- Email: [email protected]
PIM1-catalyzed CBX8 phosphorylation promotes the oncogene-induced senescence of human diploid fibroblast
Biochemical and Biophysical Research Communications 501 (2018) 779e785
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
PIM1-catalyzed CBX8 phosphorylation promotes the oncogene-induced senescence of human diploid fibroblast Xiangwen Zhan a, Jianming Yang b, Zebin Mao a, Wenhua Yu a, * a
Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, 100191, People's Republic of China b Department of Immunology, School of Basic Medical Science, Tianjing Medical University, People's Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 May 2018 Accepted 11 May 2018
The proto-oncogene PIM1 encodes Ser/Thr kinase and regulates cell growth, differentiation and apoptosis. However, more and more studies including ours have found that PIM1 can induce senescence in normal human diploid fibroblasts and behave as a tumor suppressor. But the relevant molecular mechanism of this process is not yet clear. It has been reported that Chromobox homolog 8 (CBX8) binds directly to INK4A as a transcriptional repressor, thereby suppressing stress-induced senescence. Here we report that PIM1 can phosphorylate CBX8 to promote its degradation, thereby up-regulating p16, during PIM1-induced cell senescence. Overexpression of CBX8 can inhibit PIM1-induced cell senescence. These data suggest that to promote CBX8 degradation may be an important molecular mechanism of PIM1induced cell senescence. © 2018 Elsevier Inc. All rights reserved.
Keywords: PIM1 CBX8 Phosphorylation Senescence
1. Introduction Cellular senescence is a permanent cell cycle arrest triggered by various stimuli including oncogene activation, telomere shortening, genetoxic insults and other detrimental growth conditions. About two decades ago, Serrano et al. observed normal fibroblasts with ectopic expression of RAS entered senescence different from replicative senescence without telomere shortening [1], termed oncogene-induced senescence (OIS) [2]. Subsequent research demonstrated OIS acted as a barrier preventing tumor formation. OIS was associated with signs of DNA replication stress including prematurely terminated DNA replication forks and DNA doublestrand breaks [3]. But the following molecular events involved in induction of senescence have not been elucidated very clearly. PIM1 gene was originally identified as an oncogene in murine leukemia virus-induced lymphomas, which was activated transcriptionally by the proviral insertion, encoding a constitutively active serine/threonine protein kinase. Plentiful research demonstrated PIM1 promoted tumor progression through regulation of proliferation, differentiation, survival and apoptosis cooperating with other oncogenes such as Myc [4], Bcl2 [5] and GFI-1 [6].
* Corresponding author. Department of Biochemistry and Molecular Biology Peking University Health Science Center, 38 Xue Yuan Road, Beijing, 100191, China. E-mail address: [email protected] (W. Yu). https://doi.org/10.1016/j.bbrc.2018.05.070 0006-291X/© 2018 Elsevier Inc. All rights reserved.
Interestingly, more and more studies have recently reported that PIM1 can induce cell senescence and inhibit proliferation, thus showing tumor suppressor properties. In 2008, Hogan et al. reported that overexpression of PIM1 in normal fibroblasts can induce senescence rather than enhance growth in a p53-dependent manner [7]. Our lab also reported later that PIM1 induced cellular senescence through phosphorylating UHRF1 and HP1g, which led to DNA methylation [8] and chromatin remodeling [9], respectively. Furthermore, overexpression of PIM1 can inhibit cell and tumor growth specifically in 22Rv1 human prostate cells by inducing marked increases in cellular senescence [10]. These data indicated PIM1's role in a certain cell may depend on its environment context. CBX8 is chromobox homolog 8 (CBX8) protein belonging to Polycomb Group (PcG) proteins which were originally discovered as a crucial regulator of development in Drosophila [11]. The PcG proteins form two distinct multimeric protein complexes named as polycomb repressive complexes 1 and 2 (PRC1 and PCRC2), which play significant roles in a wide variety of biological processes, including pluripotency, differentiation, senescence, carcinogenesis and so on [12,13]. Chromobox homolog 8 (CBX8) has been identified as a transcriptional repressor that functionally associate with RING1, BMI1 and MLL-ENL oncoprotein [14,15]. CBX8 is closely related to tumorigenesis and tumor progression, while the understanding of its role in cellular senescense is fewer than that in
780
X. Zhan et al. / Biochemical and Biophysical Research Communications 501 (2018) 779e785
purchased full-length PIM1 or purified mPIMK67M, and added 10 mM ATP in kinase buffer (Cell Signaling) for 30 min at 30 C. Western blot analysis was performed using p-Ser antibody.
tumors. A classic research reported that CBX8 can directly bind to and repress the INK4A-ARF locus and therefore bypassing cellular senescence in fibroblasts [16]. In addition, the relationship between CBX8 and senescence has also been reported in the inhibition of Sirtinol-induced premature aging in certain tumor cells [17,18]. Our study characterizes CBX8 as a new substrate of PIM1 kinase. CBX8 phosphorylation by PIM1 can lead to its destabilization and degradation, thereby promoting p16 expression during oncogeneinduced cell senescence. Overexpression of CBX8 shows counteractive effect on PIM1-induced cell senescence. These results indicate that CBX8 is involved in PIM1-induced cell senescence and his down-regulation is an important molecular mechanism in the process of cell senescence.
ChIP analysis was performed as previously described [20]. The antibodies used was purchased from Bethyl, while the immunoprecipitated DNA was quantified by real-time PCR. ChIP primers were designed in the region 200bp after first exon of IN4A and primer pairs only amplify one amplicon. Sequences was followed: 50 -GCCAAGGAAGAGGAATGAGGAG-30 ; 50 -CCTTCAGATCTTCTCA GCATTCG-30 .
2. Materials and methods
2.7. SA-b-gal staining
2.1. Cell culture and reagent
SA-b-gal activity staining was performed as described previously [21]. All cells were examined using fluorescence microscopy with the appropriate filters.
HEK293T cells and Human diploid fibroblasts 2BS cells were obtained from the National Institute of Biological Products, Beijing, China. These cells were cultured in complete Dulbecco's Modified Eagle Medium with 10% fetal bovine serum (FBS), streptomycin (100 mg/ml), penicillin (100 U/ml), and maintained in a humidified atmosphere of 5% CO2 at 37 C. MG132 (M8699; Sigma) was dissolved in dimethyl sulfoxide (DMSO; C6295; Sigma) and added to the culture medium at 20 mM for the indicated time periods. A corresponding volume of DMSO was added to untreated control cells. 2.2. Western blot analysis Western blot analysis was performed using standard method. Specific primary antibodies against PIM1 (3247, Cell Signaling), CBX8 (A300-882 A, Bethyl), Phosphoserine (ab9332, Abcam), p16 (sc-468, Santa Cruz), b-actin (PM053; MBL). 2.3. Immunoprecipitation, silver staining and mass spectrometry Cell lysate enriched overnight with M2 beads at 4 C. Beads were washed five times with lysis buffer and resolved on NuPAGE 4e12% Bis-Tris gel (Invitrogen), silver stained using Pierce silver stain kit (Thermo) and subjected to LCeMS/MS analysis. 2.4. Co-immunoprecipitation and in vivo phosphorylation Endogenous PIM1 or CBX8 was immunoprecipitated with antiPIM1 or anti-CBX8 and protein A-Sepharose beads. After extensive washing and heating in the sample buffer, the complexes were subjected to immunoblot [19]. Cell lysates were subjected to immunoprecipitation (IP) with CBX8 antibody (Endogenous) or gst-specific beads (Exogenous transfection, GST-CBX8). Aliquots of immunoprecipitation were subjected to Western blotting using pSer antibody. 2.5. GST pull-down and in vitro kinase assay GST and GST-CBX8 or His-PIM1 or His-mPIM1K67M were purified from Transetta (DE3) cells using glutathione Sepharose or Ni Sepharose. Co-incubate equal amounts of GST or GST-CBX8 (200 ng/each) with His-PIM1 (200 ng) in BC100 buffer overnight at 4 C, adding Glutathione-sepharose beads for 2 h. The mixture (resin and binding proteins) was washed three times by centrifugation at 500 g for 3 min to remove the unbound proteins and was analyzed by western blotting using anti-GST and anti-His. For in vitro kinase assay, Co-incubate purified GST-CBX8 with
2.6. Chromatin immunoprecipitation (ChIP)
2.8. Cell proliferation assay The CCK-8, colony formation and EdU incorporation assays were adopted to evaluate the cell proliferation. For the CCK-8 assay, CCK8 kit (CCK-8; Dojindo Laboratories, Japan) was used according to the manufacturer's instructions. 3 103 cells were planted into 96-well dish with three replicates. Then, 100 ml of serum-free cell culture medium containing 10 ml WST-8 reagent was added into each well every 24 h and the plates were incubated at standard conditions for 1 h. Optical absorbance of each well at 450 nm and 630 nm were measured with a microplate reader (Bio-Rad Laboratories, USA). Three independent experiments were performed for quantification. For colony formation assay, cells were plated in 6-well plates in triplicates (400 cells per well). After 10 days, the cells were washed with PBS, fixed with 4% formaldehyde for 10min and stained with crystal violet staining solution (Sigma-Aldrich) for 15min. The colonies were counted and photographed. For the EdU incorporation assay, EdU Apollo®567 In Vitro Imaging Kit (Ribo Bio, China) was used according to the manufacturer's instructions except that cell nuclei were stained with DAPI instead of Hoechst for 30 min. Five fields of each well were randomly chosen and observed under fluorescence microscopy. All images were processed using Image J software and the proportion of EdU incorporated cells was calculated. Three independent experiments were performed for quantification. 3. Result 3.1. PIM1 interacts with CBX8 To explore the mechanism by which PIM1 induces cell senescence, we employed immunopurification and mass spectrometry to identify proteins that are potentially associated with PIM1 in 2BS cells (human embryonic lung fibroblasts, a cell model commonly used in studies of replicative senescence [22,23]). To this end, FLAGPIM1 was transiently expressed in 2BS cells. Cellular extracts were prepared and subjected to affinity purification using anti-FLAG affinity gels. The eluted protein complex was then resolved on SDSPAGE, silver stained, and subjected to LC-MS/MS analysis (Fig. 1A). The results showed that PIM1 was co-purified with a number of proteins, including CBX8, as well as other known interacting proteins of PIM1 such as SND1 and HSP90 [24,25]. To confirm that CBX8 is physically associated with PIM1 in cultured cells, we co-transfected 293 T cells with GST-CBX8 and
X. Zhan et al. / Biochemical and Biophysical Research Communications 501 (2018) 779e785
781
Fig. 1. PIM1 interacts with CBX8. (A) Mass Spectrometry Detection of PIM1-associated Proteins. 2BS cells were infected with Flag-PIM1 using a lentiviral expression system. The cell lysates were immunoprecipitated with FLAG-M2 beads and separated by SDS-PAGE electrophoresis, silver stained, and the differential bands were sent for mass spectrometry analysis. (B) PIM1 interacts with CBX8 in cultured cells. Co-transfected GST-CBX8 and FLAG-PIM1 plasmids in 293 T cells, co-immunoprecipitation experiment was performed by using the indicated antibodies. (C) PIM1 interacts with CBX8 in vivo. In RAS-induced 2BS cells, co-immunoprecipitation was performed using antibodies against PIM1 and CBX8, respectively. Western blotting was used to detect precipitated complexes by antibodies against CBX8 and PIM1. (D) PIM1 interacts with CBX8 directly in vitro. GST pull-down experiments were performed with bacterially expressed GST-CBX8 and 6xHis-PIM1.
FLAG-PIM1 plasmids, and use the whole cell lysate for the coimmunoprecipitation (Co-IP) and western blotting analysis. The results showed that FLAG-PIM1 associated with GST-CBX8 (Fig. 1B). To further verify whether endogenous CBX8 and PIM1 interact with each other, co-IP experiments were performed in extracts of senescent cells induced by RasV12 as PIM1 expression was upregulated in RasV12-induced senescent cells [9,26]. CBX8 was observed to co-immunoprecipitate with PIM1 (Fig. 1C). In reciprocal immunoprecipitations, PIM1 was observed presenting in CBX8 immunoprecipitates (Fig. 1C). As our data provide not only exogenous but also endogenous evidences for the interaction between PIM1 and CBX8, the GST pull-down assays was used to confirm whether this interaction is direct, which was carried out by incubating the full-length of recombinant GSTeCBX8 with 6xHisPIM1. Free GST together with the full-length of recombinant GSTCBX8 and 6xHis-PIM1 were all expressed in prokaryotic cells and purified in vitro. As shown in Fig. 1D, 6xHis-PIM1 specifically interacted with full-length GST-CBX8, but not with free GST. Taken together, these results support the argument that PIM1 directly interacts with CBX8. 3.2. PIM1 phosphorylates CBX8 and promotes its degradation Since PIM1 is a constitutive kinase and we have already confirmed its direct binding with CBX8, our next step was to investigate if PIM1 could phosphorylate CBX8? To find the answer, we co-transfected GST-CBX8 with Flag-PIM1 or empty vector into 293 T cells and precipitated CBX8 with GST beads. The phosphorylation levels of the precipitated CBX8 were detected by pan-p-Ser
antibodies (Fig. 2A). Western blotting analysis showed that the phosphorylation level of Ser in CBX8 was significantly increased. To gain further support for the argument that CBX8 is a substrate of PIM1 kinase, GST-CBX8 was purified from prokaryotic cells, then incubated with PIM1 purchased from Abcam or mPIM1K67M, a catalytically inactive mutant (mPIM1K67M), purified from prokaryotic cells in the kinase assay buffer containing ATP. The result of this in vitro kinase assay showed Ser phosphorylation of CBX8 could only be detected in assays mixed with wtPIM1, GST-CBX8 and ATP and the three are indispensable for Ser phosphorylation of CBX8 (Fig. 2B), which indicate that CBX8 Ser phosphorylation is dependent on PIM1 kinase activity and the phosphoryl donor (ATP). To prove that such phosphorylation also occurs in endogenous CBX8, we infected wild-type PIM1 (wtPIM1) or a in 2BS cells by using a lentiviral expression system, then endogenous CBX8 was enriched by CBX8 antibody, and its phosphorylation level at Ser was detected by western blotting. Consistent with the in vitro results, the phosphorylation level of serine on CBX8 was significantly increased (Fig. 2C). During the investigation of the phosphorylation level of CBX8, we were surprised to find that the protein level of CBX8 gradually decreased with the overexpression of PIM1 in 2BS cells, while this decrease of CBX8 protein levels could not be observed in 2BS cells with mPIM1K67M overexpression (Fig. 2D). This result suggests that the reduction of CBX8 protein is PIM1's phosphorylation for CBX8, which is PIM1 kinase activity-dependent. To exclude the possibility that PIM1 induces CBX8 protein reduction at transcription level, realtime PCR was used to detect the mRNA level of CBX8, which showed no significant changes (Fig. 2E). This result suggest that CBX8 protein levels may be regulated by PIM1 at post-
782
X. Zhan et al. / Biochemical and Biophysical Research Communications 501 (2018) 779e785
Fig. 2. PIM1 phosphorylates CBX8 and promotes its degradation. (A) PIM1 phosphorylates CBX8 in 293 T cells. 293 T cells were Co-transfected with GST-CBX8 and Flag-PIM1 or empty vector, precipitating the cell lysate with GST-specific beads. Antibodies against p-Ser was used to detect the phosphorylation levels of CBX8. (B) In vitro kinase assay of CBX8 phosphorylation by PIM1. GST-CBX8 purified from prokaryotic cells was incubated with PIM1 purchased from Abcam or mPIM1K67M purified from prokaryotic cells in the kinase assay buffer containing ATP. Antibodies against p-Ser was used to detect the phosphorylation levels of CBX8. (C) CBX8 phosphorylation in PIM1-induced senescent cells. Immunoprecipitation was against by the antibody of CBX8, then antibodies against p-Ser was used to detect the phosphorylation levels of CBX8. (D) and (E) Changes in CBX8 in PIM1induced Cellular Senescence. Western Blot Analysis (D) and qRT-PCR (E) was used to detect the protein and mRNA levels of CBX8 on extracts of 2BS cells that were infected with wild-type PIM1 or mPIM1K67M. (F) PIM1 promotes CBX8 degradation. 2BS cells were infected with increasing amounts of wild-type PIM1 or mPIM1K67M. Changes in CBX8 were detected by immunoblotting after treatment with MG132.
translational level. To further support this proposition, MG132, which is a proteasome inhibitor and reduces the degradation of ubiquitin-conjugated proteins in mammalian cells, was added to 2BS cells infected with PIM1. As is shown in Fig. 2F, the protein level of CBX8 gradually decreased with the increase of PIM1 expression, and this decreasing of CBX8 protein was abolished when the degradation of ubiquitin-conjugated proteins was inhibited by MG132, while MG132 showed no effects on protein levels of CBX8 in 2BS cells with mPIM1K67M overexpression. These results fully demonstrate that PIM1 can phosphorylate CBX8 and hence promote its degradation.
3.3. PIM1 promotes p16 expression by down-regulating CBX8 during PIM1-induced cell senescence Previous studies have reported that CBX8 is a transcriptional repressor that binds directly to the region after first exon of INK4A (coding for p16), inhibiting the expression of p16 in human diploid fibroblasts, and knocking down CBX8 can promote cell senescence [27]. Recent studies in our laboratory show that p16 expression is up-regulated during PIM1-induced cell senescence [8,9]. Since our previous studies confirmed that PIM1 could interact directly with CBX8, catalyze its phosphorylation and hence promote its
X. Zhan et al. / Biochemical and Biophysical Research Communications 501 (2018) 779e785
degradation, it's reasonable to hypothesize that PIM1 promote the expression of p16 b y down-regulating CBX8 during PIM1-induced cell senescence? To prove this hypothesis, ChIP experiments was performed in PIM1-induced cellular senescent model of 2BS cells which was confirmed by the increased senescence-associated bgalactosidase (SA-b-gal) stain (Fig. 3A). Anti-CBX8 antibody was used for immuno-precipitating CBX8-bound-genome DNA fragments, and the enrichment of p16 gene was determined by quantitative PCR. The results of ChIP analysis show a significant reduction of the enrichment in the genomic region of p16 gene bound by CBX8 in PIM1 overexpressed cells as compared with that in control cells (Fig. 3B), while in the PIM1 kinase-dead mutant overexpressed cells, p16 gene regions enriched anti-CBX8 antibody increased obviously. At the same time, the p16 expression was further confirmed at mRNA levels and protein levels 2BS cells after exposure to PIM1 for 10 days by realtime PCR (Fig. 3C) and western blotting (Fig. 3D). Taken together, the upregulation of p16 mRNA and protein levels in PIM1-induced cell senescence may be mediated by the phosphorylation of CBX8 by PIM1.
783
3.4. CBX8 is involved in PIM1-induced cell senescence Previous studies in our laboratory have revealed that p16 expression is up-regulated during PIM1-induced cell senescence [8,9]. Dietrich et al. [16] reported that knocking down CBX8 can promote cell senescence, and that overexpression of CBX8 can bypass stress-induced cell senescence. In present studies, we have demonstrated that p16 is regulated by CBX8 during PIM1-induced senescence. However, there's still short of the direct evidence supporting for the relationship between CBX8 and the cellular phenotype of 2BS cells. To find such evidences, we co-infected PIM1 and CBX8 or the empty vector in 2BS cells, and observed the cell proliferation of these cells by CCK8, EdU incorporation and colony formation assays. PIM1 overexpression inhibited the growth of 2BS cells, while ectopic expression of CBX8 could obviously mitigate such growth inhibition by PIM1 as indicated in CCK8, EdU incorporation and colony formation assays (Fig. 4AeC). Furthermore, SAb-gal activity staining experiment showed that overexpressing CBX8 could make the percentage of stained cells decreasing as compared with that of cells with PIM1 overexpressing alone (Fig. 4D), which suggests that overexpression of CBX8 may effectively counteract PIM1-induced senescence. These data also support from the cellular level that CBX8 can mediate PIM1-induced senescence. 4. Discussion
Fig. 3. PIM1 promotes p16 expression by down-regulating CBX8 during PIM1induced cell senescence. (A) PIM1 induces cell senescence. SA-b-gal activity staining experiments showed strong staining compared to the empty vector control and mPIM1K67M groups. (B) CBX8 mediates PIM1-induced senescence. INK4A ChIP analysis in 2BS cell infected with wild-type PIM1 or mPIM1K67M using CBX8 antibodies. (C) and (D) Changes in p16 in PIM1-induced cellular senescence. Western Blot Analysis (C) and qRT-PCR (D) was used to detect the protein and mRNA levels of p16 on extracts of 2BS cells that were infected with wild-type PIM1 or mPIM1K67M.
The role of PIM1 in regulating both the growth and transformation of malignant cells is better characterized [28,29], while PIM1-induced cellular senescence was reported only a few years ago. PIM1 is a relative weak oncogene, and its functioning ways often depend on cooperating with the activation of synergistic genes in the organisms. PIM1 alone is not able to induce a massive proliferation [30]. Moreover, Zemskova et al. [10] also reported that PIM1 activates the p53 pathway and causes senescence of tumor cells in certain special prostate cancer cells. Hogan [7] and previous studies of our laboratory [8,9] also showed that the upregulation of PIM1, an oncogene, in normal fibroblasts caused cell senescence instead of promoting cell proliferation. Cellular senescence is considered to be a protective mechanism against caicinogenesis of cells [31e33]. To explore the molecular mechanism might have a potential application in clinic cancer treatment. In present study, we find that CBX8 can be phosphorylated by PIM1 kinase, and this phosphorylation promotes its degradation, which in turn upregulate p16, leading to cell senescence. That may be an important molecular mechanism in PIM1 induced cell senescence. Since PIM1 is a constitutive Ser/Thr-specific protein kinase whose function is mainly dependent on its kinase activity. Our studies discovered that CBX8 could be phosphorylated by PIM1. However, the specific phosphorylation sites of CBX8 catalyzed by PIM1 need to be determined. While serine/threonine kinases all phosphorylate serine or threonine residues in their substrates, they select specific residues to phosphorylate on the basis of residues that flank the phosphoacceptor site, which together comprise the consensus sequence. Since only the consensus sequence residues of a target substrate make contact with several key amino acids within the catalytic cleft of the kinase (usually through hydrophobic forces and ionic bonds), a kinase is usually not specific to a single substrate, but instead can phosphorylate a whole “substrate family” which share common recognition sequences. Through analysis of peptide substrates, PIM1 preferred consensus sequence have been established as R-x-R-x-x-S/T, which is further confirmed by the phosphorylation sites analysis in most known substrates of PIM1 [34e36]. We searched the amino acid sequence of CBX8 and found that the Ser311 and the residues flanking it is consistent with
784
X. Zhan et al. / Biochemical and Biophysical Research Communications 501 (2018) 779e785
Fig. 4. CBX8 is involved in PIM1-induced cell senescence. (AeC) Cell proliferation rates were rescued in 2BS cells co-expressing PIM1 and CBX8. CCK-8 (A), EdU incorporation (B) and colony formation assays (C) were used to prove 2BS cells with CBX8 overexpression exhibited higher rates of cell proliferation. (D) CBX8 can delay PIM1-induced cell senescence. SA-b-gal activity staining experiments showed that the 2bs cells that were infected with CBX8 showed weaker staining in PIM1-induced cell senescence.
the above-mentioned consensus sequence. Ser311 of CBX8 have been reported as a phosphorylation site in a characterization of human cancer cell phosphoproteome [37]. Taken together, these clues imply that Ser311 of CBX8 might the site phosphorylated by PIM1. Our experiments further proved that the addition of proteasome inhibitor MG132 can prevent the decrease of CBX8 protein induced by PIM1. This suggests that phosphorylation of CBX8 by PIM1 may destabilize CBX8 by promoting its ubiquitination. The present study found that PIM1 can down-regulate CBX8 and promote the expression of p16 in human diploid fibroblasts. It suggests that oncogene PIM1 can also play a role in suppressing cell growth in different cell environmental context. Therefore, the use of PIM1 inhibitors as a target for tumor therapy requires careful.
Conflicts of interest The authors declare no conflict of interest.
Acknowledgments This work was supported by grants 81773079, 81641183 (to W.Y.) from the National Natural Science Foundation of China. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.05.070. References [1] S. Wei, S. Wei, J.M. Sedivy, Expression of catalytically active telomerase does not prevent premature senescence caused by overexpression of oncogenic HaRas in normal human fibroblasts, Cancer Res. 59 (1999) 1539e1543. [2] M. Serrano, A.W. Lin, M.E. McCurrach, et al., Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a, Cell 88 (1997) 593e602. [3] J. Bartkova, N. Rezaei, M. Liontos, et al., Oncogene-induced senescence is part of the tumorigenesis barrier imposed by DNA damage checkpoints, Nature 444 (2006) 633e637.
X. Zhan et al. / Biochemical and Biophysical Research Communications 501 (2018) 779e785 [4] M. van Lohuizen, S. Verbeek, P. Krimpenfort, et al., Predisposition to lymphomagenesis in pim-1 transgenic mice: cooperation with c-myc and N-myc in murine leukemia virus-induced tumors, Cell 56 (1989) 673e682. [5] D. Acton, J. Domen, H. Jacobs, et al., Collaboration of pim-1 and bcl-2 in lymphomagenesis, Curr. Top. Microbiol. Immunol. 182 (1992) 293e298. [6] T. Schmidt, H. Karsunky, E. Gau, et al., Zinc finger protein GFI-1 has low oncogenic potential but cooperates strongly with pim and myc genes in T-cell lymphomagenesis, Oncogene 17 (1998) 2661e2667. [7] C. Hogan, C. Hutchison, L. Marcar, et al., Elevated levels of oncogenic protein kinase Pim-1 induce the p53 pathway in cultured cells and correlate with increased Mdm2 in mantle cell lymphoma, J. Biol. Chem. 283 (2008) 18012e18023. [8] J. Yang, K. Liu, J. Yang, et al., PIM1 induces cellular senescence through phosphorylation of UHRF1 at Ser311, Oncogene 36 (2017) 4828e4842. [9] B. Jin, Y. Wang, C.L. Wu, et al., PIM-1 modulates cellular senescence and links IL-6 signaling to heterochromatin formation, Aging Cell 13 (2014) 879e889. [10] M. Zemskova, M.B. Lilly, Y.W. Lin, et al., p53-dependent induction of prostate cancer cell senescence by the PIM1 protein kinase, Mol. Cancer Res. 8 (2010) 1126e1141. [11] E.B. Lewis, A gene complex controlling segmentation in Drosophila, Nature 276 (1978) 565e570. [12] G. Struhl, A gene product required for correct initiation of segmental determination in Drosophila, Nature 293 (1981) 36e41. [13] R. Jaenisch, R. Young, Stem cells, the molecular circuitry of pluripotency and nuclear reprogramming, Cell 132 (2008) 567e582. [14] J.I. Bardos, A.J. Saurin, C. Tissot, et al., HPC3 is a new human polycomb orthologue that interacts and associates with RING1 and Bmi1 and has transcriptional repression properties, J. Biol. Chem. 275 (2000) 28785e28792. [15] C.S. Hemenway, A.C. de Erkenez, G.C. Gould, The polycomb protein MPc3 interacts with AF9, an MLL fusion partner in t(9;11)(p22;q23) acute leukemias, Oncogene 20 (2001) 3798e3805. [16] N. Dietrich, A.P. Bracken, E. Trinh, et al., Bypass of senescence by the polycomb group protein CBX8 through direct binding to the INK4A-ARF locus, EMBO J. 26 (2007) 1637e1648. [17] S.H. Lee, S.J. Um, E.J. Kim, CBX8 suppresses Sirtinol-induced premature senescence in human breast cancer cells via cooperation with SIRT1, Cancer Lett. 335 (2013) 397e403. [18] S.H. Lee, S.J. Um, E.J. Kim, CBX8 antagonizes the effect of Sirtinol on premature senescence through the AKT-RB-E2F1 pathway in K562 leukemia cells, Biochem. Biophys. Res. Commun. 469 (2016) 884e890. [19] P. Wang, H. Gao, Y. Ni, et al., Beta-arrestin 2 functions as a G-protein-coupled receptor-activated regulator of oncoprotein Mdm2, J. Biol. Chem. 278 (2003) 6363e6370. [20] A.P. Bracken, D. Pasini, M. Capra, et al., EZH2 is downstream of the pRB-E2F pathway, essential for proliferation and amplified in cancer, EMBO J. 22 (2003) 5323e5335.
785
[21] D.X. Zhuo, X.H. Niu, Y.C. Chen, et al., Vitamin D3 up-regulated protein 1(VDUP1) is regulated by FOXO3A and miR-17-5p at the transcriptional and post-transcriptional levels, respectively, in senescent fibroblasts, J. Biol. Chem. 285 (2010) 31491e31501. [22] L. Hayflick, P.S. Moorhead, The serial cultivation of human diploid cell strains, Exp. Cell Res. 25 (1961) 585e621. [23] Z. Tang, Z. Zhang, Y. Zheng, et al., Cell aging of human diploid fibroblasts is associated with changes in responsiveness to epidermal growth factor and changes in HER-2 expression, Mech. Ageing Dev. 73 (1994) 57e67. [24] J.D. Leverson, P.J. Koskinen, F.C. Orrico, et al., Pim-1 kinase and p100 cooperate to enhance c-Myb activity, Mol. Cell 2 (1998) 417e425. [25] K.P. Shay, Z. Wang, P.X. Xing, et al., Pim-1 kinase stability is regulated by heat shock proteins and the ubiquitin-proteasome pathway, Mol. Cancer Res. 3 (2005) 170e181. [26] Z. Tu, K.M. Aird, B.G. Bitler, et al., Oncogenic RAS regulates BRIP1 expression to induce dissociation of BRCA1 from chromatin, inhibit DNA repair, and promote senescence, Dev. Cell 21 (2011) 1077e1091. [27] N. Dietrich, A.P. Bracken, E. Trinh, et al., Bypass of senescence by the polycomb group protein CBX8 through direct binding to the INK4A-ARF locus, EMBO J. 26 (2007) 1637e1648. [28] M. Narlik-Grassow, C. Blanco-Aparicio, A. Carnero, The PIM family of serine/ threonine kinases in cancer, Med. Res. Rev. 34 (2014) 136e159. [29] M.C. Nawijn, A. Alendar, A. Berns, For better or for worse: the role of Pim oncogenes in tumorigenesis, Nat. Rev. Cancer 11 (2011) 23e34. [30] M. van Lohuizen, S. Verbeek, P. Krimpenfort, et al., Predisposition to lymphomagenesis in pim-1 transgenic mice: cooperation with c-myc and N-myc in murine leukemia virus-induced tumors, Cell 56 (1989) 673e682. [31] F. Lanigan, J.G. Geraghty, A.P. Bracken, Transcriptional regulation of cellular senescence, Oncogene 30 (2011) 2901e2911. [32] L. Hoenicke, L. Zender, Immune surveillance of senescent cellsebiological significance in cancer- and non-cancer pathologies, Carcinogenesis 33 (2012) 1123e1126. [33] J. Vargas, B.C. Feltes, J.F. Poloni, et al., Senescence; an endogenous anticancer mechanism, Front. Biosci. 17 (2012) 2616e2643. [34] T.L. Aho, J. Sandholm, K.J. Peltola, et al., Pim-1 kinase promotes inactivation of the pro-apoptotic Bad protein by phosphorylating it on the Ser112 gatekeeper site, FEBS Lett. 571 (2004) 43e49. [35] A.N. Bullock, J. Debreczeni, A.L. Amos, et al., Structure and substrate specificity of the Pim-1 kinase, J. Biol. Chem. 280 (2005) 41675e41682. [36] C.K. Palaty, I. Clark-Lewis, D. Leung, et al., Phosphorylation site substrate specificity determinants for the Pim-1 protooncogene-encoded protein kinase, Biochem. Cell. Biol. 75 (1997) 153e162. [37] H. Zhou, S. Di Palma, C. Preisinger, et al., Toward a comprehensive characterization of a human cancer cell phosphoproteome, J. Proteome Res. 12 (2013) 260e271.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
PIM1-catalyzed CBX8 phosphorylation promotes the oncogene-induced senescence of human diploid fibroblast Xiangwen Zhan a, Jianming Yang b, Zebin Mao a, Wenhua Yu a, * a
Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, 100191, People's Republic of China b Department of Immunology, School of Basic Medical Science, Tianjing Medical University, People's Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 May 2018 Accepted 11 May 2018
The proto-oncogene PIM1 encodes Ser/Thr kinase and regulates cell growth, differentiation and apoptosis. However, more and more studies including ours have found that PIM1 can induce senescence in normal human diploid fibroblasts and behave as a tumor suppressor. But the relevant molecular mechanism of this process is not yet clear. It has been reported that Chromobox homolog 8 (CBX8) binds directly to INK4A as a transcriptional repressor, thereby suppressing stress-induced senescence. Here we report that PIM1 can phosphorylate CBX8 to promote its degradation, thereby up-regulating p16, during PIM1-induced cell senescence. Overexpression of CBX8 can inhibit PIM1-induced cell senescence. These data suggest that to promote CBX8 degradation may be an important molecular mechanism of PIM1induced cell senescence. © 2018 Elsevier Inc. All rights reserved.
Keywords: PIM1 CBX8 Phosphorylation Senescence
1. Introduction Cellular senescence is a permanent cell cycle arrest triggered by various stimuli including oncogene activation, telomere shortening, genetoxic insults and other detrimental growth conditions. About two decades ago, Serrano et al. observed normal fibroblasts with ectopic expression of RAS entered senescence different from replicative senescence without telomere shortening [1], termed oncogene-induced senescence (OIS) [2]. Subsequent research demonstrated OIS acted as a barrier preventing tumor formation. OIS was associated with signs of DNA replication stress including prematurely terminated DNA replication forks and DNA doublestrand breaks [3]. But the following molecular events involved in induction of senescence have not been elucidated very clearly. PIM1 gene was originally identified as an oncogene in murine leukemia virus-induced lymphomas, which was activated transcriptionally by the proviral insertion, encoding a constitutively active serine/threonine protein kinase. Plentiful research demonstrated PIM1 promoted tumor progression through regulation of proliferation, differentiation, survival and apoptosis cooperating with other oncogenes such as Myc [4], Bcl2 [5] and GFI-1 [6].
* Corresponding author. Department of Biochemistry and Molecular Biology Peking University Health Science Center, 38 Xue Yuan Road, Beijing, 100191, China. E-mail address: [email protected] (W. Yu). https://doi.org/10.1016/j.bbrc.2018.05.070 0006-291X/© 2018 Elsevier Inc. All rights reserved.
Interestingly, more and more studies have recently reported that PIM1 can induce cell senescence and inhibit proliferation, thus showing tumor suppressor properties. In 2008, Hogan et al. reported that overexpression of PIM1 in normal fibroblasts can induce senescence rather than enhance growth in a p53-dependent manner [7]. Our lab also reported later that PIM1 induced cellular senescence through phosphorylating UHRF1 and HP1g, which led to DNA methylation [8] and chromatin remodeling [9], respectively. Furthermore, overexpression of PIM1 can inhibit cell and tumor growth specifically in 22Rv1 human prostate cells by inducing marked increases in cellular senescence [10]. These data indicated PIM1's role in a certain cell may depend on its environment context. CBX8 is chromobox homolog 8 (CBX8) protein belonging to Polycomb Group (PcG) proteins which were originally discovered as a crucial regulator of development in Drosophila [11]. The PcG proteins form two distinct multimeric protein complexes named as polycomb repressive complexes 1 and 2 (PRC1 and PCRC2), which play significant roles in a wide variety of biological processes, including pluripotency, differentiation, senescence, carcinogenesis and so on [12,13]. Chromobox homolog 8 (CBX8) has been identified as a transcriptional repressor that functionally associate with RING1, BMI1 and MLL-ENL oncoprotein [14,15]. CBX8 is closely related to tumorigenesis and tumor progression, while the understanding of its role in cellular senescense is fewer than that in
780
X. Zhan et al. / Biochemical and Biophysical Research Communications 501 (2018) 779e785
purchased full-length PIM1 or purified mPIMK67M, and added 10 mM ATP in kinase buffer (Cell Signaling) for 30 min at 30 C. Western blot analysis was performed using p-Ser antibody.
tumors. A classic research reported that CBX8 can directly bind to and repress the INK4A-ARF locus and therefore bypassing cellular senescence in fibroblasts [16]. In addition, the relationship between CBX8 and senescence has also been reported in the inhibition of Sirtinol-induced premature aging in certain tumor cells [17,18]. Our study characterizes CBX8 as a new substrate of PIM1 kinase. CBX8 phosphorylation by PIM1 can lead to its destabilization and degradation, thereby promoting p16 expression during oncogeneinduced cell senescence. Overexpression of CBX8 shows counteractive effect on PIM1-induced cell senescence. These results indicate that CBX8 is involved in PIM1-induced cell senescence and his down-regulation is an important molecular mechanism in the process of cell senescence.
ChIP analysis was performed as previously described [20]. The antibodies used was purchased from Bethyl, while the immunoprecipitated DNA was quantified by real-time PCR. ChIP primers were designed in the region 200bp after first exon of IN4A and primer pairs only amplify one amplicon. Sequences was followed: 50 -GCCAAGGAAGAGGAATGAGGAG-30 ; 50 -CCTTCAGATCTTCTCA GCATTCG-30 .
2. Materials and methods
2.7. SA-b-gal staining
2.1. Cell culture and reagent
SA-b-gal activity staining was performed as described previously [21]. All cells were examined using fluorescence microscopy with the appropriate filters.
HEK293T cells and Human diploid fibroblasts 2BS cells were obtained from the National Institute of Biological Products, Beijing, China. These cells were cultured in complete Dulbecco's Modified Eagle Medium with 10% fetal bovine serum (FBS), streptomycin (100 mg/ml), penicillin (100 U/ml), and maintained in a humidified atmosphere of 5% CO2 at 37 C. MG132 (M8699; Sigma) was dissolved in dimethyl sulfoxide (DMSO; C6295; Sigma) and added to the culture medium at 20 mM for the indicated time periods. A corresponding volume of DMSO was added to untreated control cells. 2.2. Western blot analysis Western blot analysis was performed using standard method. Specific primary antibodies against PIM1 (3247, Cell Signaling), CBX8 (A300-882 A, Bethyl), Phosphoserine (ab9332, Abcam), p16 (sc-468, Santa Cruz), b-actin (PM053; MBL). 2.3. Immunoprecipitation, silver staining and mass spectrometry Cell lysate enriched overnight with M2 beads at 4 C. Beads were washed five times with lysis buffer and resolved on NuPAGE 4e12% Bis-Tris gel (Invitrogen), silver stained using Pierce silver stain kit (Thermo) and subjected to LCeMS/MS analysis. 2.4. Co-immunoprecipitation and in vivo phosphorylation Endogenous PIM1 or CBX8 was immunoprecipitated with antiPIM1 or anti-CBX8 and protein A-Sepharose beads. After extensive washing and heating in the sample buffer, the complexes were subjected to immunoblot [19]. Cell lysates were subjected to immunoprecipitation (IP) with CBX8 antibody (Endogenous) or gst-specific beads (Exogenous transfection, GST-CBX8). Aliquots of immunoprecipitation were subjected to Western blotting using pSer antibody. 2.5. GST pull-down and in vitro kinase assay GST and GST-CBX8 or His-PIM1 or His-mPIM1K67M were purified from Transetta (DE3) cells using glutathione Sepharose or Ni Sepharose. Co-incubate equal amounts of GST or GST-CBX8 (200 ng/each) with His-PIM1 (200 ng) in BC100 buffer overnight at 4 C, adding Glutathione-sepharose beads for 2 h. The mixture (resin and binding proteins) was washed three times by centrifugation at 500 g for 3 min to remove the unbound proteins and was analyzed by western blotting using anti-GST and anti-His. For in vitro kinase assay, Co-incubate purified GST-CBX8 with
2.6. Chromatin immunoprecipitation (ChIP)
2.8. Cell proliferation assay The CCK-8, colony formation and EdU incorporation assays were adopted to evaluate the cell proliferation. For the CCK-8 assay, CCK8 kit (CCK-8; Dojindo Laboratories, Japan) was used according to the manufacturer's instructions. 3 103 cells were planted into 96-well dish with three replicates. Then, 100 ml of serum-free cell culture medium containing 10 ml WST-8 reagent was added into each well every 24 h and the plates were incubated at standard conditions for 1 h. Optical absorbance of each well at 450 nm and 630 nm were measured with a microplate reader (Bio-Rad Laboratories, USA). Three independent experiments were performed for quantification. For colony formation assay, cells were plated in 6-well plates in triplicates (400 cells per well). After 10 days, the cells were washed with PBS, fixed with 4% formaldehyde for 10min and stained with crystal violet staining solution (Sigma-Aldrich) for 15min. The colonies were counted and photographed. For the EdU incorporation assay, EdU Apollo®567 In Vitro Imaging Kit (Ribo Bio, China) was used according to the manufacturer's instructions except that cell nuclei were stained with DAPI instead of Hoechst for 30 min. Five fields of each well were randomly chosen and observed under fluorescence microscopy. All images were processed using Image J software and the proportion of EdU incorporated cells was calculated. Three independent experiments were performed for quantification. 3. Result 3.1. PIM1 interacts with CBX8 To explore the mechanism by which PIM1 induces cell senescence, we employed immunopurification and mass spectrometry to identify proteins that are potentially associated with PIM1 in 2BS cells (human embryonic lung fibroblasts, a cell model commonly used in studies of replicative senescence [22,23]). To this end, FLAGPIM1 was transiently expressed in 2BS cells. Cellular extracts were prepared and subjected to affinity purification using anti-FLAG affinity gels. The eluted protein complex was then resolved on SDSPAGE, silver stained, and subjected to LC-MS/MS analysis (Fig. 1A). The results showed that PIM1 was co-purified with a number of proteins, including CBX8, as well as other known interacting proteins of PIM1 such as SND1 and HSP90 [24,25]. To confirm that CBX8 is physically associated with PIM1 in cultured cells, we co-transfected 293 T cells with GST-CBX8 and
X. Zhan et al. / Biochemical and Biophysical Research Communications 501 (2018) 779e785
781
Fig. 1. PIM1 interacts with CBX8. (A) Mass Spectrometry Detection of PIM1-associated Proteins. 2BS cells were infected with Flag-PIM1 using a lentiviral expression system. The cell lysates were immunoprecipitated with FLAG-M2 beads and separated by SDS-PAGE electrophoresis, silver stained, and the differential bands were sent for mass spectrometry analysis. (B) PIM1 interacts with CBX8 in cultured cells. Co-transfected GST-CBX8 and FLAG-PIM1 plasmids in 293 T cells, co-immunoprecipitation experiment was performed by using the indicated antibodies. (C) PIM1 interacts with CBX8 in vivo. In RAS-induced 2BS cells, co-immunoprecipitation was performed using antibodies against PIM1 and CBX8, respectively. Western blotting was used to detect precipitated complexes by antibodies against CBX8 and PIM1. (D) PIM1 interacts with CBX8 directly in vitro. GST pull-down experiments were performed with bacterially expressed GST-CBX8 and 6xHis-PIM1.
FLAG-PIM1 plasmids, and use the whole cell lysate for the coimmunoprecipitation (Co-IP) and western blotting analysis. The results showed that FLAG-PIM1 associated with GST-CBX8 (Fig. 1B). To further verify whether endogenous CBX8 and PIM1 interact with each other, co-IP experiments were performed in extracts of senescent cells induced by RasV12 as PIM1 expression was upregulated in RasV12-induced senescent cells [9,26]. CBX8 was observed to co-immunoprecipitate with PIM1 (Fig. 1C). In reciprocal immunoprecipitations, PIM1 was observed presenting in CBX8 immunoprecipitates (Fig. 1C). As our data provide not only exogenous but also endogenous evidences for the interaction between PIM1 and CBX8, the GST pull-down assays was used to confirm whether this interaction is direct, which was carried out by incubating the full-length of recombinant GSTeCBX8 with 6xHisPIM1. Free GST together with the full-length of recombinant GSTCBX8 and 6xHis-PIM1 were all expressed in prokaryotic cells and purified in vitro. As shown in Fig. 1D, 6xHis-PIM1 specifically interacted with full-length GST-CBX8, but not with free GST. Taken together, these results support the argument that PIM1 directly interacts with CBX8. 3.2. PIM1 phosphorylates CBX8 and promotes its degradation Since PIM1 is a constitutive kinase and we have already confirmed its direct binding with CBX8, our next step was to investigate if PIM1 could phosphorylate CBX8? To find the answer, we co-transfected GST-CBX8 with Flag-PIM1 or empty vector into 293 T cells and precipitated CBX8 with GST beads. The phosphorylation levels of the precipitated CBX8 were detected by pan-p-Ser
antibodies (Fig. 2A). Western blotting analysis showed that the phosphorylation level of Ser in CBX8 was significantly increased. To gain further support for the argument that CBX8 is a substrate of PIM1 kinase, GST-CBX8 was purified from prokaryotic cells, then incubated with PIM1 purchased from Abcam or mPIM1K67M, a catalytically inactive mutant (mPIM1K67M), purified from prokaryotic cells in the kinase assay buffer containing ATP. The result of this in vitro kinase assay showed Ser phosphorylation of CBX8 could only be detected in assays mixed with wtPIM1, GST-CBX8 and ATP and the three are indispensable for Ser phosphorylation of CBX8 (Fig. 2B), which indicate that CBX8 Ser phosphorylation is dependent on PIM1 kinase activity and the phosphoryl donor (ATP). To prove that such phosphorylation also occurs in endogenous CBX8, we infected wild-type PIM1 (wtPIM1) or a in 2BS cells by using a lentiviral expression system, then endogenous CBX8 was enriched by CBX8 antibody, and its phosphorylation level at Ser was detected by western blotting. Consistent with the in vitro results, the phosphorylation level of serine on CBX8 was significantly increased (Fig. 2C). During the investigation of the phosphorylation level of CBX8, we were surprised to find that the protein level of CBX8 gradually decreased with the overexpression of PIM1 in 2BS cells, while this decrease of CBX8 protein levels could not be observed in 2BS cells with mPIM1K67M overexpression (Fig. 2D). This result suggests that the reduction of CBX8 protein is PIM1's phosphorylation for CBX8, which is PIM1 kinase activity-dependent. To exclude the possibility that PIM1 induces CBX8 protein reduction at transcription level, realtime PCR was used to detect the mRNA level of CBX8, which showed no significant changes (Fig. 2E). This result suggest that CBX8 protein levels may be regulated by PIM1 at post-
782
X. Zhan et al. / Biochemical and Biophysical Research Communications 501 (2018) 779e785
Fig. 2. PIM1 phosphorylates CBX8 and promotes its degradation. (A) PIM1 phosphorylates CBX8 in 293 T cells. 293 T cells were Co-transfected with GST-CBX8 and Flag-PIM1 or empty vector, precipitating the cell lysate with GST-specific beads. Antibodies against p-Ser was used to detect the phosphorylation levels of CBX8. (B) In vitro kinase assay of CBX8 phosphorylation by PIM1. GST-CBX8 purified from prokaryotic cells was incubated with PIM1 purchased from Abcam or mPIM1K67M purified from prokaryotic cells in the kinase assay buffer containing ATP. Antibodies against p-Ser was used to detect the phosphorylation levels of CBX8. (C) CBX8 phosphorylation in PIM1-induced senescent cells. Immunoprecipitation was against by the antibody of CBX8, then antibodies against p-Ser was used to detect the phosphorylation levels of CBX8. (D) and (E) Changes in CBX8 in PIM1induced Cellular Senescence. Western Blot Analysis (D) and qRT-PCR (E) was used to detect the protein and mRNA levels of CBX8 on extracts of 2BS cells that were infected with wild-type PIM1 or mPIM1K67M. (F) PIM1 promotes CBX8 degradation. 2BS cells were infected with increasing amounts of wild-type PIM1 or mPIM1K67M. Changes in CBX8 were detected by immunoblotting after treatment with MG132.
translational level. To further support this proposition, MG132, which is a proteasome inhibitor and reduces the degradation of ubiquitin-conjugated proteins in mammalian cells, was added to 2BS cells infected with PIM1. As is shown in Fig. 2F, the protein level of CBX8 gradually decreased with the increase of PIM1 expression, and this decreasing of CBX8 protein was abolished when the degradation of ubiquitin-conjugated proteins was inhibited by MG132, while MG132 showed no effects on protein levels of CBX8 in 2BS cells with mPIM1K67M overexpression. These results fully demonstrate that PIM1 can phosphorylate CBX8 and hence promote its degradation.
3.3. PIM1 promotes p16 expression by down-regulating CBX8 during PIM1-induced cell senescence Previous studies have reported that CBX8 is a transcriptional repressor that binds directly to the region after first exon of INK4A (coding for p16), inhibiting the expression of p16 in human diploid fibroblasts, and knocking down CBX8 can promote cell senescence [27]. Recent studies in our laboratory show that p16 expression is up-regulated during PIM1-induced cell senescence [8,9]. Since our previous studies confirmed that PIM1 could interact directly with CBX8, catalyze its phosphorylation and hence promote its
X. Zhan et al. / Biochemical and Biophysical Research Communications 501 (2018) 779e785
degradation, it's reasonable to hypothesize that PIM1 promote the expression of p16 b y down-regulating CBX8 during PIM1-induced cell senescence? To prove this hypothesis, ChIP experiments was performed in PIM1-induced cellular senescent model of 2BS cells which was confirmed by the increased senescence-associated bgalactosidase (SA-b-gal) stain (Fig. 3A). Anti-CBX8 antibody was used for immuno-precipitating CBX8-bound-genome DNA fragments, and the enrichment of p16 gene was determined by quantitative PCR. The results of ChIP analysis show a significant reduction of the enrichment in the genomic region of p16 gene bound by CBX8 in PIM1 overexpressed cells as compared with that in control cells (Fig. 3B), while in the PIM1 kinase-dead mutant overexpressed cells, p16 gene regions enriched anti-CBX8 antibody increased obviously. At the same time, the p16 expression was further confirmed at mRNA levels and protein levels 2BS cells after exposure to PIM1 for 10 days by realtime PCR (Fig. 3C) and western blotting (Fig. 3D). Taken together, the upregulation of p16 mRNA and protein levels in PIM1-induced cell senescence may be mediated by the phosphorylation of CBX8 by PIM1.
783
3.4. CBX8 is involved in PIM1-induced cell senescence Previous studies in our laboratory have revealed that p16 expression is up-regulated during PIM1-induced cell senescence [8,9]. Dietrich et al. [16] reported that knocking down CBX8 can promote cell senescence, and that overexpression of CBX8 can bypass stress-induced cell senescence. In present studies, we have demonstrated that p16 is regulated by CBX8 during PIM1-induced senescence. However, there's still short of the direct evidence supporting for the relationship between CBX8 and the cellular phenotype of 2BS cells. To find such evidences, we co-infected PIM1 and CBX8 or the empty vector in 2BS cells, and observed the cell proliferation of these cells by CCK8, EdU incorporation and colony formation assays. PIM1 overexpression inhibited the growth of 2BS cells, while ectopic expression of CBX8 could obviously mitigate such growth inhibition by PIM1 as indicated in CCK8, EdU incorporation and colony formation assays (Fig. 4AeC). Furthermore, SAb-gal activity staining experiment showed that overexpressing CBX8 could make the percentage of stained cells decreasing as compared with that of cells with PIM1 overexpressing alone (Fig. 4D), which suggests that overexpression of CBX8 may effectively counteract PIM1-induced senescence. These data also support from the cellular level that CBX8 can mediate PIM1-induced senescence. 4. Discussion
Fig. 3. PIM1 promotes p16 expression by down-regulating CBX8 during PIM1induced cell senescence. (A) PIM1 induces cell senescence. SA-b-gal activity staining experiments showed strong staining compared to the empty vector control and mPIM1K67M groups. (B) CBX8 mediates PIM1-induced senescence. INK4A ChIP analysis in 2BS cell infected with wild-type PIM1 or mPIM1K67M using CBX8 antibodies. (C) and (D) Changes in p16 in PIM1-induced cellular senescence. Western Blot Analysis (C) and qRT-PCR (D) was used to detect the protein and mRNA levels of p16 on extracts of 2BS cells that were infected with wild-type PIM1 or mPIM1K67M.
The role of PIM1 in regulating both the growth and transformation of malignant cells is better characterized [28,29], while PIM1-induced cellular senescence was reported only a few years ago. PIM1 is a relative weak oncogene, and its functioning ways often depend on cooperating with the activation of synergistic genes in the organisms. PIM1 alone is not able to induce a massive proliferation [30]. Moreover, Zemskova et al. [10] also reported that PIM1 activates the p53 pathway and causes senescence of tumor cells in certain special prostate cancer cells. Hogan [7] and previous studies of our laboratory [8,9] also showed that the upregulation of PIM1, an oncogene, in normal fibroblasts caused cell senescence instead of promoting cell proliferation. Cellular senescence is considered to be a protective mechanism against caicinogenesis of cells [31e33]. To explore the molecular mechanism might have a potential application in clinic cancer treatment. In present study, we find that CBX8 can be phosphorylated by PIM1 kinase, and this phosphorylation promotes its degradation, which in turn upregulate p16, leading to cell senescence. That may be an important molecular mechanism in PIM1 induced cell senescence. Since PIM1 is a constitutive Ser/Thr-specific protein kinase whose function is mainly dependent on its kinase activity. Our studies discovered that CBX8 could be phosphorylated by PIM1. However, the specific phosphorylation sites of CBX8 catalyzed by PIM1 need to be determined. While serine/threonine kinases all phosphorylate serine or threonine residues in their substrates, they select specific residues to phosphorylate on the basis of residues that flank the phosphoacceptor site, which together comprise the consensus sequence. Since only the consensus sequence residues of a target substrate make contact with several key amino acids within the catalytic cleft of the kinase (usually through hydrophobic forces and ionic bonds), a kinase is usually not specific to a single substrate, but instead can phosphorylate a whole “substrate family” which share common recognition sequences. Through analysis of peptide substrates, PIM1 preferred consensus sequence have been established as R-x-R-x-x-S/T, which is further confirmed by the phosphorylation sites analysis in most known substrates of PIM1 [34e36]. We searched the amino acid sequence of CBX8 and found that the Ser311 and the residues flanking it is consistent with
784
X. Zhan et al. / Biochemical and Biophysical Research Communications 501 (2018) 779e785
Fig. 4. CBX8 is involved in PIM1-induced cell senescence. (AeC) Cell proliferation rates were rescued in 2BS cells co-expressing PIM1 and CBX8. CCK-8 (A), EdU incorporation (B) and colony formation assays (C) were used to prove 2BS cells with CBX8 overexpression exhibited higher rates of cell proliferation. (D) CBX8 can delay PIM1-induced cell senescence. SA-b-gal activity staining experiments showed that the 2bs cells that were infected with CBX8 showed weaker staining in PIM1-induced cell senescence.
the above-mentioned consensus sequence. Ser311 of CBX8 have been reported as a phosphorylation site in a characterization of human cancer cell phosphoproteome [37]. Taken together, these clues imply that Ser311 of CBX8 might the site phosphorylated by PIM1. Our experiments further proved that the addition of proteasome inhibitor MG132 can prevent the decrease of CBX8 protein induced by PIM1. This suggests that phosphorylation of CBX8 by PIM1 may destabilize CBX8 by promoting its ubiquitination. The present study found that PIM1 can down-regulate CBX8 and promote the expression of p16 in human diploid fibroblasts. It suggests that oncogene PIM1 can also play a role in suppressing cell growth in different cell environmental context. Therefore, the use of PIM1 inhibitors as a target for tumor therapy requires careful.
Conflicts of interest The authors declare no conflict of interest.
Acknowledgments This work was supported by grants 81773079, 81641183 (to W.Y.) from the National Natural Science Foundation of China. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.05.070. References [1] S. Wei, S. Wei, J.M. Sedivy, Expression of catalytically active telomerase does not prevent premature senescence caused by overexpression of oncogenic HaRas in normal human fibroblasts, Cancer Res. 59 (1999) 1539e1543. [2] M. Serrano, A.W. Lin, M.E. McCurrach, et al., Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a, Cell 88 (1997) 593e602. [3] J. Bartkova, N. Rezaei, M. Liontos, et al., Oncogene-induced senescence is part of the tumorigenesis barrier imposed by DNA damage checkpoints, Nature 444 (2006) 633e637.
X. Zhan et al. / Biochemical and Biophysical Research Communications 501 (2018) 779e785 [4] M. van Lohuizen, S. Verbeek, P. Krimpenfort, et al., Predisposition to lymphomagenesis in pim-1 transgenic mice: cooperation with c-myc and N-myc in murine leukemia virus-induced tumors, Cell 56 (1989) 673e682. [5] D. Acton, J. Domen, H. Jacobs, et al., Collaboration of pim-1 and bcl-2 in lymphomagenesis, Curr. Top. Microbiol. Immunol. 182 (1992) 293e298. [6] T. Schmidt, H. Karsunky, E. Gau, et al., Zinc finger protein GFI-1 has low oncogenic potential but cooperates strongly with pim and myc genes in T-cell lymphomagenesis, Oncogene 17 (1998) 2661e2667. [7] C. Hogan, C. Hutchison, L. Marcar, et al., Elevated levels of oncogenic protein kinase Pim-1 induce the p53 pathway in cultured cells and correlate with increased Mdm2 in mantle cell lymphoma, J. Biol. Chem. 283 (2008) 18012e18023. [8] J. Yang, K. Liu, J. Yang, et al., PIM1 induces cellular senescence through phosphorylation of UHRF1 at Ser311, Oncogene 36 (2017) 4828e4842. [9] B. Jin, Y. Wang, C.L. Wu, et al., PIM-1 modulates cellular senescence and links IL-6 signaling to heterochromatin formation, Aging Cell 13 (2014) 879e889. [10] M. Zemskova, M.B. Lilly, Y.W. Lin, et al., p53-dependent induction of prostate cancer cell senescence by the PIM1 protein kinase, Mol. Cancer Res. 8 (2010) 1126e1141. [11] E.B. Lewis, A gene complex controlling segmentation in Drosophila, Nature 276 (1978) 565e570. [12] G. Struhl, A gene product required for correct initiation of segmental determination in Drosophila, Nature 293 (1981) 36e41. [13] R. Jaenisch, R. Young, Stem cells, the molecular circuitry of pluripotency and nuclear reprogramming, Cell 132 (2008) 567e582. [14] J.I. Bardos, A.J. Saurin, C. Tissot, et al., HPC3 is a new human polycomb orthologue that interacts and associates with RING1 and Bmi1 and has transcriptional repression properties, J. Biol. Chem. 275 (2000) 28785e28792. [15] C.S. Hemenway, A.C. de Erkenez, G.C. Gould, The polycomb protein MPc3 interacts with AF9, an MLL fusion partner in t(9;11)(p22;q23) acute leukemias, Oncogene 20 (2001) 3798e3805. [16] N. Dietrich, A.P. Bracken, E. Trinh, et al., Bypass of senescence by the polycomb group protein CBX8 through direct binding to the INK4A-ARF locus, EMBO J. 26 (2007) 1637e1648. [17] S.H. Lee, S.J. Um, E.J. Kim, CBX8 suppresses Sirtinol-induced premature senescence in human breast cancer cells via cooperation with SIRT1, Cancer Lett. 335 (2013) 397e403. [18] S.H. Lee, S.J. Um, E.J. Kim, CBX8 antagonizes the effect of Sirtinol on premature senescence through the AKT-RB-E2F1 pathway in K562 leukemia cells, Biochem. Biophys. Res. Commun. 469 (2016) 884e890. [19] P. Wang, H. Gao, Y. Ni, et al., Beta-arrestin 2 functions as a G-protein-coupled receptor-activated regulator of oncoprotein Mdm2, J. Biol. Chem. 278 (2003) 6363e6370. [20] A.P. Bracken, D. Pasini, M. Capra, et al., EZH2 is downstream of the pRB-E2F pathway, essential for proliferation and amplified in cancer, EMBO J. 22 (2003) 5323e5335.
785
[21] D.X. Zhuo, X.H. Niu, Y.C. Chen, et al., Vitamin D3 up-regulated protein 1(VDUP1) is regulated by FOXO3A and miR-17-5p at the transcriptional and post-transcriptional levels, respectively, in senescent fibroblasts, J. Biol. Chem. 285 (2010) 31491e31501. [22] L. Hayflick, P.S. Moorhead, The serial cultivation of human diploid cell strains, Exp. Cell Res. 25 (1961) 585e621. [23] Z. Tang, Z. Zhang, Y. Zheng, et al., Cell aging of human diploid fibroblasts is associated with changes in responsiveness to epidermal growth factor and changes in HER-2 expression, Mech. Ageing Dev. 73 (1994) 57e67. [24] J.D. Leverson, P.J. Koskinen, F.C. Orrico, et al., Pim-1 kinase and p100 cooperate to enhance c-Myb activity, Mol. Cell 2 (1998) 417e425. [25] K.P. Shay, Z. Wang, P.X. Xing, et al., Pim-1 kinase stability is regulated by heat shock proteins and the ubiquitin-proteasome pathway, Mol. Cancer Res. 3 (2005) 170e181. [26] Z. Tu, K.M. Aird, B.G. Bitler, et al., Oncogenic RAS regulates BRIP1 expression to induce dissociation of BRCA1 from chromatin, inhibit DNA repair, and promote senescence, Dev. Cell 21 (2011) 1077e1091. [27] N. Dietrich, A.P. Bracken, E. Trinh, et al., Bypass of senescence by the polycomb group protein CBX8 through direct binding to the INK4A-ARF locus, EMBO J. 26 (2007) 1637e1648. [28] M. Narlik-Grassow, C. Blanco-Aparicio, A. Carnero, The PIM family of serine/ threonine kinases in cancer, Med. Res. Rev. 34 (2014) 136e159. [29] M.C. Nawijn, A. Alendar, A. Berns, For better or for worse: the role of Pim oncogenes in tumorigenesis, Nat. Rev. Cancer 11 (2011) 23e34. [30] M. van Lohuizen, S. Verbeek, P. Krimpenfort, et al., Predisposition to lymphomagenesis in pim-1 transgenic mice: cooperation with c-myc and N-myc in murine leukemia virus-induced tumors, Cell 56 (1989) 673e682. [31] F. Lanigan, J.G. Geraghty, A.P. Bracken, Transcriptional regulation of cellular senescence, Oncogene 30 (2011) 2901e2911. [32] L. Hoenicke, L. Zender, Immune surveillance of senescent cellsebiological significance in cancer- and non-cancer pathologies, Carcinogenesis 33 (2012) 1123e1126. [33] J. Vargas, B.C. Feltes, J.F. Poloni, et al., Senescence; an endogenous anticancer mechanism, Front. Biosci. 17 (2012) 2616e2643. [34] T.L. Aho, J. Sandholm, K.J. Peltola, et al., Pim-1 kinase promotes inactivation of the pro-apoptotic Bad protein by phosphorylating it on the Ser112 gatekeeper site, FEBS Lett. 571 (2004) 43e49. [35] A.N. Bullock, J. Debreczeni, A.L. Amos, et al., Structure and substrate specificity of the Pim-1 kinase, J. Biol. Chem. 280 (2005) 41675e41682. [36] C.K. Palaty, I. Clark-Lewis, D. Leung, et al., Phosphorylation site substrate specificity determinants for the Pim-1 protooncogene-encoded protein kinase, Biochem. Cell. Biol. 75 (1997) 153e162. [37] H. Zhou, S. Di Palma, C. Preisinger, et al., Toward a comprehensive characterization of a human cancer cell phosphoproteome, J. Proteome Res. 12 (2013) 260e271.