Correspondence to Sylvain Meloche: sylvain.meloche{at}umontreal.ca
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Abbreviations used in this paper: APC/C, anaphase-promoting complex/cyclosome; MEF, mouse embryonic fibroblast; siRNA, small interfering RNA.
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Introduction |
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In mice, inactivation of the Rb gene results in embryonic lethality at midgestation, associated with defects in erythropoiesis and cell death in the liver and nervous system, whereas mice lacking p107 or p130 in the same genetic background develop normally (Mulligan and Jacks, 1998; Classon and Harlow, 2002). However, when bred on a BALB/cJ background, p107 mutants display impaired growth and accelerated cell cycle (LeCouter et al., 1998a), whereas p130 mutant embryos die at day 1113 (LeCouter et al., 1998b). Double mutant mice lacking both p107 and p130 die soon after birth and exhibit defects in endochondral bone development (Cobrinik et al., 1996). Inactivation of p107 or p130 was also shown to enhance the phenotype of Rb mutation (Lee et al., 1996; Lipinski and Jacks, 1999). All these observations indicate that pRb, p107, and p130 have both overlapping and unique cellular and developmental functions.
Despite their resemblance, biochemical studies have revealed significant differences in the regulation and properties of individual pRb-family members. One clear distinction is their expression pattern during cell cycle progression (Grana et al., 1998; Nevins, 1998). Whereas the levels of pRb protein are relatively steady throughout the cell cycle and in quiescent cells, the expression of p107 and p130 vary considerably. p107 levels are low in G0 and accumulates during cell cycle reentry, whereas the levels of p130 are high in quiescent cells and drops upon growth factor stimulation. pRb-family proteins also differ in their ability to interact with the various members of the E2F family. Whereas pRb interacts with E2F1-4, p107 and p130 associate with E2F4 and E2F5 (Dyson, 1998; Trimarchi and Lees, 2002). In addition, several observations suggest that p107 and p130 are more closely related to one another than to pRb. The two proteins contain a unique motif in the spacer region that mediates binding to cyclin A and ECdk2 complexes (Classon and Dyson, 2001). The biological consequence of this interaction is unclear.
Another important regulator of the G1/S transition is the Cdk inhibitor p27, which negatively regulates the activity of cyclinCdk2 complexes (Hengst and Reed, 1998; Sherr and Roberts, 1999). Levels of p27 are high in growth-arrested cells, and decline upon mitogenic stimulation as a result of increased proteolysis. Work by several groups have implicated the SCFSkp2 E3 ligase in the ubiquitin-mediated degradation of the Cdk inhibitor p27 (Carrano et al., 1999; Sutterluty et al., 1999; Tsvetkov et al., 1999). Consistent with these findings, targeted inactivation of the Skp2 gene results in accumulation of p27 and cyclin E, and causes various cell cycle defects (Nakayama et al., 2000). More recent studies have shown that Skp2 also targets p130 for degradation (Tedesco et al., 2002; Bhattacharya et al., 2003) and participates in the regulation of Myc protein stability and activity (Kim et al., 2003; von der Lehr et al., 2003), further highlighting its central role in cell cycle control.
The generally accepted view is that pRb-family proteins negatively regulate cell proliferation by binding to E2F transcription factors, resulting in transcriptional inhibition or active repression of genes required for G1 to S phase progression. However, several studies indicate that interaction with E2F is not sufficient to explain the inhibitory action of pRb (Zhu et al., 1993; Welch and Wang, 1995) or p107/p130 (Smith and Nevins, 1995; Zhu et al., 1995; Castano et al., 1998; Gaubatz et al., 2000) on the cell cycle. Moreover, pRb-family proteins have been reported to interact with more than 100 different cellular proteins and to modulate the activity of several of these (Morris and Dyson, 2001). In the present study, we report that p107 negatively regulates expression of the F-box protein Skp2 in fibroblasts, resulting in the accumulation of p27. We provide evidence that p107 promotes the degradation of Skp2 by the proteasome. Our results identify a novel mechanism by which p107 may inhibit G1 progression through stabilization of the cell cycle inhibitor p27.
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Results |
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To establish a causal relationship between p27 stabilization and the inhibitory effect of p107 on G1 to S phase progression, we used RNA interference to reduce the expression of p27 in Rat1-p107HA cells. Exponentially proliferating cells were transfected with control or p27 small interfering RNAs (siRNAs), and the percentage of S phase cells was determined. As shown in Fig. 2 F, down-regulation of p27 increased by twofold the number of Rat1-p107HA cells that progress to S phase.
p27 is stabilized in Rat1 cells overexpressing p107
One of the key mechanisms involved in the regulation of p27 abundance is its proteolysis by the ubiquitin-proteasome pathway (Pagano et al., 1995). Therefore, we evaluated the effect of p107 overexpression on the turnover of p27 by pulse-chase experiments (Fig. 3 A). The degradation rate of labeled p27 was clearly reduced in Rat1-p107HA cells (half-life 68 h) as compared with Rat1 cells (half-life 34 h) upon exposure to serum. These results were confirmed by cycloheximide-chase experiments (Fig. 3 B). Interestingly, we observed that p27 was more stable in Rat1-p107HA cells whether cycloheximide was added to quiescent cells or after 8 or 20 h of release from growth arrest.
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p107 negatively regulates Skp2 expression
Because phosphorylation of p27 is not affected, we next analyzed the impact of p107 on the expression of SCFSkp2 subunits and its cofactor Cks1. As reported previously (Lisztwan et al., 1998; Carrano and Pagano, 2001; Rodier et al., 2001), serum stimulation of G0/G1-synchronized Rat1 cells induced the accumulation of Skp2 and Cks1 proteins as cells approached S phase (Fig. 4 A). However, the abundance of Skp2 was markedly decreased in Rat1-p107HA cells, whereas the levels of Cks1 and Cul1 remained similar. To exclude the possibility that the observed changes in Skp2 expression were an indirect consequence of p107 effect on cell cycle progression, Rat1 and Rat1-p107HA cells were first sorted at the different phases of the cell cycle by flow cytometry before measurement of Skp2 levels. This experiment clearly showed that the expression of Skp2 is considerably decreased in all phases of the cell cycle in Rat1-p107HA cells, as compared with parental Rat1 cells (Fig. 4 B). These results suggest that Skp2 is directly regulated by p107. To determine if the effect of p107 is specific to Skp2, we monitored the expression of ßTrCP, another F-box member of SCF E3 ligase complexes. The SCFßTrCP ligase is implicated in the phosphorylation-dependent ubiquitination and degradation of IB
in response to TNF and other signals (Rothwarf and Karin, 1999). No difference in the levels of ßTrCP (Fig. 4 C) or in the turnover rate of I
B
in response to TNF
(unpublished data) was observed between Rat1 cells and cells overexpressing p107. To confirm that the reduction in Skp2 expression is not an indirect consequence of the long-term stable overexpression of p107, we infected populations of Rat1 cells with a retrovirus encoding p107. Acute overexpression of p107 similarly resulted in the accumulation of p27 and cyclin E1 proteins, concomitant with down-regulation of Skp2 expression (Fig. 4 D).
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Skp2 is a physiological target of p107 that is important for its negative control of G1 to S phase progression
To evaluate the physiological relevance of the aforementioned findings, we examined the regulation of Skp2 expression in p107/ MEFs. These cells display a twofold acceleration in doubling time (LeCouter et al., 1998a). Fig. 5 A shows that induction of Skp2 protein by serum was much greater in p107/ MEFs as compared with wild-type p107+/+ cells. This observation, together with the findings described in Fig. 4, strongly suggests that p107 is a negative regulator of Skp2 expression. Next, we wanted to determine if Skp2 is essential for the delay in S phase entry induced by p107. To test this idea, we monitored the incorporation of BrdU in wild-type and Skp2/ MEFs (Nakayama et al., 2000) transfected with a control plasmid or p107HA. In exponentially proliferating cells, the ability of p107 to decrease the number of S phase cells was completely suppressed in Skp2/ MEFs (Fig. 5 B). These results argue that Skp2 is a target of p107 that mediates, at least in part, its negative regulatory effect on cell cycle progression.
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Role of Cul1 and Cdh1 in the regulation of Skp2 turnover by p107
To get some insight into the mechanism by which p107 regulates Skp2 proteolysis, we performed experiments to determine whether or not p107 expression impacts on known Skp2 degradation pathways. Early work has suggested that degradation of Skp2 in G0/G1 cells is mediated in part by an autocatalytic mechanism involving a Cul1-based ubiquitin ligase (Wirbelauer et al., 2000). To test the role of a Cul1-based E3 ligase pathway, we used a 293 cell line conditionally expressing a Cul1 deletion mutant (Cul1-N252) that lacks the binding site for Roc1 and Cdc34 (Piva et al., 2002). Expression of this mutant is predicted to sequestrate Skp1-F box protein complexes and interfere with SCF-dependent degradation. Indeed, doxycycline induction of Cul1-N252 expression significantly increases the steady-state abundance of p27 in these cells (Fig. 7 A). We found that overexpression of Cul1-N252 leads to a modest accumulation of ectopically expressed HA-Skp2 (Fig. 7 A), associated with an increase in the half-life of the protein (not depicted). Enforced expression of p107 markedly down-regulated ectopic HA-Skp2, whether or not the cells were induced to express Cul1-N252 mutant (Fig. 7 A). Cycloheximide-chase experiments confirmed that the reduction in Skp2 levels was correlated with an accelerated degradation of the protein (Fig. 7 B). These results argue against the involvement of a Cul1-based ubiquitin ligase in mediating the effect of p107 on Skp2 turnover.
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Skp2 does not target p107 for degradation
Skp2 mediates the ubiquitin-dependent degradation of various cell cycle regulatory proteins, including the pRb-family member p130 (Tedesco et al., 2002). Interestingly, we found that p107 and Skp2 physically associate upon coexpression in NIH 3T3 cells (unpublished data). To determine if p107 is a target of the SCFSkp2 complex, we monitored the expression of p107 in wild-type and Skp2/ cells during cell cycle progression. No difference in p107 protein levels was observed between the two cell lines (Fig. 8).
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Discussion |
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The precise mechanisms underlying the regulation of Skp2 expression during the cell cycle are not fully understood. The levels of Skp2 protein are undetectable or low in G0/early G1, increase gradually during progression to S and G2 phases, and then decline abruptly in late M phase (Lisztwan et al., 1998; Wirbelauer et al., 2000; Carrano and Pagano, 2001; Rodier et al., 2001; Bashir et al., 2004; Wei et al., 2004). The induction of Skp2 is associated with the cell cycledependent accumulation of Skp2 mRNA (Zhang et al., 1995; Carrano and Pagano, 2001; Imaki et al., 2003) and the stabilization of the protein (Wirbelauer et al., 2000; Bashir et al., 2004; Wei et al., 2004). It has been suggested that the transcriptional induction of Skp2 mRNA results from the cell cycledependent binding of GA-binding protein to the Skp2 promoter (Imaki et al., 2003). Cell adhesion to the extracellular matrix is also required for the accumulation of Skp2 mRNA (Carrano and Pagano, 2001). Two recent studies have provided an important insight into the cell cycle dependence of Skp2 degradation, by showing that Skp2 is targeted for ubiquitination and destruction by the APC/CCdh1 ubiquitin ligase (Bashir et al., 2004; Wei et al., 2004). Earlier work also suggested that the rapid turnover of Skp2 in G0/G1 cells involves a Skp2-bound Cul1-based core ubiquitin ligase (Wirbelauer et al., 2000). Moreover, Skp2 was found to self-ubiquitinate in the presence of SCF components in in vitro ubiquitination studies (Wirbelauer et al., 2000; Wang et al., 2004). Thus, expression of Skp2 appears to be controlled by multiple transcriptional and posttranscriptional mechanisms.
In this work, we found that overexpression of p107 partially suppresses the transient increase in Skp2 mRNA detected as cells progress into G1 phase. p107 is known to bind specifically to E2F4, which is primarily involved in the repression of E2F-responsive genes (Dyson, 1998; Trimarchi and Lees, 2002). Of note, the Skp2 gene was recently shown to be up-regulated upon E2F induction in U-2 OS cells (Vernell et al., 2003), although other DNA microarray experiments failed to identify Skp2 as an E2F target. Here, we showed that complete silencing of E2F4 has no impact on Skp2 levels in p107-overexpressing Rat1 cells, thereby suggesting that Skp2 is not a bona fide E2F4 target gene. Consistent with these findings, microarray analysis of anti-E2F4 chromatin immunoprecipitates failed to identify the Skp2 promoter among the target genes (Ren et al., 2002; Weinmann et al., 2002).
Our results suggest that repression of Skp2 transcription is unlikely to be the principal mechanism by which p107 down-regulates Skp2 expression. We found that increasing the levels of p107 completely inhibits the stabilization of Skp2 protein that occurs during G1 to S phase progression. This depends on a direct effect of p107 on Skp2 turnover as the ectopically expressed Skp2 was also found to be less stable in p107-overexpressing cells. We also observed that the L19 mutant of p107 (Zhu et al., 1995), which is defective in E2F binding, effectively promotes the degradation of Skp2, demonstrating the lack of involvement of E2F transcriptional activity in this process (unpublished data). Various mechanisms can be envisaged to explain the effect of p107 on Skp2 degradation. We have shown that Cul1 and Cdh1 levels are unchanged in p107-overexpressing cells. We also provide evidence that inactivation of Cul1-based ubiquitin ligases by overexpression of Cul1-N252 mutant or inactivation of APC/CCdh1 by silencing of Cdh1 expression is not sufficient to render Skp2 immune to p107 overexpression, suggesting that p107 may regulate Skp2 proteolysis, at least in part, by a novel pathway. In further support of this idea, we observed that the half-life of Skp2 is much shorter in p107-overexpressing cells when measured at 18 h after serum stimulation, a time where a large proportion of cells are in S and G2/M phases and Cdh1 is inactive (Fig. 6 C, bottom panel). The steady-state levels of Skp2 were also found to be significantly lower in S and G2/M phase extracts of Rat1-p107HA cells, as compared with Rat1 cells (Fig. 4 B). Intriguingly, we found that p107 can be coprecipitated with Skp2 in vivo upon coexpression of the two proteins (unpublished data), but the physiological significance of this observation remains to be established. Our results do not exclude the possibility that p107 contributes in some way to the regulation of Skp2 degradation by APC/CCdh1. Indeed, we observed that the impact of p107 overexpression on Skp2 turnover was somewhat less pronounced in Cdh1-silenced cells. Additional studies are clearly warranted to clarify these issues.
The expression of Skp2 is elevated in many transformed cells (Zhang et al., 1995) as well as in various human cancers, including lymphomas (Latres et al., 2001), oral epithelial carcinomas (Gstaiger et al., 2001), and breast cancers (Signoretti et al., 2002). The overexpression of Skp2 correlates with increasing grade of the tumor. Importantly, Skp2 can cooperate with activated Ras to transform primary cells in vitro and in vivo, demonstrating its oncogenic potential (Gstaiger et al., 2001; Latres et al., 2001). These observations underscore the importance of elucidating the mechanisms that control Skp2 abundance in normal and cancer cells. Our work identifies p107 as a novel regulator of Skp2 expression. p107 levels normally increase in late G1 to reach a maximum in S and G2/M phases of the cell cycle. Interestingly, we found that inactivation of p107 in fibroblasts leads to the inappropriate expression of Skp2 during G1 to S phase progression. From these observations, it is tempting to speculate that p107 may act as a modulator that limits Skp2 accumulation in G1 to prevent premature S phase entry. p107 may also act later to control the correct timing of S phase and mitosis (Nakayama et al., 2004).
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Materials and methods |
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Antibodies and DNA constructs
The p27 phospho-Ser10 specific antibody was generated in collaboration with Cell Signaling Technology. Polyclonal antibodies to p107 (C18), Cks1 (FL79), p57Kip2 (H91), ßTrCP (H300), Myc (A14), and HA (Y11) were obtained from Santa Cruz Biotechnology, Inc. mAbs to cyclin A (E-23), Cdk4 (DCS-35), E2F4 (Ab-4) and Cdh1 (DH-01), and polyclonal anti-cyclin D1 (Ab-4) were obtained from Neomarkers. The source of other antibodies has been described previously (Servant et al., 2000; Rodier et al., 2001).
The human HA-tagged p107 cDNA (Zhu et al., 1995; provided by L. Zhu, Albert Einstein College of Medicine, Bronx, NY) was ligated into the BamHI and XhoI sites of pBabe-puro. The human Skp2 cDNA (provided by H. Zhang, Yale University School of Medicine, New Haven, CT) was ligated into the EcoRI site of pcDNA3-Myc6, pBabe-puro-Myc6, and pcDNA3-HA expression vectors (Coulombe et al., 2004). pcDNA3-Myc6-GFP was constructed by subcloning the PCR-amplified GFP sequence into the EcoRIXbaI sites of pcDNA3-Myc6. pcDNA3-HA-GFP has been described previously (Coulombe et al., 2003).
Immunoblot analysis and Cdk2 kinase assays
Cell lysis, immunoprecipitation, and immunoblot analysis were performed as described previously (Servant et al., 2000). For coimmunoprecipitation studies, cell lysates (500 µg of protein) were incubated for 4 h at 4°C with anti-Cdk2 or anti-HA antibodies cross-linked to protein ASepharose beads, and the precipitated proteins were analyzed by immunoblotting with p27, p107, cyclin E1, or Myc specific antibodies. The phosphotransferase activity of Cdk2 was measured by immune complex kinase assay using histone H1 as substrate (Servant et al., 2000).
Pulse-chase experiments
Pulse-chase analysis of p27 turnover was performed as described previously (Rodier et al., 2001). For cycloheximide chase, cells at different stages of the cell cycle were incubated with 100 µg/ml of cycloheximide for the indicated times. Cell lysates were prepared and the expression of p27 or Skp2 was analyzed by immunoblotting.
Cell cycle analysis and cell sorting
For BrdU incorporation studies, cells plated on glass coverslips were serum starved and then stimulated with 10% serum for the times indicated in the presence of 10 µM BrdU. The cells were fixed as described previously (Rodier et al., 2001). DNA was denatured by treatment with 2 N HCl for 10 min and staining was performed by incubating cells for 1 h at 37°C with anti-BrdU antibody, followed by incubation with FITC-conjugated antimouse IgG as secondary reagent. DAPI staining was performed to visualize the nuclei. Cell samples were analyzed by fluorescence microscopy (model DM RB; Leica), and the percentage of cells showing nuclear labeling for BrdU was calculated. For transfection studies, the transfected cells were first detected by immunostaining with anti-Myc or anti-HA as primary antibody and FITC-conjugated antirabbit IgG as secondary reagent. The DNA was then denatured and BrdU was stained as described, except that the secondary reagent was a Rhodamine-conjugated antimouse IgG. A minimum of 100 cells was scored for each coverslip. The results are expressed as the percentage of transfected cells showing nuclear BrdU labeling.
For cell cycle analysis, exponentially proliferating or G0-synchronized cells allowed to reenter the cell cycle were scraped in 1 mM PBS/EDTA, fixed in cold 70% ethanol, and kept at 20°C until flow cytometry analysis. The cells were washed with PBS and incubated on ice for 30 min in PI buffer (0.1% sodium citrate, 50 µg/ml propidium iodide, and 0.2 mg/ml RNase) in the dark. Fluorescence was recorded on an flow cytometer (model epics XL; Beckman Coulter) and the cell cycle profiles were determined using the Multicycle AV software. For cell sorting experiments, the cells were stained with 7.5 µg/ml of Hoescht 33342 for 30 min at 37°C, and then sorted in the G0/G1, S, and G2/M phases of the cell cycle using the MoFlo high speed cell sorter (DakoCytomation). Once sorted, the cells were lysed and analyzed as described previously (Servant et al., 2000).
RNA interference
siRNAs were purchased from Dharmacon and transfected into Rat1 or HeLa cells using Oligofectamine reagent (Invitrogen). The siRNA target sequences were 5'-CUGCCGAGAUAUGGAAGAAdTdT-3' (p27), 5'-CGAGAGUGAAGGUGUCUGUdTdT-3' (E2F4), and 5'-UGAGAAGUCUCCCAGUCAGdTdT-3' (Cdh1).
Northern blot analysis
RNA isolation and Northern blot analysis was performed as described previously (Servant et al., 2000). The probe used was a 1-kb EcoRI fragment of the rat Skp2 cDNA (provided by G. Baffet, Hôpital Pontchaillou, Rennes, France).
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Acknowledgments |
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G. Rodier is recipient of fellowships from the National Cancer Institute of Canada and the American Association for Cancer Research (Anna D. Barker Fellowship), and P. Coulombe is recipient of a studentship from the National Cancer Institute of Canada. S. Meloche holds a Canada Research Chair in Cellular Signaling. This work was supported by a grant from the Canadian Institutes for Health Research (MOP-14168).
Submitted: 26 April 2004
Accepted: 9 November 2004
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References |
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