Skp2 regulates the antiproliferative function of the tumor ... - Nature

Oncogene (2008) 27, 3176–3185

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ORIGINAL ARTICLE

Skp2 regulates the antiproliferative function of the tumor suppressor RASSF1A via ubiquitin-mediated degradation at the G1–S transition MS Song1,4, SJ Song1,4, SJ Kim1, K Nakayama2, KI Nakayama3 and D-S Lim1 1 Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, Korea; 2Division of Developmental Genetics, Center for Translational and Advanced Animal Research on Human Diseases, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan and 3Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan

The tumor suppressor RASSF1A is inactivated in many human cancers and is implicated in regulation of microtubule stability, cell cycle progression and apoptosis. However, the precise mechanisms of RASSF1A action and their regulation remain unclear. Here we show that Skp2, an oncogenic subunit of the Skp1–Cul1–F–box ubiquitin ligase complex, interacts with, ubiquitinates, and promotes the degradation of RASSF1A at the G1–S transition of the cell cycle. This Skp2-dependent destruction of RASSF1A requires phosphorylation of the latter on serine-203 by cyclin D–cyclin-dependent kinase 4. Interestingly, mutation of RASSF1A-phosphorylation site Ser203 to alanine results in a delay in cell cycle progression from G1 to S phase. Moreover, enforced expression of Skp2 abolishes the inhibitory effect of RASSF1A on cell proliferation. Finally, the delay in G1–S progression after Skp2 removal is normalized by depletion of RASSF1A. These findings suggest that the Skp2-mediated degradation of RASSF1A plays an important role in cell proliferation and survival. Oncogene (2008) 27, 3176–3185; doi:10.1038/sj.onc.1210971; published online 10 December 2007 Keywords: Skp2; RASSF1A; cyclin D-Cdk4; ubiquitination; G1–S transition

Introduction Two major types of ubiquitin ligase contribute to regulation of cell cycle progression: the Skp1–Cul1– F–box protein (SCF) complex is implicated in regulation of the G1–S transition, and the anaphase-promoting complex (APC) is required both for separation of sister chromatids at anaphase and for exit from M phase into G1 (Zachariae and Nasmyth, 1999). The SCF complex Correspondence: Professor D-S Lim, Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1 Guseong-D. Yuseong-Gu, Daejeon 305-701, Korea. E-mail: [email protected] 4 These authors contributed equally to this work. Received 11 July 2007; revised 15 October 2007; accepted 5 November 2007; published online 10 December 2007

consists of the essential components Skp1, Cul1, Rbx1 and an F-box protein, the latter of which binds to Skp1 through its F-box motif and serves to recognize and recruit target proteins (Cardozo and Pagano, 2004). The F-box protein Skp2 has been implicated in the ubiquitin-dependent degradation of p27, E2F-1, free cyclin E and FOXO1 (Marti et al., 1999; Nakayama et al., 2000; Huang et al., 2005). The APC–Cdh1 complex degrades Skp2 at the onset of G1 phase, after which Skp2 begins to accumulate late in G1 and its abundance peaks during S and G2 phases (Bashir et al., 2004; Wei et al., 2004). Cell cycle progression from G1 to S phase is governed by cyclin-dependent kinase (Cdk) 4 as well as Cdk2. Cdk4 is activated by D-type cyclins at early to mid-G1 phase, whereas Cdk2 is activated by E- and A-type cyclins during late G1 and S phase, respectively. The activities of Cdk4 and Cdk2 are constrained by the p16 Ink4a family and the p21 Cip/Kip family inhibitors, respectively (Sherr and Roberts, 1999). Cyclin D–Cdk4 phosphorylates the retinoblastoma protein pRb, pRbrelated proteins p107 and p130, and Smad3 (Sherr and Roberts, 1999; Matsuura et al., 2004). In addition, cyclin D–Cdk4 titrates p27 and p21 to cyclin D–Cdk4, thereby triggering the activity of the cyclin E–Cdk2 holoenzyme (Sherr and Roberts, 1999). Consistent with their growth-promoting functions, the cyclin D1 and the Cdk4 gene were found to be amplified in many human cancers (Bartkova et al., 1995; An et al., 1999). RASSF1A is a tumor suppressor gene on chromosome 3p21 that is inactivated in many types of carcinoma (Dammann et al., 2000). Moreover, knockout mice defective for RASSF1A are prone to tumor development (Tommasi et al., 2005; van der Weyden et al., 2005), further confirming that RASSF1A functions as a tumor suppressor. Apparently RASSF1A appears to function in diverse series of critical cellular processes involved in tumor suppression. RASSF1A has been shown to regulate mitotic progression by inhibiting the activity of APCCdc20 (Song et al., 2004) and affect microtubule dynamics (Vos et al., 2004). It binds the p120 E4F transcription factor that forms a complex with both the Rb and p53 tumor suppressors (Fenton et al., 2004). Furthermore, RASSF1A induces a G1 arrest by

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engaging the Rb family G1–S checkpoint through inhibition of cyclin D1 (Shivakumar et al., 2002). RASSF1A is also involved in apoptosis by linking death receptor signaling to the apoptotic machinery (Baksh et al., 2005; Oh et al., 2006; Matallanas et al., 2007). However, the precise mechanism by which RASSF1A is regulated remains unclear. We now show that Skp2 interacts with and promotes the ubiquitinmediated degradation of RASSF1A, thereby inhibiting tumor suppressor function of RASSF1A.

Results The tumor suppressor RASSF1A is degraded at the G1–S transition Previous studies have suggested a growth-inhibitory function of RASSF1A at G1 (Shivakumar et al., 2002; Rong et al., 2004) and during mitosis (Song et al., 2004; Vos et al., 2004). Thus, we reexamined the ability of RASSF1A to affect the cell cycle progression in various cell lines (U2OS, SaoS2, H1299, A549, MCF7, HeLa and 293T). Interestingly, we found that ectopic expression of RASSF1A induced a delay in G1–S cell cycle progression in U2OS, A549 and MCF7 cells, whereas enforced expression of RASSF1A in SaoS2, H1299, HeLa and 293T cells increased the cell population with a G2–M (data not shown). Consistent with a previous report (Rong et al., 2004), RASSF1A likely modulates both the G1–S and mitotic cell cycle regulation by two independent mechanisms in a cell line context-dependent manner. To further examine the contribution of RASSF1A regulation at G1–S cell cycle progression, we monitored RASSF1A expression during the cell cycle

by immunoblot analysis. The abundance of endogenous RASSF1A in U2OS cells decreased markedly during S phase and increased again as the cells approached the G2–M transition (Figure 1a). Analysis of G1–S cell cycle progression after serum deprivation and restimulation in NIH3T3 cells also showed the change in the level of RASSF1A protein at the G1–S transition (Supplementary Figure 1). We next determined whether this downregulation of RASSF1A expression in S phase is due to protein destabilization by measuring the half-life of RASSF1A during mitosis and at the G1–S transition. Endogenous RASSF1A was more unstable at G1–S, with a half-life substantially less than 1 h, than during mitosis (Figure 1b). In addition, the proteasome inhibitor MG132 prevented the downregulation of RASSF1A expression at G1–S (Figure 1c). These observations thus indicated that RASSF1A is degraded by the proteasome beginning at the G1–S transition. Skp2 interacts with and promotes ubiquitination and degradation of RASSF1A The changes in RASSF1A abundance during the cell cycle were similar to those of the Cdk inhibitor p27 and opposite to those of Skp2 (Figure 1a). We thus investigated the possible role of Skp2 in the degradation of RASSF1A. To address this issue, we first assessed whether Skp2 interacts with RASSF1A with the use of a co-immunoprecipitation assay. Skp2 was the only F-box protein among those tested (Skp2, b-TrCP1, b-TrCP2, Fbw7, Fbl5, Fbl12) that interacted with RASSF1A in vivo and in vitro (Figure 2a; data not shown). Forced expression of Skp2 resulted in a marked decrease in the steady-state amount of RASSF1A in U2OS cells, whereas overexpression of other F-box proteins (data

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Figure 1 RASSF1A is degraded at the G1–S transition of the cell cycle. (a) U2OS cells were released from nocodazole block (400 ng ml1, 18 h) at indicated time point and subjected to immunoblot analysis with antibodies to the indicated proteins. (b) U2OS cells synchronized in mitosis by nocodazole block or at G1–S by thymidine block were released for the indicated times in the presence of cycloheximide (CHX, 50 mg ml1) and then subjected to immunoblot analysis with anti-RASSF1A and -b-actin. (c) U2OS cells synchronized at G1–S were released from nocodazole block for 9 h in the absence or presence of 1, 5 or 10 mM MG132 and then subjected to immunoblot analysis with anti-RASSF1A and -b-actin. Oncogene


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Figure 2 Skp2 interacts with and induces ubiquitination and degradation of RASSF1A. (a) Lysate from HeLa cells was subjected to immunoprecipitation (IP) with anti-Skp2 or control immunoglobulin G (IgG), and the resulting precipitates as well as cell lysate were subjected to immunoblot analysis with anti-RASSF1A and anti-Skp2 (top). Immunoprecipitates with anti-Skp2 from the mixture of in vitro translated (IVT) Skp2 and RASSF1A were subjected together with the input proteins to immunoblot analysis with antibodies to RASSF1A or to Skp2 (bottom). (b) U2OS cells were transfected with a vector for Myc-tagged wild-type Skp2 or for an Myc-tagged Skp2 mutant (DF) that lacks the F box. The cells were subsequently synchronized at G1–S and subjected to immunoblot analysis with antibodies to RASSF1A, Myc or b-actin. Asterisk is nonspecific band. (c) Lysates from 293T cells transfected with a vector for Flag-RASSF1A were immunoprecipitated with anti-Flag, and the resulting precipitates were subjected to immunoblotting with anti-Cul1, anti-Skp2, anti-Skp1 and anti-Flag. Asterisks are nonspecific bands. (d) Ubiquitination reactions were performed with in vitro translated Cul1, Rbx1, Skp1 and Skp2 together with ubiquitin (Ub) and E1 and E2 enzymes. The ubiquitination of 35S-labeled RASSF1A was detected by SDS–PAGE and autoradiography. (e) 293T cells transfected with a vector for His-tagged ubiquitin and Flag-RASSF1A were synchronized with or without thymidine (G1–S) and then incubated with MG132 (20 mM) for 6 h before harvest. Cell lysates were immunoprecipitated with anti-Flag. The ubiquitination of RASSF1A was detected by immunoblotting with anti-Ub. An asterisk represents heavy chain. (f) U2OS cells were transfected for the indicated times with a vector for Skp2 small interfering RNA (siRNA) or for a control green fluorescent protein (GFP) siRNA, after which the abundance of the indicated proteins was examined by immunoblot analysis. (g) Skp2 þ / þ or Skp2–/– MEFs were synchronized at G1–S by thymidine block and released in the presence of cycloheximide (50 mg ml1) for the indicated times, after which the abundance of RASSF1A was examined by immunoblot analysis. Oncogene


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not shown) or expression of the mutant Skp2(DF), which lacks the F-box domain, had no such effect (Figure 2b). A co-immunoprecipitation assay revealed that some portions of endogenous SCF complex including Cul1 and Skp1, as well as endogenous Skp2 were co-precipitated with RASSF1A (Figure 2c). These results thus supported the notion that RASS1A degradation depends on a functional F box of Skp2. We next determined whether the SCF–Skp2 complex directly ubiquitinates RASSF1A. Addition of in vitro translated Skp2 to an in vitro reconstituted Skp1–Cul1– Rbx1 complex stimulated ubiquitination of 35S-labeled RASSF1A (Figure 2d). Expectedly, an in vivo ubiquitination assay revealed that the ubiquitination of RASSF1A was evident during G1–S transition (Figure 2e). To further confirm that Skp2 regulates RASSF1A stability, we depleted U2OS cells of endogenous Skp2 by RNA interference (RNAi) with a small interfering RNA (siRNA) specific for Skp2 mRNA. Skp2-depleted cells exhibited an increased level of endogenous RASSF1A and p27 (Figure 2f). Consistently, the stability of RASSF1A was greater in Skp2–/– MEFs than in wildtype cells (Figure 2g). Taken together, these results thus

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Cyclin D–cdk4 phosphorylates RASSF1A Given that the SCF complex recognizes most substrates in a phosphorylation-dependent manner (Harper, 2002), we screened several Cdks for the ability to phosphorylate RASSF1A. Among the various Cdks examined, only the cyclin D–Cdk4 phosphorylated RASSF1A (Figure 3a). We then determined the region of RASSF1A that is phosphorylated by cyclin D–Cdk4 in vitro with the use of purified RASSF1A fragments fused to glutathione S-transferase (GST). Only the RASSF1A fragment comprising residues 180–239 was phosphorylated by cyclin D–Cdk4 (Supplementary Figure 2a). Replacement of each of six serine, threonine or tyrosine residues in this fragment of RASSF1A with a nonphosphorylatable alanine residue revealed that Ser203 is the phosphorylation site for cyclin D–Cdk4, because phosphorylation of the fragment with the S203A mutation was undetectable, whereas all other mutant fragments were still phosphorylated (Supplementary Figure 2b). Next, we prepared antibodies specific for the

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Figure 3 Cyclin D–Cdk4 phosphorylates RASSF1A. (a) Immunoprecipitates prepared from HeLa cells with antibodies to the indicted cyclins were incubated with GST-RASSF1A and [g-32P]ATP, after which the reaction mixtures were subjected to SDS–PAGE and autoradiography for the detection of 32P-labeled GST-RASSF1A as well as to immunoblot analysis with antibodies to GST or to the corresponding cyclins. (b) A purified GST-cyclin D–Cdk4 complex was incubated with GST or GST-RASSF1A (left) or with GST fusion proteins of wild-type (WT) or S203A mutant forms of RASSF1A (right) in the presence of [g-32P]ATP. The reaction mixtures were then subjected to SDS–PAGE and autoradiography for the detection of 32P-labeled GST-RASSF1A as well as to immunoblot analysis with antibodies specific for GST or RASSF1A or those specific for Ser203-phosphorylated RASSF1A (p-RASSF1A) that had been preincubated or not with the corresponding phosphopeptide antigen (p-peptide). (c) Human foreskin fibroblasts were synchronized in G0–G1 by serum deprivation, then restimulated with serum for the indicated times and subjected to immunoblot analysis with antibodies to the indicated proteins. Oncogene


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Ser203-phosphorylated form of RASSF1A. These antibodies reacted with a GST-RASSF1A fusion protein only when it was phosphorylated by cyclin D–Cdk4, the reactivity was blocked by prior incubation of the antibodies with the phosphopeptide antigen, and the antibodies did not recognize the GST-RASSF1A (S203A) mutant (Figure 3b). To determine whether endogenous RASSF1A is phosphorylated in vivo, we synchronized human foreskin fibroblasts at the G0–G1 phase by serum deprivation. Cells were then released from growth arrest by incubation with fresh medium containing complete serum and, at different time points, were subjected to immunoblot analysis with the phospho-specific RASSF1A antibodies and flow cytometric analysis. Consistently, the maximal phosphorylation of RASSF1A occurred at the G1–S transition (Figure 3c). Taken together, these data indicated that cyclin D–Cdk4 phosphorylates RASSF1A at the G1–S transition.

Phosphorylation and degradation of RASSF1A are required for cell cycle progression To examine the effect of RASSF1A regulation on cell proliferation, we analysed the cell cycle progression in RASSF1A þ / þ and RASSF1A–/– MEFs. The entry into S phase after release from serum deprivation occurred much earlier in RASSF1A–/– MEFs than in wild-type cells (Figure 5). Of consistent with this, induction of cyclin A was seen earlier in RASSF1A–/– MEFs than in RASSF1A þ / þ . We next reintroduced a vector for wildtype or S203A mutant forms of RASSF1A into RASSF1A–/– MEFs. Expression of the RASSF1A(S203A) mutant lead to a significant delay in G1–S progression, whereas wild-type RASSF1A induced only a slight delay (Figure 5). These observations thus suggested that phosphorylation and degradation of RASSF1A are required for G1–S cycle progression after serum stimulation.

Interaction and degradation of RASSF1A by Skp2 require phosphorylation of RASSF1A at Ser203 We then evaluated the possible relationship between RASSF1A phosphorylation and its degradation. The reduction in Cdk4 activity induced by overexpression of the Cdk4-specific inhibitor p16 Ink4a or expression of a Cdk4 siRNA, but not a Cdk2 siRNA in U2OS cells, resulted in the decrease in both the phosphorylation and degradation of RASSF1A (Supplementary Figure 3 and data not shown). Given that Cdk4-dependent phosphorylation plays an important role in the degradation of RASSF1A and Skp2 preferentially recognize the phosphorylated substrates, we assessed whether mutation of RASSF1A-phosphorylation site Ser203 to alanine affects the interaction of RASSF1A with Skp2. Co-immunoprecipitation analysis revealed that replacement of Ser203 with alanine abolished the interaction of RASSF1A with Skp2 (Figure 4a). Furthermore, an in vitro binding assay revealed that an RASSF1A peptide (residues 197–209) containing a phosphoserine at position 203 interacted with Skp2 to a much greater extent than did the corresponding nonphosphorylated peptide (Figure 4b). Pulse-chase analysis showed that the RASSF1A(S203A) mutant was more stable than was wild-type RASSF1A in 293T cells (Supplementary Figure 4). Consistent with this observation, the abundance of ectopic wild-type RASSF1A was markedly decreased, whereas that of the RASSF1A(S203A) mutant remained stable in RASSF1A-deficient H1299 cells transfected with a vector for Skp2 (Figure 4c). In addition, MG132 blocked the degradation of wild-type RASSF1A in these cells but had little effect on the abundance of the S203A mutant (data not shown). Finally, we examined whether phosphorylation of RASSF1A at Ser203 might be required for Skp2-mediated ubiquitination. An in vitro ubiquitination assay revealed that ubiquitination of RASSF1A was abolished by the S203A mutation (Figure 4d). Taken together, these data indicated that phosphorylation of RASSF1A on Ser203 by cyclin D–Cdk4 is required for interaction and degradation by Skp2.

Skp2 regulates the antiproliferative and antisurvival function of RASSF1A at the G1–S transition Given that RASSF1A has a tumor suppressor function by inhibiting cell proliferation and survival (Dammann et al., 2000; Shivakumar et al., 2002; Song et al., 2004; Baksh et al., 2005), we thus examined whether Skp2 inhibits the tumor suppression function of RASSF1A. Overexpression of wild-type and S203A mutant forms of RASSF1A resulted in a significant loss of viability in U2OS cells (Figures 6a and b). Importantly, the inhibitory effect of wild-type RASSF1A was significantly abolished by expression of Skp2, whereas no effect on viability was observed when cells were cotransfected with Skp2 and the RASSF1A(S203A) mutant (Figures 6a and b). Moreover, overexpression of RASSF1A resulted in a G1 arrest and this effect was also inhibited by the additional Skp2 expression in U2OS cells (data not shown). Together, these observations thus suggested that Skp2 inhibits the tumor suppressor activity of RASSF1A. Skp2 regulates entry into S phase by mediating degradation of the Cdk inhibitor p27 (Reed, 2003). However, the observation that the phenotype of Skp2–/–p27–/– mice is similar but not identical to that of p27–/– mice (Nakayama et al., 2004) suggests that, although p27 might be the main target of Skp2, Skp2 likely also mediates the ubiquitination of other substrates. We therefore investigated whether G1–S progression might be affected by Skp2-mediated degradation of RASSF1A. Analysis of cell cycle progression after serum deprivation and restimulation revealed that both Skp2 þ / þ p27–/– MEFs transfected with a vector for RASSF1A and Skp2–/–p27–/– MEFs entered S phase B3 h later than did Skp2 þ / þ p27–/– MEFs (Supplementary Figure 5). We also examined the effects of RNAi-mediated depletion of Skp2 on cell cycle progression in human cells. Similar to previous findings (Bashir et al., 2004; Wei et al., 2004), loss of Skp2 resulted in a marked delay in G1–S progression in U2OS cells (Figure 6c). This delay was accompanied by accumulation of RASSF1A and p27 (Figure 6c). To

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Figure 4 Interaction and ubiquitination of RASSF1A by Skp2 depend on phosphorylation of RASSF1A at Ser203. (a) 293 T cells transiently expressing the indicated proteins were subjected to immunoprecipitation (IP) with anti-Flag, and the resulting precipitates as well as cell lysates were subjected to immunoblot analysis with anti-HA and -Flag. The asterisk marks a nonspecific band. (b) In vitro translated 35S-Skp2 was incubated with beads conjugated with a RASSF1A peptide (residues 197–209) containing phosphorylated (203p) or nonphosphorylated (203n) Ser203. The beads were then washed, and bound 35S-Skp2 was detected by SDS–PAGE and autoradiography. (c) RASSF1A-deficient H1299 cells transfected with vectors for HA-tagged wild-type or S203A mutant forms of RASSF1A in the presence or absence of Flag-Skp2, as indicated, were subjected to immunoblot analysis with antibodies to the indicated proteins. (d) Bead-immobilized immunoprecipitates prepared with anti-HA from 293T cells expressing HA-tagged wild-type or S203A mutant forms of RASSF1A were incubated with Flag-Skp2 immunoprecipitates from transfected 293T cells in a reaction mixture containing 20 mM MG132 and rabbit reticulocyte lysate, which was depleted of Skp2 with anti-Skp2. The reaction mixture was subjected to immunoblot analysis with antibodies to HA or to Flag.

examine whether the accumulation of p27 or that of RASSF1A is mainly responsible for the delay in progression from G1 to S phase, we depleted cells of both Skp2 and RASSF1A. The delay in G1–S progression induced by Skp2 removal was normalized by depletion of RASSF1A even in the presence of a high level of p27, supporting the notion that G1–S progression depends, at least in part, on degradation of RASSF1A (Figure 6c). Collectively, these results demonstrated that the Skp2-mediated degradation of RASSF1A plays an important role in cell proliferation and survival. Discussion Although molecular mechanisms by which RASSF1A participates in tumorigenesis are not fully elucidated,

RASSF1A has been implicated in various cellular processes involved in tumor suppression. RASSF1A regulates the mitotic progression, microtubule dynamics (Song et al., 2004; Vos et al., 2004) and G1–S transition (Fenton et al., 2004). The frequently observed downregulation of RASSF1A expression in human tumors has in many cases been attributed to promoter methylation (Agathanggelou et al., 2005), however, other potential causes have not been revealed yet. There are many evidence, that aberrant protein degradation of cell cycle regulators is an important factor contributing to tumorigenesis (Bashir and Pagano, 2003; Pagano and Benmaamar, 2003). Skp2 is the substrate-recognition component of the SCFSkp2 ubiquitin ligase complex and is a key regulator of S-phase entry (Nakayama et al., 2000; Harper, 2002; Cardozo and Pagano, 2004). Overexpression or Oncogene


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Figure 5 Phosphorylation and degradation of RASSF1A are required for cell cycle progression. RASSF1A–/– MEFs were infected with retrovirus vectors for empty, wild-type or S203A mutant forms of RASSF1A. Infected cells were rendered quiescent by serum deprivation and then restimulated with serum for the indicated times before analysis of cell-cycle progression by flow cytometry (a) and immunoblot analysis of cell lysates with antibodies to the indicated proteins (b).

amplification of Skp2 has been reported in a large number of human cancers (Yokoi et al., 2002) and transgenic expression of Skp2 in mice leads to tumor formation, suggesting that Skp2 is oncogenic (Gstaiger et al., 2001; Latres et al., 2001). Skp2 targets p27, cyclin E and FOXO1 for degradation in a phosphorylationdependent manner (Marti et al., 1999; Nakayama et al., 2000; Huang et al., 2005). Here we provide several lines of evidence that RASSF1A is also targeted by Skp2 at the G1–S transition: (1) the level of RASSF1A during cell cycle progression is inversely related to that of Skp2; (2) Skp2 directly interacts with and ubiquitinates RASSF1A in a manner dependent on phosphorylation of RASSF1A on Ser203 in G1–S transition; (3) overexpression of Skp2 results in a decrease in the cellular abundance of RASSF1A, thereby elevating cell viability and proliferation; (4) deletion of Skp2 or depletion of Skp2 increases both the level and half-life of RASSF1A as well as induces a delay in G1–S progression; and (5) entry into S phase is accelerated in RASSF1A–/– MEFs. The phenotype of Skp2–/– mice is rescued in large part by knockout of p27. However, the phenotype of Skp2–/–p27–/– mice is not identical to that of p27–/– mice. Although accumulation of p27 mainly contributes to the proliferation defect of Skp2–/– MEFs, Skp2 appears to prevent the accumulation of p27 during S–G2 rather than at the G1–S transition (Nakayama et al., 2004). We have now shown that the delay in G1–S progression induced by Skp2 depletion was normalized by the additional depletion of RASSF1A even in the presence Oncogene

of a high level of p27 (Figure 6c). Thus it is likely that Skp2 targets RASSF1A to allow entry of cells into S phase and targets p27 to regulate progression into mitosis. The ubiquitination of RASSF1A is regulated by the interaction of Skp2 with RASSF1A in a phosphorylation-dependent manner (Figures 4a and b). Phosphorylation of RASSF1A on Ser203 by cyclin D–Cdk4 appears to be the key step required for Skp2 binding. Except for the retinoblastoma protein family, Smad3 has been the only Cdk4 substrate demonstrated so far (Matsuura et al., 2004). Here we have shown that RASSF1A is also phosphorylated by cyclin D–Cdk4 (Figure 3 ; Supplementary Figure 3). Mutation of this cyclin D–Cdk4 phosphorylation site in RASSF1A increases its abundance and antiproliferative function (Figures 4c, d and 5). Cyclin D–Cdk4 thus likely inhibits the accumulation of RASSF1A, thus facilitating cell cycle progression from G1 to S phase. Recently, it has been reported that Aurora A appears to phosphorylate RASSF1A at Thr-202 and/or Ser-203, although further studies should be required for determination of which one of two is predominant phosphoresidue in RASSF1A (Rong, 2007). Indeed, we also found that Aurora kinases A and B primarily phosphorylate RASSF1A on Ser-203 for early and late mitosis, respectively (SJ Song and D-S Lim, unpublished data). Thus, phosphorylation of RASSF1A Ser203 by Aurora kinases and Cdk4 appears to have clearly distinct roles in cell cycle control at different stages of the cell cycle.


3183 120

Vector

Vector

Cell Survival (%)

100

Vector

Flag-Skp2

Skp2

80 60 40

HA-RASSF1A (WT)

20

HA-RASSF1A (S203A)

Flag-Skp2: HA-RASSF1A:

-

SF (W 1A T) R AS (S SF 20 1 3A A )

R AS

Ve

ct or

0

-

-

+

+

+

- WT SA - WT SA HA-RASSF1A Flag-Skp2 β-Actin

GFP siRNA

Skp2 siRNA

Skp2 siRNA + RASSF1A siRNA

13 12

4N

13

2N 12

4N

9 7

GFP siRNA

13

2N 12

4N

11

11

11

Nocodazole release (h):

14

14

14 2N


9 7

Skp2 siRNA


9 7

Skp2 siRNA + RASSF1A siRNA Cyclin A p27 Skp2 RASSF1A

Nocodazole release (h) :

β-Actin 7 9 11 12 13 14

7 9 11 12 13 14

7 9 11 12 13 14

Figure 6 Skp2-mediated degradation of RASSF1A is required for cell survival and proliferation. (a and b) U2OS cells were cotransfected with pEGFP vector and vectors for HA-tagged wild-type or S203A mutant form of RASSF1A in the presence or absence of Flag-Skp2, as indicated. At 48 h after transfection, transfected viable cells were photographed under both UV and transmitted light (a) and quantified by using bromophenol blue (b, top) and subjected to immunoblot analysis with antibodies to the indicated proteins (b, bottom). (c) U2OS cells transfected with a vector for Skp2 small interfering RNA (siRNA) or a control green fluorescent protein (GFP) siRNA in the presence or absence of a vector for RASSF1A siRNA were released from nocodazole block for the indicated times before analysis of cell cycle progression by flow cytometry (top) or immunoblot analysis of cell lysates with antibodies to the indicated proteins (bottom).

Since human cancers often show overexpression of Aurora A, Skp2, cyclin D1 and high levels of Cdk4 activity, the diminishing RASSF1A activity by Aurora A, Skp2 or cyclin D–Cdk4 may contribute to tumorigenesis.

Materials and methods Cell culture U2OS, HeLa, 293T, human foreskin fibroblasts, H1299, RASSF1A–/– MEFs, Skp2–/– MEFs, p27–/– MEFs and Skp2–/– Oncogene


3184 p27–/– MEFs were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. Cycloheximide or MG132 (EMD Biosciences, San Diego, CA, USA) was added at a final concentration of 50 mg ml1 or 10 mM, respectively. Cells were synchronized in G0–G1, mitosis or G1–S by incubation for 36 h with 0.1% serum-containing medium, 16 h with nocodazole (100 ng ml1; Sigma-Aldrich, St Louis, MO, USA) or thymidine (2 mM), respectively. Transfections were done with the Effectene transfection reagent (Qiagen, Valencia, CA, USA), and transfection efficiencies of 70–90% in cells were achieved. For retrovirus production, retroviral RASSF1A plasmid was transfected into 293T packaging cells to produce retroviruses as described previously (Lim et al., 2000). RASSF1A–/– MEFs were infected at greater than 90% efficiency. Plasmids and protein generation The vectors for HA-RASSF1A, GST-RASSF1A and retroviral RASSF1A plasmid were previously described (Song et al., 2004). The plasmids pcDNA3-Flag or -Myc-Skp2 and –Flag or -Myc-Skp2(DF) were kindly provided by M Pagano, and plasmids encoding other F-box proteins were kindly provided by JW Harper. The plasmids pcDNA3-HA-Cul1, pcDNA3-Rbx1 and pcDNA3-Skp1 were kindly provided by JB Yoon, and pcDNA3-HA-p16 was kindly provided by HW Lee. The RASSF1A(S203A) mutant was generated with the use of a site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). GST fusion proteins were purified by standard methods. Cyclin D–Cdk4 kinase was from Cell Signaling Technology Inc. (Danvers, MA, USA). Antibodies Guinea pig polyclonal antibodies to RASSF1A were prepared with a GST fusion protein containing an N-terminal fragment of human RASSF1A (residues 1–119) as the antigen. Rabbit polyclonal antibodies specific for Ser203-phosphorylated RASSF1A were generated with a synthetic peptide containing phospho-Ser203 (CSVRRRTpSFYLPKD). Specific antibodies were affinity purified with the appropriate antigen. Other antibodies included HA (Roche Applied Science, Indianapolis, IN, USA); Flag or b-actin (Sigma); p27 (BD Biosciences, San Jose, CA, USA); p21, cyclin A, cyclin B, cyclin D, cyclin E, Skp2, Myc, GST or Cdk4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); Skp2 (Zymed, Carlsbad, CA, USA) and p-pRb (S780, Cell Signaling Technology Inc.). RNA interference The sequences of siRNAs for Skp2 and RASSF1A were previously described (Bashir et al., 2004; Song et al., 2004). The DNA sequences for these siRNAs were inserted into the plasmid pSUPER (Oligoengine, Seattle, WA, USA) and introduced into cells by transfection with the use of the Effectene transfection reagent (Qiagen). The plasmid pSHAG1–Cdk4 or –Cdk2 RNAi for a Cdk4 or Cdk2 siRNA was kindly provided by F Liu. Cell cycle analysis Analysis of DNA content during cell cycle progression was performed as described (Oh et al., 2006). Cell cycle

distributions were determined by a FACSCalibur flow cytometer (Becton Dickinson and Co., Franklin Lakes, NJ, USA) and analysed with MODFIT II software (Verity Software House Inc., Topsham, ME, USA). Immunofluorescence was also performed as described (Song et al., 2004). Immunoblot analysis, immunoprecipitation, in vitro binding assays and kinase assays Immunoblot analysis and immunoprecipitation were performed as described (Song et al., 2004). For in vitro binding assays, in vitro translated 35S-Skp2 was incubated with beadimmobilized RASSF1A peptides in binding buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 0.1% Triton X-100, phosphatase/protease inhibitors). The beads were washed four times with binding buffer and bound 35S-Skp2 was detected by SDS–PAGE and autoradiography. In vitro kinase assays were performed as described (Oh et al., 2006). In vitro ubiquitination assays To examine the ability of Skp2 to ubiquitinate RASSF1A in vitro, SCFSkp2 immunopurified from a mixture of in vitro translated Cul1, Rbx1, Skp1 and Skp2 was incubated for 1 h at 30 1C with ubiquitin (1.25 mg ml1; Sigma), rabbit E1 (EMD Biosciences), Ubc5a (EMD Biosciences), an ATP-regenerating system, HeLa cell extract and in vitro translated 35S-RASSF1A. In vitro ubiquitination assays using rabbit reticulocyte lysate were performed as described (Carrano et al., 1999). In vivo ubiquitination assays Cells transfected with a plasmid encoding His-tagged human ubiquitin and Flag-RASSF1A were subjected to thymidine block (2 mM) for 26 h or asynchronized. Cells were incubated with MG132 (20 mM) for 6 h before harvest and then analysed for in vivo ubiquitination. Cells were lysed by incubation for 10 min at 95 1C with two volumes of tris-buffered saline TBS (10 mM Tris–HCl (pH 7.5); 150 mM NaCl) containing 2% SDS. After the addition of eight volumes of 1% Triton X-100 in TBS, the lysates were sonicated for 2 min and then incubated with protein G agarose. The beads were removed by centrifugation, and the lysates were then subjected to immunoprecipitation with anti-Flag coupled to protein G agarose. The beads were washed first with 0.5 M LiCl in TBS and then twice with TBS, boiled and subjected to immunoblot analysis with anti-ubiquitin. Cell lysates were subjected to immunoblotting with anti-Flag. Acknowledgements We are grateful to M Pagano, JW Harper, JB Yoon, HW Lee, F Liu for DNAs and to G Pfeifer for RASSF1A wild-type and null MEFs. This study was supported by a grant from the Korea Research Foundation and the 21st Century Frontier Functional Human Genome Project of KISTEP (Ministry of Science and Technology of Korea), and the National Research Laboratory Program of Korea.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

Oncogene