Cyclin B1 Represses Its DNA-Binding Activity During Mitosis in Cancer Cells
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Oncogene (2012) 31, 4946–4959 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc ORIGINAL ARTICLE Sp1 phosphorylation by cyclin-dependent kinase 1/cyclin B1 represses its DNA-binding activity during mitosis in cancer cells J-Y Chuang1, S-A Wang1, W-B Yang1, H-C Yang1, C-Y Hung2, T-P Su3, W-C Chang2,4,5,6 and J-J Hung1,2,4,5 1Institute of Bioinformatics and Biosignal Transduction, College of Bioscience and Biotechnology, Tainan, Taiwan; 2Institute of Medicine, College of Medicine, Tainan, Taiwan; 3National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD, USA; 4Department of Pharmacology, Tainan, Taiwan; 5Center for Infectious Disease and Signal Transduction, National Cheng-Kung University, Tainan, Taiwan and 6Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan Sp1 is important for the transcription of many genes. Our Introduction previous studies have shown that Sp1 is degraded in normal cell, but it is preserved in cancer cells during During interphase of the cell cycle, the transcription mitosis and exists a priori in the daughter cells, ready to factor Sp1 has an important role in regulating the engage in gene transcription and thereby contributes to expression of genes involved in many cellular processes the proliferation and survival of cancer cells. The by binding to the promoter regions of its target genes. mechanism by which Sp1 is preserved in cancer cells Sp1 binds specifically to the GC-rich promoter elements during mitosis remains unknown. In this study, we via 3 C2H2-type zinc finger regions at the C-terminus of observed that Sp1 strongly colocalized with cyclin- the protein (Li and Davie, 2010) and regulates the dependent kinase 1 (CDK1)/cyclin B1 during mitosis. transcriptional activity of target genes by using two Moreover, we showed that Sp1 is a novel mitotic substrate major glutamine-rich transactivation domains localized of CDK1/cyclin B1 and is phosphorylated by it at Thr 739 at the N-terminus and the medial region (Li and Davie, before the onset of mitosis. Phospho-Sp1 reduced its 2010). Recent studies have shown that the DNA-binding DNA-binding ability and facilitated the chromatin con- affinity, transactivational activity, and protein stability densation process during mitosis. Mutation of Thr739 to of Sp1 may be regulated by posttranslational modifica- alanine resulted in Sp1 remaining in the chromosomes, tions such as glycosylation, ubiquitination, sumoylation, delayed cell-cycle progression, and eventually led to acetylation, and phosphorylation (Hung et al., 2006; apoptosis. Screening of Sp1-associated proteins during Chuang et al., 2008; Wang et al., 2008; Tan and mitosis by using liquid chromatography/mass spectro- Khachigian, 2009; Li and Davie, 2010; Chuang and metry indicated the tethering of Sp1 to myosin/F-actin. Hung, 2011). Among the posttranslational modifica- Furthermore, phospho-Sp1 and myosin/F-actin appeared tions of Sp1, phosphorylation is one of the most studied, to exist as a congregated ring at the periphery of the especially with regard to its role during interphase of the chromosome. However, at the end of mitosis and the cell cycle. For example, Sp1 serine or threonine residues beginning of interphase, Sp1 was dephosphorylated by are phosphorylated by different kinases, including PP2A and returned to the chromatin. These results DNA-dependent protein kinase, protein kinase A, and indicate that cancer cells use CDK1 and PP2A to regulate protein kinase C-z, and these phosphorylations cause an the movement of Sp1 in and out of the chromosomes increase in the transcriptional activity of Sp1 by the during cell-cycle progression, which may benefit cancer- enhancement of Sp1 binding to DNA (Chu and Ferro, cell proliferation. 2005; Tan and Khachigian, 2009). On the other hand, Oncogene (2012) 31, 4946–4959; doi:10.1038/onc.2011.649; some kinases can inhibit Sp1 function. During terminal published online 23 January 2012 liver differentiation, for example, casein kinase II modifies Thr579 on Sp1 and downregulates the DNA- Keywords: Sp1; CDK1; PP2A; myosin; mitosis binding ability of Sp1 (Chu and Ferro, 2005). Previous studies have indicated that Sp1 accumulates in most types of cancer cells (Abdelrahim et al., 2004; Mertens-Talcott et al., 2007; Wang et al., 2008; Chuang et al., 2009; Li and Davie, 2010). Our recent study showing that Sp1 is modified by c-Jun NH2-terminal protein kinase 1 (JNK1) in mitosis, and phospho-Sp1 Correspondence: Professor J-J Hung, Institute of Bioinformatics and increases its protein stability, and is more present in Biosignal Transduction, National Cheng-Kung University, Tainan cancer cells than that in primary noncancerous cells 701, Taiwan. raised the possibility that the phosphorylation of Sp1 E-mail: [email protected] Received 3 April 2011; revised 3 November 2011; accepted 16 December has an important role in the stability of Sp1 during 2011; published online 23 January 2012 mitosis (Chuang et al., 2008). Sp1 is known to be Myosin/F-actin determines Sp1 position in mitosis J-Y Chuang et al 4947 displaced from chromatin and remain stable in mitotic (Figure 1d), suggesting that CDK1/cyclin B1 might cancer cells, and then reserve Sp1 to daughter cells, thus phosphorylate Sp1 during mitosis. We examined the apparently facilitating a quick start and the execution of localization of Sp1, cyclin B1, and CDK1 and found cell growth (He and Davie, 2006; Chuang et al., 2008). that Sp1 colocalized with cyclin B1 and CDK1 during However, the exact mechanism of how Sp1 modulates its mitosis (Figures 2a and b, Supplementary Figure S1 and DNA-binding activity to shuttle in and out of the Figure S4). In addition, we used thymidine (Figure 2c) chromosome during cell-cycle progression remains unknown. and nocodazole (Figure 2d) to synchronize the cells in Mitosis in vertebrates is triggered by cyclin-dependent the G1/S phase and mitosis in order to observe the kinase 1 (CDK1). In G2 stage, cyclin B1 is accumulated activation of CDK1 and the phosphorylation of Sp1 in cytoplasm, forms complex with CDK1, and then during cell-cycle progression, respectively. Approxi- phosphorylated at Thr14 and Thr15, which causes the mately 10 h after removing thymidine, the levels of inactivation of cyclin B1 (Strebhardt, 2010). Until late cyclin B1 and active CDK1 (hypophosphorylated G2 phase, CDK1 activation begins when it is depho- CDK1) were increased along with an increase in Sp1 sphorylated by phosphatase CDC25, and most of the phosphorylation (Figure 2c). Furthermore, at the cyclin B1/Cdk1 complexes are translocated rapidly from initiation of nocodazole release, the cells remained in the cytoplasm into the nucleus while the nuclear early mitosis and showed high levels of cyclin B1, envelope breaks down (Strebhardt, 2010). In this study, activation CDK1, and phospho-Sp1 (Figure 2d). Con- we discovered that CDK1 is a novel kinase to versely, the levels of both activated CDK1 and phospho- phosphorylate Sp1 at Thr739 during mitosis, and Sp1 decreased as the cells entered interphase (Figure 2d). consequently, Sp1 DNA-binding ability is repressed. These results imply that CDK1 is responsible for In addition, myosin/F-actin was found to interact with phosphorylating Sp1 during mitosis. phospho-Sp1 and thereby results in its displacement For further addressing the interaction between Sp1 from the chromatin, which is required for chromosome and CDK1 in mitosis, an immunoprecipitation assay was packaging during mitosis. Moreover, at the end of performed (Figure 3a and Supplementary Figure S2). The mitosis, PP2A was shown to dephosphorylate Sp1 to results showed that Sp1 interacted with cyclin B1/CDK1 restore its DNA-binding activity. Finally, in N-methyl- in mitosis but not in interphase. Three truncated Sp1 N-nitrosourea (MNU)-induced mammary tumors, we fragments were subsequently used for the CDK1 found strong CDK1 and Sp1 accumulation. Therefore, immunoprecipitation assay (Figure 3b). The data the full phosphorylation of Sp1 is essential for cell-cycle revealed that only the C-terminal DNA-binding domain progression of cancer cells. of Sp1 could interact with CDK1. Furthermore, when we used the active CDK1/cyclin B1 to perform in vitro kinase assays, we found that CDK1 could phosphor- ylate the C-terminus of Sp1 at Thr739 (Figures 3c and Results d). Next, we used a phospho-peptide of Sp1, EGSG- TAT(p)PSALIT, as an antigen to generate an antibody Phosphorylation of Sp1 in mitosis reduces its that could recognize the phosphorylated Thr739 motif. DNA-binding affinity The specificity of the antibody was verified by its To further investigate the localization of Sp1 during recognition of CDK1-phosphorylated Sp1 and mitotic different phases of the cell cycle, we investigated Sp1 Sp1 (Figure 3e and Supplementary Figure S3). Immuno- distribution pattern in asynchronized cells by fluoromi- fluorescence data showed that Sp1 was evenly distrib- croscopy. Sp1 was dissociated from the chromosomes uted within the nucleus during interphase but no during mitosis (Figure 1a). The results from chromatin phospho-Sp1 signal was evident (Figure 3f). When the immunoprecipitation (ChIP) assays confirmed that Sp1 cells entered early prophase, CDK1 and cyclin B1 significantly lost its DNA-binding activity during entered into nucleus, and the phospho-Sp1 signal mitosis (Figure 1b). As the percentage of cells in mitosis increased in a parallel manner (Figures 3g and h, increased during nocodazole treatment, the Sp1 signal Supplementary Figure S5 and Figure S6). As the shifted from the lower band to the upper one, as shown phospho-Sp1 signal became evident, it showed almost in western blotting (Figure 1c). Moreover, the DNA- no colocalization with the DNA (Figure 3f, Supplemen- binding ability of the shifted Sp1 was repressed tary Figure S5 and Figure S6). Moreover, the phospho- (Figure 1c). After an alkaline phosphatase treatment, Sp1 signal continued to be detected but not colocalized the upper band for the mitotic Sp1 shifted to the lower with the DNA during the entire course of mitosis.