Ubiquitin- and ATP-Independent Proteolytic Turnover of p21 by the REGγ-Proteasome Pathway

Molecular Cell

Article Ubiquitin- and ATP-Independent Proteolytic Turnover of p21 by the REGg-Proteasome Pathway Xiaotao Li,1 Larbi Amazit,1 Weiwen Long,1 David M. Lonard,1 John J. Monaco,2 and Bert W. O’Malley1,* 1

Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, Cincinnati, OH 45267, USA *Correspondence: [email protected] DOI 10.1016/j.molcel.2007.05.028 2

SUMMARY

We previously demonstrated that the proteasome activator REGg directs degradation of the steroid receptor coactivator SRC-3 by the 20S proteasome in an ATP- and ubiquitinindependent manner. Our efforts to identify additional endogenous direct targets of the REGg proteasome revealed that p21Waf/Cip1, a central cyclin-dependent kinase inhibitor, is another endogenous target. Gain-of-function analysis, RNAi knockdown, REGg-deficient MEF analysis, and pulse-chase experiments substantiate that REGg promotes degradation of unbound p21. Cell-free proteasome proteolysis assays using purified REGg, p21, and the 20S proteasome confirm that REGg directly mediates degradation of free p21 in an ATPand ubiquitin-independent manner. Depletion of REGg in a thyroid carcinoma cell line results in cell-cycle and proliferative alterations. Our study reveals that, in addition to degrading the SRC-3 growth coactivator, REGg also has a role in the regulation of the cell cycle through its ability to influence the level of a cell-cycle regulator(s). INTRODUCTION The proteasome, a large multisubunit protease, is the primary protease responsible for the degradation of short-lived regulatory proteins. Generally, destruction of intact cellular proteins is orchestrated in an ATP- and ubiquitin-dependent manner by the 26S proteasome (Varshavsky, 2005). However, a growing number of ubiquitin-independent proteasome degradation pathways also have been characterized. Recently, REGg-mediated proteolysis has been added to this group, degrading the oncogenic protein SRC-3 (Li et al., 2006). REGg (also known as PA28, PSME3, and Ki antigen) belongs to the REG or 11S family of proteasome activator ‘‘caps’’ that have been shown to bind and activate the 20S proteasome (Dubiel et al., 1992; Ma et al., 1992). Originally

identified as the Ki antigen, REGg is the target of an autoantibody appearing in the serum of systemic lupus erythematosus patients, but the relationship between REGg’s role in the proteasome and this autoimmune syndrome is not clear (Nikaido et al., 1990). Although REGg shares only about 25% identity with REGa and REGb, the overall secondary structure of REG proteins is quite similar with all proteins composed of four long a helixes of 33–45 residues in length (Li and Rechsteiner, 2001). The linker sequence between helix 2 and 3, designated the ‘‘activation loop,’’ is highly conserved in all three REG species. Mutagenesis studies demonstrated that this region is particularly important for proteasome activation (Zhang et al., 1998); a single point mutation in this loop (N151Y) results in a REGg mutant that is unable to activate the proteasome (Li et al., 2006; Zhang et al., 1998). Two individual groups have generated REGg-deficient mice (Barton et al., 2004; Murata et al., 1999). Both studies found that loss of REGg expression results in reduced body size and cell-specific mitotic defects, indicating a role for REGg in cell-cycle regulation and proliferation. For quite a long time, REGg has been only characterized in terms of its ability to degrade small peptide model substrates in vitro. Recently, we found that REGg is a component of certain nuclear receptor (NR) coactivator complexes. Our recent discovery that the proteasome activator REGg directs degradation of the steroid receptor coactivator SRC-3 by the 20S proteasome in an ATP- and ubiquitin-independent manner has been a challenge to this traditional notion that REGg is not involved in the destruction of intact endogenous proteins (Zhou, 2006). We believed that the role of REGg in SRC-3 degradation was likely to be only the first of a number of yet-to-beidentified direct targets of the REGg proteasome in human cells. To seek further insight into REGg biological function, we have searched for additional REGg targets. By using a combination of 2D electrophoresis and mass spectrometry, we discovered that p21Waf/Cip1 (abbreviated as p21 hereafter) is another important REGg target protein. As a broad-acting cyclin-dependent kinase inhibitor, p21 occupies a central position in the regulation of the cell cycle in many tissues (Weinberg and Denning, 2002). The effects of targeted deletion of p21 in mice and its expression patterns in some human cancers are consistent with a role for p21 as both a tumor suppressor and an oncogene (Roninson, 2002). Induction of p21 has

Molecular Cell 26, 831–842, June 22, 2007 ª2007 Elsevier Inc. 831

Molecular Cell p21 Degradation by the REGg Proteasome

been known to ensue through both p53-dependent and -independent pathways (Weinberg and Denning, 2002). The dual role of p21 in both the suppression and promotion of cell growth suggests that its biological role is complex and may be related to a delicate balance in the steady-state levels and/or activity of the p21 protein. Although p21 is subjected to proteasome-dependent degradation, the details of how this occurs are not fully characterized. There is evidence suggesting that degradation of p21 requires its ubiquitination (Bendjennat et al., 2003; Bloom et al., 2003; Coulombe et al., 2004). However, other reports indicate that proteasome degradation does not require that p21 be ubiquitinated (Chen et al., 2004; Sheaff et al., 2000; Touitou et al., 2001). Also, a recent study (Chen et al., 2004) demonstrated that endogenous cellular p21 can be completely acetylated at its amino terminus and is therefore not a substrate for N-end rule ubiquitination (Varshavsky et al., 2000). These studies indicate that ubiquitination of endogenous p21 either at internal lysines or on the N terminus is not entirely essential for its destruction by the proteasome and that, at least in part, a ubiquitin-independent means is involved in its destruction. Here we report the identification of p21 as another direct biological target of the REGg proteasome in vivo and in vitro. Furthermore, in select cell contexts, the effect of REGg on the cell-cycle progression is likely due to its influence on p21 protein stability. Our results suggest that REGg plays a major cell-specific and ubiquitinindependent role in regulating proteasomal turnover of key cellular growth control proteins.

RESULTS Identification of Additional REGg Protein Targets We generated two tetracycline/doxycycline-inducible 293 cell lines, one that overexpresses REGg (293-REGg) and another that expresses a mutant form of REGg (293mut-REGg) that is unable to activate the proteasome (Li et al., 2006; Zhang et al., 1998). Lysates from both cell types were resolved by 2D gel electrophoresis followed by silver staining. Spots that were significantly diminished in 293-REGg cells when cultured in the presence of doxycycline compared to untreated cells were subjected to further scrutiny by mass spectrometry. Also, control spots that demonstrated no change or higher levels of expression in the mutant REGg cell line were also chosen for mass spectrometric identification. One of these proteins is shown in Figure S1 (in the Supplemental Data available with this article online) where overexpression of REGg (arrows on the right panel) resulted in a reduced level of an 20 kDa protein (circled on the right panel) when doxycycline was present. However, the level of this protein was slightly elevated in doxycycline-treated 293-mut-REGg cells (data not shown). Mass spectrometric analysis identified this protein as p21.

REGg Is Involved in p21 Degradation To validate our data suggesting that REGg may be involved in p21 degradation, we examined p21 expression levels by western analysis in 293-REGg and 293-mutREGg cell lines. Overexpression of REGg resulted in a reduction of p21 in the 293-REGg cell line in the presence of doxycycline (Figure 1A, left panel). However, doxycycline induction of the mutant REGg in the 293-mutREGg cell did not reduce the level of the p21 protein; rather, it resulted in an increase (Figure 1A, right panel). Since the mutant REGg can still bind to the 20S proteasome, it is possible that it blocks p21 degradation by competing with the endogenous REGg. In the 293-REGg cell line, we observed a reduction in p21 protein levels as early as 5 hr after induction of REGg (data not shown), indicating that a prompt regulatory process was responsible. By pulse-chase experiments, we confirmed that the REGg-mediated regulation of p21 is due to accelerated degradation of the p21 protein. In the presence of doxycycline, the rate of p21 degradation is doubled (Figures 1B and 1C). To exclude the possible role of a transcriptional mechanism, we performed real-time RT-PCR using total RNA extracted from 293-REGg cell lines with or without doxycycline induction. The mRNA level of p21 (Figure 1D) was not decreased after treatment with doxycycline, indicating that REGg-mediated reduction of p21 protein content is not controlled at the transcriptional level. These experiments point to the likelihood that REGg is involved in proteasome-mediated p21 protein degradation. To further characterize REGg-mediated turnover of p21, we used RNA interference to reduce REGg expression in a series of cancer cell lines. We chose a thyroid cancer cell line (TPC) based on a previous report of abnormally high expression of REGg in thyroid neoplasms from which these cells are derived (Okamura et al., 2003). Reduction of REGg expression resulted in a significant increase of p21 in this cell line by either a pooled siRNA or an individual siRNA (Figure 2A and data not shown). This effect is specific since the protein level of another cell-cycle regulator, p27, was not changed by knocking down REGg, which is known to be degraded in a ubiquitindependent manner (Tsvetkov et al., 1999). Increased p21 protein levels were observed when REGg was knocked down in cervical carcinoma-derived HeLa cells and prostate cancer-derived LNCaP cells, indicating a role for REGg in controlling p21 levels in multiple tissue contexts (Figure 2A). In addition, overexpression of REGg resulted in a reduction of p21 in the 293-REGg cell line, but not in 293-mut-REGg cells (Figure S2). To determine the effect of RNA interference on p21 levels more quantitatively, we performed high-throughput imaging analysis of average nuclear p21 intensity before and after depletion of REGg in TPC cells. Quantitative analysis of more than 200 cells indicates that depletion of REGg results in approximately a 10-fold increase in p21 protein level (Figure 2B). A representative immunofluorescence study following RNA interference in the TPC cells is shown in Figure 2C; cells depleted of REGg

832 Molecular Cell 26, 831–842, June 22, 2007 ª2007 Elsevier Inc.

Molecular Cell p21 Degradation by the REGg Proteasome

Figure 1. Overexpression of REGg Reduced p21 Level (A) The 293-REGg or 293-mut-REGg cells were treated with vehicle (DMSO) or doxycycline for 24 hr. Total cell lysates were prepared for western blot analysis of REGg and p21 levels. b-actin was probed to assess protein loading. Relative protein levels were quantitated by NIH ImageJ software and listed below each panel. (B) The 293-REGg cells were transfected with 1 mg of pCR3.1-p21. About 12 hr posttransfection, cells were induced with 1 mg/ml of doxycycline or with vehicle for 24 hr. Pulse-chase experiments were performed as described (Li et al., 2006). Total cell lysates were extracted for immunoprecipitation, SDS-PAGE, and autoradiography. (C) Quantitated results of (B) were plotted against the indicated time course. Error bars refer to standard deviation of the average quantitated results. (D) Total RNA extracted from 293-REGg cells treated with vehicle or doxycycline for 24 hr was prepared for real-time RT-PCR analysis. Results from three repeats were averaged and plotted as relative p21 mRNA levels. A no-RT control was included to validate this experimental procedure. Error bars refer to standard deviation of the average quantitated results.

revealed high levels of p21. Interestingly, REGg-dependent p21 regulation seems also to be cell type specific. Knocking down REGg had only a relatively slight effect on p21 levels in MCF-7 cells and no effect in HepG2 and 3T3-L1 cells (data not shown). To verify the role of REGg in the regulation of p21 protein levels in vivo, we utilized REGg knockout mice (Barton et al., 2004). Primary mouse embryonic fibroblast (MEF) cells were generated from REGg-deficient and wild-type mice. At the third passage, MEF cells were examined for their expression of REGg and p21 protein levels. Consistent with our expectation, REGg-deficient MEF cells do not express REGg and display a higher expression of p21 than wild-type MEF cells (Figure 2D). Based on the high homology between mouse and human REGg gene sequences, we introduced human REGg cDNA by transient transfection to the REGg/ MEFs to test its ability to enhance p21 degradation. We found that human

REGg can successfully induce mouse p21 degradation in the transfected MEF cells (Figure S3A, lower panel), while transfection of an empty vector or mutant REGg had no effect on endogenous p21 levels in the REGg/ MEFs (Figure S3A, upper and middle panels). We manually assessed p21 protein expression levels in about 70 cells transfected with wild-type or mutant REGg. The results in Figure S3B indicate that 86% of mutant REGgtransfected cells were p21 positive, while only 24% of wild-type REGg-transfected cells were p21 positive. These results are supportive of our other experimental results and substantiate that p21 is an authentic biological target of the REGg proteasome. Proteolytic Turnover of p21 by the REGg Proteasome In Vitro We next wished to determine whether the effect of REGg on p21 turnover was direct or indirect. Since the REGg

Molecular Cell 26, 831–842, June 22, 2007 ª2007 Elsevier Inc. 833

Molecular Cell p21 Degradation by the REGg Proteasome

Figure 2. Depletion of REGg Enhanced the p21 Level in Multiple Cell Lines (A) TPC, HeLa, or LNCaP cells were treated with 20 nM siREGg or a negative control (siNeg). Endogenous REGg, p21, p27, or b-actin was detected by western blotting. (B) High-throughput analysis of nuclear p21 levels following RNAi in TPC cells. Automated quantitation of the average nuclear intensity of p21 was performed as described in the Experimental Procedures. The difference of nuclear accumulation of p21 between the siRNA control and siRNAREGg is statistically significant (p < 0.01). Error bars refer to standard deviation of the average quantitated results. (C) A representative immunofluorescence of cells treated with siREGg. Cells successfully ablated with endogenous REGg (pointed with white arrows) resulted in a significant increase of p21 (cells with green color). Untransfected cells displayed a high level of REGg staining (cells with red color) and undetected p21 (pointed with red arrows). (D) Primary MEF cells were generated as described in the Experimental Procedures. Relative protein levels were quantitated and listed. Primary MEFs from the third passage were used for western blot analysis.

mutant used in this study possesses only a single amino acid substitution in its activation loop and failed to promote p21 degradation, we reasoned that the wildtype REGg degrades p21 in a direct manner. To test whether p21 interacts with REGg in vitro, GST-p21 was used to pull down in vitro-translated, 35S-labeled REGg. When compared to the input control, GST-p21 was found to bind a significant amount of REGg (Figure 3A, upper panel), indicating a direct interaction between p21 and REGg in vitro. In reciprocal experiments, purified REGg was incubated with in vitro-translated p21 and an antibody against REGg. In this case, the REGg antibody can successfully coimmunoprecipitate p21, while an IgG control antibody does not (Figure S4), again indicating that p21 and REGg can directly interact with each other.

We next assessed whether a physical association could be observed between endogenous p21 and REGg in a coimmunoprecipitation assays. Using HeLa whole-cell lysates, a p21 antibody immunoprecipitated a significant amount of endogenous REGg, while an IgG control antibody did not (Figure 3A lower panel). These interaction data indicate that p21 and REGg directly interact with each other and associate with one another in cell culture. To gain more definitive insight into the mechanism of REGg-mediated p21 degradation, we determined the capacity of REGg to direct cell-free proteolysis. We used a rabbit reticulocyte-coupled transcription/translation system (Promega) to generate an entirely wild-type p21 protein bearing no tags. Incubation of latent 20S

834 Molecular Cell 26, 831–842, June 22, 2007 ª2007 Elsevier Inc.

Molecular Cell p21 Degradation by the REGg Proteasome

Figure 3. REGg Mediates Direct Degradation of p21 (A) REGg and p21 interaction in vitro and in vivo. In the upper panel, GST-p21 and 35S-labeled, in vitro-translated REGg were incubated for 4 hr at 4 C. The amount of REGg proteins pulled down by GST-p21 was detected by SDS-PAGE and autoradiography. In the lower panel, cell lysates from HeLa cells were immunoprecipitated with anti-p21 or control IgG and detected by western blotting as described in the Experimental Procedures. (B) In the upper panel, purified REGg, 20S proteasome and in vitro-translated p21 were incubated as indicated for 40 min at 30 C. The reaction in lane 5 contained 200 nM ATPgS, and the reaction in lane 6 was in the presence of 50 nM of epoxomicin (Epox). A fraction of the reaction was analyzed by western blotting with anti-p21 or anti-b-actin. In the lower panel, purified in vitro-translated p21 was used as a substrate for the proteolytic analysis as in the upper panel. Note that a nonspecific band (indicated by an asterisk), possibly a byproduct of GST-p21, was not degraded by the REGg proteasome.

proteasome or REGg alone with p21 for 40 min did not have a significant effect on p21 degradation, indicating that neither of these components was able to degrade p21 by itself (Figure 3B, upper panel). In the same assay, a combination of REGg and 20S proteasome promoted complete degradation of p21, which could be inhibited by the proteasome inhibitor epoxomicin but still proceeded in the presence of ATPgS (Figure 3B, upper panel). This experiment shows that REGg-dependent protein degradation by the 20S core proteasome does not require ATP. To confirm that REGg-proteasome degradation of p21 does not require other components that are present in reticulocyte lysates, we immunoprecipitated the in vitro-transcribed/-translated p21 and then eluted it with an excess of purified GST-p21 protein. Using the purified p21 as substrate, we performed a similar in vitro proteolytic assay. We achieved essentially the same results, except this approach was slightly less efficient in degrading p21, perhaps due to residual GSTp21 competing with the natural p21 for the REGg proteasome (Figure 3B, lower panel). Also, the ‘‘nonspecific’’ band indicated by the asterisks reinforces the fact that REGg proteasome is specific for p21 in this cell-free system. Taken together, this analysis of the physical interaction between REGg and p21 and the in vitro proteolytic degradation assays employed herein indicate that p21 is directly degraded by the REGg proteasome.

Free p21 Is Targeted for Degradation by the REGg Proteasome Endogenous p21 has been known to interact with many different proteins such as CDK2/cyclin complexes and PCNA. We further investigated the mechanism of REGgdependent degradation of p21 under more physiological conditions. Using our established in vitro proteolytic degradation assay system, we found that adding purified Cdk2/cyclin E protein complex into the REGg-20S proteasome-p21 mixture significantly delayed the REGg-dependent degradation of p21 in vitro, while an equal amount of BSA protein added into the mixture did not impede the rate of p21 degradation over the same time course (Figure 4A). The result from this in vitro study is consistent with previous reports that the stability of p21 is increased when it is bound to cyclins (Bloom et al., 2003; Chen et al., 2004; Clurman et al., 1996). We next asked whether the increased stability of p21 in the presence of Cdk2/cyclin E might be due to a decreased pool of free p21 that is available to interact with the REGg proteasome and be targeted for degradation. It turned out to be the case when we examined the interactions between p21 and REGg by immunoprecipitation using the REGg-p21 mixture with or without purified Cdk2/cyclin E protein complex (similar molar ratio of each component to that used in the in vitro degradation assays). The results in Figure S4 demonstrated

Molecular Cell 26, 831–842, June 22, 2007 ª2007 Elsevier Inc. 835

Molecular Cell p21 Degradation by the REGg Proteasome

Figure 4. Cdk/Cyclin Complexes Can Delay REGg-Dependent Degradation of p21 (A) In vitro proteolytic assays were performed as described in Figure 3B, except that the Cdk2/cyclin E complex or BSA was added along with p21 at the molar ratio of 2:1. Reactions were stopped at indicated time points by adding 53 SDS sample buffer. (B) 293-REGg cells seated in 12-well plates and transfected with 250 ng of pCR3.1-p21 plus 1 mg of SRa296-Cdk2 and 2 mg of SRa296-cyclin A or an equal amount of control vector SRa296. Twelve hours posttransfection, cells were treated with or without 1 mg/ml doxycycline for 24 hr. Cells were then treated with 100 mg/ml cycloheximide for different periods of time as indicated. Numbers below the p21 blot indicate the relative p21 protein level of each sample relative to that at ‘‘0’’ time point in each individual group. Since the Cdk2 blot has indicated equal loading of individual samples, the results of the b-actin blot are not shown.

that anti-REGg cannot precipitate significant amounts of p21 when Cdk2/cyclin E is added in an 2:1 (cyclin: p21) ratio to the REGg-p21 mixture, while the control experiment using BSA had no influence on the interaction between p21 and REGg, as demonstrated by a significant amount of p21 precipitated by anti-REGg, but not by a control IgG. We did not observe coimmunoprecipitation of Cdk2 along with p21 in any of the three repeating experiments (Figure S4), suggesting that REGg does not interact with Cdk2/cyclin complexes. These experimental results indicate that REGg interacts preferentially with unbound p21 and that the REGg proteasome mainly targets free p21 for degradation. To further validate this mechanism, we examined the intracellular effect of the Cdk/cyclin complex on REGg-dependent p21 degradation. Since intracellular cyclin A was shown to comigrate with p21 in gel filtra-

tion experiments (Bloom et al., 2003), we coexpressed p21 along with Cdk2/cyclin A or control vectors in the 293-REGg cell lines incubated with or without doxycycline, followed by cycloheximide treatment for different periods of time. The results in Figure 4B demonstrate that, in the presence of doxycycline which induced overexpression of REGg (data not shown) as previously described (Figure 1A), overexpression of Cdk2/cyclin A significantly reduced the rate of p21 degradation 2- to 3-fold (comparing 1–2 hr of CHX treatment with or without Cdk2/cyclin A), consistent with our observation from in vitro degradation experiments (Figure 4A). However, in the absence of doxycycline, overexpression of Cdk2/cyclin A only slightly delayed p21 degradation, indicating that degradation pathways other than a REGg-dependent mechanism also can be involved in p21 turnover.

836 Molecular Cell 26, 831–842, June 22, 2007 ª2007 Elsevier Inc.

Molecular Cell p21 Degradation by the REGg Proteasome

Figure 5. REGg-Dependent Degradation of p21 Is Not Dependent on a Functional Ubiquitin-Activating Enzyme The ubiquitin-activating enzyme temperature-sensitive cells, ts85, were transfected with control or siREGg at 30 C. Twenty-four hours posttransfection, cells were shifted to 37 C for 20 hr followed by treatment with 100 mg/ml cycloheximide for different periods of time as indicated. An equal amount of total protein was loaded for western blotting analysis, and the relative protein levels were quantitated as described in Figure 1.

In Vivo Degradation of p21 by the REGg Proteasome Is Not Dependent on a Functional UbiquitinActivating Enzyme It has been reported previously that p21 degradation can be mediated by ubiquitin-dependent and ubiquitinindependent pathways (Bloom et al., 2003; Chen et al., 2004). The observation of REG-20S-19S hybrid proteasomes in electron microscopic studies (Cascio et al., 2002) leads us to ask whether the REGg proteasome is capable of degrading p21 independent of a functional cellular ubiquitination system. We have previously demonstrated that a variety of coactivators are targets of the proteasome in the ts85 cell line, which harbors a thermolabile ubiquitin-activating enzyme that abolishes the transfer of ubiquitin to target proteins at nonpermissive temperatures, thereby disabling the ubiquitin-proteasome protein degradation pathway (Lonard et al., 2004; Yan et al., 2003). It can be seen in Figure 5 that the steady-state levels for both p21 and REGg decay at a slower rate at the restrictive temperature. Since the large increase of p21 level at the restrictive temperature has been shown to be due to p53 mediated-transcription in the ts85 cell line (Zhu et al., 2007), we believe that the ubiquitindependent turnover of p21, if it has a role, plays a minor role in this cell line. When REGg was knocked down in ts85 cells, degradation of p21 was further delayed, indicating that intracellular degradation of p21 by the REGg proteasome does not require a functional ubiquitinactivating enzyme.

Modulation of Cell Cycle by REGg-Mediated Regulation of p21 A previous study suggests that REGg can promote cell growth in thyroid cancer cells (Okamura et al., 2003). To understand whether REGg’s effect on cell growth is mediated by altering p21 protein stability, we performed cell-cycle analysis of the thyroid carcinoma-derived TPC cells (Ouyang et al., 2006) after RNA interference of REGg, of p21 alone, and the two in combination. Figure 6 shows representative DNA content graphs along with the average percentage of cell populations in different cellcycle stages calculated from three individual experiments. Upon treatment with siREGg, DNA content analysis demonstrated a decrease of cells in G0/G1 and an increase in the G2/M population (Figure 6B) compared with that treated with a control siRNA (Figure 6A). The pattern is quite similar to that following g-irradiation (data not shown), indicating that upregulation of p21 may be in part the reason for G2/M phase arrest. Knocking down p21 resulted in only a slight increase in the proportion of S phase cells; this result was reproducible in all experiments in this cell line (Figure 6C). Interestingly, a combination of siREGg and sip21 reversed the effect observed in the presence of siREGg alone (Figure 4D and compare with Figure 4B), indicating that the major effect of depleting REGg is likely dependent upon elevated levels of p21. Although we have tested both pooled and individual siRNAs that target REGg, to further exclude the possibility

Molecular Cell 26, 831–842, June 22, 2007 ª2007 Elsevier Inc. 837

Molecular Cell p21 Degradation by the REGg Proteasome

Figure 6. Effect of REGg on Cell Cycle (A–D) A representative result of propidium iodide staining/FACScan analysis of TPC cells treated with control siRNA (A), siREGg (B), sip21 (C), or a combination of siREGg and sip21 (D) following RNA interference for 3 days was displayed. The averaged cell percentage and standard deviation (in parenthesis) in each phase were summarized from three individual experiments. Statistically significant differences (p < 0.05) in G2/M content (indicated by an asterisk), but not G0/G1 content, were observed between (A) and (B). (E) The BrdU incorporation of the TPC cells treated with different siRNA from three individual experiments were plotted for comparison. The data are represented as mean ± standard deviation. The statistical analysis of the results between siNeg and siREGg, or between siNeg and siREGg/sip21 (indicated by *), were all significant by paired t test (p < 0.05). (F) Western blotting demonstrates the efficient knockdown of target proteins.

of off-target effects by RNAi in our cell-cycle analysis, we performed rescue experiments. TPC cells were transfected with a control siRNA or siREGg along with either a REGg expression vector or an empty vector. Twentyfour hours after transfection, the cells were collected and processed for cell-cycle or western blot analysis. The results in Figures S5A and S5B demonstrate a decrease of cells in G0/G1 and an increase in the G2/M population following treatment with siREGg (compare Figures S5A and S5B), with the expected changes of REGg and p21 protein levels by western blotting (Figure S5E). The noticeable increase of S phase cells in the rescue experiments, compared with that in Figure 6, is likely due to the effects of the different transfection reagents on cell growth. When REGg was overexpressed along with the control siRNA, we observed a significant reduction in p21 protein levels and a slight increase in the S phase population (compare Figures S5A and S5C), consistent with the results of siRNA against p21. Simulta-

neous cotransfection of optimal amounts of a REGg expression vector with siREGg successfully prevented accumulation of p21 in TPC cells (Figure S5E), and cellcycle analysis revealed that the DNA content of these cells is close to those treated with control siRNA and an empty vector (Figure S5A). Taken together, these results support the fact that the impact of REGg alteration on the cell cycle in RNAi experiments was due to REGg’s influence on p21 and was not due to off-target effects. To exclude the possibility that overexpression of REGg may lead cells to accumulate at a point in the cell cycle where p21 might be more unstable, we performed flow cytometry of HeLa cells with or without overexpressed REGg. The results in Figure S6 indicate no significant alteration of cell-cycle profiles with or without overexpression of REGg, except for a slight change in S phase content, which should not indirectly affect p21 turnover. In the same experiments in Figure 6, we measured BrdU incorporation following RNAi treatment. Knocking down

838 Molecular Cell 26, 831–842, June 22, 2007 ª2007 Elsevier Inc.

Molecular Cell p21 Degradation by the REGg Proteasome

REGg reduced BrdU incorporation about 50%. Interestingly, treatment with sip21 had little effect on BrdU incorporation in this cell type. However, combined RNA interference using both REGg and p21 together attenuated the effect when compared to using siREGg alone, indicating that p21 mediates, at least in part, the function of REGg in cell-cycle regulation (Figure 6E). The efficiency of RNA interference was assessed by western analysis, revealing greater than 90% reductions for either protein (Figure 6F). We also examined BrdU incorporation in HeLa cells, which showed that, although the effect of siREGg on BrdU incorporation is less dramatic than in TPC, similar results were obtained (data not shown). Our results suggest that elimination of REGg results in upregulation of p21, which in turn prevents normal cell-cycle progression and results in reduced BrdU incorporation and G2/M phase arrest. This interpretation is consistent with previous reports indicating that overexpression of p21 induces cell-cycle arrest at the G2/M phase (Bunz et al., 1998). DISCUSSION In this study, we present evidence that p21 is a second direct biological target of the REGg proteasome, which previously was shown to be involved in the degradation of SRC-3 (Li et al., 2006) and the exogenous hepatitis C virus core protein (Moriishi et al., 2003). It has been shown recently that SRC-3 also can carry along the growth regulatory protein PTTG1 in complex to the REGg proteasome for degradation (Ying et al., 2006). Consistent with the mechanism that we previously reported concerning SRC-3 degradation by the REGg proteasome, we see that it can also degrade p21 in an ATP- and ubiquitinindependent manner. The present study provides a biological role for REGg in the regulation of a molecule governing regulation of the cell cycle and reveals the potential for REGg to directly target a broader range of growth regulatory substrates. In recent years, there has been significant debate as to whether the p21 protein is degraded through a ubiquitinindependent or ubiquitin-dependent process for its destruction. Although we have focused our study on the ubiquitin-independent, REGg-dependent destruction of p21, we believe that it can be degraded through a ubiquitin-dependent pathway as well. For instance, many important molecules such as p53, p73, and SRC-3 are subject to destruction by both ubiquitin-dependent and ubiquitin-independent proteasome pathways (Asher et al., 2005; Li et al., 2006). We posit that the ubiquitindependent degradation pathway may serve to degrade p21 when complexed with CDK2 in some cells, while the REGg-dependent mechanism targets free p21 for degradation (Bloom et al., 2003). We also believe that the contribution of each proteasome-dependent degradation pathway to p21 turnover could be cell type specific, as REGg was not able to influence p21 protein content or cell-cycle progression in certain cell lines. Additionally, the interplay

of these two degradation pathways may be regulated by extracellular signaling systems triggered by distinct environmental stimuli (Bendjennat et al., 2003). Although REGg has been reported to be a target of phosphorylation (Hagemann et al., 2003), regulation of the proteolytic activity of the REGg proteasome via posttranslational modification has not been explored. Despite the evidence for both ubiquitin-dependent and ubiquitin-independent degradation of p21 from different groups, the precise molecular mechanism of this mode of degradation is unclear. Nevertheless, it has been agreed that its ubiquitin-independent degradation is due to an interaction with a core 20S proteasome subunit (Touitou et al., 2001). Our results clearly establish a key role for REGg in the ubiquitin-independent degradation of p21 in a cell type-specific manner. Depletion of REGg resulted in significant elevation of p21 in multiple cell lines as well as in primary MEF cells, indicating that this mode of p21 degradation applies to a variety of cell types. Consistent with our observation, Barton et al. observed proliferative defects in REGg-deficient MEFs, but not in lymphocytes (B cells or T cells), suggesting a cell type-specific effect of REGg on mitotic control (Barton et al., 2004). Our results also indicate that the REGg proteasome mainly targets free p21 for destruction, consistent with our previous prediction that REGg proteasome targets unstructured, free SRC-3 for degradation in a cell typespecific manner. Although most endogenous proteins are associated with specific complexes, the pool of free proteins is dynamically equilibrated with those in the protein complexes. Abnormally high levels of REGg proteasome will eventually decrease the total amount of substrate proteins in a cell. We believe that REGg acts as a sensor to maintain cellular levels of potent regulators. In this way, REGg can control the cellular ‘‘potential’’ for activity of the regulatory proteins, while the levels of the actively engaged proteins are controlled more by the ubiquitin-dependent turnover system. In other studies that characterized REGg-mediated turnover of SRC-3 and the hepatitis C virus core protein, both proteins were demonstrated to physically interact with REGg similar to that reported here for p21. It is therefore likely that the physical interaction of REGg with its protein targets plays an important role in determining which proteins are degraded and that REGg likely does not indiscriminately degrade substrates with disordered elements. It is possible that REGg-proteasome-mediated degradation is mechanistically linked to its reported ubiquitin-independent degradation through interaction with the core C8 a subunit protein of 20S proteasome (Touitou et al., 2001). Because C8 is an integral part of the REGg proteasome, this fact suggests that a complex association between p21, REGg, and C8 may be required for the turnover of p21. As we have speculated previously, it is possible that REGg and C8 function as anchors for proteins such as p21, increasing their temporal residency with the proteasome, so that subsequent Brownian ratchet forces can drive substrates through the central

Molecular Cell 26, 831–842, June 22, 2007 ª2007 Elsevier Inc. 839

Molecular Cell p21 Degradation by the REGg Proteasome

pore of the proteasome. Related to this mechanism, REGg proteasome-substrate complex formation could facilitate the cleavage at internal peptide bonds of naturally unfolded or unstructured proteins because ATP-dependent energy is not required to unfold these types of proteins for entry into proteasome (Liu et al., 2003). The requirement for precise control of p21 abundance is underscored by its role in cell-cycle regulation. In parallel with the accumulation of p21 levels upon treatment of cells with siRNA against REGg, we observed cell-cycle arrest and reduction of BrdU incorporation in a thyroid cancer cell line. Interestingly, p21-mediated cell-cycle arrest occurs mainly at the G2/M phase of the cell cycle. Although overexpression of p21 has been known to cause cell-cycle arrest at G0/G1, there are numerous reports that accumulation of p21 can lead to cell-cycle arrest in the G2/M phase as well (Cayrol et al., 1998; Niculescu et al., 1998). Similar cell-cycle arrest was observed previously in mouse embryonic fibroblast cells derived from REGg-deficient mice (Barton et al., 2004; Murata et al., 1999). Finally, the discovery of p21 as a REGg-proteasome target reveals a potential biological role of REGg. Given that the REGg proteasome is a basic proteasome-species (along with the 26S proteasome and other 11S proteasome species), it is expected that it will be shown to degrade a spectrum of protein substrates. Our study in the thyroid cancer cell line suggests that REGg may have an oncogenic role in some cancers. The biological function of p21 is complex, as p21 appears to have both tumor suppressor and oncogenic roles, depending upon the cellular context. Because of this, REGg may also play either tumor-promoting or tumor-suppressive roles. In support of its oncogenic role, p21 was reported to be a positive regulator of IGF-1-induced cell proliferation in MCF-7 breast cancer cells, and abrogation of p21 expression decreased cell proliferation (Dupont et al., 2003). The overall biological outcome of REGg activity is complicated by the fact that it also degrades SRC-3, another wellestablished oncogenic protein. This is illustrated by the fact that we have previously demonstrated a potential tumor-suppressive role of REGg in a breast cancer cell line as a result of its ability to reduce the cellular levels of the SRC-3 protein. Even the oncogenic action of SRC-3 protein must be placed in context, as SRC-3 is able to act as a selective tumor suppressor in lymphocytes (Coste et al., 2006). In spite of the myriad of positive and negative context-specific functions influenced by these two targets of REGg, it is evident that REGg has a dynamic and dominant role in regulating the cellular levels of select important growth control proteins, and it may represent a new cell type-specific target for pharmacologic growth modulators. EXPERIMENTAL PROCEDURES Cell Culture and Reagents HeLa and LNCaP cells were obtained by ATCC and maintained as described previously (Li et al., 2006). The thyroid cancer cell line TPC was provided by Dr. El Naggar at the University of Texas MD

Anderson Cancer Center and maintained in DMEM with 5% serum supplemented with sodium pyruvate. The ubiquitin-activating enzyme temperature-sensitive cell line, ts85, was maintained at a permissive (30 C) temperature in McCoy’s medium supplemented with 5% calf serum. REGg antibody was purchased from Zymed (catalogue number 38-3800). Anti-p21 antibodies were purchased from BD Pharmingen (catalogue number 556431) and Santa Cruz (catalogue number SC-397), and anti-p27 antibody was purchased from Santa Cruz (catalogue number sc-1641). All the experiments in this study have been repeated at least three to five times. 2D Gel Electrophoresis and Mass Spectrometry The first dimension isoelectric focusing (IEF) separations were carried out following the manufacturer’s instructions for PROTEAN IEF cell (Bio-Rad). The 7 cm IPG (immobilized pH gradient) ReadyStrips (pH 3–10, Bio-Rad catalogue number 163-2000) were chosen in order to arrange two strips side by side on the same SDS-PAGE. Rehydration/sample buffer and 1003 Bio-Lyte 3-10 Ampholyte (1% final concentration) were mixed with 25 mg of samples to make a total volume of 125 ml per sample. Rehydrations were performed passively in an equilibration tray overnight at 20 C. For IEF, the setting of 20,000 vhours is programmed following the rapid slope to reach the maximum voltage. Following equilibration, the ReadyStrips were loaded for SDS-PAGE separation at 200 constant voltage. The results were visualized by silver staining. Mass spectrometry was performed as previously described (Li et al., 2006). Construct Generation To generate GST-p21, a PCR product was generated using the following primers: 50 -ATGGGATCCGAAAACCTGTATTTTCAGGGCATGT CAGAACCGGCTGGGGAT-30 and 50 -GTGCTCGAGTTAGGGCTTCCT CTTGGAGAAGAT-30 . A purified DNA fragment, following digestion by BamHI and HindIII restriction enzymes, was cloned into properly digested pGEX 4T-1 (Amersham Biosciences). The mammalian expression vector PCR3.1-p21 was generated by inserting a digested PCR fragment into HindIII/XhoI of PCR3.1 vector. All the constructs were verified by sequence analysis. GST Pull-Down, RNA Interference, and Western Blot Analysis GST-REGg and GST-p21 fusion proteins were expressed and purified as described previously (Li et al., 2006). All siRNAs were purchased from Dharmacon (Lafayette, CO). RNA interference and western blot analysis were performed as previously described (Li et al., 2006). For experiments using the E1 temperature-sensitive mutant cell line, the ts85 cells were maintained at 30 C and seeded at 9 3 105 cells per well and transfected with control siRNA or siREGg (20 nM). Twentyfour hours thereafter, cells were transferred to a 37 C incubator (nonpermissive temperature). Twenty hours later, cells in either temperature regimen were treated with cycloheximide and harvested at the indicated times thereafter. For rescue experiments, DharmaFect Duo transfection reagent (Dharmacon, catalogue number T-2010-02) was used to cotransfect 30 nM of siRNA and 5 mg of DNA into TPC cells following instructions by the manufacturer. Protein Purification and In Vitro Proteolytic Analysis The REGg protein complex used herein was purified as described (Li et al., 2006). The p21 substrate in Figure 4B (upper panel) was generated by in vitro translation using the PCR3.1-p21 vector. To further purify natural p21 from the in vitro translation mixture, 400 ml of total translation mixture was diluted in one volume of GST binding buffer (see above) with additional 300 mM NaCl and incubated with 10 mg of anti-p21 (Santa Cruz) overnight followed by 1 hr incubation with protein G plus beads (Santa Cruz). The first two washes were performed with binding buffer containing 500 mM NaCl for 5 min each. The third wash was performed using PBS buffer. The last wash was carried out in proteolytic reaction buffer (10 mM Tris [pH 7.5], 10 mM KCl, 10% glycerol). To elute immunoprecipitated

840 Molecular Cell 26, 831–842, June 22, 2007 ª2007 Elsevier Inc.

Molecular Cell p21 Degradation by the REGg Proteasome

p21 from the beads, 40 mg of GST-p21 dialyzed with proteolytic reaction buffer was incubated with the beads at room temperature with gentle rotation for 1.5 hr. Excess glutathione beads were then added to the eluate to remove the GST-p21. The supernatant was aliquoted and saved for subsequent proteolytic assays. The proteolytic analysis was performed using 5 ml of in vitro-translated p21 substrate or 10 ml of purified p21, 0.25 mg of 20S proteasome (BioMol International LP, catalogue number PW8720, batch Z06003), and 1 mg of REGg heptamers for the indicated times in 50 ml reaction volume at 30 C. An aliquot of the reaction was analyzed by western blotting. For in vitro proteolysis in the presence of cyclin complex, we added 1 mg of purified Cdk2/cyclin E complex (stock 1 mg/ml, purchased from Millipore, catalogue number 14-475, and verified by an SDS-PAGE with Coomassie blue stain) into 50 ml of in vitro-translated p21 lysate (estimated molar ratio of Cdk/cyclin to p21 is 2:1), and 5 ml of the p21/Cdk2/cyclin E was used in each reaction. For in vitro immunoprecipitation assays, 50 ml of the p21/Cdk2/cyclin E or p21/ BSA mixture was added to 1 mg of purified REGg, diluted with GST binding buffer, and divided into two fractions for immunoprecipitation by anti-REGg or a control IgG. BrdU Staining and Flow Cytometry Analysis TPC cells or HeLa cells treated with siRNA for 3 days were exposed to BrdU at 30 mM for 4 hr. Collected cells were fixed with 70% ice-cold ethanol for 30 min on ice followed by 4 N HCl denaturation and neutralization with 0.1 M sodium tetraborate. Direct immunofluoresence staining was performed by adding 20 ml of anti-BrdU FITC and incubated for 30 min at room temperature. Propidium iodide (10 mg/ml) and DNase-free RNase (1 ml of 100mg/ml) were added prior to flow cytometry. Samples were analyzed on a Beckman-Coulter Epics XLMCL apparatus.

REFERENCES Asher, G., Tsvetkov, P., Kahana, C., and Shaul, Y. (2005). A mechanism of ubiquitin-independent proteasomal degradation of the tumor suppressors p53 and p73. Genes Dev. 19, 316–321. Barton, L.F., Runnels, H.A., Schell, T.D., Cho, Y., Gibbons, R., Tevethia, S.S., Deepe, G.S., Jr., and Monaco, J.J. (2004). Immune defects in 28-kDa proteasome activator gamma-deficient mice. J. Immunol. 172, 3948–3954. Bendjennat, M., Boulaire, J., Jascur, T., Brickner, H., Barbier, V., Sarasin, A., Fotedar, A., and Fotedar, R. (2003). UV irradiation triggers ubiquitin-dependent degradation of p21(WAF1) to promote DNA repair. Cell 114, 599–610. Bloom, J., Amador, V., Bartolini, F., DeMartino, G., and Pagano, M. (2003). Proteasome-mediated degradation of p21 via N-terminal ubiquitinylation. Cell 115, 71–82. Bunz, F., Dutriaux, A., Lengauer, C., Waldman, T., Zhou, S., Brown, J.P., Sedivy, J.M., Kinzler, K.W., and Vogelstein, B. (1998). Requirement for p53 and p21 to sustain G2 arrest after DNA damage. Science 282, 1497–1501. Cascio, P., Call, M., Petre, B.M., Walz, T., and Goldberg, A.L. (2002). Properties of the hybrid form of the 26S proteasome containing both 19S and PA28 complexes. EMBO J. 21, 2636–2645. Cayrol, C., Knibiehler, M., and Ducommun, B. (1998). p21 binding to PCNA causes G1 and G2 cell cycle arrest in p53-deficient cells. Oncogene 16, 311–320. Chen, X., Chi, Y., Bloecher, A., Aebersold, R., Clurman, B.E., and Roberts, J.M. (2004). N-acetylation and ubiquitin-independent proteasomal degradation of p21(Cip1). Mol. Cell 16, 839–847.

Preparation of Primary MEF Cells Eleven day postcoitum embryos were separated from maternal tissues, and the yolk sac and internal organs, such as the liver, were removed. Then the embryos were incubated at 4 C for 16 hr in 0.25% (wt/vol) trypsin-EDTA solution (Invitrogen) followed by incubation at 37 C for 1 hr. The embryo tissues were then broken down by vigorous pipetting and were grown in DMEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U of penicillin/ml, and 100 mg of streptomycin/ml at 37 C in a 5% CO2 atmosphere. MEFs were grown to confluence in tissue culture flasks at 37 C in a 5% CO2 atmosphere and then saved for future studies.

Clurman, B.E., Sheaff, R.J., Thress, K., Groudine, M., and Roberts, J.M. (1996). Turnover of cyclin E by the ubiquitin-proteasome pathway is regulated by cdk2 binding and cyclin phosphorylation. Genes Dev. 10, 1979–1990.

Immunofluorescence, High-Throughput Microscopy, and Statistical Analysis Immunofluorescence was carried out as described (Li et al., 2006). High-throughput microscopy was performed as described previously (Marcelli et al., 2006). All statistical analysis was performed using GraphPad Prism 5 software.

Dubiel, W., Pratt, G., Ferrell, K., and Rechsteiner, M. (1992). Purification of an 11 S regulator of the multicatalytic protease. J. Biol. Chem. 267, 22369–22377.

Supplemental Data Supplemental Data include six figures and can be found with this article online at http://www.molecule.org/cgi/content/full/26/6/831/ DC1/.

Coulombe, P., Rodier, G., Bonneil, E., Thibault, P., and Meloche, S. (2004). N-terminal ubiquitination of extracellular signal-regulated kinase 3 and p21 directs their degradation by the proteasome. Mol. Cell. Biol. 24, 6140–6150.

Dupont, J., Karas, M., and LeRoith, D. (2003). The cyclin-dependent kinase inhibitor p21CIP/WAF is a positive regulator of insulin-like growth factor I-induced cell proliferation in MCF-7 human breast cancer cells. J. Biol. Chem. 278, 37256–37264. Hagemann, C., Patel, R., and Blank, J.L. (2003). MEKK3 interacts with the PA28 gamma regulatory subunit of the proteasome. Biochem. J. 373, 71–79. Li, J., and Rechsteiner, M. (2001). Molecular dissection of the 11S REG (PA28) proteasome activators. Biochimie 83, 373–383.

ACKNOWLEDGMENTS We acknowledge Drs. Jianming Xu, Lan Liao, James Fagin, Meabh McCurtin, and James Harper for their assistance/obtaining materials for this study. This work is supported by National Institutes of Health (NIH)-NICHD R01, NURSA-NIDDK, and Welch Foundation grants to B.W.O. Received: November 30, 2006 Revised: April 11, 2007 Accepted: May 14, 2007 Published: June 21, 2007

Coste, A., Antal, M.C., Chan, S., Kastner, P., Mark, M., O’Malley, B.W., and Auwerx, J. (2006). Absence of the steroid receptor coactivator-3 induces B-cell lymphoma. EMBO J. 25, 2453–2464.

Li, X., Lonard, D.M., Jung, S.Y., Malovannaya, A., Feng, Q., Qin, J., Tsai, S.Y., Tsai, M.J., and O’Malley, B.W. (2006). The SRC-3/AIB1 coactivator is degraded in a ubiquitin- and ATP-independent manner by the REGgamma proteasome. Cell 124, 381–392. Liu, C.W., Corboy, M.J., DeMartino, G.N., and Thomas, P.J. (2003). Endoproteolytic activity of the proteasome. Science 299, 408–411. Lonard, D.M., Tsai, S.Y., and O’Malley, B.W. (2004). Selective estrogen receptor modulators 4-hydroxytamoxifen and raloxifene impact the stability and function of SRC-1 and SRC-3 coactivator proteins. Mol. Cell. Biol. 24, 14–24.

Molecular Cell 26, 831–842, June 22, 2007 ª2007 Elsevier Inc. 841

Molecular Cell p21 Degradation by the REGg Proteasome

Ma, C.P., Slaughter, C.A., and DeMartino, G.N. (1992). Identification, purification, and characterization of a protein activator (PA28) of the 20 S proteasome (macropain). J. Biol. Chem. 267, 10515–10523.

Sheaff, R.J., Singer, J.D., Swanger, J., Smitherman, M., Roberts, J.M., and Clurman, B.E. (2000). Proteasomal turnover of p21Cip1 does not require p21Cip1 ubiquitination. Mol. Cell 5, 403–410.

Marcelli, M., Stenoien, D.L., Szafran, A.T., Simeoni, S., Agoulnik, I.U., Weigel, N.L., Moran, T., Mikic, I., Price, J.H., and Mancini, M.A. (2006). Quantifying effects of ligands on androgen receptor nuclear translocation, intranuclear dynamics, and solubility. J. Cell. Biochem. 98, 770–788.

Touitou, R., Richardson, J., Bose, S., Nakanishi, M., Rivett, J., and Allday, M.J. (2001). A degradation signal located in the C-terminus of p21WAF1/CIP1 is a binding site for the C8 alpha-subunit of the 20S proteasome. EMBO J. 20, 2367–2375.

Moriishi, K., Okabayashi, T., Nakai, K., Moriya, K., Koike, K., Murata, S., Chiba, T., Tanaka, K., Suzuki, R., Suzuki, T., et al. (2003). Proteasome activator PA28gamma-dependent nuclear retention and degradation of hepatitis C virus core protein. J. Virol. 77, 10237–10249. Murata, S., Kawahara, H., Tohma, S., Yamamoto, K., Kasahara, M., Nabeshima, Y., Tanaka, K., and Chiba, T. (1999). Growth retardation in mice lacking the proteasome activator PA28gamma. J. Biol. Chem. 274, 38211–38215. Niculescu, A.B., 3rd, Chen, X., Smeets, M., Hengst, L., Prives, C., and Reed, S.I. (1998). Effects of p21(Cip1/Waf1) at both the G1/S and the G2/M cell cycle transitions: pRb is a critical determinant in blocking DNA replication and in preventing endoreduplication. Mol. Cell. Biol. 18, 629–643. Nikaido, T., Shimada, K., Shibata, M., Hata, M., Sakamoto, M., Takasaki, Y., Sato, C., Takahashi, T., and Nishida, Y. (1990). Cloning and nucleotide sequence of cDNA for Ki antigen, a highly conserved nuclear protein detected with sera from patients with systemic lupus erythematosus. Clin. Exp. Immunol. 79, 209–214.

Tsvetkov, L.M., Yeh, K.H., Lee, S.J., Sun, H., and Zhang, H. (1999). p27(Kip1) ubiquitination and degradation is regulated by the SCF(Skp2) complex through phosphorylated Thr187 in p27. Curr. Biol. 9, 661–664. Varshavsky, A. (2005). Regulated protein degradation. Trends Biochem. Sci. 30, 283–286. Varshavsky, A., Turner, G., Du, F., and Xie, Y. (2000). Felix HoppeSeyler Lecture 2000. The ubiquitin system and the N-end rule pathway. Biol. Chem. 381, 779–789. Weinberg, W.C., and Denning, M.F. (2002). P21Waf1 control of epithelial cell cycle and cell fate. Crit. Rev. Oral Biol. Med. 13, 453–464. Yan, F., Gao, X., Lonard, D.M., and Nawaz, Z. (2003). Specific ubiquitin-conjugating enzymes promote degradation of specific nuclear receptor coactivators. Mol. Endocrinol. 17, 1315–1331. Ying, H., Furuya, F., Zhao, L., Araki, O., West, B.L., Hanover, J.A., Willingham, M.C., and Cheng, S.Y. (2006). Aberrant accumulation of PTTG1 induced by a mutated thyroid hormone beta receptor inhibits mitotic progression. J. Clin. Invest. 116, 2972–2984.

Okamura, T., Taniguchi, S., Ohkura, T., Yoshida, A., Shimizu, H., Sakai, M., Maeta, H., Fukui, H., Ueta, Y., Hisatome, I., and Shigemasa, C. (2003). Abnormally high expression of proteasome activator-gamma in thyroid neoplasm. J. Clin. Endocrinol. Metab. 88, 1374–1383.

Zhang, Z., Clawson, A., Realini, C., Jensen, C.C., Knowlton, J.R., Hill, C.P., and Rechsteiner, M. (1998). Identification of an activation region in the proteasome activator REGalpha. Proc. Natl. Acad. Sci. USA 95, 2807–2811.

Ouyang, B., Knauf, J.A., Smith, E.P., Zhang, L., Ramsey, T., Yusuff, N., Batt, D., and Fagin, J.A. (2006). Inhibitors of Raf kinase activity block growth of thyroid cancer cells with RET/PTC or BRAF mutations in vitro and in vivo. Clin. Cancer Res. 12, 1785–1793.

Zhou, P. (2006). REGgamma: a shortcut to destruction. Cell 124, 256– 257.

Roninson, I.B. (2002). Oncogenic functions of tumour suppressor p21(Waf1/Cip1/Sdi1): association with cell senescence and tumourpromoting activities of stromal fibroblasts. Cancer Lett. 179, 1–14.

Zhu, Q., Wani, G., Yao, J., Patnaik, S., Wang, Q.E., El-Mahdy, M.A., Praetorius-Ibba, M., and Wani, A.A. (2007). The ubiquitin-proteasome system regulates p53-mediated transcription at p21(waf1) promoter. Oncogene. Published online January 15, 2007. 10.1038/sj.onc. 1210191.

842 Molecular Cell 26, 831–842, June 22, 2007 ª2007 Elsevier Inc.