Proteasome involvement in agonist-induced down-regulation of mu and delta opioid receptors

This study investigated the mechanism of agonist-induced opioid receptor down-regulation. Incubation of HEK 293 cells expressing FLAG-tagged delta and mu receptors with agonists caused a time-dependent decrease in opioid receptor levels assayed by immunoblotting. Pulse-chase experiments using [(35)S]methionine metabolic labeling indicated that the turnover rate of delta receptors was accelerated 5-fold following agonist stimulation. Inactivation of functional G(i) and G(o) proteins by pertussis toxin-attenuated down-regulation of the mu opioid receptor, while down-regulation of the delta opioid receptor was unaffected. Pretreatment of cells with inhibitors of lysosomal proteases, calpain, and caspases had little effect on mu and delta opioid receptor down-regulation. In marked contrast, pretreatment with proteasome inhibitors attenuated agonist-induced mu and delta receptor down-regulation. In addition, incubation of cells with proteasome inhibitors in the absence of agonists increased steady-state mu and delta opioid receptor levels. Immunoprecipitation of mu and delta opioid receptors followed by immunoblotting with ubiquitin antibodies suggested that preincubation with proteasome inhibitors promoted accumulation of polyubiquitinated receptors. These data provide evidence that the ubiquitin/proteasome pathway plays a role in agonist-induced down-regulation and basal turnover of opioid receptors.     

Rapid down-regulation of the type I inositol 1,4,5-trisphosphate receptor and desensitization of gonadotropin-releasing hormone-mediated Ca2+ responses in alpha T3-1 gonadotropes

Despite no evidence for desensitization of phospholipase C-coupled gonadotropin-releasing hormone (GnRH) receptors, we previously reported marked suppression of GnRH-mediated Ca(2+) responses in alphaT3-1 cells by pre-exposure to GnRH. This suppression could not be accounted for solely by reduced inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) responses, thereby implicating uncoupling of Ins(1,4,5)P(3) production and Ca(2+) mobilization (McArdle, C. A., Willars, G. B., Fowkes, R. C., Nahorski, S. R., Davidson, J. S., and Forrest-Owen, W. (1996) J. Biol. Chem. 271, 23711-23717). In the current study we demonstrate that GnRH causes a homologous and heterologous desensitization of Ca(2+) signaling in alphaT3-1 cells that is coincident with a rapid (t((12)) < 20 min), marked, and functionally relevant loss of type I Ins(1,4,5)P(3) receptor immunoreactivity and binding. Furthermore, using an alphaT3-1 cell line expressing recombinant muscarinic M(3) receptors we show that the unique resistance of the GnRH receptor to rapid desensitization contributes to a fast, profound, and sustained loss of Ins(1,4,5)P(3) receptor immunoreactivity. These data highlight a potential role for rapid Ins(1,4,5)P(3) receptor down-regulation in homologous and heterologous desensitization and in particular suggest that this mechanism may contribute to the suppression of the reproductive system that is exploited in the major clinical applications of GnRH analogues.     

Identification of Ser153 in ICL2 of the gonadotropin-releasing hormone (GnRH) receptor as a phosphorylation-independent site for inhibition of Gq coupling

Type I gonadotropin-releasing hormone (GnRH) receptor (GnRHR) is unique among mammalian G-protein-coupled receptors (GPCRs) in lacking a C-terminal tail, which is involved in desensitization in GPCRs. Therefore, we searched for inhibitory sites in the intracellular loops (ICLs) of the GnRHR. Synthetic peptides corresponding to the three ICLs were inserted into permeabilized alphaT3-1 gonadotrope cells, and GnRH-induced inositol phosphate (InsP) formation was determined. GnRH-induced InsP production was potentiated by ICL2 > ICL3 but not by the ICL1 peptides, suggesting they are acting as decoy peptides. We examined the effects of six peptides in which only one of the Ser or Thr residues was substituted with Ala or Glu. Only substitution of Ser153 with Ala or Glu ablated the potentiating effect upon GnRH-induced InsP elevation. ERK activation was enhanced, and the rate of GnRH-induced InsP formation was about 6.5-fold higher in the first 10 min in COS-1 cells that were transfected with mutants of the GnRHR in which the ICL2 Ser/Thr residues (Ser151, Ser153, and Thr142) or only Ser153 was mutated to Ala as compared with the wild type GnRHR. The data indicate that ICL2 harbors an inhibitory domain, such that exogenous ICL2 peptide serves as a decoy for the inhibitory site (Ser153) of the GnRHR, thus enabling further activation. GnRH does not induce receptor phosphorylation in alphaT3-1 cells. Because the phosphomimetic ICL2-S153E peptide did not mimic the stimulatory effect of the ICL2 peptide, the inhibitory effect of Ser153 operates through a phosphorylation-independent mechanism.     

Estrogen receptor-alpha hinge-region lysines 302 and 303 regulate receptor degradation by the proteasome

Cellular levels of estrogen receptor-alpha (ERalpha) protein are regulated primarily by the ubiquitin-proteasome pathway. Dynamic interactions between ERalpha and the protein degradation machinery facilitate the down-regulation process by targeting receptor lysine residues for polyubiquitination. To date, the lysines that control receptor degradation have not been identified. Two receptor lysines, K302 and K303, located in the hinge-region of ERalpha, serve multiple regulatory functions, and we examined whether these might also regulate receptor polyubiquitination, turnover, and receptor-protein interactions. We used ERalpha-negative breast cancer C4-12 cells to generate cells stably expressing wild-type (wt)ERalpha or ERalpha with lysine-to-alanine substitutions at K302 and K303 (ERalpha-AA). In the unliganded state, ERalpha-AA displayed rapid polyubiquitination and enhanced basal turnover, as compared with wtERalpha, due to its elevated association with the ubiquitin ligase carboxy terminus of Hsc70-interacting protein (CHIP) and the proteasome-associated cochaperone Bag1. Treatment of C4-12 cells with either 17beta-estradiol (E2) or the pure antiestrogen ICI 182,780 (ICI) induced rapid degradation of wtERalpha via the ubiquitin-proteasome pathway; however, in the presence of these ligands, ERalpha-AA was less efficiently degraded. Furthermore, ERalpha-AA was resistant to ICI-induced polyubiquitination, suggesting that these lysines are polyubiquitinated in response to the antiestrogen and demonstrate a novel role for these two lysines in the mechanism of action of ICI-induced receptor down-regulation. The reduced stability of ERalpha-AA in the unliganded state and the increased stability of ERalpha-AA in the liganded state were concordant with reporter gene assays demonstrating that ERalpha-AA has lower basal activity but higher E2 inducibility than wtERalpha. These data provide the first evidence that K302/303 protect ERalpha from basal degradation and are necessary for efficient E2- and ICI-induced turnover in breast cancer cells.     

Activation of translation in pituitary gonadotrope cells by gonadotropin-releasing hormone

The neuropeptide GnRH is a central regulator of mammalian reproductive function produced by a dispersed population of hypothalamic neurosecretory neurons. The principal action of GnRH is to regulate release of the gonadotropins, LH and FSH, by the gonadotrope cells of the anterior pituitary. Using a cultured cell model of mouse pituitary gonadotrope cells, alphaT3-1 cells, we present evidence that GnRH stimulation of alphaT3-1 cells results in an increase in cap-dependent mRNA translation. GnRH receptor activation results in increased protein synthesis through a regulator of mRNA translation initiation, eukaryotic translation initiation factor 4E-binding protein, known as 4EBP or PHAS (protein, heat, and acid stable). Although the GnRH receptor is a member of the rhodopsin-like family of G protein-linked receptors, we show that activation of translation proceeds through a signaling pathway previously described for receptor tyrosine kinases. Stimulation of translation by GnRH is protein kinase C and Ras dependent and sensitive to rapamycin. Furthermore, GnRH may also regulate the cell cycle in alphaT3-1 cells. The activation of a signaling pathway that regulates both protein synthesis and cell cycle suggests that GnRH may have a significant role in the maintenance of the pituitary gonadotrope population in addition to directing the release of gonadotropins.     

SPFH2 mediates the endoplasmic reticulum-associated degradation of inositol 1,4,5-trisphosphate receptors and other substrates in mammalian cells

Inositol 1,4,5-trisphosphate (IP(3)) receptors are endoplasmic reticulum (ER) membrane calcium channels that, upon activation, become substrates for the ER-associated degradation (ERAD) pathway. Although it is clear that IP(3) receptors are polyubiquitinated upon activation and are transferred to the proteasome by a p97-based complex, currently nothing is known about the proteins that initially select activated IP(3) receptors for ERAD. Here, we sought to identify novel proteins that associate with and mediate the ERAD of endogenous activated IP(3) receptors. SPFH2, an uncharacterized SPFH domain-containing protein, rapidly associated with IP(3) receptors in a manner that preceded significant polyubiquitination and the association of p97 and related proteins. SPFH2 was found to be an ER membrane protein largely residing within the ER lumen and in resting and stimulated cells was linked to ERAD pathway components, apparently via endogenous substrates undergoing degradation. Suppression of SPFH2 expression by RNA interference markedly inhibited IP(3) receptor polyubiquitination and degradation and the processing of other ERAD substrates. Overall, these studies identify SPFH2 as a key ERAD pathway component and suggest that it may act as a substrate recognition factor.     

Matrix metalloproteinases 2 and 9 mediate epidermal growth factor receptor transactivation by gonadotropin-releasing hormone

Rapid engagement of the extracellular signal-regulated kinase (ERK) cascade via the Gq/11-coupled GnRH receptor (GnRHR) is mediated by transactivation of the epidermal growth factor receptor (EGFR). Here we show that the cross-talk between GnRHR and EGFR in gonadotropic cells is accomplished via gelatinases A and B (matrix metalloproteinases (MMPs) 2 and 9), identifying gelatinases as the first distinct members of the MMP family mediating EGFR transactivation by G protein-coupled receptors. Using a specific MMP2 and MMP9 inhibitor, Ro28-2653, GnRH-dependent EGFR transactivation was abrogated. Proving the specificity of the effect, transient transfection of alphaT3-1 cells with ribozymes directed against MMP2 or MMP9 specifically blocked EGFR tyrosine phosphorylation in response to GnRH stimulation. GnRH challenge of alphaT3-1 cells furthered the release of active MMP2 and MMP9 and increased their gelatinolytic activities within 5 min. Rapid release of activated MMP2 or MMP9 was inhibited by ribozyme-targeted down-regulation of MT1-MMP or MMP2, respectively. We found that GnRH-induced Src, Ras, and ERK activation were also gelatinase-dependent. Thus, gelatinase-induced EGFR transactivation was required to engage the extracellular-signal regulated kinase cascade. Activation of c-Jun N-terminal kinase and p38 MAPK by GnRH was unaffected by EGFR or gelatinase inhibition that, however, suppressed GnRH induction of c-Jun and c-Fos. Our findings suggest a novel role for gelatinases in the endocrine regulation of pituitary gonadotropes.     

The importance of nuclear import in protection of the vitamin D receptor from polyubiquitination and proteasome‐mediated degradation

Others and we previously showed that the vitamin D receptor (VDR) is subject to degradation by the 26S proteasome and that treatment with 1,25‐dihydroxyvitamin D₃ (1,25D₃) inhibited this degradation. In the present study, we found that in osteoblasts, but not in intestinal epithelial cells, the VDR was susceptible to degradation by the 26S proteasome. The subcellular site for degradation of the VDR in osteoblasts is the cytoplasm and the site for ligand‐dependent protection of the VDR from the 26S proteasome is the chromatin. These direct relationships between nuclear localization and protection of the VDR from 26S proteasome degradation led us to hypothesize that the unoccupied cytoplasmic VDR is a substrate for polyubiquitination, which targets VDR for degradation by the 26S proteasome, and that nuclear localization has the ability to protect the VDR from polyubiquitination and degradation. To test these hypotheses, we used Cos‐1 cells transfected with human VDR and histidine‐tagged ubiquitin expression vectors. We found that unoccupied VDR was polyubiquitinated and that 1,25D₃ inhibited this modification. Mutations in the nuclear localization signal of VDR (R49W/R50G and K53Q/R54G/K55E) or in the dimerization interface of VDR with retinoid X receptor (M383G/Q385A) abolished the ability of 1,25D₃ to protect the VDR from polyubiquitination, although these mutations had no effect on the ligand‐binding activity of VDR. Therefore, we concluded that in some cellular environments unoccupied cytoplasmic VDR is susceptible to polyubiquitination and proteasome degradation and that ligand‐dependent heterodimerization and nuclear localization protect the VDR from these modifications. J. Cell. Biochem. 110: 926–934, 2010. © 2010 Wiley‐Liss, Inc.     

Involvement of the ubiquitin-proteasome pathway in the degradation of nontyrosine kinase-type cytokine receptors of IL-9, IL-2, and erythropoietin

The ubiquitin-dependent proteasome-mediated (Ub-Pr) degradation pathway has been shown to regulate a large variety of substrates, including nuclear, cytosolic, and membrane proteins. In mammalian systems, polyubiquitin modification has been identified in a number of cell surface receptors for more than a decade; however, its biological significance has remained unclear until recently. For growth factor receptors with intrinsic tyrosine kinase domains, polyubiquitination is believed to trigger the internalization and subsequent degradation via the lysosomal pathway. In this study we provide the first evidence that non-tyrosine kinase-type cytokine surface receptors, IL-9R alpha-chain, IL-2 receptor ss-chain, and erythropoietin receptor, can be polyubiquitinated and degraded by proteasomes. The Ub-Pr pathway regulates both the basal level turnover and the ligand-induced degradation of the receptors. A previously identified putative molecular chaperon, valosin-containing protein, undergoes tyrosine phosphorylation in a cytokine-dependent manner and associates with the receptor complexes following receptor engagement, suggesting that valosin-containing protein may target the ubiquitinated receptors to the proteasome for degradation.     

Alternative effects of the ubiquitin-proteasome pathway on glucocorticoid receptor down-regulation and transactivation are mediated by CHIP, an E3 ligase

The ubiquitin/proteasome-dependent protein degradation pathway (UPP) is responsible for the accelerated down-regulation of glucocorticoid receptor (GR) levels in cells subjected to chronic glucocorticoid exposure. Whereas hormone-dependent down-regulation of GR operates in most cells, the receptor is not down-regulated after long-term glucocorticoid treatment of either cultured embryonic hippocampal neurons or the HT22 hippocampal cell line. In this report, we show that stable overexpression of the carboxy terminus of heat shock protein 70-interacting protein (CHIP) E3 ligase can restore hormone-dependent down-regulation of GR in HT22 cells. Proteasome inhibitor studies establish that ubiquitylated GR can be efficiently engaged with the proteasome upon CHIP overexpression, unlike the case in parental HT22 cells. In addition to its impact on GR down-regulation, CHIP overexpression alters the coupling between the UPP and GR transactivation. Unlike other steroid receptors whose transactivation properties are typically reduced upon proteasome inhibition, GR transactivation in HT22 cells and other cell lines is enhanced upon proteasome inhibition. However, in HT22 cells overexpressing CHIP, proteasome inhibition leads to a reduction in GR transactivation activity. Thus, the divergent response of a single transactivator (i.e. GR) to the UPP can be dictated by CHIP, an E3 ligase that also functions as a proteasome-targeting factor.     

Gonadotropin-releasing hormone receptor initiates multiple signaling pathways by exclusively coupling to G(q/11) proteins

The agonist-bound gonadotropin-releasing hormone (GnRH) receptor engages several distinct signaling cascades, and it has recently been proposed that coupling of a single type of receptor to multiple G proteins (G(q), G(s), and G(i)) is responsible for this behavior. GnRH-dependent signaling was studied in gonadotropic alphaT3-1 cells endogenously expressing the murine receptor and in CHO-K1 (CHO#3) and COS-7 cells transfected with the human GnRH receptor cDNA. In all cell systems studied, GnRH-induced phospholipase C activation and Ca(2+) mobilization was pertussis toxin-insensitive, as was GnRH-mediated extracellular signal-regulated kinase activation. Whereas the G(i)-coupled m2 muscarinic receptor interacted with a chimeric G(s) protein (G(s)i5) containing the C-terminal five amino acids of Galpha(i2), the human GnRH receptor was unable to activate the G protein chimera. GnRH challenge of alphaT3-1, CHO#3 and of GnRH receptor-expressing COS-7 cells did not result in agonist-dependent cAMP formation. GnRH challenge of CHO#3 cells expressing a cAMP-responsive element-driven firefly luciferase did not result in increased reporter gene expression. However, coexpression of the human GnRH receptor and adenylyl cyclase I in COS-7 cells led to clearly discernible GnRH-dependent cAMP formation subsequent to GnRH-elicited rises in [Ca(2+)](i). In alphaT3-1 and CHO#3 cell membranes, addition of [alpha-(32)P]GTP azidoanilide resulted in GnRH receptor-dependent labeling of Galpha(q/11) but not of Galpha(i), Galpha(s) or Galpha(12/13) proteins. Thus, the murine and human GnRH receptors exclusively couple to G proteins of the G(q/11) family. Multiple GnRH-dependent signaling pathways are therefore initiated downstream of the receptor/G protein interface and are not indicative of a multiple G protein coupling potential of the GnRH receptor.     

Adenylate cyclase regulation via proteasome-mediated modulation of Galphas levels

The adenylate cyclase (AC)/cyclic AMP (cAMP)/cAMP-dependent protein kinase pathway controls many biological phenomena. The ubiquitin/proteasome system, controlling the levels of many proteins, modulates important cellular processes such as cell cycle and cell growth. Here we describe a novel mechanism for AC regulation by proteasome pathway. Pharmacological inhibition of proteasome function in human osteosarcoma U2OS cells results in up-regulation of AC activity, increase of levels of alpha subunit of heterotrimeric stimulatory GTP-binding proteins (alphas) and, remarkably, also in preventing of beta-adrenergic receptor-mediated down-regulation of alphas protein levels. Accumulation of alphas protein is also accompanied by the appearance of polyubiquitinated alphas species. Our results: (1) identify alphas protein as a novel proteasome substrate in mammalian cells; (2) indicate that proteasome might play a physiological role in controlling AC/cAMP mediated pathways by modulating the levels of Galphas protein; (3) suggest a role for the proteasome also in controlling alphas-mediated signaling pathways other than those affecting AC complex.     

Cytosolic components are required for proteasomal degradation of newly synthesized apolipoprotein B in permeabilized HepG2 cells.

Recent studies have proposed that post-translational degradation of apolipoprotein B100 (apoB) involves the cytosolic ubiquitin-proteasome pathway. In this study, immunocytochemistry indicated that endoplasmic reticulum (ER)-associated proteasome molecules were concentrated in perinuclear regions of digitonin-permeabilized HepG2 cells. Signals produced by antibodies that recognize both alpha- and beta-subunits of the proteasome co-localized in the ER with specific domains of apoB. The mechanism of apoB degradation in the ER by the ubiquitin-proteasome pathway was studied using pulse-chase labeling and digitonin-permeabilized cells. ApoB in permeabilized cells incubated at 37 degrees C in buffer alone was relatively stable. When permeabilized cells were incubated with both exogenous ATP and rabbit reticulocyte lysate (RRL) as a source of ubiquitin-proteasome factors, >50% of [3H]apoB was degraded in 30 min. The degradation of apoB in the intact ER of permeabilized cells was much more rapid than that of extracted [3H]apoB incubated with RRL and ATP in vitro. The degradation of apoB was reduced by clasto-lactacystin beta-lactone, a potent proteasome inhibitor, and by ubiquitin K48R mutant protein, an inhibitor of polyubiquitination. ApoB in HepG2 cells was ubiquitinated, and polyubiquitination of apoB was stimulated by incubation of permeabilized cells with RRL. These results suggest that newly synthesized apoB in the ER is accessible to the cytoplasmic ubiquitin-proteasome pathway and that factors in RRL stimulate polyubiquitination of apoB, leading to rapid degradation of apoB in permeabilized cells.     

Involvement of the betaTrCP in the ubiquitination and stability of the HIV-1 Vpu protein

The human immunodeficiency virus type 1 (HIV-1) Vpu protein binds to the CD4 receptor and targets it to the proteasome for degradation. This process requires the recruitment of human betaTrCP, a component of the Skp1-Cullin-F box (SCF) ubiquitin ligase complex, that interacts with phosphorylated Vpu molecules. Vpu, unlike other ligands of betaTrCP, has never been reported to be degraded. We provide evidence that Vpu, itself, is ubiquitinated and targeted for degradation by the proteasome. We demonstrate that the mutant Vpu2.6, which cannot interact with betaTrCP, is stable and, unlike wild-type Vpu, is not polyubiquitinated. These results suggest that betaTrCP is involved in Vpu polyubiquitination.     

Real-time analysis of the activities of GnRH and GnRH analogs in alphaT3-1 cells by the Cytosensor microphysiometer

Gonadotropin-releasing hormone (GnRH), acting via the GnRH receptor, elicited rapid extracellular acidification responses in mouse gonadotrope-derived alphaT3-1 cells as measured by the Cytosensor microphysiometer, which indirectly monitors cellular metabolic rates. GnRH increased the extracellular acidification rate of the cells in a dose-dependent manner (EC(50) = 1.81 +/- 0.24 nM). The GnRH-stimulated acidification rate could be attenuated by protein kinase C (PKC) down-regulation, extracellular Ca2+ depletion, and the voltage-sensitive Ca2+ channel (VSCC) blocker nifedipine, indicating that the acidification response is activated by both Ca2+ and PKC-mediated pathways. Upon continuous exposure to 100 nM GnRH or periodic stimulation by 10 nM GnRH at 40 min intervals, homologous desensitization was more pronounced in the absence of extracellular Ca2+, suggesting that desensitization of GnRH activity may be mediated via depletion of intracellular Ca2+ stores. We have also compared the potency of eight GnRH analogs on alphaT3-1 cells. No acidification response was detected for GnRH free acid, consistent with the idea that the C-terminal amide is a critical structural determinant for GnRH activity. Replacement of Gly-NH(2) at the C-terminus by N-ethylamide dramatically reduced the EC(50) value, suggesting that substitution of the Gly-NH(2) moiety by N-ethylamide increases the potency of GnRH analogs. Substitution of Gly at position 6 by D-Trp significantly reduced the EC(50) value, whereas D-Lys at the same position slightly increased the EC(50) value, implying that either an aromatic amino acid or a non-basic amino acid at position 6 may be essential for potent GnRH agonists. In summary, our results demonstrate that the Cytosensor microphysiometer can be used to evaluate the actions of GnRH and GnRH analogs in alphaT3-1 cells in a real-time and noninvasive manner. This silicon-based microphysiometric system should provide new information on the structure-function studies of GnRH and is an invaluable tool for the screening of new GnRH agonists and antagonists in the future.     

Steroid receptor coactivator-3 is required for progesterone receptor trans-activation of target genes in response to gonadotropin-releasing hormone treatment of pituitary cells

Regulation of gonadotropin production involves interplay between steroids and neuropeptides, and we have examined the effects of gonadotropin-releasing hormones (GnRH I and GnRH II) on progesterone receptor (PR) activation in alphaT3-1 pituitary cells. Treatment with GnRHs activated a progester-one response element (PRE)-luciferase reporter gene, and this was blocked by protein kinase C and protein kinase A inhibitors but not by RU486. Treatment with GnRHs phosphorylated the PR at Ser(294) and increased PR translocation to the nucleus within 1 h. Interactions between the PR and several coactivators were examined, and treatment with GnRHs specifically induced PR-steroid receptor coactivator-3 (SRC-3) interactions within 8 h. In chromatin immunoprecipitation assays, recruitment of PR and SRC-3 by the PREs of the luciferase reporter gene or the gonadotopin alpha-subunit gene promoter was also increased by GnRHs within 8 h, while progesterone-induced recruitment of PR to the PREs occurred in association with much less SRC-3. A small interfering RNA knockdown of type I GnRH receptor levels reduced PR activation by GnRHs, while progesterone-dependent PR activation was unaffected. Moreover, small interfering RNA knockdown of SRC-3 abolished PRE-luciferase trans-activation by the PR in response to GnRHs. Collectively, these data indicate that PR activation by GnRHs in alphaT3-1 cells is type I GnRH receptor-mediated and that trans-activation of PR-responsive genes requires SRC-3 in this context.     

X-linked inhibitor of apoptosis functions as ubiquitin ligase toward mature caspase-9 and cytosolic Smac/DIABLO

Members of the IAP (inhibitor of apoptosis) family function as anti-apoptotic proteins by binding directly to caspase-3, -7, and -9 to inhibit their activities. During apoptosis, the activities of IAPs are relieved by a second mitochondria-derived caspase activator, named Smac/DIABLO. Some IAPs have a C-terminal RING finger domain that has been identified as the essential motif for the activity of ubiquitin ligase (E3). Here we show that X-linked IAP (XIAP) mediates the polyubiquitination of caspase-9 and Smac. The large subunit of mature caspase-9 was polyubiquitinated by XIAP in vitro, while procaspase-9 was not. Furthermore, the polyubiquitinated form of caspase-9 accumulated in an XIAP-dependent manner in intact cells. The ubiquitination of caspase-9 was significantly inhibited in the presence of mature Smac, whereas XIAP was also found to promote the polyubiquitination of cytosolic Smac both in vitro and in intact cells. These ubiquitination reactions require the RING finger domain of XIAP. These findings suggest that XIAP functions as ubiquitin ligase toward mature caspase-9 and Smac to inhibit apoptosis.