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DNAJB1 stabilizes MDM2 and contributes to cancer cell proliferation in a p53-dependent manner
Biochimica et Biophysica Acta 1839 (2014) 62–69
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DNAJB1 stabilizes MDM2 and contributes to cancer cell proliferation in a p53-dependent manner☆ Min Qi b, Jianglin Zhang a, Weiqi Zeng a,⁎, Xiang Chen a,⁎⁎ a b
Department of Dermatology, Xiangya Hospital, Central South University, Changsha, Hunan, PR China Department of Plastic Surgery, Xiangya Hospital, Central South University, Changsha, Hunan, PR China
a r t i c l e
i n f o
Article history: Received 20 October 2013 Received in revised form 29 November 2013 Accepted 12 December 2013 Available online 19 December 2013 Keywords: DNAJB1 MDM2 p53 p21 HSP70
a b s t r a c t Both MDM2 and MDMX regulate p53, but these proteins play different roles in this process. To clarify the difference, we performed a yeast 2 hybrid (Y2H) screen using the MDM2 acidic domain as bait. DNAJB1 was found to specifically bind to MDM2, but not MDMX, in vitro and in vivo. Further investigation revealed that DNAJB1 stabilizes MDM2 at the post-translational level. The C-terminus of DNAJB1 is essential for its interaction with MDM2 and for MDM2 accumulation. MDM2 was degraded faster by a ubiquitin-mediated pathway when DNAJB1 was depleted. DNAJB1 inhibited the MDM2-mediated ubiquitination and degradation of p53 and contributed to p53 activation in cancer cells. Depletion of DNAJB1 in cancer cells inhibited activity of the p53 pathway, enhanced the activity of the Rb/E2F pathway, and promoted cancer cell growth in vitro and in vivo. This function was p53 dependent, and either human papillomavirus (HPV) E6 protein or siRNA against p53 was able to block the contribution caused by DNAJB1 depletion. In this study, we discovered a new MDM2 interacting protein, DNAJB1, and provided evidence to support its p53-dependent tumor suppressor function. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The transcription factor p53 responds to various stresses, including DNA damage, overexpressed oncogenes and various metabolic limitations, and it regulates many target genes that can induce cell-cycle arrest, apoptosis, senescence and DNA repair or that can alter metabolism [1,2]. One well-known p53 target, p21, is upregulated upon p53 activation [3]. p21 is able to bind and inhibit the activity of the cyclin/CDK complexes, thereby reducing RB/E2F1 transcription ability [4]. In this way, the p53–p21 axis regulates the cell cycle and can arrest damaged cells at the G1/S checkpoint. In most cancer cells, this pathway can be inactivated through several different mechanisms. p53 mutation is the most common mechanism, and mutated p53 has been found in more than 50% of cancer patients [5]. In many tumors that express wild-type p53, the function of p53 can be compromised by viral oncogenes, such as the papillomavirus E6 and adenovirus E1B proteins, that induce p53 degradation [6,7]. Similarly, MDM2, an endogenous E3 ligase for p53 that is also responsible for the degradation of p53, is clearly a clinically relevant cellular oncogene. Within 5 years of its discovery as an amplified gene in a transformed murine cell line, it was found to be amplified in 7.2% of 3889 ☆ Conflict of interest: The authors declare no conflict of interest. ⁎ Correspondence to: W. Zeng, 87 Xiangya Road, Department of Dermatology, Xiangya Hospital, Central South University, Changsha, Hunan, PR China. Tel.: +86 158 7498 3697; fax: +86 731 8432 8478. ⁎⁎ Correspondence to: X. Chen, 87 Xiangya Road, Department of Dermatology, Xiangya Hospital, Central South University, Changsha, Hunan, PR China. Tel.: +86 731 8432 7128; fax: +86 731 8432 8478. E-mail addresses: [email protected] (W. Zeng), [email protected] (X. Chen). 1874-9399/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbagrm.2013.12.003
human tumors that lacked p53 mutations. MDM2 amplification was more recently reported in approximately 10.5% of 7711 tumors [5]. Mouse models established MDM2 and MDMX as essential p53 regulators [8–12]. MDM2 and MDMX protein sequences harbor three regions of high identity, dubbed CR1, CR2, and CR3. In MDM2, CR1 (residues 42 to 94) is responsible for binding to p53 and inhibiting its transactivation function. CR2 (residues 301 to 329) encodes a putative zinc-binding domain and partially overlaps with a region required for binding to the retinoblastoma tumor suppressor protein (RB). CR3 (residues 444 to 483) encodes the ring finger domain (RFD), which binds two Zn atoms and contains a cysteine residue (residue 464) required for ubiquitin conjugation to p53. In the region between CR1 and CR2 of MDM2, there is a nuclear localization/export sequence (NLS/NES) and an acidic domain (AD) that are not highly conserved in MDMX. The MDM2 acidic domain consists of 81 amino acids; a previous study from Dr. Yuan's group revealed that the MDM2 AD is unique and essential for p53 ubiquitination, as this function cannot be rescued by the MDMX AD domain [13]. DnaJ/Hsp40 (heat shock protein 40) proteins are important for protein translation, folding, unfolding, translocation, and degradation, primarily by stimulating the ATPase activity of the chaperone proteins, Hsp70s. Because ATP hydrolysis is essential for the activity of the Hsp70s, DnaJ/Hsp40 proteins actually determine the activity of the Hsp70s by stabilizing their interaction with the substrate proteins [14]. The human DNAJ family has over 40 members [14,15], and can be subdivided into three subfamilies, DNAJA, DNAJB and DNAJC. Recent reports have described the involvement of some Hsp40 family members of distinct classes, such as hTid I (class DNAJA3) and HLJ1 (class
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DNAJB4), in the modulation of tumor growth, and these findings are opening new frontiers for studies on this family [16–22]. DNAJB1 has been linked to several cellular processes, such as the proteasome pathway [23], endoplasmic reticulum (ER) stress [24], and virus infection [25], while few publications mention its function in cancer progression. In the current study, DNAJB1 was first identified as an MDM2 AD binding protein through an Y2H screen. Further experiments revealed its function in p53 ubiquitination and degradation, p53-dependent cell cycle regulation, and tumor progression.
The transformants with non-interacting protein pairs were able to grow on media lacking leucine and tryptophan (SD/-Leu/-Trp, -2 SD), while only positive clones containing interacting prey and bait proteins were able to grow on dropout medium lacking tryptophan, leucine, histidine, and adenine (SD/-Leu/-Trp/-His/-Ade, -4 SD). The positive colonies were lysed and subjected to the ortho-nitrophenyl-β-Dgalactopyranoside (ONPG) assay to verify the interaction. Finally, the confirmed positive colonies were selected, and the individual plasmids were amplified, purified, and analyzed by sequencing.
2. Materials and methods
2.2. PCR, quantitative-PCR and cloning
2.1. Yeast two hybrid screen
Oligonucleotides were synthesized by Invitrogen and are listed in Table S1. Quantitative-PCR (Q-PCR) was performed using the SYBR® Green Master Mix kit (4367659, Invitrogen) and an ABI 7500 instrument. All plasmids were constructed using restriction enzyme digestion.
A human fetal cDNA library and yeast two-hybrid system were purchased from Clontech (Cat. 637242). The MDM2 acidic domain was amplified and inserted into the pGBKT7 plasmid to yield pGBKT7-MDM2 AD. This recombinant plasmid was transformed into the AH109 yeast strain and was transformed with the human fetal brain cDNA library plasmids. After screening according to the manufacturer's instructions, the prey plasmids were purified from the positive clones, and the yeast was co-transformed with pGBKT7-BSG and the prey plasmid to verify the interaction. These transformants containing both bait and prey plasmids were plated on synthetic defined dropout medium.
2.3. Cell culture, transfection and reagents MCF7, U2OS and 293T cells were purchased from the American Type Culture Collection (ATCC) and were maintained in their corresponding media as previously described [13]. MCF7-E6 cells were created and maintained following the published methods [38]. All cell culture reagents were purchased from Gibco, and all other reagents were
Fig. 1. The C-terminus of DNAJB1 binds and stabilizes MDM2. A. MDM2 and DNAJB1 protein domain information. For MDM2, CR1 and CR2 denote conserved regions found in both MDM2 and MDMX; NLS/NES denotes nuclear localization/export sequence; AD is the acidic domain; L23 is a linker region; and RFD denotes the ring finger domain. For DNAJB1, J domain is an identical domain found in all DNAJ members; SBD denotes the substrate-binding domain. The region of DNAJB1 used in the Y2H screen is indicated. B. DNAJB1 binds MDM2 in vitro. The DNAJB1-BD (GAL4 binding domain) recombinant protein expression plasmid was co-transformed with the empty vector (EV-AD, AD denotes GAL4 activation domain), MDMXAD or MDM2-AD. In -2 SD, the appearance of a colony indicates that the transformants harbor both plasmids. In -4 SD, the appearance of a colony indicates that there was a positive interaction between both proteins. The ONPG assay is a method of β-galactosidase detection. A darker yellow color indicates that more β-galactosidase was present and reflects a stronger protein interaction. C. IP results revealed the endogenous DNAJB1–MDM2 interaction. D. DNAJB1 co-localized with MDM2 in the nucleus. DNAJB1 was labeled with Alexa Fluor 594 (red), and MDM2 was labeled with Alexa Fluor 488 (green). 4′,6-Diamidino-2-phenylindole (DAPI) was used for nuclear staining (blue). E. Exogenous DNAJB1 interacted with MDM2 but not with MDMX. WL denotes whole lysate. The molecular weight of flag-tagged DNAJB is similar to that of IgG; thus, the bands are indicated with arrowheads. F. The C-terminus, but not the N-terminus, of DNAJB1 binds and stabilizes MDM2.
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purchased from Sigma except indicated. Transfections were performed with Lipofectamine 2000 (Invitrogen) according to the standard instructions. In each experiment, the amounts of the transfected plasmids were consistent, and an empty vector was used to compensate for any remaining amount. Each experiment was repeated three times.
2.4. Western blot analysis, IPs, IF and antibodies Western blot analysis, IPs and IF were performed according to described procedures [39]. The MDM2 (SC-965, 1:500), p53 (SC-126, 1:1000), MYC (SC-40, 1:2000), and DNAJB1 (sc-135943, 1:500) antibodies and Protein A/G PLUS agarose beads (SC-2003) were purchased from Santa Cruz Biotechnology. Flag (F4042, 1:5000, mouse), Flag (F2555, 1:1000, rabbit), and actin (A4700, 1:5000) antibodies and Anti-FLAG® M2 Magnetic Beads (M8823) were purchased from Sigma. The p21 (2946L, 1:300) antibody was purchased from Cell Signaling Technology. The MDMX (A300-287A, 1:1000) antibody was purchased from Bethyl Laboratories. The goat anti-mouse (SC-2005, 1:500) and goat anti-rabbit (SC-2004, 1:500) secondary antibodies were purchased from Santa Cruz Biotechnology. The secondary antibodies used for IF were purchased from Invitrogen, including Alexa Fluor 594 (A11020, 1:500) and Alexa Fluor 488 (A11034, 1:500). The blot images were taken using a ChemiDoc XRS+ system (BioRad). IF images were taken using a microscope (CX41-32RFL, OLYMPUS).
2.5. Cancer 10-pathway reporter arrays The Cancer 10-pathway Reporter kit (CCA-001L) was purchased from SABiosciences. The screening process was performed following the standard manual. In brief, MCF7 cells were seeded into 96-well plates at a density of 1 × 104 cells per well. Reverse transfections were performed when the cells were seeded. After 48 h, the cells were harvested and subjected to dual luciferase analysis, which was performed using the Dual-Luciferase® Reporter Assay System (E1960, Promega). 2.6. Virus packaging and infection The pLKO1 plasmid and helper vectors, VSVG and delta8.2, were purchased from Addgene (www.addgene.org). Virus packaging and MCF7 cell infection were performed following standard protocols from Addgene. Stable cell lines were created by selection with 1 μg/ml puromycin (P9620, Sigma) for 6 days. 2.7. Xenograft mouse models Xenograft tumor models were established according to a previously described procedure [40]. In brief, 5 × 106 MCF7 cells were subcutaneously injected into the hind limbs region of 6-week-old female BALB/C mice. To stimulate tumor growth, estradiol supplementation was provided in the form of 90-day release 0.25 mg estradiol pellets (Innovative
Fig. 2. DNAJB1 stabilizes MDM2 at a post-translational level. A. Depletion of DNAJB1 by shRNA in MCF7 cells decreased MDM2 protein levels. shGFP is a sequence that does not target endogenous transcripts in the mammalian genome and was used as a control. Both sh1 and sh2 were designed against the human DNAJB1 mRNA at different sites. B. DNAJB1 and MDM2 mRNA levels in the indicated cells as detected by RT-PCR. The error bars were calculated from three replicates. C. MDM2 (1 μg) was co-transfected with 0.5 μg of the indicated plasmids in each 35-mm culture dish. EV denotes an empty vector; FL, N or C denotes full length, N-terminus or C-terminus of DNAJB1, respectively; H32Q is a J domain function null mutation. D. Hsp70 only weakly stabilized MDM2. MDM2 (1 μg) was co-transfected with 0.5 μg of DNAJB1, 0.5 μg of Hsp70 or both. E. DNAJB1 stabilized MDM2 in a dose-dependent manner. MDM2 (1 μg) was co-transfected with increasing amounts of DNAJB1 (0, 0.1, 0.2, 0.5, or 1 μg). F. MG132 recovers MDM2 in DNAJB1 depletion cells. The shDNAJB1 cell is infected with both sh1 and sh2 virus. Cells were treated with 10 μm MG132 for the indicated time and then harvested.
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Research of America, Sarasota, FL) placed in the interscapular region of the mice. The surgical incision was closed with a staple. After 52 days, the mice were euthanized for analysis. 3. Results 3.1. DNAJB1 binds to MDM2 in vitro and in vivo through its C-terminus and stabilizes MDM2 DNAJB1 was identified as a positive candidate through the Y2H screen. The sequencing results revealed that the C-terminus of DNAJB1 (from amino acids 167–340) interacted with the MDM2 acidic domain (Fig. 1A). This interaction was unique because the C-terminus of DNAJB1 could not interact with either the empty vector, which expressed the GAL4 activation domain (AD), or with MDMX-AD, which expressed the MDMX protein fused to the GAL4-AD (Fig. 1B). To verify the interaction in vivo, immunoprecipitations (IPs) were performed. MDM2 was precipitated with a DNAJB1 antibody, and DNAJB1 was also precipitated using an MDM2 antibody. p53 was precipitated in both reactions (Fig. 1C). Immunofluorescence (IF) was performed and revealed that DNAJB1 co-localized with MDM2 in the nucleus (Fig. 1D). Interestingly, when MDM2 was co-expressed with DNAJB1, it significantly
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accumulated (Fig. S4). Western blot analysis showed results consistent with the IF analysis, mainly that the level of exogenous MDM2 protein co-transfected with DNAJB1 was much higher than the expression of MDM2 alone. The stabilization of MDM2 by DNAJB1 was specific, as the MDMX protein level did not change in the presence or absence of DNAJB1 (Fig. 1E, WL panel). We performed immunoprecipitation experiments to test the exogenous MDM2 and DNAJB1 interaction and found that only MDM2 could be precipitated by the M2 beads, whereas MDMX could not (Fig. 1E). Another immunoprecipitation experiment showed that MDM2 was interacting with the C-terminus of DNAJB1, which was consistent with the Y2H results (Fig. 1F). 3.2. DNAJB1 stabilizes MDM2 at the post-translational level and slows MDM2-mediated degradation by ubiquitin pathway To investigate the stability of endogenous MDM2 protein in the presence or absence of DNAJB1, we depleted cells of DNAJB1 using a lentivirus-based small hairpin RNA (shRNA). Two shRNA constructs that targeted different sites on the DNAJB1 mRNA reduced the levels of DNAJB1 protein by over 70%, and MCF7 cells that were depleted of DNAJB1 had lower levels of MDM2 compared to the control shGFP cells (Fig. 2A). The same experiment was performed in U2OS cells, and
Fig. 3. DNAJB1 stabilized p53 in an MDM2-dependent manner. A. Exogenous DNAJB1 stabilized MDM2, MDMX and p53 and led to an increase in p21 levels. Increasing amounts of DNAJB1 (0, 0.1, 0.2, 0.5, 1, or 2 μg/35-mm dish) were transfected into MCF7 cells. B. MCF7 cells were treated with cycloheximide (10 μg/ml) for the indicated times. Cell lysates were subjected to Western blot analysis,half-life time of p53 was labeled with dark arrows. The p53 blot was scanned and analyzed. (C) and (D) were performed as in (B), with the addition of the nutlin-3 or siMDM2 treatments, respectively.
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the MDM2 level consistently decreased (Fig. S1). Next, real-time PCR was performed to examine the levels of MDM2 mRNA. MDM2 mRNA levels did not change with or without DNAJB1 (Fig. 2B). This result suggested that DNAJB1 regulates MDM2 at a post-translational level. DNAJB1 has a J domain at its N-terminus that harbors the “HPD” amino acid sequence, which is essential for DNAJB1's co-chaperone function, as this is its binding site with Hsp70 [14]. The H32Q mutant of DNAJB1 cannot activate Hsp70. Hsp70 interaction was dispensable for the DNAJB1-mediated accumulation of MDM2; the C-terminus of DNAJB1 alone was sufficient to stabilize MDM2 (Fig. 2C). MDM2 was co-transfected with DNAJB1 only, with Hsp70 only, or with both. Hsp70 could only weakly stabilize MDM2. Interestingly, the level of MDM2 was lower when both DNAJB1 and Hsp70 were co-transfected compared to co-transfection with DNAJB1 only (Fig. 2D). A dose-dependent effect was observed when MDM2 was cotransfected with increasing amounts of DNAJB1 (Fig. 2E). Because MDM2 is mainly degraded through the ubiquitin pathway, the proteasome inhibitor MG132 was added to culture media to block ubiquitin– proteasome dependent protein degradation. MDM2 protein levels did not change in the shGFP group, but MDM2 levels were fully rescued in the DNAJB1 depleted group after 60 min. This result suggested that MDM2 was degraded faster when DNAJB1 was depleted. Overexpression of exogenous DNAJB1 in MCF7 cells also stabilized MDM2, as well as its ubiquitinated forms (Fig. 3A). 3.3. DNAJB1 stabilizes p53 in an MDM2-dependent manner The classical role of MDM2 is a p53 regulator; therefore, investigating the level and activity of p53 is important to understanding
DNAJB1. MCF7 cells were transiently transfected with increasing amounts of DNAJB1. In addition to the stabilization of MDM2, MDMX and p53 were also stabilized. The accumulation of p21 showed that the transcriptional activity of p53 also increased (Fig. 3A). Cycloheximide (CHX), an mRNA translation inhibitor, was added to the culture media to block protein synthesis. Both MDM2 and p53 protein levels decreased much faster in the DNAJB1-depleted group. In the control group, the p53 half-life was approximately 1 h, while its half-life was only 30 min in the shDNAJB1 group (Fig. 3B). To clarify whether the faster degradation of p53 was MDM2-dependent, two methods were used to block MDM2 E3 ligase activity. First, nutlin-3, which is a small compound that specifically interrupts the interaction between MDM2 and p53, was added to the culture media along with CHX. In both groups, p53 protein showed similar degradation rates (Fig. 3C). Second, small interfering RNA (siRNA) against MDM2 was used to deplete MCF7 cells of MDM2. When MDM2 protein was eliminated, DNAJB1 depletion did not affect p53 degradation (Fig. 3D). 3.4. DNAJB1 decreases MDM2-mediated p53 ubiquitination and enhances p53 transcriptional activity To investigate the status of p53 when DNAJB1 is bound to MDM2, immunoprecipitation experiments were performed. Flag-tagged p53 was used to pull down DNAJB1, and increasing amounts of DNAJB1 were precipitated when more DNAJB1 was transfected (Fig. 4A). This result suggested that DNAJB1 binds to MDM2, but MDM2 does not release p53. Even the binding affinity of MDM2 to p53 did not change, but the degradation of p53 was slower. Therefore, it is possible that DNAJB1 reduces MDM2 E3 ligase activity. MDM2, MDMX, DNAJB1 and p53
Fig. 4. DNAJB1 attenuated p53 ubiquitination and increased p21 levels in a p53-dependent manner. A. MDM2 plasmids (2 μg) and 2 μg of Flag-p53 were co-transfected with the indicated amounts of DNAJB1 (0, 0, 0.2, 0.5, or 1 μg) into 293T cells. IPs were performed using M5 beads. B. MYC-p53 (0.5 μg), MDM2 (2 μg), Flag-MDMX (1 μg) and DNAJB1 (0.5 μg) were transfected into 293T cells, and the protein levels of p53, ubiquitinated p53 and DNAJB1 were examined. C. MCF7 wild-type cells and MCF7-E6 cells were infected with a lentivirus to deplete endogenous DNAJB1. The levels of the indicated proteins were examined. D. MCF wild-type cells and MCF7-E6 cells were transfected with DNAJB1 expression plasmids. The levels of the indicated proteins were examined. E. DNAJB1 was introduced into MCF7 cells with or without siRNA against p53.
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were co-transfected in the indicated combinations, and p53 was efficiently degraded by the MDM2/MDMX complex (Fig. 4B). When DNAJB1 was introduced, the levels of p53 protein partially recovered, and the levels of ubiquitinated p53 were attenuated (Fig. 4B). To understand how DNAJB1 function is mediated by the MDM2–p53 axis, p21 levels were investigated in p53 wild-type (WT) or inactivated (E6 protein overexpression) MCF7 cells. p21 protein was reduced in p53 WT cells, but not E6 cells, when DNAJB1 was depleted (Fig. 4C). Correspondingly, overexpression of DNAJB1 increased p21 levels in p53 WT cells but not in E6 cells (Fig. 4D). Depletion of p53 completely blocked p21 accumulation (Fig. 4D). 3.5. DNAJB1 contributes to cancer cell growth in vitro and in vivo in a p53-dependent manner The activities of 10 pathways that are commonly associated with cancer progression were tested. Depletion of DNAJB1 in MCF7 cells altered the activity of 4 pathways. The reduction of p53 and the induction of the RB/E2F pathway were interesting results (Fig. 5A). As mentioned previously, the RB/E2F pathway works downstream of p53 to regulate the cell cycle. Thus, we checked the proliferation of both the
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MCF7-WT and MCF7-E6 cells with or without endogenous DNAJB1. DNAJB1-depleted MCF7 cells grew faster than the control cells when WT p53 was present (Fig. 5B, upper panel) but showed a similar growth rate as the control cells when p53 was inactivated (Fig. 5B, lower panel). To verify the data in vivo, nude mice were used to test xenograft tumor formation. The tumor masses of the DNAJB1-depleted cells were 2.5 folds larger than those formed by the control cells (Fig. 5C, D and E). 4. Discussion The MDM2/MDMX/p53 pathway has been extensively investigated, and it is known that MDM2 stabilization is important for the p53 response to oncogene stress. Dr. Zhang's group developed the MDM2 C462A mouse strain, which has an inactive MDM2 E3 ligase, and reported 2 observations [26]. The first observation was that MDM2's E3 function was not required for MDM2 degradation. Thus, MDM2 was degraded by an unidentified pathway. Two groups reported that the MDM2 AD domain contributed to the regulation of MDM2 stability and provided evidence that casein kinase I phosphorylation regulated MDM2 turnover by the SCFβ-TRCP ubiquitin ligase [27,28]. Our data support this model. We showed that DNAJB1 binds to MDM2
Fig. 5. DNAJB1 inhibited MCF7 growth in vitro and in vivo. A. The cancer-10 pathway array was performed according to the manufacturer's protocol. The p53 and RB/E2F pathways are labeled with triangles. One-way ANOVA was down to assess the statistical significance. Single asterisk (*) means p b 0.05, and double asterisk (**) means p b 0.01. B. MCF7 and MCF7-E6 cells were infected with the shDNAJB1 lentivirus and selected by puromycin to create a stable cell line. Cells were seeded into 96-well plates at a density of 1000 cells/well. Each day, three wells were counted. The experiments were repeated three times. C. Xenograft mouse models were created as described. For each mouse, the control cells were injected on the left side, and the DNAJB1-depleted cells were injected on the right side. Tumors were cycled by red line. D. Xenografts were isolated and arranged. Each horizontal line is from the same mouse. E. Relative tumor volume. To eliminate individual differences, we set each shGFP tumor volume as “1”, and compare shDNAJB1 tumor volume with shGFP tumor from same mouse. Average folds were present. Error bar was drawn by Excel “STDEV” method. Tumor volumes were measured (Table S2) and analyzed by one way ANOVA. Double asterisk (**) means p b 0.01.
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and inhibits its E3 ligase activity against p53 (Fig. 4B), but MDM2 ubiquitination was not interrupted (Fig. 3A). The second observation from the MDM2 C462A mice was that the Mdm2–p53 physical interaction, in the absence of Mdm2-mediated p53 ubiquitination, could not sufficiently control p53 activity and allow early mouse embryonic development. Our data show that DNAJB1 stabilizes p53 but does not suppress its transcriptional activity, as DNAJB1 also attenuated the MDM2-mediated ubiquitination of p53. Recently, it was reported that MDM2 binding partners also serve as substrates of MDM2 and competitively inhibit MDM2 E3 ligase activity, such as ribosomal protein S7 [29,30]. When co-transfected with MDM2, DNAJB1 showed clear ubiquitinated forms (Fig. S2). When co-transfected with MDM2/MDMX and p53, DNAJB1 competed with p53 as a substrate of MDM2 (Fig. 4B). Another potential possibility is that DNAJB1 inhibits MDM2 E3 ligase activity by changing the conformation of MDM2. It was reported that peptide ligands that compete for binding to the acidic domain of MDM2 can inhibit MDM2 E3 ligase activity both in vitro and in cells [31]. The binding of DNAJB1 to the acidic domain of MDM2 might change the conformation of MDM2, thereby inhibiting its E3 ligase activity. Due to technological constraints, we were unable to examine this possibility. One of the functions of heat shock proteins is to bind to unfolded proteins and refold them or promote their degradation. DNAJ members work with Hsp70 proteins, and binding and release by Hsp70 are coordinated via an array of co-chaperones that tightly regulate the Hsp70 ATP hydrolytic cycle [32–34]. Once released, the polypeptide can fold into its native conformation or remain misfolded and reenter the cycle for subsequent rounds of refolding. However, in some instances, Hsp70-bound clients are recognized by the E3 ubiquitin ligase CHIP (carboxyl terminus of Hsp70-interacting protein) and are targeted for degradation by the ubiquitin–proteasome system [35]. This process is initiated when a non-native polypeptide is bound by an Hsp40 cochaperone. Without Hsp70, DNAJ members can stabilize substrates, but they cannot refold or degrade them [36,37]. Our data support this model. DNAJB1 stabilizes MDM2 in Hsp70-independent manner. Surprisingly, comparing to DNAJB1 only co-transfection, MDM2 protein level is lower when Hsp70 is present (Fig. 2D). An MDM2 mutant, in which all of the lysine residues were replaced with arginine, was co-transfected with DNAJB1 and Hsp70. This mutant could not be degraded by the DNAJB1-Hsp70 system because of the ubiquitination site mutations (Fig. S3). In this study, DNAJB1 was identified as a new regulator of MDM2 stability. p53 and its downstream pathways were investigated as well. Ongoing studies in primary cells should help us to understand how the DNAJB1/Hsp70 system works with MDM2/p53 during cancer initiation. Acknowledgements The Authors thank Prof. Zhimin Yuan for project design and insightful comments. This work was supported by grant 81201240 from the National Natural Science Foundation of China (Weiqi Zeng), and the National Natural Science Funds 81225013 for Distinguished Young Scholar (Xiang Chen). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bbagrm.2013.12.003. References [1] K. Harms, S. Nozell, X. Chen, The common and distinct target genes of the p53 family transcription factors, Cell. Mol. Life Sci. 61 (2004) 822–842. [2] D.R. Green, J.E. Chipuk, p53 and metabolism: inside the TIGAR, Cell 126 (2006) 30–32.
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Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbagrm
DNAJB1 stabilizes MDM2 and contributes to cancer cell proliferation in a p53-dependent manner☆ Min Qi b, Jianglin Zhang a, Weiqi Zeng a,⁎, Xiang Chen a,⁎⁎ a b
Department of Dermatology, Xiangya Hospital, Central South University, Changsha, Hunan, PR China Department of Plastic Surgery, Xiangya Hospital, Central South University, Changsha, Hunan, PR China
a r t i c l e
i n f o
Article history: Received 20 October 2013 Received in revised form 29 November 2013 Accepted 12 December 2013 Available online 19 December 2013 Keywords: DNAJB1 MDM2 p53 p21 HSP70
a b s t r a c t Both MDM2 and MDMX regulate p53, but these proteins play different roles in this process. To clarify the difference, we performed a yeast 2 hybrid (Y2H) screen using the MDM2 acidic domain as bait. DNAJB1 was found to specifically bind to MDM2, but not MDMX, in vitro and in vivo. Further investigation revealed that DNAJB1 stabilizes MDM2 at the post-translational level. The C-terminus of DNAJB1 is essential for its interaction with MDM2 and for MDM2 accumulation. MDM2 was degraded faster by a ubiquitin-mediated pathway when DNAJB1 was depleted. DNAJB1 inhibited the MDM2-mediated ubiquitination and degradation of p53 and contributed to p53 activation in cancer cells. Depletion of DNAJB1 in cancer cells inhibited activity of the p53 pathway, enhanced the activity of the Rb/E2F pathway, and promoted cancer cell growth in vitro and in vivo. This function was p53 dependent, and either human papillomavirus (HPV) E6 protein or siRNA against p53 was able to block the contribution caused by DNAJB1 depletion. In this study, we discovered a new MDM2 interacting protein, DNAJB1, and provided evidence to support its p53-dependent tumor suppressor function. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The transcription factor p53 responds to various stresses, including DNA damage, overexpressed oncogenes and various metabolic limitations, and it regulates many target genes that can induce cell-cycle arrest, apoptosis, senescence and DNA repair or that can alter metabolism [1,2]. One well-known p53 target, p21, is upregulated upon p53 activation [3]. p21 is able to bind and inhibit the activity of the cyclin/CDK complexes, thereby reducing RB/E2F1 transcription ability [4]. In this way, the p53–p21 axis regulates the cell cycle and can arrest damaged cells at the G1/S checkpoint. In most cancer cells, this pathway can be inactivated through several different mechanisms. p53 mutation is the most common mechanism, and mutated p53 has been found in more than 50% of cancer patients [5]. In many tumors that express wild-type p53, the function of p53 can be compromised by viral oncogenes, such as the papillomavirus E6 and adenovirus E1B proteins, that induce p53 degradation [6,7]. Similarly, MDM2, an endogenous E3 ligase for p53 that is also responsible for the degradation of p53, is clearly a clinically relevant cellular oncogene. Within 5 years of its discovery as an amplified gene in a transformed murine cell line, it was found to be amplified in 7.2% of 3889 ☆ Conflict of interest: The authors declare no conflict of interest. ⁎ Correspondence to: W. Zeng, 87 Xiangya Road, Department of Dermatology, Xiangya Hospital, Central South University, Changsha, Hunan, PR China. Tel.: +86 158 7498 3697; fax: +86 731 8432 8478. ⁎⁎ Correspondence to: X. Chen, 87 Xiangya Road, Department of Dermatology, Xiangya Hospital, Central South University, Changsha, Hunan, PR China. Tel.: +86 731 8432 7128; fax: +86 731 8432 8478. E-mail addresses: [email protected] (W. Zeng), [email protected] (X. Chen). 1874-9399/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbagrm.2013.12.003
human tumors that lacked p53 mutations. MDM2 amplification was more recently reported in approximately 10.5% of 7711 tumors [5]. Mouse models established MDM2 and MDMX as essential p53 regulators [8–12]. MDM2 and MDMX protein sequences harbor three regions of high identity, dubbed CR1, CR2, and CR3. In MDM2, CR1 (residues 42 to 94) is responsible for binding to p53 and inhibiting its transactivation function. CR2 (residues 301 to 329) encodes a putative zinc-binding domain and partially overlaps with a region required for binding to the retinoblastoma tumor suppressor protein (RB). CR3 (residues 444 to 483) encodes the ring finger domain (RFD), which binds two Zn atoms and contains a cysteine residue (residue 464) required for ubiquitin conjugation to p53. In the region between CR1 and CR2 of MDM2, there is a nuclear localization/export sequence (NLS/NES) and an acidic domain (AD) that are not highly conserved in MDMX. The MDM2 acidic domain consists of 81 amino acids; a previous study from Dr. Yuan's group revealed that the MDM2 AD is unique and essential for p53 ubiquitination, as this function cannot be rescued by the MDMX AD domain [13]. DnaJ/Hsp40 (heat shock protein 40) proteins are important for protein translation, folding, unfolding, translocation, and degradation, primarily by stimulating the ATPase activity of the chaperone proteins, Hsp70s. Because ATP hydrolysis is essential for the activity of the Hsp70s, DnaJ/Hsp40 proteins actually determine the activity of the Hsp70s by stabilizing their interaction with the substrate proteins [14]. The human DNAJ family has over 40 members [14,15], and can be subdivided into three subfamilies, DNAJA, DNAJB and DNAJC. Recent reports have described the involvement of some Hsp40 family members of distinct classes, such as hTid I (class DNAJA3) and HLJ1 (class
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DNAJB4), in the modulation of tumor growth, and these findings are opening new frontiers for studies on this family [16–22]. DNAJB1 has been linked to several cellular processes, such as the proteasome pathway [23], endoplasmic reticulum (ER) stress [24], and virus infection [25], while few publications mention its function in cancer progression. In the current study, DNAJB1 was first identified as an MDM2 AD binding protein through an Y2H screen. Further experiments revealed its function in p53 ubiquitination and degradation, p53-dependent cell cycle regulation, and tumor progression.
The transformants with non-interacting protein pairs were able to grow on media lacking leucine and tryptophan (SD/-Leu/-Trp, -2 SD), while only positive clones containing interacting prey and bait proteins were able to grow on dropout medium lacking tryptophan, leucine, histidine, and adenine (SD/-Leu/-Trp/-His/-Ade, -4 SD). The positive colonies were lysed and subjected to the ortho-nitrophenyl-β-Dgalactopyranoside (ONPG) assay to verify the interaction. Finally, the confirmed positive colonies were selected, and the individual plasmids were amplified, purified, and analyzed by sequencing.
2. Materials and methods
2.2. PCR, quantitative-PCR and cloning
2.1. Yeast two hybrid screen
Oligonucleotides were synthesized by Invitrogen and are listed in Table S1. Quantitative-PCR (Q-PCR) was performed using the SYBR® Green Master Mix kit (4367659, Invitrogen) and an ABI 7500 instrument. All plasmids were constructed using restriction enzyme digestion.
A human fetal cDNA library and yeast two-hybrid system were purchased from Clontech (Cat. 637242). The MDM2 acidic domain was amplified and inserted into the pGBKT7 plasmid to yield pGBKT7-MDM2 AD. This recombinant plasmid was transformed into the AH109 yeast strain and was transformed with the human fetal brain cDNA library plasmids. After screening according to the manufacturer's instructions, the prey plasmids were purified from the positive clones, and the yeast was co-transformed with pGBKT7-BSG and the prey plasmid to verify the interaction. These transformants containing both bait and prey plasmids were plated on synthetic defined dropout medium.
2.3. Cell culture, transfection and reagents MCF7, U2OS and 293T cells were purchased from the American Type Culture Collection (ATCC) and were maintained in their corresponding media as previously described [13]. MCF7-E6 cells were created and maintained following the published methods [38]. All cell culture reagents were purchased from Gibco, and all other reagents were
Fig. 1. The C-terminus of DNAJB1 binds and stabilizes MDM2. A. MDM2 and DNAJB1 protein domain information. For MDM2, CR1 and CR2 denote conserved regions found in both MDM2 and MDMX; NLS/NES denotes nuclear localization/export sequence; AD is the acidic domain; L23 is a linker region; and RFD denotes the ring finger domain. For DNAJB1, J domain is an identical domain found in all DNAJ members; SBD denotes the substrate-binding domain. The region of DNAJB1 used in the Y2H screen is indicated. B. DNAJB1 binds MDM2 in vitro. The DNAJB1-BD (GAL4 binding domain) recombinant protein expression plasmid was co-transformed with the empty vector (EV-AD, AD denotes GAL4 activation domain), MDMXAD or MDM2-AD. In -2 SD, the appearance of a colony indicates that the transformants harbor both plasmids. In -4 SD, the appearance of a colony indicates that there was a positive interaction between both proteins. The ONPG assay is a method of β-galactosidase detection. A darker yellow color indicates that more β-galactosidase was present and reflects a stronger protein interaction. C. IP results revealed the endogenous DNAJB1–MDM2 interaction. D. DNAJB1 co-localized with MDM2 in the nucleus. DNAJB1 was labeled with Alexa Fluor 594 (red), and MDM2 was labeled with Alexa Fluor 488 (green). 4′,6-Diamidino-2-phenylindole (DAPI) was used for nuclear staining (blue). E. Exogenous DNAJB1 interacted with MDM2 but not with MDMX. WL denotes whole lysate. The molecular weight of flag-tagged DNAJB is similar to that of IgG; thus, the bands are indicated with arrowheads. F. The C-terminus, but not the N-terminus, of DNAJB1 binds and stabilizes MDM2.
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purchased from Sigma except indicated. Transfections were performed with Lipofectamine 2000 (Invitrogen) according to the standard instructions. In each experiment, the amounts of the transfected plasmids were consistent, and an empty vector was used to compensate for any remaining amount. Each experiment was repeated three times.
2.4. Western blot analysis, IPs, IF and antibodies Western blot analysis, IPs and IF were performed according to described procedures [39]. The MDM2 (SC-965, 1:500), p53 (SC-126, 1:1000), MYC (SC-40, 1:2000), and DNAJB1 (sc-135943, 1:500) antibodies and Protein A/G PLUS agarose beads (SC-2003) were purchased from Santa Cruz Biotechnology. Flag (F4042, 1:5000, mouse), Flag (F2555, 1:1000, rabbit), and actin (A4700, 1:5000) antibodies and Anti-FLAG® M2 Magnetic Beads (M8823) were purchased from Sigma. The p21 (2946L, 1:300) antibody was purchased from Cell Signaling Technology. The MDMX (A300-287A, 1:1000) antibody was purchased from Bethyl Laboratories. The goat anti-mouse (SC-2005, 1:500) and goat anti-rabbit (SC-2004, 1:500) secondary antibodies were purchased from Santa Cruz Biotechnology. The secondary antibodies used for IF were purchased from Invitrogen, including Alexa Fluor 594 (A11020, 1:500) and Alexa Fluor 488 (A11034, 1:500). The blot images were taken using a ChemiDoc XRS+ system (BioRad). IF images were taken using a microscope (CX41-32RFL, OLYMPUS).
2.5. Cancer 10-pathway reporter arrays The Cancer 10-pathway Reporter kit (CCA-001L) was purchased from SABiosciences. The screening process was performed following the standard manual. In brief, MCF7 cells were seeded into 96-well plates at a density of 1 × 104 cells per well. Reverse transfections were performed when the cells were seeded. After 48 h, the cells were harvested and subjected to dual luciferase analysis, which was performed using the Dual-Luciferase® Reporter Assay System (E1960, Promega). 2.6. Virus packaging and infection The pLKO1 plasmid and helper vectors, VSVG and delta8.2, were purchased from Addgene (www.addgene.org). Virus packaging and MCF7 cell infection were performed following standard protocols from Addgene. Stable cell lines were created by selection with 1 μg/ml puromycin (P9620, Sigma) for 6 days. 2.7. Xenograft mouse models Xenograft tumor models were established according to a previously described procedure [40]. In brief, 5 × 106 MCF7 cells were subcutaneously injected into the hind limbs region of 6-week-old female BALB/C mice. To stimulate tumor growth, estradiol supplementation was provided in the form of 90-day release 0.25 mg estradiol pellets (Innovative
Fig. 2. DNAJB1 stabilizes MDM2 at a post-translational level. A. Depletion of DNAJB1 by shRNA in MCF7 cells decreased MDM2 protein levels. shGFP is a sequence that does not target endogenous transcripts in the mammalian genome and was used as a control. Both sh1 and sh2 were designed against the human DNAJB1 mRNA at different sites. B. DNAJB1 and MDM2 mRNA levels in the indicated cells as detected by RT-PCR. The error bars were calculated from three replicates. C. MDM2 (1 μg) was co-transfected with 0.5 μg of the indicated plasmids in each 35-mm culture dish. EV denotes an empty vector; FL, N or C denotes full length, N-terminus or C-terminus of DNAJB1, respectively; H32Q is a J domain function null mutation. D. Hsp70 only weakly stabilized MDM2. MDM2 (1 μg) was co-transfected with 0.5 μg of DNAJB1, 0.5 μg of Hsp70 or both. E. DNAJB1 stabilized MDM2 in a dose-dependent manner. MDM2 (1 μg) was co-transfected with increasing amounts of DNAJB1 (0, 0.1, 0.2, 0.5, or 1 μg). F. MG132 recovers MDM2 in DNAJB1 depletion cells. The shDNAJB1 cell is infected with both sh1 and sh2 virus. Cells were treated with 10 μm MG132 for the indicated time and then harvested.
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Research of America, Sarasota, FL) placed in the interscapular region of the mice. The surgical incision was closed with a staple. After 52 days, the mice were euthanized for analysis. 3. Results 3.1. DNAJB1 binds to MDM2 in vitro and in vivo through its C-terminus and stabilizes MDM2 DNAJB1 was identified as a positive candidate through the Y2H screen. The sequencing results revealed that the C-terminus of DNAJB1 (from amino acids 167–340) interacted with the MDM2 acidic domain (Fig. 1A). This interaction was unique because the C-terminus of DNAJB1 could not interact with either the empty vector, which expressed the GAL4 activation domain (AD), or with MDMX-AD, which expressed the MDMX protein fused to the GAL4-AD (Fig. 1B). To verify the interaction in vivo, immunoprecipitations (IPs) were performed. MDM2 was precipitated with a DNAJB1 antibody, and DNAJB1 was also precipitated using an MDM2 antibody. p53 was precipitated in both reactions (Fig. 1C). Immunofluorescence (IF) was performed and revealed that DNAJB1 co-localized with MDM2 in the nucleus (Fig. 1D). Interestingly, when MDM2 was co-expressed with DNAJB1, it significantly
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accumulated (Fig. S4). Western blot analysis showed results consistent with the IF analysis, mainly that the level of exogenous MDM2 protein co-transfected with DNAJB1 was much higher than the expression of MDM2 alone. The stabilization of MDM2 by DNAJB1 was specific, as the MDMX protein level did not change in the presence or absence of DNAJB1 (Fig. 1E, WL panel). We performed immunoprecipitation experiments to test the exogenous MDM2 and DNAJB1 interaction and found that only MDM2 could be precipitated by the M2 beads, whereas MDMX could not (Fig. 1E). Another immunoprecipitation experiment showed that MDM2 was interacting with the C-terminus of DNAJB1, which was consistent with the Y2H results (Fig. 1F). 3.2. DNAJB1 stabilizes MDM2 at the post-translational level and slows MDM2-mediated degradation by ubiquitin pathway To investigate the stability of endogenous MDM2 protein in the presence or absence of DNAJB1, we depleted cells of DNAJB1 using a lentivirus-based small hairpin RNA (shRNA). Two shRNA constructs that targeted different sites on the DNAJB1 mRNA reduced the levels of DNAJB1 protein by over 70%, and MCF7 cells that were depleted of DNAJB1 had lower levels of MDM2 compared to the control shGFP cells (Fig. 2A). The same experiment was performed in U2OS cells, and
Fig. 3. DNAJB1 stabilized p53 in an MDM2-dependent manner. A. Exogenous DNAJB1 stabilized MDM2, MDMX and p53 and led to an increase in p21 levels. Increasing amounts of DNAJB1 (0, 0.1, 0.2, 0.5, 1, or 2 μg/35-mm dish) were transfected into MCF7 cells. B. MCF7 cells were treated with cycloheximide (10 μg/ml) for the indicated times. Cell lysates were subjected to Western blot analysis,half-life time of p53 was labeled with dark arrows. The p53 blot was scanned and analyzed. (C) and (D) were performed as in (B), with the addition of the nutlin-3 or siMDM2 treatments, respectively.
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the MDM2 level consistently decreased (Fig. S1). Next, real-time PCR was performed to examine the levels of MDM2 mRNA. MDM2 mRNA levels did not change with or without DNAJB1 (Fig. 2B). This result suggested that DNAJB1 regulates MDM2 at a post-translational level. DNAJB1 has a J domain at its N-terminus that harbors the “HPD” amino acid sequence, which is essential for DNAJB1's co-chaperone function, as this is its binding site with Hsp70 [14]. The H32Q mutant of DNAJB1 cannot activate Hsp70. Hsp70 interaction was dispensable for the DNAJB1-mediated accumulation of MDM2; the C-terminus of DNAJB1 alone was sufficient to stabilize MDM2 (Fig. 2C). MDM2 was co-transfected with DNAJB1 only, with Hsp70 only, or with both. Hsp70 could only weakly stabilize MDM2. Interestingly, the level of MDM2 was lower when both DNAJB1 and Hsp70 were co-transfected compared to co-transfection with DNAJB1 only (Fig. 2D). A dose-dependent effect was observed when MDM2 was cotransfected with increasing amounts of DNAJB1 (Fig. 2E). Because MDM2 is mainly degraded through the ubiquitin pathway, the proteasome inhibitor MG132 was added to culture media to block ubiquitin– proteasome dependent protein degradation. MDM2 protein levels did not change in the shGFP group, but MDM2 levels were fully rescued in the DNAJB1 depleted group after 60 min. This result suggested that MDM2 was degraded faster when DNAJB1 was depleted. Overexpression of exogenous DNAJB1 in MCF7 cells also stabilized MDM2, as well as its ubiquitinated forms (Fig. 3A). 3.3. DNAJB1 stabilizes p53 in an MDM2-dependent manner The classical role of MDM2 is a p53 regulator; therefore, investigating the level and activity of p53 is important to understanding
DNAJB1. MCF7 cells were transiently transfected with increasing amounts of DNAJB1. In addition to the stabilization of MDM2, MDMX and p53 were also stabilized. The accumulation of p21 showed that the transcriptional activity of p53 also increased (Fig. 3A). Cycloheximide (CHX), an mRNA translation inhibitor, was added to the culture media to block protein synthesis. Both MDM2 and p53 protein levels decreased much faster in the DNAJB1-depleted group. In the control group, the p53 half-life was approximately 1 h, while its half-life was only 30 min in the shDNAJB1 group (Fig. 3B). To clarify whether the faster degradation of p53 was MDM2-dependent, two methods were used to block MDM2 E3 ligase activity. First, nutlin-3, which is a small compound that specifically interrupts the interaction between MDM2 and p53, was added to the culture media along with CHX. In both groups, p53 protein showed similar degradation rates (Fig. 3C). Second, small interfering RNA (siRNA) against MDM2 was used to deplete MCF7 cells of MDM2. When MDM2 protein was eliminated, DNAJB1 depletion did not affect p53 degradation (Fig. 3D). 3.4. DNAJB1 decreases MDM2-mediated p53 ubiquitination and enhances p53 transcriptional activity To investigate the status of p53 when DNAJB1 is bound to MDM2, immunoprecipitation experiments were performed. Flag-tagged p53 was used to pull down DNAJB1, and increasing amounts of DNAJB1 were precipitated when more DNAJB1 was transfected (Fig. 4A). This result suggested that DNAJB1 binds to MDM2, but MDM2 does not release p53. Even the binding affinity of MDM2 to p53 did not change, but the degradation of p53 was slower. Therefore, it is possible that DNAJB1 reduces MDM2 E3 ligase activity. MDM2, MDMX, DNAJB1 and p53
Fig. 4. DNAJB1 attenuated p53 ubiquitination and increased p21 levels in a p53-dependent manner. A. MDM2 plasmids (2 μg) and 2 μg of Flag-p53 were co-transfected with the indicated amounts of DNAJB1 (0, 0, 0.2, 0.5, or 1 μg) into 293T cells. IPs were performed using M5 beads. B. MYC-p53 (0.5 μg), MDM2 (2 μg), Flag-MDMX (1 μg) and DNAJB1 (0.5 μg) were transfected into 293T cells, and the protein levels of p53, ubiquitinated p53 and DNAJB1 were examined. C. MCF7 wild-type cells and MCF7-E6 cells were infected with a lentivirus to deplete endogenous DNAJB1. The levels of the indicated proteins were examined. D. MCF wild-type cells and MCF7-E6 cells were transfected with DNAJB1 expression plasmids. The levels of the indicated proteins were examined. E. DNAJB1 was introduced into MCF7 cells with or without siRNA against p53.
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were co-transfected in the indicated combinations, and p53 was efficiently degraded by the MDM2/MDMX complex (Fig. 4B). When DNAJB1 was introduced, the levels of p53 protein partially recovered, and the levels of ubiquitinated p53 were attenuated (Fig. 4B). To understand how DNAJB1 function is mediated by the MDM2–p53 axis, p21 levels were investigated in p53 wild-type (WT) or inactivated (E6 protein overexpression) MCF7 cells. p21 protein was reduced in p53 WT cells, but not E6 cells, when DNAJB1 was depleted (Fig. 4C). Correspondingly, overexpression of DNAJB1 increased p21 levels in p53 WT cells but not in E6 cells (Fig. 4D). Depletion of p53 completely blocked p21 accumulation (Fig. 4D). 3.5. DNAJB1 contributes to cancer cell growth in vitro and in vivo in a p53-dependent manner The activities of 10 pathways that are commonly associated with cancer progression were tested. Depletion of DNAJB1 in MCF7 cells altered the activity of 4 pathways. The reduction of p53 and the induction of the RB/E2F pathway were interesting results (Fig. 5A). As mentioned previously, the RB/E2F pathway works downstream of p53 to regulate the cell cycle. Thus, we checked the proliferation of both the
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MCF7-WT and MCF7-E6 cells with or without endogenous DNAJB1. DNAJB1-depleted MCF7 cells grew faster than the control cells when WT p53 was present (Fig. 5B, upper panel) but showed a similar growth rate as the control cells when p53 was inactivated (Fig. 5B, lower panel). To verify the data in vivo, nude mice were used to test xenograft tumor formation. The tumor masses of the DNAJB1-depleted cells were 2.5 folds larger than those formed by the control cells (Fig. 5C, D and E). 4. Discussion The MDM2/MDMX/p53 pathway has been extensively investigated, and it is known that MDM2 stabilization is important for the p53 response to oncogene stress. Dr. Zhang's group developed the MDM2 C462A mouse strain, which has an inactive MDM2 E3 ligase, and reported 2 observations [26]. The first observation was that MDM2's E3 function was not required for MDM2 degradation. Thus, MDM2 was degraded by an unidentified pathway. Two groups reported that the MDM2 AD domain contributed to the regulation of MDM2 stability and provided evidence that casein kinase I phosphorylation regulated MDM2 turnover by the SCFβ-TRCP ubiquitin ligase [27,28]. Our data support this model. We showed that DNAJB1 binds to MDM2
Fig. 5. DNAJB1 inhibited MCF7 growth in vitro and in vivo. A. The cancer-10 pathway array was performed according to the manufacturer's protocol. The p53 and RB/E2F pathways are labeled with triangles. One-way ANOVA was down to assess the statistical significance. Single asterisk (*) means p b 0.05, and double asterisk (**) means p b 0.01. B. MCF7 and MCF7-E6 cells were infected with the shDNAJB1 lentivirus and selected by puromycin to create a stable cell line. Cells were seeded into 96-well plates at a density of 1000 cells/well. Each day, three wells were counted. The experiments were repeated three times. C. Xenograft mouse models were created as described. For each mouse, the control cells were injected on the left side, and the DNAJB1-depleted cells were injected on the right side. Tumors were cycled by red line. D. Xenografts were isolated and arranged. Each horizontal line is from the same mouse. E. Relative tumor volume. To eliminate individual differences, we set each shGFP tumor volume as “1”, and compare shDNAJB1 tumor volume with shGFP tumor from same mouse. Average folds were present. Error bar was drawn by Excel “STDEV” method. Tumor volumes were measured (Table S2) and analyzed by one way ANOVA. Double asterisk (**) means p b 0.01.
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and inhibits its E3 ligase activity against p53 (Fig. 4B), but MDM2 ubiquitination was not interrupted (Fig. 3A). The second observation from the MDM2 C462A mice was that the Mdm2–p53 physical interaction, in the absence of Mdm2-mediated p53 ubiquitination, could not sufficiently control p53 activity and allow early mouse embryonic development. Our data show that DNAJB1 stabilizes p53 but does not suppress its transcriptional activity, as DNAJB1 also attenuated the MDM2-mediated ubiquitination of p53. Recently, it was reported that MDM2 binding partners also serve as substrates of MDM2 and competitively inhibit MDM2 E3 ligase activity, such as ribosomal protein S7 [29,30]. When co-transfected with MDM2, DNAJB1 showed clear ubiquitinated forms (Fig. S2). When co-transfected with MDM2/MDMX and p53, DNAJB1 competed with p53 as a substrate of MDM2 (Fig. 4B). Another potential possibility is that DNAJB1 inhibits MDM2 E3 ligase activity by changing the conformation of MDM2. It was reported that peptide ligands that compete for binding to the acidic domain of MDM2 can inhibit MDM2 E3 ligase activity both in vitro and in cells [31]. The binding of DNAJB1 to the acidic domain of MDM2 might change the conformation of MDM2, thereby inhibiting its E3 ligase activity. Due to technological constraints, we were unable to examine this possibility. One of the functions of heat shock proteins is to bind to unfolded proteins and refold them or promote their degradation. DNAJ members work with Hsp70 proteins, and binding and release by Hsp70 are coordinated via an array of co-chaperones that tightly regulate the Hsp70 ATP hydrolytic cycle [32–34]. Once released, the polypeptide can fold into its native conformation or remain misfolded and reenter the cycle for subsequent rounds of refolding. However, in some instances, Hsp70-bound clients are recognized by the E3 ubiquitin ligase CHIP (carboxyl terminus of Hsp70-interacting protein) and are targeted for degradation by the ubiquitin–proteasome system [35]. This process is initiated when a non-native polypeptide is bound by an Hsp40 cochaperone. Without Hsp70, DNAJ members can stabilize substrates, but they cannot refold or degrade them [36,37]. Our data support this model. DNAJB1 stabilizes MDM2 in Hsp70-independent manner. Surprisingly, comparing to DNAJB1 only co-transfection, MDM2 protein level is lower when Hsp70 is present (Fig. 2D). An MDM2 mutant, in which all of the lysine residues were replaced with arginine, was co-transfected with DNAJB1 and Hsp70. This mutant could not be degraded by the DNAJB1-Hsp70 system because of the ubiquitination site mutations (Fig. S3). In this study, DNAJB1 was identified as a new regulator of MDM2 stability. p53 and its downstream pathways were investigated as well. Ongoing studies in primary cells should help us to understand how the DNAJB1/Hsp70 system works with MDM2/p53 during cancer initiation. Acknowledgements The Authors thank Prof. Zhimin Yuan for project design and insightful comments. This work was supported by grant 81201240 from the National Natural Science Foundation of China (Weiqi Zeng), and the National Natural Science Funds 81225013 for Distinguished Young Scholar (Xiang Chen). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bbagrm.2013.12.003. References [1] K. Harms, S. Nozell, X. Chen, The common and distinct target genes of the p53 family transcription factors, Cell. Mol. Life Sci. 61 (2004) 822–842. [2] D.R. Green, J.E. Chipuk, p53 and metabolism: inside the TIGAR, Cell 126 (2006) 30–32.
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