TY - JOUR
AU - Chen,, Yan
AB - Abstract CDK4 is crucial for G1-to-S transition of cell cycle. It is well established that ubiquitin-mediated degradations of CDK inhibitors and cyclins are pivotal for the timely and unidirectional progression of cell cycle. However, how CDK4 itself is modulated by ubiquitin-mediated degradation has been elusive. Here we report that the steady-state level of CDK4 is controlled by PAQR4, a member of the progestin and adipoQ receptor family, and SKP2, an E3 ubiquitin ligase. Knockdown of PAQR4 leads to reduction of cell proliferation, accompanied by reduced protein level of CDK4. PAQR4 reduces polyubiquitination and degradation of CDK4. PAQR4 interacts with the C-terminal lobe of CDK4. On the other hand, SKP2 also interacts with the C-terminal lobe of CDK4 and enhances polyubiquitination and degradation of CDK4. Importantly, PAQR4 and SKP2 bind to the same region in CDK4, and PAQR4 competes with SKP2 for the binding, thereby abrogating SKP2-mediated ubiquitination of CDK4. Using a two-stage DMBA/TPA-induced skin cancer model, we find that PAQR4-deleted mice are resistant to chemical carcinogen-induced tumor formation. Collectively, our findings reveal that the steady-state level of CDK4 is controlled by the antagonistic actions between PAQR4 and SKP2, contributing to modulation of cell proliferation and tumorigenesis. CDK4, PAQR4, SKP2, ubiquitination, protein degradation, tumorigenesis Introduction An important feature of the cell cycle regulatory program is that the key cell cycle proteins are present and active during specific stages but are later removed or inhibited to maintain timely progression (Teixeira and Reed, 2013). The ubiquitin–proteasome system has emerged as an important mechanism to target cell cycle proteins for degradation at critical time points during cell division. Two key E3 ubiquitin ligase complexes that target cell cycle proteins are the Skp1–Cul1–F-box (SCF) complex and the anaphase-promoting complex/cyclosome (APC/C) (Harper et al., 2002; Vodermaier, 2004). Although the SCF and APC/C complexes are both important for temporally controlled degradation of key cell cycle proteins and evolutionarily related, they have distinct functions. The SCF complex mainly controls G1-to-S and G2-to-M transitions, whereas the APC/C complex functions primarily at mitosis and during the entry into the G1 phase (Harper et al., 2002). Cyclin-dependent kinase 4 (CDK4) is an essential protein important for normal cell proliferation as a G1 serine/threonine kinase (Matsushime et al., 1992; Rane et al., 1999). CDK4 activation requires binding to the regulatory cyclin subunits cyclins D1, D2 or D3. The CDK4/cyclin D complexes phosphorylate and inactivate a tumor suppressor protein retinoblastoma (RB), leading to release of E2F transcription factors that regulate expression of genes necessary for entry and progression through the S phase (Sherr and Roberts, 2004). It has long been thought that CDK4 functions as an oncogene (Zou et al., 2002; de Marval et al., 2004; Yu et al., 2006). Some types of tumors such as liposarcoma and B-cell lymphoma are present with amplified CDK4 or cyclin D1 (Korz et al., 2002; Italiano et al., 2009). Melanoma cells are also highly reliant on CDK4, or the related kinase CDK6 (Anders et al., 2011). In addition, the CDK4/6 inhibitor could induce cell cycle arrest and significantly inhibit cell proliferation both in vitro and in vivo (Rivadeneira et al., 2010; Barton et al., 2013). Moreover, it was found that CDK4 and cyclin D1 are required for breast cancer tumorigenesis (Yu et al., 2001, 2006; Reddy et al., 2010). Intriguingly, little is known about the E3 ubiquitin ligase that regulates the degradation of CDK4. There is only a piece of evidence indicating that C/EBP could reduce the protein level of CDK4 by disrupting the CDK4–CDC37–HSP90 chaperone complex and enhancing the formation of CDK4-ubiquitin conjugates (Wang et al., 2002). It is known that the SCF family of E3 ubiquitin ligases promotes ubiquitination of phosphorylated substrates and typically targets mediators for G1-to-S phase transition (Skowyra et al., 1997). The F-box proteins within the SCF family are divided into three classes: Fbws containing WD-40 domains, Fbls containing leucine-rich repeats, and Fbxs containing either different protein–protein interaction modules or no recognizable motifs. Cyclin D1 is degraded by FBX4 and FBXO31 and both of them are in the Fbxs subfamily. SKP2, a member of the Fbls subfamily, mainly directs degradation of p27Kip1, a key inhibitor of G1 CDKs (Frescas and Pagano, 2008). Many other cell cycle regulators are also substrates of SKP2, such as p21Cip1, p57Kip2, E2F-1, p130/RB2, cyclin E, and c-Myc (Frescas and Pagano, 2008), although the physiological significance of these regulations remains to be determined. On the other hand, FBW7 and β-Trcp/BTRC within the Fbws family are reported to regulate degradation of other cell cycle proteins (Teixeira and Reed, 2013). The PAQR (progestin and adipoQ receptor) family was first identified in 2005, defined by an ancient seven transmembrane pass motif and consists of 11 members (PAQR1 to PAQR11) in the human genome (Tang et al., 2005). Although the functions of the members of PAQR family have begun to be recognized such as their regulations on metabolism and oncogenesis (Yamauchi et al., 2003; Zhu et al., 2003; Tang et al., 2005; Feng et al., 2007; Jin et al., 2012), the function of PAQR4 is unknown. In this study, we identified that PAQR4 has an essential role in cell proliferation. Furthermore, we found that PAQR4 is able to regulate ubiquitination and degradation of CDK4. We also identified that SKP2 functions as an E3 ubiquitin ligase of CDK4 and that PAQR4 prevents CDK4 from SKP2-mediated ubiquitination. At the animal level, PAQR4-deleted mice are resistant to chemical carcinogen-induced tumor formation. Therefore, our findings not only uncover a biological function of PAQR4, but also unravel that the steady-state level of CDK4 is controlled by PAQR4 and SKP2 and related to tumorigenesis. Results PAQR4 regulates cell proliferation Our previous studies have revealed that PAQR3 functions as a tumor suppressor both in vitro and in vivo (Fan et al., 2008; Xie et al., 2008; Jiang et al., 2011; Ling et al., 2014). To explore the potential function of PAQR4, a close member of PAQR3 (Tang et al., 2005), we investigated whether PAQR4 also played a role in cell proliferation. We analyzed the effect of PAQR4 knockdown in NIH3T3 cells using PAQR4-specific shRNAs. Two PAQR4 shRNAs could significantly reduce the mRNA level of PAQR4 in these cells as analyzed by real-time RT-PCR (Figure 1A). PAQR4 knockdown could inhibit the cell proliferation rate in NIH3T3 cells (Figure 1B). Then we used a colony formation assay to further analyze the effect of PAQR4 on cell proliferation. Silencing of PAQR4 by two shRNAs could significantly reduce the cell growth in this assay (Figure 1C). Expression of a shRNA-resistant PAQR4 could partially abrogate the inhibitory effect of PAQR4-specific shRNA on cell proliferation (Supplementary Figure S1). In RKO colon cancer cells, overexpression of PAQR4 could also enhance cell proliferation by both MTT and colony formation assays (Supplementary Figure S2). Then we used FACS assay to analyze if PAQR4 had an effect on cell cycle progression. NIH3T3 cells were arrested in G0/G1 phase by serum starvation for 48 h and then released by addition of 10% bovine calf serum. Knockdown of PAQR4 caused slight cell cycle delay in G1-to-S phase transition, shown as reduced cell numbers in the S phase after release from serum starvation for 14–16 h (Supplementary Figure S3). Consistently, FACS analysis using unsynchronized NIH3T3 cells revealed that the percentage of cells in G0/G1 phase was significantly elevated by PAQR4 knockdown, while the percentage of cells in S phase was significantly reduced by PAQR4 knockdown (Supplementary Figure S4), further indicating that PAQR4 downregulation is associated with a delay in G1-to-S transition. Figure 1 Open in new tabDownload slide PAQR4 positively regulates cell proliferation. (A) The result of RT-PCR to determine the effect of knocking down endogenous PAQR4. NIH3T3 cells were infected with the control lentivirus (mock) and the lentivirus containing PAQR4-specific shRNA. The mRNA was isolated and used in real-time RT-PCR to determine the mRNA level of PAQR4. The data are shown as mean ± SD. ** indicates P < 0.01 compared to the mock. (B) MTT assay was used to analyze the cell proliferation rate in NIH3T3 cells as in A. Three independent experiments were performed with similar results. The data are shown as mean ± SD. * and *** indicate P < 0.05 and P < 0.001 between mock and shPAQR4-1. ^^ and ^^^ indicate P < 0.01 and P < 0.001 between mock and shPAQR4-2. (C) Colony formation is reduced by PAQR4 knockdown. The cells as in A were allowed to grow for 7 days before staining. The data are shown as mean ± SD. ** indicates P < 0.01 compared to the mock. (D–F) The protein level but not the mRNA level of CDK4 is decreased by PAQR4 knockdown. Unsynchronized NIH3T3 cells were infected with either control or PAQR4 shRNA lentivirus, followed by immunoblotting (D) or RT-PCR (E). Synchronized NIH3T3 cells were also used in immunoblotting (F). For synchronization, the cells were treated by serum deprivation (DMEM with 0.1% BCS) for 48 h, and then stimulated with 10% BCS to induce synchronous cell cycle entry for different hours as indicated. Figure 1 Open in new tabDownload slide PAQR4 positively regulates cell proliferation. (A) The result of RT-PCR to determine the effect of knocking down endogenous PAQR4. NIH3T3 cells were infected with the control lentivirus (mock) and the lentivirus containing PAQR4-specific shRNA. The mRNA was isolated and used in real-time RT-PCR to determine the mRNA level of PAQR4. The data are shown as mean ± SD. ** indicates P < 0.01 compared to the mock. (B) MTT assay was used to analyze the cell proliferation rate in NIH3T3 cells as in A. Three independent experiments were performed with similar results. The data are shown as mean ± SD. * and *** indicate P < 0.05 and P < 0.001 between mock and shPAQR4-1. ^^ and ^^^ indicate P < 0.01 and P < 0.001 between mock and shPAQR4-2. (C) Colony formation is reduced by PAQR4 knockdown. The cells as in A were allowed to grow for 7 days before staining. The data are shown as mean ± SD. ** indicates P < 0.01 compared to the mock. (D–F) The protein level but not the mRNA level of CDK4 is decreased by PAQR4 knockdown. Unsynchronized NIH3T3 cells were infected with either control or PAQR4 shRNA lentivirus, followed by immunoblotting (D) or RT-PCR (E). Synchronized NIH3T3 cells were also used in immunoblotting (F). For synchronization, the cells were treated by serum deprivation (DMEM with 0.1% BCS) for 48 h, and then stimulated with 10% BCS to induce synchronous cell cycle entry for different hours as indicated. We next investigated a series of CDKs and cyclins required for G1-to-S phase transition. We found that knockdown of PAQR4 had no effect on the protein levels of CDK2, cyclin A, cyclin D1 and cyclin E (Figure 1D). However, silencing of PAQR4 could reduce the protein level of CDK4 (Figure 1D). In addition, knockdown of PAQR4 could not decrease the mRNA level of CDK4 (Figure 1E), indicating that only CDK4 protein is regulated by PAQR4. We next investigated the expression of these CDKs and cyclins using synchronized NIH3T3 cells. As expected, the CDKs and cyclins required for G1-to-S phase transition began to accumulate after release from G0 block (Figure 1F). We found that the accumulation of CDK4, but not CDK2, cyclin A, and cyclin D1 was slowed down by PAQR4 knockdown (Figure 1F). Consistently, knockdown of CDK4 was also able to inhibit the proliferation rate in NIH3T3 cells (Supplementary Figure S5). Furthermore, simultaneous knockdown of PAQR4 and CDK4 had a synergistic effect to inhibit cell proliferation in these cells (Supplementary Figure S6). To rule out the possibility that PAQR4 might alter cell proliferation via changes of apoptosis, we analyzed the effect of PAQR4 knockdown on cleavage of poly(ADP-ribose) polymerase (PARP), a marker of apoptosis. We found that PARP cleavage was not affected by PAQR4 knockdown in NIH3T3 cells (Supplementary Figure S7). These results, collectively, suggested that PAQR4 has an effect on cell proliferation in association with alteration of CDK4 protein. PAQR4 reduces ubiquitination and degradation of CDK4 The observed alteration of CDK4 protein by PAQR4 knockdown led us to explore the possibility that ubiquitin-mediated degradation of the protein is modulated by PAQR4, as most cell cycle-related proteins are controlled by ubiquitination. We analyzed whether ubiquitination of G1 cyclins and CDKs was affected by PAQR4. In HEK293T cells, polyubiquitination of CDK2, cyclin D1 and cyclin E was not apparently influenced by PAQR4 overexpression (Figure 2A). However, polyubiquitination of CDK4 was markedly reduced by PAQR4 overexpression (Figure 2A). These data, therefore, provided a clue that PAQR4 is likely involved in regulating ubiquitination of CDK4. Figure 2 Open in new tabDownload slide PAQR4 reduces ubiquitination and degradation of CDK4. (A) PAQR4 reduces polyubiquitination of CDK4 but not CDK2, cyclin E, or cyclin D1. HEK293T cells were transfected with the plasmids as indicated. MG132 (10 μM) was added for 6 h before the cells were harvested. The cell lysates were used in immunoprecipitation (IP) and immunoblotting (IB) with the antibodies as indicated. (B and C) PAQR4 negatively regulates CDK4 polyubiquitination in the presence of MG132. HEK293T cells were overexpressed with PAQR4 (B) or infected with the shRNA-containing lentivirus (C) as indicated, and then treated with or without MG132 (10 μM) for 6 h before IP and IB. (D–F) PAQR4 negatively regulates degradation of CDK4. HEK293T cells were transfected with the plasmids (D and E) or infected with the shRNA-containing lentivirus (F) as indicated. At 24 h after the transfection, the cells were treated with 100 μg/ml CHX for various times and then harvested for IB. The IB results (repeated at least three times) were quantified and shown in the lower panels. The data are shown as mean ± SD. For D, * indicates P < 0.05 between the two groups. For F, * and ** indicate P < 0.05 and P < 0.01 between mock and shPAQR4-1; ^, ^^, and ^^^ indicate P < 0.05, P < 0.01, and P < 0.001 between mock and shPAQR4-2. Figure 2 Open in new tabDownload slide PAQR4 reduces ubiquitination and degradation of CDK4. (A) PAQR4 reduces polyubiquitination of CDK4 but not CDK2, cyclin E, or cyclin D1. HEK293T cells were transfected with the plasmids as indicated. MG132 (10 μM) was added for 6 h before the cells were harvested. The cell lysates were used in immunoprecipitation (IP) and immunoblotting (IB) with the antibodies as indicated. (B and C) PAQR4 negatively regulates CDK4 polyubiquitination in the presence of MG132. HEK293T cells were overexpressed with PAQR4 (B) or infected with the shRNA-containing lentivirus (C) as indicated, and then treated with or without MG132 (10 μM) for 6 h before IP and IB. (D–F) PAQR4 negatively regulates degradation of CDK4. HEK293T cells were transfected with the plasmids (D and E) or infected with the shRNA-containing lentivirus (F) as indicated. At 24 h after the transfection, the cells were treated with 100 μg/ml CHX for various times and then harvested for IB. The IB results (repeated at least three times) were quantified and shown in the lower panels. The data are shown as mean ± SD. For D, * indicates P < 0.05 between the two groups. For F, * and ** indicate P < 0.05 and P < 0.01 between mock and shPAQR4-1; ^, ^^, and ^^^ indicate P < 0.05, P < 0.01, and P < 0.001 between mock and shPAQR4-2. We further analyzed the effect of PAQR4 on CDK4 ubiquitination in the presence and absence of MG132, a proteasome inhibitor. Addition of MG132 apparently enhanced ubiquitination of CDK4 (Figure 2B), supporting that CDK4 is degraded via the ubiquitin-mediated degradation in proteasome. Consistently, overexpression of PAQR4 could abrogate polyubiquitination of CDK4 (Figure 2B). On the other hand, knockdown of PAQR4 by two PAQR4-specific shRNAs greatly accelerated polyubiquitination of CDK4 in the presence of MG132 (Figure 2C). We also investigated the protein degradation rate of CDK4. Overexpression of PAQR4 could drastically slow down protein degradation of CDK4 (Figure 2D). As a control, the degradation rate of cyclin D1 was not affected by PAQR4 overexpression (Figure 2E). In contrast, silencing of PAQR4 accelerated CDK4 degradation (Figure 2F). We also analyzed the potential effect of PAQR4 on ubiquitination of CDK6, a close family member of CDK4. Overexpression of PAQR4 could not alter the polyubiquitination level of endogenous CDK6 protein (Supplementary Figure S8). These data, collectively, indicated that PAQR4 is able to modulate ubiquitination and degradation of CDK4 protein. PAQR4 interacts with CDK4 As PAQR4 is implicated in the ubiquitination of CDK4, we next asked whether these two proteins could interact with each other. By a co-immunoprecipitation assay, precipitation of CDK4 could pull down PAQR4 (Figure 3A). Likewise, immunoprecipitation of PAQR4 could pull down CDK4 (Figure 3B). Immunoprecipitation with endogenous CDK4 could also pull down ectopically expressed PAQR4 (Figure 3C). However, PAQR3, a close family member of PAQR4, could not interact with CDK4 (Supplementary Figure S9). Next, we investigated the domain(s) of CDK4 and PAQR4 involved in such interaction. CDK4 contained two structural domains, the N-terminal lobe and the C-terminal lobe (Day et al., 2009). We constructed two constructs of CDK4 that contained these two domains respectively, i.e. amino acid (aa) residues 1–96 for the N-terminal lobe and 97–303 for the C-terminal lobe. We found that PAQR4 efficiently bound the full-length CDK4 and the 97–303aa region (Figure 3D). We also analyzed which domain of PAQR4 is involved in its interaction with CDK4. For PAQR4 is a membrane protein defined by an ancient 7-transmembrane pass motif (Tang et al., 2005), we cloned several possible non-transmembrane helices including amino acid residues 1–55, 95–123, 165–188, and 220–248. We demonstrated that the third non-transmembrane domain of PAQR4 (95–123aa) was sufficient to interact with CDK4 (Figure 3E). Collectively, these data revealed that the C-terminal lobe of CDK4 is able to specifically interact with the third non-transmembrane domain of PAQR4 (Figure 3F). Figure 3 Open in new tabDownload slide Interaction of PAQR4 with CDK4. (A–C) Interaction of CDK4 with PAQR4. HEK293T cells were transfected with the plasmids as indicated. At 24 h post-transfection, the cell lysates were used in immunoprecipitation (IP) and immunoblotting (IB) using the antibodies as indicated. (D and E) Identification of the domains of CDK4 and PAQR4 involved in their interaction. Different deletion mutants of CDK4 (D) and PAQR4 (E) were overexpressed in HEK293T cells as indicated. The cell lysate was used in IP and IB. (F) A diagram depicting the domains of CDK4 and PAQR4 involved in the interaction. The interaction regions are marked with green colors. Figure 3 Open in new tabDownload slide Interaction of PAQR4 with CDK4. (A–C) Interaction of CDK4 with PAQR4. HEK293T cells were transfected with the plasmids as indicated. At 24 h post-transfection, the cell lysates were used in immunoprecipitation (IP) and immunoblotting (IB) using the antibodies as indicated. (D and E) Identification of the domains of CDK4 and PAQR4 involved in their interaction. Different deletion mutants of CDK4 (D) and PAQR4 (E) were overexpressed in HEK293T cells as indicated. The cell lysate was used in IP and IB. (F) A diagram depicting the domains of CDK4 and PAQR4 involved in the interaction. The interaction regions are marked with green colors. SKP2 interacts with CDK4 and regulates CDK4 ubiquitination/degradation To further investigate the molecular mechanism underlying the regulation of CDK4 ubiquitination by PAQR4, we attempted to identify the E3 ubiquitin ligase of CDK4. We analyzed an array of reported E3 ubiquitin ligases involved in degradation of cell cycle-related proteins. The SCF and APC/C complexes have been proposed to be the major players regulating ubiquitination of cell cycle-related proteins (Vodermaier, 2004; Teixeira and Reed, 2013). In brief, among these E3 ligases, Cul3 targets cyclin E (Singer et al., 1999); FBXW7 also targets cyclin E (Koepp et al., 2001); BTRC (β-Trcp) targets Cdc25A, Emi1, Wee1 (Teixeira and Reed, 2013); SKP2 targets Geminin, Cdc6, p21Cip1, p27KIP1, p57Kip2, p130/RB2, Cdt1, and Orc1 (Teixeira and Reed, 2013); FBXO4 (FBX4) targets cyclin D1 (Lin et al., 2006); CDC20 targets cyclin A/B, Securin and Aurora (Teixeira and Reed, 2013); DTL (CDT2) targets p21Cip1 (Hall et al., 2014); and RNF114 (ZNF313) targets p21Cip1 (Han et al., 2013). Using a co-immunoprecipitation assay, we found that CDK4 could interact with SKP2 and FBXO4 among the known E3 ubiquitin ligases that regulate cell cycle proteins (Figure 4A). To analyze whether SKP2 and FBXO4 regulate the degradation of CDK4, we measured whether the half-life of CDK4 was altered by overexpression of either SKP2 or FBXO4. While the half-life of endogenous CDK4 was not altered by FBXO4 overexpression, it was greatly shortened upon SKP2 overexpression (Figure 4B). Based on these results, we hypothesized that CDK4 might also be a substrate of SKP2. To address this hypothesis, we investigated the interaction between CDK4 and SKP2. By a co-immunoprecipitation assay, CDK4 was able to bind SKP2 (Figure 4C). To investigate the domain(s) of CDK4 implicated with its interaction with SKP2, we co-expressed SKP2 with the CDK4 mutants and discovered that the C-terminal lobe of CDK4 was mainly responsible for its interaction with SKP2 (Figure 4D), which was also required for its interaction with PAQR4 (Figure 3D). Figure 4 Open in new tabDownload slide SKP2 interacts with CDK4 and regulates CDK4 degradation. (A) Possible interaction of CDK4 with an array of reported E3 ubiquitin ligases that target cell cycle-related proteins. HEK293T cells were transfected with the plasmids as indicated. At 24 h post-transfection, the cell lysates were used in immunoprecipitation (IP) and immunoblotting (IB) using the antibodies as indicated. (B) SKP2 but not FBXO4 affects the degradation of CDK4. HEK293T cells were transfected with the plasmids as indicated. At 24 h after transfection, the cells were treated with CHX (100 μg/ml) for various times and then harvested for IB. The IB results (repeated at least three times) were quantified and shown in the lower panels. The data are shown as mean ± SD. * and ** indicate P < 0.05 and P < 0.01. (C) Interaction of CDK4 with SKP2. HEK293T cells were transfected with the plasmids as indicated. At 24 h post-transfection, the cell lysates were used in IP and IB. The interaction of CDK4 with PAQR4 was used as a positive control (last lane). (D) Identification of the domains of CDK4 involved in its interaction SKP2. Different deletion mutants of CDK4 and full-length SKP2 were expressed in HEK293T cells, followed by IP and IB. (E) SKP2 enhances CDK4 ubiquitination. HEK293T cells were transfected with the plasmids as indicated and then treated with or without MG132 (10 μM) for 6 h. The cell lysates were used in IP and IB. (F–H) SKP2 knockdown delays degradation of CDK4. HEK293T cells were infected with lentivirus containing control or SKP2 shRNAs. The knockdown efficiency was determined by RT-PCR using mRNA isolated from these cells (F). Immunoblotting was also used to analyze SKP2 protein (G). The cells were treated with CHX for various times and harvested for IB (H). (I) SKP2 knockdown reduces ubiquitination of CDK4. HEK293T cells infected with lentivirus containing control shRNA or SKP2 shRNAs were transfected with plasmids as indicated. The cell lysate was used in IP and IB. Figure 4 Open in new tabDownload slide SKP2 interacts with CDK4 and regulates CDK4 degradation. (A) Possible interaction of CDK4 with an array of reported E3 ubiquitin ligases that target cell cycle-related proteins. HEK293T cells were transfected with the plasmids as indicated. At 24 h post-transfection, the cell lysates were used in immunoprecipitation (IP) and immunoblotting (IB) using the antibodies as indicated. (B) SKP2 but not FBXO4 affects the degradation of CDK4. HEK293T cells were transfected with the plasmids as indicated. At 24 h after transfection, the cells were treated with CHX (100 μg/ml) for various times and then harvested for IB. The IB results (repeated at least three times) were quantified and shown in the lower panels. The data are shown as mean ± SD. * and ** indicate P < 0.05 and P < 0.01. (C) Interaction of CDK4 with SKP2. HEK293T cells were transfected with the plasmids as indicated. At 24 h post-transfection, the cell lysates were used in IP and IB. The interaction of CDK4 with PAQR4 was used as a positive control (last lane). (D) Identification of the domains of CDK4 involved in its interaction SKP2. Different deletion mutants of CDK4 and full-length SKP2 were expressed in HEK293T cells, followed by IP and IB. (E) SKP2 enhances CDK4 ubiquitination. HEK293T cells were transfected with the plasmids as indicated and then treated with or without MG132 (10 μM) for 6 h. The cell lysates were used in IP and IB. (F–H) SKP2 knockdown delays degradation of CDK4. HEK293T cells were infected with lentivirus containing control or SKP2 shRNAs. The knockdown efficiency was determined by RT-PCR using mRNA isolated from these cells (F). Immunoblotting was also used to analyze SKP2 protein (G). The cells were treated with CHX for various times and harvested for IB (H). (I) SKP2 knockdown reduces ubiquitination of CDK4. HEK293T cells infected with lentivirus containing control shRNA or SKP2 shRNAs were transfected with plasmids as indicated. The cell lysate was used in IP and IB. We next analyzed the effect of SKP2 on ubiquitination of CDK4. Overexpression of SKP2 could increase polyubiquitination of CDK4 in the presence of MG132 (Figure 4E). In contrast, knockdown of SKP2 could reduce the degradation rate of endogenous CDK4 (Figure 4F–H). Consistently, downregulation of SKP2 also decreased polyubiquitination of CDK4 in the presence of MG132 (Figure 4I). Collectively, these data indicated that proteasome-mediated degradation of CDK4 is, at least partially, mediated by the E3 ubiquitin ligase SKP2. PAQR4 prevents CDK4 from interaction with SKP2 and ubiquitination by SKP2 As we found that PAQR4 interacts with CDK4 and protects CDK4 from ubiquitin-mediated degradation (Figures 2 and 3), we hypothesized that PAQR4 might shield CDK4 from SKP2-mediated ubiquitination. To address this hypothesis, we analyzed whether PAQR4 could interact with SKP2. In co-immunoprecipitation assays, when SKP2 and PAQR4 were co-expressed, they could interact with each other (Figure 5A and B). Next, we investigated whether SKP2 also regulated ubiquitination of PAQR4 due to their interaction. Surprisingly, overexpression of SKP2 could not alter ubiquitination of PAQR4 in the presence of MG132 (Figure 5C). Figure 5 Open in new tabDownload slide PAQR4 shields CDK4 from ubiquitination by SKP2. (A and B) Interaction of PAQR4 with SKP2. HEK293T cells were transfected with the plasmids as indicated. At 24 h post-transfection, the cell lysates were used in immunoprecipitation (IP) and immunoblotting (IB) using the antibodies as indicated. (C) SKP2 does not influence PAQR4 ubiquitination. HEK293T cells were transfected with the plasmids as indicated and treated with or without MG132 (10 μM) for 6 h. The cell lysate was then used in IP and IB. (D) PAQR4 decreases the interaction of CDK4 with SKP2. HEK293T cells were transfected with the plasmids as indicated. At 24 h post-transfection, the cell lysates were used in IP and IB. (E) PAQR4 reduces SKP2-mediated CDK4 ubiquitination. HEK293T cells were transfected with the plasmids as indicated. As 24 h post-transfection, the cells were treated with or without MG132 (10 μM) for 6 h. The cell lysates were used in IP and IB. (F) PAQR4 knockdown abrogates SKP2-stimuated ubiquitination of CDK4. HEK293T cells infected with shRNA-containing lentivirus were transfected with the plasmids as indicated. Analysis of CDK4 ubiquitination was performed as in E. (G) A model depicting how PAQR4 affects the function of CDK4. PAQR4 competes with SKP2 from binding CDK4, thereby preventing SKP2-mediated ubiquitination and degradation of CDK4 and resulting in acceleration of cell progression. The pink area in the CDK4 protein indicates the region interacting with both SKP2 and PAQR4. Figure 5 Open in new tabDownload slide PAQR4 shields CDK4 from ubiquitination by SKP2. (A and B) Interaction of PAQR4 with SKP2. HEK293T cells were transfected with the plasmids as indicated. At 24 h post-transfection, the cell lysates were used in immunoprecipitation (IP) and immunoblotting (IB) using the antibodies as indicated. (C) SKP2 does not influence PAQR4 ubiquitination. HEK293T cells were transfected with the plasmids as indicated and treated with or without MG132 (10 μM) for 6 h. The cell lysate was then used in IP and IB. (D) PAQR4 decreases the interaction of CDK4 with SKP2. HEK293T cells were transfected with the plasmids as indicated. At 24 h post-transfection, the cell lysates were used in IP and IB. (E) PAQR4 reduces SKP2-mediated CDK4 ubiquitination. HEK293T cells were transfected with the plasmids as indicated. As 24 h post-transfection, the cells were treated with or without MG132 (10 μM) for 6 h. The cell lysates were used in IP and IB. (F) PAQR4 knockdown abrogates SKP2-stimuated ubiquitination of CDK4. HEK293T cells infected with shRNA-containing lentivirus were transfected with the plasmids as indicated. Analysis of CDK4 ubiquitination was performed as in E. (G) A model depicting how PAQR4 affects the function of CDK4. PAQR4 competes with SKP2 from binding CDK4, thereby preventing SKP2-mediated ubiquitination and degradation of CDK4 and resulting in acceleration of cell progression. The pink area in the CDK4 protein indicates the region interacting with both SKP2 and PAQR4. Keeping our observation in mind that the C-terminal lobe of CDK4 is required for the interaction with both PAQR4 and SKP2 (Figures 3D and 4D), we postulated that PAQR4 and SKP2 might compete with each other for their interaction with CDK4. To test this hypothesis, we analyzed whether the interaction of CDK4 with SKP2 was altered by PAQR4. In a co-immunoprecipitation assay, the interaction of CDK4 with SKP2 was dose-dependently decreased by PAQR4 overexpression (Figure 5D), suggesting that PAQR4 and SKP2 may functionally compete with each other for their interaction with CDK4. However, here we could not rule out the possibility that different amino acid residues of CDK4 at its C-terminal might be responsible for the interaction with PAQR4 and SKP2, respectively. In addition, increasing PAQR4 might result in decreasing interaction between SKP2 and CKD4 due to the interaction between PAQR4 and SKP2, so that the interface of SKP2 responsible for its CDK4 interaction is blocked by PAQR4. However, we found that PAQR4 could not affect the CDK4-SKP2 interaction (Supplementary Figure S10), thus excluding this possibility. We next analyzed the effect of PAQR4 on SKP2-mediated ubiquitination of CDK4. Consistent with our previous observation (Figure 4E), overexpression of SKP2 greatly enhanced polyubiquitination of CDK4 (Figure 5E, the second lane). However, such enhancement by SKP2 was abrogated by PAQR4 overexpression in a dose-dependent manner (Figure 5E). Furthermore, the reduction of CDK4 polyubiquitination by SKP2 knockdown was partially abrogated by PAQR4 knockdown (Figure 5F). Collectively, these results indicated PAQR4 stabilizes CDK4 via protecting CDK4 from SKP2-mediated ubiquitination and degradation (Figure 5G). Paqr4 deletion suppresses chemical carcinogen-induced skin carcinogenesis in mouse To explore the potential function of PAQR4 in vivo, we constructed a global Paqr4 knockout mouse through replacing the second and third exons of the mouse Paqr4 gene by a neomycin-selectable cassette (Figure 6A). Deletion of Paqr4 was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) using total RNA isolated from the skin, liver, and lung (Figure 6B). Next, we analyzed the potential role of PAQR4 in skin carcinogenesis. Skin tumors were induced in wild type and Paqr4-deficient mice by topical treatment with a single dose of 7,12-dimethylbenz(a)anthracene (DMBA) followed by repeated administrations of phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) for 27 weeks (Abel et al., 2009). It is known that animals subjected to this experimental protocol evolve through different stages of cancer progression, including the ‘initiation’, ‘promotion’, and ‘progression’ stage (Abel et al., 2009). Interestingly, Paqr4-deleted mice developed significantly less and smaller papillomas per animal than their wild type littermates (Figure 6C and D), with the mean tumor number 7.8 in wild type mice vs. 4.0 in Paqr4-deleted mice (Figure 6E). In addition, the overall size of papillomas was smaller in the Paqr4-deleted mice than the wild type controls (Figure 6F). Histopathological analysis also revealed that the papillomas in the Paqr4 knockout mice were less deformed than the controls (Figure 6G). We analyzed the CDK4 protein level with the skin samples from wild type and Paqr4-deleted mice. Deletion of Paqr4 led to an apparent reduction of CDK4 protein level (Figure 6H). In addition, we analyzed Ki67 staining with the tumor samples. We found that the intensity of Ki67 staining was slightly reduced by Paqr4 deletion (Supplementary Figure S11), further indicative of the modulatory role of PAQR4 on cell proliferation. These data, collectively, indicated that deletion of Paqr4 could significantly suppress chemical carcinogen-induced skin carcinogenesis in vivo. In other word, PAQR4 appeared to possess an oncogenic activity as deletion of the gene reduced tumor formation. Figure 6 Open in new tabDownload slide PAQR4 deletion suppresses chemical carcinogen-induced skin carcinogenesis in mouse. (A) A diagram depicting the Paqr4 knockout construct. The second and third exons of the mouse Paqr4 gene were replaced by a neomycin-selectable cassette by genetic recombination. (B) Confirmation of Paqr4 deletion in the knockout mice. The mRNA level of PAQR4 in the skin, liver and lung of wild type and Paqr4-deleted mice was analyzed by RT-PCT using total RNA isolated from the mouse tissues. A representative result is shown here with two wild type animals and two knockout mouse lines (#194 and #200). β-actin was used as a loading control. (C) Wild type and Paqr4-deleted mice were topically treated with a single dose of DMBA followed by repeated applications of TPA for 27 weeks. Representative images of wild type (left panel) and Paqr4-deleted mice (right panel) bearing papillomas are shown. (D) The numbers of mice having carcinoma and different sizes of papilloma at the time of sacrifice (n = 16 for wild type and n = 14 for Paqr4 knockout mice). (E) Tumor multiplicity, expressed as number of papilloma and carcinoma per mouse, is shown at various time points upon carcinogen treatment. (F) Papilloma size distribution of the mice was classified according to their diameter size in millimeters. The average number of carcinomas and papilloma belonging to each size classification per mouse was shown at the time of sacrifice. (G) Representative H&E staining of normal skin and skins with papilloma/carcinoma in wild type mice (left panel) and Paqr4 knockout mice (right panel). (H) CDK4 protein level is reduced by PAQR4 deletion. The skin samples from wild type and Paqr4–/– mice were analyzed by immunoblotting. The data are shown as mean ± SD. * and ** indicate P < 0.05 and P < 0.01. Figure 6 Open in new tabDownload slide PAQR4 deletion suppresses chemical carcinogen-induced skin carcinogenesis in mouse. (A) A diagram depicting the Paqr4 knockout construct. The second and third exons of the mouse Paqr4 gene were replaced by a neomycin-selectable cassette by genetic recombination. (B) Confirmation of Paqr4 deletion in the knockout mice. The mRNA level of PAQR4 in the skin, liver and lung of wild type and Paqr4-deleted mice was analyzed by RT-PCT using total RNA isolated from the mouse tissues. A representative result is shown here with two wild type animals and two knockout mouse lines (#194 and #200). β-actin was used as a loading control. (C) Wild type and Paqr4-deleted mice were topically treated with a single dose of DMBA followed by repeated applications of TPA for 27 weeks. Representative images of wild type (left panel) and Paqr4-deleted mice (right panel) bearing papillomas are shown. (D) The numbers of mice having carcinoma and different sizes of papilloma at the time of sacrifice (n = 16 for wild type and n = 14 for Paqr4 knockout mice). (E) Tumor multiplicity, expressed as number of papilloma and carcinoma per mouse, is shown at various time points upon carcinogen treatment. (F) Papilloma size distribution of the mice was classified according to their diameter size in millimeters. The average number of carcinomas and papilloma belonging to each size classification per mouse was shown at the time of sacrifice. (G) Representative H&E staining of normal skin and skins with papilloma/carcinoma in wild type mice (left panel) and Paqr4 knockout mice (right panel). (H) CDK4 protein level is reduced by PAQR4 deletion. The skin samples from wild type and Paqr4–/– mice were analyzed by immunoblotting. The data are shown as mean ± SD. * and ** indicate P < 0.05 and P < 0.01. Discussion Ubiquitin-mediated degradation is crucial to control CDKs and cyclins required for cell cycle progression. Interestingly, how CDK4 itself is regulated by ubiquitin-mediated degradation has been elusive in the past. CDK4 is of uttermost importance for G1-to-S transition. The correct fluctuations in CDK activity during cell cycle are essential for the precise timing in the process (Morgan, 1997; Inze and De Veylder, 2006). The activity of CDK is negatively regulated by a group of CDK inhibitor (CKIs). However, previous studies were mainly focused on the timely degradation of CKIs to potentiate G1-to-S transition (Teixeira and Reed, 2013). On the other hand, the protein level of CDK4 is considered to be relatively constant during the cell cycle. Our study reveals that CDK4 itself is degraded by SKP2-mediated ubiquitination in a PAQR4-dependent manner. What is the functional significance? We propose that PAQR4 is able to control the steady-state level of CDK4 via its regulation on SKP2-mediated ubiquitination of CDK4. In this way, PAQR4 may set up a tone for the maximal level of CKD4 kinase activity. In cells with high expression level of PAQR4 such as in cancer cells, the steady-state CDK4 level would be elevated due to reduced degradation, leading to an overall high activity of CDK4 that would facilitate G1-to-S transition. Therefore, it will be of great importance to investigate whether PAQR4 possess an oncogenic activity in certain types of human cancers in the future. One of the intriguing discoveries in this study is that SKP2 can also target a protein that promotes cell cycle transition. The SCF family of E3 ubiquitin ligases promotes ubiquitination of phosphorylated substrates and typically targets mediators of the G1-to-S phase transition (Skowyra et al., 1997). SKP2 acts by using its F-box to recruit substrates for polyubiquitination by the SCF E3 ligase, leading to proteasome-mediated degradation of substrate proteins (Frescas and Pagano, 2008). SKP2 promotes cell proliferation mainly by directing the degradation of p27Kip1, a key CKI for G1 phase (Carrano et al., 1999; Sutterluty et al., 1999; Tsvetkov et al., 1999; Frescas and Pagano, 2008). Due to the importance of SKP2 in the degradation of p27Kip1, SKP2 has been proposed as a promising target for tumor therapy as inhibition of its function would lead to upregulation of p27Kip1 and cell cycle arrest (Chan et al., 2014). However, considering that SKP2 also targets CDK4, inhibition of SKP2 function could lead to upregulation of CDK4 and result in a faster G1-to-S phase transition. Keeping this idea in mind, certain caution should be casted on the beneficial effect of SKP2 inhibitor for cancer therapy. It is noteworthy that CDK4 is not vital for all type of cells in early development. The CDK4-deleted mice are viable with a major phenotype of insulin-deficient diabetes caused reduced number of pancreatic β-cells (Rane et al., 1999; Tsutsui et al., 1999). However, CDK4 is required for the oncogenic transformation and development of certain type of cancers (Zou et al., 2002; de Marval et al., 2004; Yu et al., 2006). Some types of tumors such as liposarcoma and B-cell lymphoma are present with amplified CDK4 or cyclin D1 (Korz et al., 2002; Italiano et al., 2009). Melanoma cells are also highly reliant on CDK4, or the related kinase CDK6 (Anders et al., 2011). CDK4/6 inhibitor could induce cell cycle arrest and significantly inhibit cell proliferation both in vitro and in vivo (Rivadeneira et al., 2010; Barton et al., 2013). In addition, CDK4 and cyclin D1 are required for breast cancer tumorigenesis (Yu et al., 2001, 2006; Reddy et al., 2010). In our study, we found that PAQR4 has an oncogenic effect as deletion of the gene reduces chemical carcinogen-induced skin tumor formation. This result is consistent with the findings that CDK4 deletion is able to inhibit skin tumor development in the mouse (Rodriguez-Puebla et al., 2002); and CDK4 overexpression can enhance skin tumorigenesis (Miliani de Marval et al., 2004). Due to the importance of PAQR4 in the regulation of the steady-state level of CDK4, we postulate that PAQR4 may possess an oncogenic function in human cancers. In the future, this important issue can be addressed by analyzing the association of altered PAQR4 expression with development of human cancers. In addition, it is noteworthy that this study is an initial report about the function of PAQR4 and it is likely that PAQR4 has other biological activities or physiological functions that await to be discovered in the future. In particular, we cannot rule out the possibility that PAQR4 may affect other cellular functions. Considering that knockdown of both PAQR4 and CDK4 had a synergistic effect on cell proliferation (Supplementary Figure S6), we cannot rule out the possibility that PAQR4 may regulate cell proliferation through other mechanisms. Nevertheless, the initial characterization of PAQR4 as reported here would herald the biological importance of this previously unknown protein. Materials and methods RNA isolation, RT-PCR, and real-time PCR The cells were lysed in TRIzol reagent (Invitrogen). Total RNA was purified according to the manufacturer’s instructions, reverse transcribed, and synthesized to complementary DNA using FastQuant RT Kit (with gDNase) (Tiangen Biotech Co., Ltd). Real-time PCR was conducted with ABI Prism 7900 sequence detection system following the manufacturer’s recommendations (Applied Biosystems). The primers used in RT-PCR are listed as follows: 5′-CTGCAGCCTCTGTGCTGTAT-3′ and 5′-CCACACATATCCAAGGCAAG-3′ for mouse PAQR4; 5′-GATCATTGCTCCTCCTGAGC-3′ and 5′-ACTCCTGCTTGCTGATCCAC-3′ for mouse β-actin; 5′-GGTCTGCCTTGTCAACACC-3′ and 5′-AACACAGTGTAGCCCACCAG-3′ for human PAQR4; 5′-TTAGGATCCGGTTGGACTCT-3′ and 5′-GGCTGGACTTGAGTTTGGA-3′ for human SKP2; 5′-CCTCACGCCTGTGGTGGTTA-3′ and 5′-CCCAACTGGTCGGCTTCAGA-3′ for mouse CDK4. Plasmid constructions The cDNAs of full-length PAQR4 (Gene ID: 124222, on NCBI), CDK4, SKP2, CUL3, FBXW7, BTRC, FBXO4, CDC20, DTL, and RNF114 were isolated from HEK293T cells by RT-PCR and confirmed by DNA sequencing. PAQR4 and its deletion mutants as well as SKP2 were subcloned into the mammalian expression vector pCS2 + MT with six Myc tags at the N terminus. PAQR4 and CDK4 were cloned into p3XFLAG-CMV-10 expression vector. PAQR4 was also cloned into pLVXm-N-His-IRES-Puro vector. Deletion mutants of CDK4 were cloned into pEGFP-C1 vector. Cell culture, lentivirus production, and transfection HEK293T and NIH3T3 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS (BCS for NIH3T3) and 1% penicillin/streptomycin at 37°C, under 5% CO2. Synchronization in G1 phase was made by serum deprivation (DMEM with 0.1% BCS) for 48 h, and then stimulation with 10% BCS for inducing synchronous cell cycle entry. Short hairpin RNA (shRNA) constructs for PAQR4 and SKP2 were inserted into the hU6-MCS-Ubiquitin-EGFP-IRES-puromycin vector. Lentivirus with different shRNA was generated by co-transfection in HEK293T cells as previously described (Qin et al., 2003). The annealed shRNA cassette targets sequences of 5′-GTGCTCTATCACCTCTTTA-3′ and 5′-AGTTCAATAAGTTCGTGCT-3′ for human PAQR4, 5′-GTGCTGTATCACCTCTTCA-3′ and 5′-AGTTCAATAAGTTCGTATT-3′ for mouse PAQR4, 5′-TAGTGTCATGCTAAAGAAT-3′ and 5′-CTAAGCTAAATCGAGAGAA-3′ for human SKP2, 5′-CATTGGATTGCCTCCAGAA-3′ and 5′-TAGCTAGAATCTACAGCTA-3′ for mouse CDK4. Transient transfection was performed with the polyethylenimine (PEI) method for HEK293T and NIH3T3 cells. MTT assay The MTT (Thiazolyl Blue Tetrazolium Bromide, from Sigma) assay was performed according to the method described by Mosmann (1983). NIH3T3 cells (5 × 103 cells/well) were seeded in a 96-well culture plate and incubated for different length of time. At the time of experiment, the culture medium was discarded and the plate was incubated at 37°C for 4 h with MTT (5 mg/ml). The MTT solution was then discarded, and dimethyl sulfoxide was added to dissolve the formazan product. The optical density was measured at 490 nm using a Versa max microplate reader (Molecular Devices). Colony formation assay NIH3T3 cells were inoculated into 6-well culture plate with 300 cells per well and cultured for 7 days. Then the cells were stained with Crystal Violet and colonies containing. Antibodies, immunoprecipitation, and immunoblotting The antibodies were purchased as follows: The antibodies against PARP, cyclin E and CKD2 were from Cell Signaling Technology. The antibodies against c-Myc, GFP, cyclin A, and HA were from Santa Cruz Biotechnology. The antibodies against Flag and α-tubulin were from Sigma-Aldrich. The antibodies against CDK4 and cyclin D1 were from UpState Biotechnology Incorporated. The antibody against His was from TransGen Biotech. The antibody against SKP2 was from Abways Technology. The protocols for immunoprecipitation and immunoblotting have been described previously (Feng et al., 2007). Protein degradation analysis For CHX (cycloheximide, from Sigma) assay, HEK293T cells transfected with different plasmids after 24 h, as well as cells infected with control or PAQR4 siRNA lentivirus were treated with 100 μg/ml CHX, and harvested at various time points. The cell lysates were subjected to immunoblotting. And the results were quantified by Quantityone software. For ubiquitination assay, HEK293T cells were transfected with vector expressing HA-tagged ubiquitin and other plasmids as mentioned in the research and treated with or without 10 μM MG132 (BD Biosciences) for 6 h before cells were harvested in IP buffer. The lysates were immunoprecipated with antibody as indicated and then immunoblotted. Generation of Paqr4 knockout mouse The PAQR4 knockout construct was made with a bacteria-based homologous recombination method (Cotta-De-Almeida et al., 2003) in which the second and third exons of the mouse PAQR4 gene were replaced by a neomycin-selectable cassette. To acquire homozygous Paqr4–/– mice in C57BL/6J background, we crossed chimeric founders with C57BL/6J mice for at least five generations. For genotyping, mouse-tail DNA was amplified with primers 5′-ACTGAGCTGCCTCCTAGCTG-3′ and 5′-GTCAGGAAGCAGGGTCAGAG-3′ for the wild type allele, 5′-GGGAGGATTGGGAAGACAAT-3′ and 5′-GTCAGGAAGCAGGGTCAGAG-3′ for the knockout allele. All animals were maintained and used in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Institute for Nutritional Sciences. All of the experimental procedures were carried out in accordance with the CAS ethics commission with an approval number 2010-AN-8. Skin treatment with chemical carcinogens and histological analysis For the two-stage carcinogenesis experiments, tumors were induced on the shaved dorsal skin of wild type (n = 16) and Paqr4 knockout mice (n = 14) by topical application of a single dose of DMBA (100 nmol/L, in 200 μl of acetone, from Sigma), followed by twice-weekly applications of TPA (16 nM) in 200 μl of acetone (Sigma) for 27 weeks. The number of papillomas was recorded weekly starting at 10 weeks after TPA promotion. At the end of the experiments, the skin connected with papilloma/carcinoma and the normal skin were excised promptly after euthanasia, placed in 4% paraformaldehyde, fixed overnight, dehydrated in graded alcohols and normal butanol and then embedded in paraffin. The paraffin-embedded sections were de-paraffinized in xylenes and rehydrated in graded alcohols and then in ddH2O for 5 min twice, and then applied to hematoxylin and eosin staining. Both male and female mice were used for the experiments in this study. Statistical analysis Statistical analysis was performed using the Student’s t-test for all the data. Supplementary material Supplementary material is available at Journal of Molecular Cell Biology online. Funding This work was supported by research grants from the National Natural Science Foundation of China (31630036 and 81390350 to Y.C.), Ministry of Science and Technology of China (2016YFA0500103 to Y.C.), Chinese Academy of Sciences (XDA12010102, QYZDJ-SSW-SMC008, and ZDRW-ZS-2016-8 to Y.C.). Conflict of interest: none declared. References Abel , E.L. , Angel , J.M., Kiguchi , K., et al. . ( 2009 ). Multi-stage chemical carcinogenesis in mouse skin: fundamentals and applications . Nat. Protoc. 4 , 1350 – 1362 . Google Scholar Crossref Search ADS PubMed WorldCat Anders , L. , Ke , N., Hydbring , P., et al. . ( 2011 ). 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TI - The steady-state level of CDK4 protein is regulated by antagonistic actions between PAQR4 and SKP2 and involved in tumorigenesis
JO - Journal of Molecular Cell Biology
DO - 10.1093/jmcb/mjx028
DA - 2017-10-01
UR - https://www.deepdyve.com/lp/oxford-university-press/the-steady-state-level-of-cdk4-protein-is-regulated-by-antagonistic-x63TTTb4el
SP - 409
VL - 9
IS - 5
DP - DeepDyve
ER -