283,1325-1328[Abstract/Free Full Text]
  12. Kishino, T., Lalande, M., Wagstaff, J. (1997) UBE3A/E6-AP mutations cause Angelman syndrome. Nat. Genet. 15,70-73[CrossRef][Medline]
  13. Rotin, D., Kanelis, V., Schild, L. (2001) Trafficking and cell surface stability of ENaC. Am. J. Physiol. 281,F391-F399
  14. Haupt, Y., Maya, R., Kazaz, A., Oren, M. (1997) Mdm2 promotes the rapid degradation of p53. Nature (London) 387,296-299[CrossRef][Medline]
  15. Kubbutat, M. H., Jones, S. N., Vousden, K. H. (1997) Regulation of p53 stability by Mdm2. Nature (London) 387,299-303[CrossRef][Medline]
  16. Joazeiro, C. A., Weissman, A. M. (2000) RING finger proteins: mediators of ubiquitin ligase activity. Cell 102,549-552[CrossRef][Medline]
  17. Zheng, N., Wang, P., Jeffrey, P. D., Pavletich, N. P. (2000) Structure of a c-Cbl-UbcH7 complex: RING domain function in ubiquitin-protein ligases. Cell 102,533-539[CrossRef][Medline]
  18. Zheng, N., Schulman, B. A., Song, L., Miller, J. J., Jeffrey, P. D., Wang, P., Chu, C., Koepp, D. M., Elledge, S. J., Pagano, M., et al (2002) Structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF ubiquitin ligase complex. Nature (London) 416,703-709[CrossRef][Medline]
  19. Hatakeyama, S., Yada, M., Matsumoto, M., Ishida, N., Nakayama, K. I. (2001) U box proteins as a new family of ubiquitin-protein ligases. J. Biol. Chem. 276,33111-33120[Abstract/Free Full Text]
  20. Coscoy, L., Sanchez, D. J., Ganem, D. (2001) A novel class of herpesvirus-encoded membrane-bound E3 ubiquitin ligases regulates endocytosis of proteins involved in immune recognition. J. Cell Biol. 155,1265-1273[Abstract/Free Full Text]
  21. Lu, Z., Xu, S., Joazeiro, C., Cobb, M. H., Hunter, T. (2002) The PHD domain of MEKK1 acts as an E3 ubiquitin ligase and mediates ubiquitination and degradation of ERK1/2. Mol. Cell 9,945-956[CrossRef][Medline]
  22. Capili, A. D., Schultz, D. C., Rauscher, I. F., Borden, K. L. (2001) Solution structure of the PHD domain from the KAP-1 corepressor: structural determinants for PHD, RING and LIM zinc-binding domains. EMBO J. 20,165-177[CrossRef][Medline]
  23. Aravind, L., Koonin, E. V. (2000) The U box is a modified RING finger—a common domain in ubiquitination. Curr. Biol. 10,R132-R134[CrossRef][Medline]
  24. Connell, P., Ballinger, C. A., Jiang, J., Wu, Y., Thompson, L. J., Hohfeld, J., Patterson, C. (2001) The co-chaperone CHIP regulates protein triage decisions mediated by heat-shock proteins. Nat. Cell Biol. 3,93-96[CrossRef][Medline]
  25. Kim, K. I., Baek, S. H., Chung, C. H. (2002) Versatile protein tag, SUMO: its enzymology and biological function. J. Cell. Physiol. 191,257-268[CrossRef][Medline]
  26. Buchberger, A., Howard, M. J., Proctor, M., Bycroft, M. (2001) The UBX domain: a widespread ubiquitin-like module. J. Mol. Biol. 307,17-24[CrossRef][Medline]
  27. Ryu, S. W., Kim, E. (2001) Apoptosis induced by human Fas-associated factor 1, hFAF1, requires its ubiquitin homologous domain, but not the Fas-binding domain. Biochem. Biophys. Res. Commun. 286,1027-1032[CrossRef][Medline]
  28. Schwartz, L. M., Myer, A., Kosz, L., Engelstein, M., Maier, C. (1990) Activation of polyubiquitin gene expression during developmentally programmed cell death. Neuron 5,411-419[CrossRef][Medline]
  29. Orlowski, R. Z. (1999) The role of the ubiquitin-proteasome pathway in apoptosis. Cell Death Differ. 6,303-313[CrossRef][Medline]
  30. Delic, J., Morange, M., Magdelenat, H. (1993) Ubiquitin pathway involvement in human lymphocyte gamma-irradiation-induced apoptosis. Mol. Cell. Biol. 13,4875-4883[Abstract/Free Full Text]
  31. Grimm, L. M., Goldberg, A. L., Poirier, G. G., Schwartz, L. M., Osborne, B. A. (1996) Proteasomes play an essential role in thymocyte apoptosis. EMBO J. 15,3835-3844[Medline]
  32. Sadoul, R., Fernandez, P. A., Quiquerez, A. L., Martinou, I., Maki, M., Schroter, M., Becherer, J. D., Irmler, M., Tschopp, J., Martinou, J. C. (1996) Involvement of the proteasome in the programmed cell death of NGF-deprived sympathetic neurons. EMBO J. 15,3845-3852[Medline]
  33. Hirsch, T., Dallaporta, B., Zamzami, N., Susin, S. A., Ravagnan, L., Marzo, I., Brenner, C., Kroemer, G. (1998) Proteasome activation occurs at an early, premitochondrial step of thymocyte apoptosis. J. Immunol. 161,35-40[Abstract/Free Full Text]
  34. Oren, M. (1999) Regulation of the p53 tumor suppressor protein. J. Biol. Chem. 274,36031-36034[Free Full Text]
  35. Balint, E. E., Vousden, K. H. (2001) Activation and activities of the p53 tumour suppressor protein. Br. J. Cancer 85,1813-1823[CrossRef][Medline]
  36. Sherr, C. J. (2001) The INK4a/ARF network in tumour suppression. Nat. Rev. Mol. Cell Biol. 2,731-737[CrossRef][Medline]
  37. Parant, J., Chavez-Reyes, A., Little, N. A., Yan, W., Reinke, V., Jochemsen, A. G., Lozano, G. (2001) Rescue of embryonic lethality in Mdm4-null mice by loss of Trp53 suggests a nonoverlapping pathway with MDM2 to regulate p53. Nat. Genet. 29,92-95[CrossRef][Medline]
  38. Finch, R. A., Donoviel, D. B., Potter, D., Shi, M., Fan, A., Freed, D. D., Wang, C. Y., Zambrowicz, B. P., Ramirez-Solis, R., Sands, A. T., et al (2002) MdmX is a negative regulator of p53 activity in vivo. Cancer Res. 62,3221-3225[Abstract/Free Full Text]
  39. Migliorini, D., Denchi, E. L., Danovi, D., Jochemsen, A., Capillo, M., Gobbi, A., Helin, K., Pelicci, P. G., Marine, J. C. (2002) Mdm4 (Mdmx) regulates p53-induced growth arrest and neuronal cell death during early embryonic mouse development. Mol. Cell. Biol. 22,5527-5538[Abstract/Free Full Text]
  40. Gu, J., Kawai, H., Nie, L., Kitao, H., Wiederschain, D., Jochemsen, A. G., Parant, J., Lozano, G., Yuan, Z. M. (2002) Mutual dependence of MDM2 and MDMX in their functional inactivation of p53. J. Biol. Chem. 277,19251-19254[Abstract/Free Full Text]
  41. Riemenschneider, M. J., Buschges, R., Wolter, M., Reifenberger, J., Bostrom, J., Kraus, J. A., Schlegel, U., Reifenberger, G. (1999) Amplification and overexpression of the MDM4 (MDMX) gene from 1q32 in a subset of malignant gliomas without TP53 mutation or MDM2 amplification. Cancer Res. 59,6091-6096[Abstract/Free Full Text]
  42. Ryan, K. M., Phillips, A. C., Vousden, K. H. (2001) Regulation and function of the p53 tumor suppressor protein. Curr. Opin. Cell Biol. 13,332-337[CrossRef][Medline]
  43. Cory, S., Adams, J. M. (2002) The bcl2 family: regulators of the cellular life-or-death switch. Nat. Rev. Cancer 2,647-656[CrossRef][Medline]
  44. Fesik, S. W. (2000) Insights into programmed cell death through structural biology. Cell 103,273-282[CrossRef][Medline]
  45. Huang, D. C., Strasser, A. (2000) BH3-only proteins—essential initiators of apoptotic cell death. Cell 103,839-842[CrossRef][Medline]
  46. Lindsten, T., Ross, A. J., King, A., Zong, W. X., Rathmell, J. C., Shiels, H. A., Ulrich, E., Waymire, K. G., Mahar, P., Frauwirth, K., et al (2000) The combined functions of proapoptotic Bcl-2 family members bak and bax are essential for normal development of multiple tissues. Mol. Cell 6,1389-1399[CrossRef][Medline]
  47. Wei, M. C., Zong, W. X., Cheng, E. H., Lindsten, T., Panoutsakopoulou, V., Ross, A. J., Roth, K. A., MacGregor, G. R., Thompson, C. B., Korsmeyer, S. J. (2001) Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death. Science 292,727-730[Abstract/Free Full Text]
  48. Wei, M. C., Lindsten, T., Mootha, V. K., Weiler, S., Gross, A., Ashiya, M., Thompson, C. B., Korsmeyer, S. J. (2000) tBID, a membrane-targeted death ligand, oligomerizes BAK to release cytochrome c. Genes Dev. 14,2060-2071[Abstract/Free Full Text]
  49. Dimmeler, S., Breitschopf, K., Haendeler, J., Zeiher, A. M. (1999) Dephosphorylation targets Bcl-2 for ubiquitin-dependent degradation: a link between the apoptosome and the proteasome pathway. J. Exp. Med. 189,1815-1822[Abstract/Free Full Text]
  50. Breitschopf, K., Haendeler, J., Malchow, P., Zeiher, A. M., Dimmeler, S. (2000) Posttranslational modification of Bcl-2 facilitates its proteasome-dependent degradation: molecular characterization of the involved signaling pathway. Mol. Cell. Biol. 20,1886-1896[Abstract/Free Full Text]
  51. Li, B., Dou, Q. P. (2000) Bax degradation by the ubiquitin/proteasome-dependent pathway: involvement in tumor survival and progression. Proc. Natl. Acad. Sci. USA 97,3850-3855[Abstract/Free Full Text]
  52. Marshansky, V., Wang, X., Bertrand, R., Luo, H., Duguid, W., Chinnadurai, G., Kanaan, N., Vu, M. D., Wu, J. (2001) Proteasomes modulate balance among proapoptotic and antiapoptotic Bcl-2 family members and compromise functioning of the electron transport chain in leukemic cells. J. Immunol. 166,3130-3142[Abstract/Free Full Text]
  53. Breitschopf, K., Zeiher, A. M., Dimmeler, S. (2000) Ubiquitin-mediated degradation of the proapoptotic active form of bid. A functional consequence on apoptosis induction. J. Biol. Chem. 275,21648-21652[Abstract/Free Full Text]
  54. Okura, T., Gong, L., Kamitani, T., Wada, T., Okura, I., Wei, C. F., Chang, H. M., Yeh, E. T. (1996) Protection against Fas/APO-1- and tumor necrosis factor-mediated cell death by a novel protein, sentrin. J. Immunol. 157,4277-4281[Abstract]
  55. Becker, K., Schneider, P., Hofmann, K., Mattmann, C., Tschopp, J. (1997) Interaction of Fas (Apo-1/CD95) with proteins implicated in the ubiquitination pathway. FEBS Lett. 412,102-106[CrossRef][Medline]
  56. Ryu, S. W., Chae, S. K., Kim, E. (2000) Interaction of Daxx, a Fas binding protein, with sentrin and Ubc9. Biochem. Biophys. Res. Commun. 279,6-10[CrossRef][Medline]
  57. Lalioti, V. S., Vergarajauregui, S., Pulido, D., Sandoval, I. V. (2002) The insulin-sensitive glucose transporter, GLUT4, interacts physically with Daxx. Two proteins with capacity to bind Ubc9 and conjugated to SUMO1. J. Biol. Chem. 277,19783-19791[Abstract/Free Full Text]
  58. Thome, M., Tschopp, J. (2001) Regulation of lymphocyte proliferation and death by FLIP. Nat Rev Immunol 1,50-58[CrossRef][Medline]
  59. Yeh, W. C., Itie, A., Elia, A. J., Ng, M., Shu, H. B., Wakeham, A., Mirtsos, C., Suzuki, N., Bonnard, M., Goeddel, D. V., et al (2000) Requirement for Casper (c-FLIP) in regulation of death receptor-induced apoptosis and embryonic development. Immunity 12,633-642[CrossRef][Medline]
  60. Griffith, T. S., Chin, W. A., Jackson, G. C., Lynch, D. H., Kubin, M. Z. (1998) Intracellular regulation of TRAIL-induced apoptosis in human melanoma cells. J. Immunol. 161,2833-2840[Abstract/Free Full Text]
  61. Kim, Y., Suh, N., Sporn, M., Reed, J. C. (2002) An inducible pathway for degradation of FLIP protein sensitizes tumor cells to TRAIL-induced apoptosis. J. Biol. Chem. 277,22320-22329[Abstract/Free Full Text]
  62. Bonavida, B., Ng, C. P., Jazirehi, A., Schiller, G., Mizutani, Y. (1999) Selectivity of TRAIL-mediated apoptosis of cancer cells and synergy with drugs: the trail to non-toxic cancer therapeutics (review). Int. J. Oncol. 15,793-802[Medline]
  63. Krebs, D. L., Hilton, D. J. (2001) SOCS proteins: negative regulators of cytokine signaling. Stem Cells 19,378-387[Abstract/Free Full Text]
  64. Kamura, T., Sato, S., Haque, D., Liu, L., Kaelin, W. G., Jr, Conaway, R. C., Conaway, J. W. (1998) The Elongin BC complex interacts with the conserved SOCS-box motif present in members of the SOCS, ras, WD-40 repeat, and ankyrin repeat families. Genes Dev. 12,3872-3881[Abstract/Free Full Text]
  65. Zhang, J. G., Farley, A., Nicholson, S. E., Willson, T. A., Zugaro, L. M., Simpson, R. J., Moritz, R. L., Cary, D., Richardson, R., Hausmann, G., et al (1999) The conserved SOCS box motif in suppressors of cytokine signaling binds to elongins B and C and may couple bound proteins to proteasomal degradation. Proc. Natl. Acad. Sci. USA 96,2071-2076[Abstract/Free Full Text]
  66. Frantsve, J., Schwaller, J., Sternberg, D. W., Kutok, J., Gilliland, D. G. (2001) Socs-1 inhibits TEL-JAK2-mediated transformation of hematopoietic cells through inhibition of JAK2 kinase activity and induction of proteasome-mediated degradation. Mol. Cell. Biol. 21,3547-3557[Abstract/Free Full Text]
  67. Kamizono, S., Hanada, T., Yasukawa, H., Minoguchi, S., Kato, R., Minoguchi, M., Hattori, K., Hatakeyama, S., Yada, M., Morita, S., et al (2001) The SOCS box of SOCS-1 accelerates ubiquitin-dependent proteolysis of TEL-JAK2. J. Biol. Chem. 276,12530-12538[Abstract/Free Full Text]
  68. De Sepulveda, P., Ilangumaran, S., Rottapel, R. (2000) Suppressor of cytokine signaling-1 inhibits VAV function through protein degradation. J. Biol. Chem. 275,14005-14008[Abstract/Free Full Text]
  69. Deng, L., Wang, C., Spencer, E., Yang, L., Braun, A., You, J., Slaughter, C., Pickart, C., Chen, Z. J. (2000) Activation of the IkappaB kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain. Cell 103,351-361[CrossRef][Medline]
  70. Wang, C., Deng, L., Hong, M., Akkaraju, G. R., Inoue, J., Chen, Z. J. (2001) TAK1 is a ubiquitin-dependent kinase of MKK and IKK. Nature (London) 412,346-351[CrossRef][Medline]
  71. Karin, M., Ben-Neriah, Y. (2000) Phosphorylation meets ubiquitination: the control of NF{kappa}B activity. Annu. Rev. Immunol. 18,621-663[CrossRef][Medline]
  72. Crook, N. E., Clem, R. J., Miller, L. K. (1993) An apoptosis-inhibiting baculovirus gene with a zinc finger-like motif. J. Virol. 67,2168-2174[Abstract/Free Full Text]
  73. Deveraux, Q. L., Reed, J. C. (1999) IAP family proteins–suppressors of apoptosis. Genes Dev. 13,239-252[Free Full Text]
  74. Richter, B. W., Mir, S. S., Eiben, L. J., Lewis, J., Reffey, S. B., Frattini, A., Tian, L., Frank, S., Youle, R. J., Nelson, D. L., et al (2001) Molecular cloning of ILP-2, a novel member of the inhibitor of apoptosis protein family. Mol. Cell. Biol. 21,4292-4301[Abstract/Free Full Text]
  75. Vucic, D., Stennicke, H. R., Pisabarro, M. T., Salvesen, G. S., Dixit, V. M. (2000) ML-IAP, a novel inhibitor of apoptosis that is preferentially expressed in human melanomas. Curr. Biol. 10,1359-1366[CrossRef][Medline]
  76. Goyal, L. (2001) Cell death inhibition: keeping caspases in check. Cell 104,805-808[CrossRef][Medline]
  77. Yang, Y. L., Li, X. M. (2000) The IAP family: endogenous caspase inhibitors with multiple biological activities. Cell Res. 10,169-177[CrossRef][Medline]
  78. Li, F., Ambrosini, G., Chu, E. Y., Plescia, J., Tognin, S., Marchisio, P. C., Altieri, D. C. (1998) Control of apoptosis and mitotic spindle checkpoint by survivin. Nature (London) 396,580-584[CrossRef][Medline]
  79. Fraser, A. G., James, C., Evan, G. I., Hengartner, M. O. (1999) Caenorhabditis elegans inhibitor of apoptosis protein (IAP) homologue BIR-1 plays a conserved role in cytokinesis. Curr. Biol. 9,292-301[CrossRef][Medline]
  80. Uren, A. G., Beilharz, T., O’Connell, M. J., Bugg, S. J., van Driel, R., Vaux, D. L., Lithgow, T. (1999) Role for yeast inhibitor of apoptosis (IAP)-like proteins in cell division. Proc. Natl. Acad. Sci. USA 96,10170-10175[Abstract/Free Full Text]
  81. Verhagen, A. M., Coulson, E. J., Vaux, D. L. (2001) Inhibitor of apoptosis proteins and their relatives: IAPs and other BIRPs. Genome Biol. 2REVIEWS3009
  82. Yang, Y., Fang, S., Jensen, J. P., Weissman, A. M., Ashwell, J. D. (2000) Ubiquitin protein ligase activity of IAPs and their degradation in proteasomes in response to apoptotic stimuli. Science 288,874-877[Abstract/Free Full Text]
  83. Clem, R. J., Sheu, T. T., Richter, B. W., He, W. W., Thornberry, N. A., Duckett, C. S., Hardwick, J. M. (2001) c-IAP1 is cleaved by caspases to produce a proapoptotic C-terminal fragment. J. Biol. Chem. 276,7602-7608[Abstract/Free Full Text]
  84. Huang, H., Joazeiro, C. A., Bonfoco, E., Kamada, S., Leverson, J. D., Hunter, T. (2000) The inhibitor of apoptosis, cIAP2, functions as a ubiquitin-protein ligase and promotes in vitro monoubiquitination of caspases 3 and 7. J. Biol. Chem. 275,26661-26664[Abstract/Free Full Text]
  85. Suzuki, Y., Nakabayashi, Y., Takahashi, R. (2001) 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. Proc. Natl. Acad. Sci. USA 98,8662-8667[Abstract/Free Full Text]
  86. Harlin, H., Reffey, S. B., Duckett, C. S., Lindsten, T., Thompson, C. B. (2001) Characterization of XIAP-deficient mice. Mol. Cell. Biol. 21,3604-3608[Abstract/Free Full Text]
  87. Hay, B. A., Wassarman, D. A., Rubin, G. M. (1995) Drosophila homologs of baculovirus inhibitor of apoptosis proteins function to block cell death. Cell 83,1253-1262[CrossRef][Medline]
  88. Vucic, D., Kaiser, W. J., Miller, L. K. (1998) Inhibitor of apoptosis proteins physically interact with and block apoptosis induced by Drosophila proteins HID and GRIM. Mol. Cell. Biol. 18,3300-3309[Abstract/Free Full Text]
  89. Wang, S. L., Hawkins, C. J., Yoo, S. J., Muller, H. A., Hay, B. A. (1999) The Drosophila caspase inhibitor DIAP1 is essential for cell survival and is negatively regulated by HID. Cell 98,453-463[CrossRef][Medline]
  90. Goyal, L., McCall, K., Agapite, J., Hartwieg, E., Steller, H. (2000) Induction of apoptosis by Drosophila reaper, hid and grim through inhibition of IAP function. EMBO J. 19,589-597[CrossRef][Medline]
  91. Palaga, T., Osborne, B. (2002) The 3D’s of apoptosis: death, degradation and DIAPs. Nat. Cell Biol. 4,E149-E151[CrossRef][Medline]
  92. Hays, R., Wickline, L., Cagan, R. (2002) Morgue mediates apoptosis in the Drosophila melanogaster retina by promoting degradation of DIAP1. Nat. Cell Biol. 4,425-431[CrossRef][Medline]
  93. Ryoo, H. D., Bergmann, A., Gonen, H., Ciechanover, A., Steller, H. (2002) Regulation of Drosophila IAP1 degradation and apoptosis by reaper and ubcD1. Nat. Cell Biol. 4,432-438[CrossRef][Medline]
  94. Holley, C. L., Olson, M. R., Colon-Ramos, D. A., Kornbluth, S. (2002) Reaper eliminates IAP proteins through stimulated IAP degradation and generalized translational inhibition. Nat. Cell Biol. 4,439-444[CrossRef][Medline]
  95. Yoo, S. J., Huh, J. R., Muro, I., Yu, H., Wang, L., Wang, S. L., Feldman, R. M., Clem, R. J., Muller, H. A., Hay, B. A. (2002) Hid, Rpr and Grim negatively regulate DIAP1 levels through distinct mechanisms. Nat. Cell Biol. 4,416-424[CrossRef][Medline]
  96. Wilson, R., Goyal, L., Ditzel, M., Zachariou, A., Baker, D. A., Agapite, J., Steller, H., Meier, P. (2002) The DIAP1 RING finger mediates ubiquitination of Dronc and is indispensable for regulating apoptosis. Nat. Cell Biol. 4,445-450[CrossRef][Medline]
  97. Wing, J. P., Schreader, B. A., Yokokura, T., Wang, Y., Andrews, P. S., Huseinovic, N., Dong, C. K., Ogdahl, J. L., Schwartz, L. M., White, K., et al (2002) Drosophila morgue is an F box/ubiquitin conjugase domain protein important for grim-reaper mediated apoptosis. Nat. Cell Biol. 4,451-456[CrossRef][Medline]
  98. Tenev, T., Zachariou, A., Wilson, R., Paul, A., Meier, P. (2002) Jafrac2 is an IAP antagonist that promotes cell death by liberating Dronc from DIAP1. EMBO J. 21,5118-5129[CrossRef][Medline]
  99. Du, C., Fang, M., Li, Y., Li, L., Wang, X. (2000) Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition. Cell 102,33-42[CrossRef][Medline]
  100. Verhagen, A. M., Ekert, P. G., Pakusch, M., Silke, J., Connolly, L. M., Reid, G. E., Moritz, R. L., Simpson, R. J., Vaux, D. L. (2000) Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins. Cell 102,43-53[CrossRef][Medline]
  101. Chai, J., Du, C., Wu, J. W., Kyin, S., Wang, X., Shi, Y. (2000) Structural and biochemical basis of apoptotic activation by Smac/DIABLO. Nature (London) 406,855-862[CrossRef][Medline]
  102. Liu, Z., Sun, C., Olejniczak, E. T., Meadows, R. P., Betz, S. F., Oost, T., Herrmann, J., Wu, J. C., Fesik, S. W. (2000) Structural basis for binding of Smac/DIABLO to the XIAP BIR3 domain. Nature (London) 408,1004-1008