Myeloid leukemia factor 1 regulates p53 by suppressing COP1 via COP9 signalosome subunit 3
2005; Springer Nature; Volume: 24; Issue: 9 Linguagem: Inglês
10.1038/sj.emboj.7600656
ISSN1460-2075
AutoresNoriko Yoneda‐Kato, Kiichiro Tomoda, Mari Umehara, Yukinobu Arata, Jun‐ya Kato,
Tópico(s)Nuclear Structure and Function
ResumoArticle21 April 2005free access Myeloid leukemia factor 1 regulates p53 by suppressing COP1 via COP9 signalosome subunit 3 Noriko Yoneda-Kato Corresponding Author Noriko Yoneda-Kato Department of Animal Molecular Genetics, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Takayama, Ikoma, Nara, Japan Search for more papers by this author Kiichiro Tomoda Kiichiro Tomoda Department of Animal Molecular Genetics, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Takayama, Ikoma, Nara, Japan Search for more papers by this author Mari Umehara Mari Umehara Department of Animal Molecular Genetics, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Takayama, Ikoma, Nara, Japan Search for more papers by this author Yukinobu Arata Yukinobu Arata Department of Animal Molecular Genetics, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Takayama, Ikoma, Nara, Japan Search for more papers by this author Jun-ya Kato Jun-ya Kato Department of Animal Molecular Genetics, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Takayama, Ikoma, Nara, Japan Search for more papers by this author Noriko Yoneda-Kato Corresponding Author Noriko Yoneda-Kato Department of Animal Molecular Genetics, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Takayama, Ikoma, Nara, Japan Search for more papers by this author Kiichiro Tomoda Kiichiro Tomoda Department of Animal Molecular Genetics, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Takayama, Ikoma, Nara, Japan Search for more papers by this author Mari Umehara Mari Umehara Department of Animal Molecular Genetics, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Takayama, Ikoma, Nara, Japan Search for more papers by this author Yukinobu Arata Yukinobu Arata Department of Animal Molecular Genetics, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Takayama, Ikoma, Nara, Japan Search for more papers by this author Jun-ya Kato Jun-ya Kato Department of Animal Molecular Genetics, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Takayama, Ikoma, Nara, Japan Search for more papers by this author Author Information Noriko Yoneda-Kato 1, Kiichiro Tomoda1, Mari Umehara1, Yukinobu Arata1 and Jun-ya Kato1 1Department of Animal Molecular Genetics, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Takayama, Ikoma, Nara, Japan *Corresponding author. Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan. Tel.: +81 743 72 5514; Fax: +81 743 72 5519; E-mail: [email protected] The EMBO Journal (2005)24:1739-1749https://doi.org/10.1038/sj.emboj.7600656 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Myeloid leukemia factor 1 (MLF1) was first identified as the leukemic fusion protein NPM-MLF1 generated by the t(3;5)(q25.1;q34) chromosomal translocation. Although MLF1 expresses normally in a variety of tissues including hematopoietic stem cells and the overexpression of MLF1 correlates with malignant transformation in human cancer, little is known about how MLF1 is involved in the regulation of cell growth. Here we show that MLF1 is a negative regulator of cell cycle progression functioning upstream of the tumor suppressor p53. MLF1 induces p53-dependent cell cycle arrest in murine embryonic fibroblasts. This action requires a novel binding partner, subunit 3 of the COP9 signalosome (CSN3). A reduction in the level of CSN3 protein with small interfering RNA abrogated MLF1-induced G1 arrest and impaired the activation of p53 by genotoxic stress. Furthermore, ectopic MLF1 expression and CSN3 knockdown inversely affect the endogenous level of COP1, a ubiquitin ligase for p53. Exogenous expression of COP1 overcomes MLF1-induced growth arrest. These results indicate that MLF1 is a critical regulator of p53 and suggest its involvement in leukemogenesis through a novel CSN3–COP1 pathway. Introduction Chromosomal translocations in human leukemia often create aberrant fusion proteins, which act as an initiating event in tumorigenesis by disrupting the normal hematopoietic control of proliferation, differentiation and cell death. Molecular functional analysis of the fusion proteins frequently leads to the discovery of novel regulatory pathways essential for both normal cell growth and tumorigenesis (for reviews, see Rabbitts, 1994; Look, 1997). The t(3;5)(q25.1;q34) chromosomal translocation is associated with myelodysplastic syndrome (MDS) often prior to acute myeloid leukemia (AML) (Raimondi et al, 1989) and generates a fusion protein consisting of nucleophosmin (NPM)/B23 and cytoplasmic protein, myeloid leukemia factor 1 (MLF1) (Yoneda-Kato et al, 1996). NPM is a ubiquitously expressed multifunctional nucleolar phosphoprotein, which plays a part in nuclear–cytoplasmic shuttling of ribonucleoproteins during the ribosomal assembly (Yung et al, 1985; Borer et al, 1989; Olson et al, 2000). NPM is a direct regulator of the Arf–Mdm2–p53 tumor suppressor protein pathway (Colombo et al, 2002; Itahana et al, 2003; Bertwistle et al, 2004; Kurki et al, 2004). NPM binds to Arf and recruits it to the nucleoli, which affects the regulation of the cell cycle. NPM also interacts with Mdm2 and prevents the degradation of p53 mediated by Mdm2. By contrast, the biochemical activity of MLF1 remains unknown because of a lack of significant homology between MLF1 and any previously identified protein, but clues as to the biological function have been obtained. In clinical studies, MLF1 expression was preferentially detected in CD34+ primitive cells and declined in more lineage-committed cells during normal hematopoiesis (Matsumoto et al, 2000). MLF1 is aberrantly overexpressed in over 25% of patients with MDS-associated AML and the malignant transformation phase of MDS (Matsumoto et al, 2000). In addition, enforced expression of murine MLF1 disturbed the development of erythroid colonies in normal hematopoietic precursors and differentiation to erythrocytes in an erythropoietin-dependent cell line (Williams et al, 1999). These findings suggest that MLF1 normally functions as a regulatory factor in the development of primitive hematopoietic cells and its deregulation contributes to hematopoietic dysplasia and leukemogenesis. However, endogenous expression of MLF1 is not restricted in hematopoietic cells (Yoneda-Kato et al, 1996) and the aberrant overexpression of MLF1 is also reported in lung squamous cell carcinoma (Sun et al, 2004), suggesting that MLF1 is involved in a common regulatory pathway related to tumorigenesis. We show here that MLF1 inhibits cell cycle progression through a p53-dependent mechanism. MLF1 binds directly to the third component of the COP9 signalosome complex (CSN3) and activates the p53/p21 pathway via a novel CSN3–COP1-mediated pathway. The COP9 signalosome (CSN) and COP1 were initially defined as repressors of photomorphogenesis in plants. CSN is placed upstream of COP1, which promotes the degradation of the transcription factor HY5, a positive regulator of photomorphogenesis, in the dark (Schwechheimer and Deng, 2001). Although COP1 was recently discovered to exhibit E3 ubiquitin ligase activity toward p53 (Dornan et al, 2004), the existence of the CSN–COP1–p53 pathway and its upstream regulator in mammalian cells has never been demonstrated before. The expression of MLF1 results in the downregulation of COP1 through CSN3 and consequent stabilization of p53. Importantly, knockdown of CSN3 protein by the small interfering RNA (siRNA) method not only impairs MLF1-induced cell cycle arrest but also interferes with the activation of p53 by genotoxic stress despite the presence of MLF1, implying that CSN3 is a critical factor mediating genotoxic stress leading to p53 activation. Identification of the novel pathway of p53 activation mediated by CSN3–COP1 in response to MLF1 signaling and DNA damage will provide new insight into the regulation of mammalian cell proliferation and tumorigenesis. Results MLF1 induces cell cycle arrest in G1 phase To elucidate the function of MLF1, we ectopically expressed MLF1 protein in murine fibroblasts and examined its effect on cell proliferation. Human MLF1 has a single nucleotide polymorphism at the 226th amino-acid codon, which produces proline and threonine at a ratio of 3:1 in normal genomic alleles (Figure 1A). We designated the proteins P-MLF1 and T-MLF1, respectively, and introduced expression vectors encoding P-MLF1, T-MLF1 and NPM-MLF1 fused with green fluorescent protein (GFP) and a puromycin resistance gene into mouse NIH3T3 fibroblasts. The number of colonies that appeared after selection in puromycin was much smaller for MLF1-transfected cells (ca. 10%) than for the cells transfected with control GFP and NPM-MLF1. The colonies expressing P-MLF1 and T-MLF1 were smaller, consisted of fewer cells and exhibited lower cellular density (Figure 1B). T-MLF1 seemed to have a stronger suppressive effect on cell growth than did P-MLF1 because the number of colonies and the cell density were significantly lower on transfection of T-MLF1 than P-MLF1. In addition, cells expressing T-MLF1 ceased to proliferate much earlier than those expressing P-MLF1. Therefore, we performed the analyses mainly with the P-MLF1 transfectants. As expected, the cells expressing P-MLF1 grew slower than those expressing GFP alone and NPM-MLF1 (Figure 1C). A similar retardation of growth was observed in several independent single cell-derived clones as well as in mixed populations of GFP-positive cells, and in cells transfected with intact MLF1 and HA-tagged MLF1 under the control of several different promoters (data not shown), indicating that the phenotype seen in this experiment was not due to the clonal variation of the cells or to the fusion with the GFP molecule. Examination of the cell cycle distribution of these cell clones revealed that P-MLF1-expressing cells exhibited a markedly increased G1-phase population (77%) at the expense of a decrease in the S and G2/M phases (14 and 9%, respectively), while the control cell population contained 45% G1-phase cells, 35% S-phase cells and 19% G2/M-phase cells (Figure 1D). Importantly, these cell clones expressed comparable amounts of MLF1 and NPM-MLF1 proteins (Figure 1E). Taken together, these results indicate that ectopically expressed MLF1 induced cell cycle arrest at G1 phase and the fusion protein comprising NPM and MLF1 generated in leukemia cells lost this activity. Figure 1.MLF1 induces cell cycle arrest in G1 phase. (A) A single nucleotide polymorphism at the 226th amino-acid sequence of human MLF1 produces proline (P) and threonine (T) at a ratio of 3:1 in normal genomic alleles. (B, C) MLF1 suppresses cell growth. NIH3T3 (p53 wild type, Arf null) mouse fibroblasts were transfected with GFP empty vector and GFP-fused P-MLF1(226 Proline), T-MLF1(226 Threonine) and NPM-MLF1. After selection with puromycin, GFP-positive single-cell clones expressing P-MLF1 and T-MLF1 exhibited a lower cell density and growth rate than the clones expressing GFP alone or NPM-MLF1. (D) The cell cycle distributions of the GFP-positive cells were analyzed using a flow cytometer after staining with propidium iodide. (E) Cell lysates from GFP-positive single-cell clones were immunoblotted with antibodies to MLF1 and anti-γ-tubulin. Download figure Download PowerPoint To test whether the MLF1-associated growth suppressing phenotype is not restricted to fibroblasts, we introduced MLF1 expression vectors into several hematopoietic and adherent cell lines. As a result, we found that a stable expression of MLF1 in cells depends on the status of p53: we failed to establish clones expressing either type of MLF1 protein from 32D immature myeloid (p53 wild type) cells and U2OS (p53 wild type) cells, whereas MLF1 expression was stable in M1 (p53 null), K562 (p53 mutation) and 293T (p53 inactivated) cells. In addition, the NIH3T3 cells (provided by Drs CJ Sherr and MF Roussel) used in Figure 1 lack the ARF locus but retain the intact p53 allele. MLF1-induced growth arrest is dependent on p53 To assess the molecular mechanism involved in MLF1-induced cell cycle arrest, we examined the effect of ectopic expression of MLF1 on the growth of genetically altered murine primary embryonic fibroblasts (MEFs). We infected MEF cells (wild type, p53−/− and p27−/−) with retroviruses that express GFP alone and GFP-(P-type and T-type)-MLF1 fusion proteins and selected them in puromycin. Growth curves show that wild-type MEF cells expressing either type of MLF1 protein grew markedly slower than those expressing GFP alone (Figure 2A), consistent with the results obtained in NIH3T3 cells (Figure 1C). In this experiment, the proliferation rate of T-MLF1-expressing cells was only marginally lower than that of cells expressing P-MLF1, but Western blot analysis revealed that expression levels of the protein were significantly lower in T-MLF1-expressing cells (Figure 2D, lanes 1–3), confirming the result that T-MLF1 was a more potent growth suppressor in murine fibroblasts. Protein analysis also shows that, in these growth-retarded cells, the expression of Cdk inhibitors, p21 and p27, and the tumor suppressor p53 was upregulated (Figure 2D, lanes 1–3). The growth curves of p53- or p27-deficient MEF cells infected with MLF1 retroviruses show that p53−/− MEF cells were refractory to the MLF1 action (Figure 2B), while ectopic MLF1 induced growth arrest in p27−/− MEF cells as effectively as in wild-type cells (Figure 2C). Protein analysis shows that MLF1 expression resulted in induction of p53 and its target protein p21 expression in p27−/−, but not p53−/−, MEF cells as expected. However, we did not detect upregulated p27 expression in p53−/− cells for unknown reasons (Figure 2D). Thus, consistent with the findings obtained from various cell lines with the different p53 status, these results demonstrate that MLF1-induced growth arrest depended on the integrity of the p53 allele. Figure 2.MLF1 induces G1 arrest in a p53-dependent manner. Growth curves of wild-type (A), p53−/− (B) and p27−/− (C) MEFs infected with retroviruses that expressed GFP alone, GFP-P-MLF1 and GFP-T-MLF1. (D) Cell lysates from MEFs were analyzed by immunoblotting using antibodies against MLF1, p21, p53, p27 and γ-tubulin. Download figure Download PowerPoint MLF1 specifically interacts with CSN3 To search for factors involved in the MLF1-mediated signaling pathway, we utilized the yeast two-hybrid system to screen murine T-cell lymphoma and human K562 erythroleukemia cDNA libraries to identify genes whose products directly interact with MLF1. We eventually isolated four types of cDNA inserts as classified by sequence, one of which contained sequences 100% identical to those of the mouse and human COP9 signalosome subunit 3 (CSN3). We tested the specificity of the interaction between MLF1 and CSN3 in living cells. In Cos7 cells transiently transfected with HA-tagged CSN3 and MLF1, a substantial amount of MLF1 protein was detected in an anti-CSN3 immunoprecipitate (Figure 3A, middle panel). In a reciprocal experiment, we found a significant portion of CSN3 in an anti-MLF1 immunoprecipitate (Figure 3A, bottom panel). Furthermore, we successfully detected the interaction between endogenous CSN3 and MLF1 proteins in human leukemia K562 cells (Figure 3B). The majority of endogenous and ectopically expressed MLF1 protein is located in the cytoplasm, concentrated around the nucleus, with a small fraction in the nucleus in cultured mammalian cells (Yoneda-Kato et al, 1996; Matsumoto et al, 2000). By contrast, the endogenous CSN3 protein was detected in both the nucleus and cytoplasm, with the highest level in the nucleoplasm in murine fibroblasts (Figure 3C, the upper right panel). Ectopic MLF1 was colocalized with endogenous CSN3 in the perinuclear region of the cytoplasm with minimal changes in the subcellular distribution of CSN3 (Figure 3C, the lower panels). Taken together, these results indicate that MLF1 and CSN3 interact with each other in vivo. Figure 3.CSN3 specifically interacts with MLF1 in vivo. (A) CSN3 specifically interacts with MLF1 in ectopically overexpressed cells. Cos7 cells were transfected with the expression vectors shown at the top. Total expression was analyzed by anti-HA immunoblotting (upper panel). Cell lysates were analyzed by sequential immunoprecipitation and immunoblotting with antibodies to CSN3 and MLF1 as shown at the left of the panels (middle and lower panels). NRS, preimmune normal rabbit serum. (B) Specific interaction between endogenous CSN3 and MLF1 proteins. Endogenous CSN3 and MLF1 proteins were immunoprecipitated from the K562 cell lysate shown at the top, and analyzed by immunoblotting with antibodies to CSN3 (upper panel) and MLF1 (lower panel). (C) Subcellular localization of MLF1 and CSN3. NIH3T3 cells were transfected with the GFP-control (upper panel) and GFP-MLF1 (lower panel) expression vectors, stained with an antibody to CSN3 and viewed using fluorescence (GFP for GFP-MLF1 and Texas red for endogenous CSN3) microscopy. The merged panel is also shown. Download figure Download PowerPoint CSN3 is required for the MLF1-induced growth arrest To investigate whether CSN3 is involved in MLF1-mediated growth arrest and activation of the p53 pathway, we used an RNA interference (RNAi) technique to reduce the amount of endogenous CSN3 protein in mammalian cells, and examined its effect on the MLF1 action. NIH3T3 cells were transfected with expression vectors containing GFP alone and GFP-P-MLF1 fusion cDNA together with an siRNA vector specific for mouse CSN3 (CSN3 siRNA) and a luciferase (Luc siRNA, used as a negative control). The cells were selected in puromycin for 5 days and then allowed to form colonies for an additional 9 days. The flat morphology and low cell density induced by MLF1 was markedly recovered in cells cotransfected with CSN3 siRNA, but not with Luc siRNA (Figure 4A). We chose several independent colonies, which uniformly expressed GFP proteins, expanded them, and used them for further analyses. The results from two representative clones that express different levels of CSN3 are shown in Figure 4B and C, but similar results were obtained in the experiment using different clones. The introduction of MLF1 into mouse fibroblasts activated the p53 pathway and induced the expression of p21 as in Figure 2D, but a decrease in the intracellular level of CSN3 protein prevented activation of p53 and resultant induction of p21 (Figure 4B). Surprisingly, a moderate reduction of CSN3 was sufficient to prevent the full induction of p21 and suppression of cell proliferation (Figure 4B and C, clone #2). Importantly, reduction of the intracellular level of CSN3 did not affect the expression level or the intracellular localization of MLF1 protein. The growth curve of each clone shows that a reduction of endogenous CSN3 protein resulted in a marked increase in the rate of proliferation regardless of the ectopic MLF1 expression (Figure 4C). As expected from the above result (Figure 4B), even a moderate reduction of CSN3 protein was sufficient to abrogate MLF1-induced growth suppression (Figure 4C). Thus, maintenance of CSN3 expression is required for the growth suppressive function of MLF1, suggesting that CSN3 is one of the key downstream effectors of the MLF1-mediated signaling pathway. Figure 4.CSN3 is required for the MLF1-induced G1 arrest. NIH3T3 cells were transfected with control luciferase (Luc) siRNA and CSN3 siRNA expression vectors together with the GFP-control and GFP-MLF1 expression vectors and selected in puromycin for 5 days. (A) At 14 days post-transfection, representative GFP-positive single clones were photographed. PC, phase-contrast. (B) Two representative clones with extensive and moderate reductions of CSN3 were chosen from each transfectant and analyzed by Western blotting using antibodies to CSN3, p53, p21 and γ-tubulin. (C) Growth curves of each clone. (D) Cell lysates were separated by native-PAGE (COP9 complex and small complex) and by SDS–PAGE (total CSN3 and γ-tubulin) and analyzed by immunoblotting using an antibody to CSN3. (E) Endogenous MLF1 was immunoprecipitated from the K562 cell lysate and analyzed by immunoblotting with antibodies to CSN1 (upper panel) and CSN3 (lower panel). Download figure Download PowerPoint Although Arabidopsis CSN3 is exclusively found in the CSN complex (Peng et al, 2001), mammalian CSN3 forms a unique smaller subcomplex besides CSN in murine fibroblasts (Fukumoto et al, 2005). To investigate the effect of CSN3 knockdown on the formation of the CSN3-containing complexes, we performed the nondenaturing polyacrylamide gel electrophoresis (native-PAGE) analysis (Seeger et al, 1998; Fukumoto et al, 2005; Tomoda et al, 2005), and found that the small CSN3 subcomplex, rather than CSN, was predominantly reduced (Figure 4D). Furthermore, to know the specificity of interaction in vivo, we immunoprecipitated endogenous MLF1 protein from the K562 cell lysate and found that MLF1 forms a complex with CSN3 but not with CSN1 (Figure 4E). Because CSN1 is found only in CSN in Arabidopsis (Wei et al, 1994; Staub et al, 1996) and in murine fibroblasts (Fukumoto et al, 2005), the target of MLF1 seems to be specific to CSN3 rather than CSN. In addition, we used the Jab1/CSN5 siRNA in an attempt to rescue the growth suppression mediated by MLF1, but were not successful (data not shown), supporting the notion that MLF1 is involved in the CSN3-specific function, such as the small CSN3 subcomplex. However, we do see some reduction of CSN in CSN3 siRNA-treated cells (Figure 4D), and do not know the exact relationship between the small CSN3 subcomplex and CSN yet. Therefore, we do not exclude the possibility that CSN plays an important role in the MLF1-mediated signaling pathway, and it will be most adequate to say that both CSN and the small CSN3 subcomplex are involved in the regulation of p53 by MLF1. Residues 50–125 of MLF1 are required for both CSN3 binding and growth inhibition To map the region of MLF1 required for association with CSN3, a series of MLF1 deletion mutants were generated (Figure 5A). Glutathione S-transferase (GST)-tagged recombinant MLF1 proteins were tested for binding to CSN3 in the lysate prepared from Cos7 cells transfected with CSN3 cDNA. An MLF1 mutant lacking the N-terminal 49 amino acids (MLF1/50–268), but not the one lacking 125 amino acids (MLF1/126–268), bound to CSN3 in vitro (Figure 5B), suggesting that residues 50–125 are required for interaction with CSN3. To determine the domain of MLF1 critical for growth suppression, we transfected NIH3T3 cells with expression vectors containing GFP-fused MLF1 mutants and enumerated the number of GFP-positive colonies formed after selection in puromycin. Again, the mutant lacking the N-terminal 125 amino acids (MLF1/126–268) lost the ability to suppress cell growth (Figure 5C). Thus, as summarized in Figure 5A, only the mutant unable to bind to CSN3 lost the growth inhibitory activity, indicating that the interaction with CSN3 is essential for the MLF1-induced growth arrest. Figure 5.Mapping the domain of MLF1 required for CSN3 binding and growth inhibition. (A) Schematic representation of MLF1 deletion mutants. The results of CSN3 binding and growth inhibition are summarized on the right. (B) The region of MLF1 involved in binding to CSN3 was determined by the GST pull-down assay. Beads coated with GST-control or the GST-MLF1 mutant fusion proteins shown at the top of the panel were incubated with Cos7 cell lysates containing HA-tagged CSN3 proteins. Bound proteins were detected by immunoblotting with antibody to CSN3. The amounts of GST proteins absorbed on the beads were evaluated by anti-GST immunoblotting. The positions of full-length GST-MLF1 mutant fusion proteins are indicated with arrowheads in lanes 3–5. (C) NIH3T3 cells were transfected with GFP-control and GFP-MLF1 mutant expression vectors and selected in puromycin for 5 days. At 14 days post-transfection, GFP-positive colonies were enumerated. Upper right: Expression levels of GFP-MLF1 mutant proteins before puromycin selection are shown by Western blotting with antibodies to GFP and γ-tubulin. Download figure Download PowerPoint CSN3 mediates MLF1 and genotoxic stress signaling in p53 activation via COP1 In plants, it was demonstrated that the COP9 signalosome complex is required for the proper functioning of the RING-finger-type E3 ubiquitin ligase COP1, which promotes the degradation of the transcription factor HY5, a positive regulator of photomorphogenesis, in the dark (Schwechheimer and Deng, 2001). Recently, COP1 was reported to serve as an E3 ubiquitin ligase for p53 in mammalian cells (Dornan et al, 2004). Therefore, we addressed whether COP1 is a downstream target leading to p53 activation in the MLF1 signaling. To avoid clonal deviation of the transfected cells, we isolated the GFP-positive population of cells by cell sorting from the mixed population of NIH3T3 cells transfected with GFP and siRNA vectors as shown in Figure 6A. We found that ectopic MLF1 expression resulted in a marked decrease in the endogenous COP1 protein level, whereas a reduction of CSN3 led to an increase of COP1 regardless of the MLF1 expression (Figure 6A), suggesting that MLF1 modulates COP1 expression via CSN3 to activate the p53 pathway. Quantitative RT–PCR analysis revealed that the relative levels of p53 and COP1 mRNA in cells transfected with MLF1 or CSN3 siRNA remained unchanged (Figure 6B), whereas the half-life of p53 in MLF1-transfected cells (ca. 30 min) determined by the pulse-chase analysis was significantly longer than that in control cells (ca. 18 min) (Figure 6C). Thus, MLF1 upregulates p53 through inhibition of protein degradation. Figure 6.Effects of CSN3 siRNA on the MLF1-induced G1 arrest and p53 stability. NIH3T3 cells were transfected with control luciferase (Luc) siRNA and CSN3 siRNA expression vectors together with the GFP-control and GFP-MLF1 expression vectors. Multiclonal GFP-positive cells were isolated by cell sorting. (A) Cells were lysed and analyzed by Western blotting using antibodies against MLF1, CSN3, p53, p21, COP1, Mdm2 and Pirh2 and γ-tubulin. (B) Total RNAs were extracted from cells and used for the quantitative RT–PCR analysis to determine the relative amounts of p53 and COP1 transcripts. (C) GFP-control and GFP-MLF1 cells were metabolically labeled with [35S]methionine, and chased for the indicated periods of time. Labeled p53 protein was immunoprecipitated with an antibody to p53 and quantified using a phosphorimager. (D) Cells were treated with 25 J/m2 of UV irradiation for the indicated period of time before harvest. Cells were lysed and analyzed by Western blotting using antibodies against p53, p21, COP1, Mdm2 and γ-tubulin. Results shown in (A, D) are representative of three independent experiments. Results in (B, C) represent the average of triplicate experiments. Download figure Download PowerPoint To know whether CSN3 is more commonly involved in the regulation of p53, we examined the effect of the knockdown of CSN3 on a genotoxic stress-induced activation of p53. We treated the siRNA-transfected cells used in Figure 6A with ultraviolet light (UV, 25 J/m2) and analyzed the kinetics of endogenous p53, p21, COP1 and Mdm2 protein expression. Treatment with UV resulted in a marked increase of p53 and p21 and a subsequent decrease of COP1 in control cells and a more enhanced and prolonged increase of p53 and p21 in MLF1-transfected cells, but failed to induce full upregulation of p53 and p21 in cells transfected with a CSN3-specific siRNA vector regardless of the MLF1 expression. Importantly, a relatively large amount of COP1 was maintained in these cells after exposure to UV irradiation (Figure 6D). These results show that CSN3 is an essential mediator needed to suppress COP1 and activate p53 in the genotoxic stress-induced signaling pathway as well as in MLF1-mediated signaling. COP1 interferes with the growth inhibitory activity of MLF1 To assess whether COP1 can counteract the action of MLF1 in p53 activation, we investigated the effect of ectopic COP1 expression on the growth arrest induced by MLF1. NIH3T3 cells were transfected with a constant amount of GFP-MLF1 cDNA together with an increasing amount of HA-tagged COP1 expression vector. First, transfected cells were harvested before selection and analyzed for the protein expression by immunoblotting with antibodies specifically recognizing MLF1 and COP1. Figure 7A shows that an equivalent amount of MLF1 protein was expressed in each transfectant regardless of the COP1 expression. Next, transfected cells were selected in puromycin for 5 days and allowed to form colonies for an additional 9 days in the absence of the drug. Introduction of the MLF1 cDNA alone did not affect survival much as expected. However, cotransfection with a COP1 expression vector substantially increased the number of GFP-positive colonies in a dose-dependent manner (Figure 7B). Importantly, the single cell-derived clones maintained an equivalent level of MLF1 and COP1 expression after colony formation, and the level of p53 was markedly lower in HA-COP1-transfected c
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