Dual-Specificity Phosphatase 5 (DUSP5) As a Direct Transcriptional
Total Page：16
File Type：pdf, Size：1020Kb
Oncogene (2003) 22, 5586–5591 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc Dual-specificity phosphatase 5 (DUSP5) as a direct transcriptional target of tumor suppressor p53 Koji Ueda1, Hirofumi Arakawa1,2 and Yusuke Nakamura*,1 1Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan Dual-specificity phosphatase 5 (DUSP5), a VH1-like dozens of such targets have been reported, but many enzyme that hydrolyses nuclear substrates phosphorylated remain to be identified. on both tyrosine and serine/threonine residues, has a Earlier we used two different methods to isolate p53- potential role in deactivation of mitogen- or stress- target genes. One was differential display, using a cell activated protein kinases. Using cDNA-microarray tech- line in which the expression of an exogenous wild-type nology, we found that the expression of DUSP5 mRNA p53 gene can be regulated under the control of the was dramatically increased by exogenous p53 in lactose operon. In this manner, we isolated five genes, U373MG, a p53-mutant glioblastoma cell line. Transcrip- TP53TG1 (Takei et al., 1998), TP53TG3 (Ng et al., tion of DUSP5 was also remarkably activated by 1999), fractalkine (Shiraishi et al., 2000), p53R2 endogenous p53 in response to DNA damage in colon- (Tanaka et al., 2000), and p53DINP1 (Okamura et al., cancer cells (p53 þ / þ ) that contained wild-type p53, but 2001). The other method was a yeast enhancer-trap not in p53À/À cells. Chromatin-immunoprecipitation system that allowed direct cloning of functional p53- (ChIP) and reporter assays demonstrated that endogenous binding sequences from human genomic DNA (Tokino p53 protein would bind directly to the promoter region of et al., 1994). By isolating and sequencing cosmid clones the DUSP5 gene, implying p53-dependent transcriptional containing every p53-binding site obtained in that way, activity. Overexpression of DUSP5 suppressed the growth we identified four novel p53-target genes from genomic of several types of human cancer cells, in which Erk1/2 regions surrounding the binding sequences: GML was significantly dephosphorylated. If, as the results (Furuhata et al., 1996), P2XM (Urano et al., 1997), suggest, DUSP5 is a direct target of p53, it represents a BAI1 (Nishimori et al., 1997), and p53AIP1 (Oda et al., novel mechanism by which p53 might negatively regulate 2000). cell-cycle progression by downregulating mitogen- or The VH1 phosphatase encoded by a gene of vaccinia stress-activated protein kinases. virus is a dual-specificity protein-tyrosine phosphatase Oncogene (2003) 22, 5586–5591. doi:10.1038/sj.onc.1206845 (PTPase) that hydrolyses substrates phosphorylated on both tyrosine and serine/threonine residues. VH1-like Keywords: DUSP5; p53; Erk1/2 PTPases have since been identified in humans and other organisms. Screening a human genomic library with a partial DUSP1 cDNA yielded several novel PTPase genes, including one designated HVH3 (human VH1- like PTPase-3) (Martell et al., 1994). HVH3, also called Introduction as DUSP5, was upregulated by TALL-1, a member of the family of tumor necrosis factors (Kovanen et al., Mutations in the p53 gene are found in about half of all 2002); this observation implied that induction of DUSP5 human cancers examined (Hollstein et al., 1991). The might deactivate mitogen- or stress-activated protein p53 gene product, a transcription factor, binds to its kinases. A recombinant protein containing the catalytic target genes in a sequence-specific manner. By regulating domain of DUSP5 has in fact displayed phosphatase those transcriptional targets, p53 protein can protect activity toward ERK1 (MAPK3) in vitro (Ishibashi et al., cells from malignant transformation arising from severe 1994). DNA damage or critical cellular stress, by inducing As a third approach to the search for p53 targets, we either cell-cycle arrest or apoptosis (el-Deiry et al., 1992; have been applying cDNA-microarray technology; such Oren, 1994; Ko and Prives, 1996; Levine, 1997). To date, experiments have allowed us to document p53-depen- dent transcription of genes encoding Semaphorin3B (Ochi et al., 2002), CABC1 (Iiizumi et al., 2002), IRF5 (Mori et al., 2002b), and CyclinK (Mori et al., 2002a). *Correspondence: Y Nakamura; E-mail: [email protected] Here, we report that the DUSP5 gene is yet another 2Current address: Cancer Medicine and Biophysics Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo transcriptional target of p53, and show that it might 104-0045, Japan be involved in p53-dependent suppression of cell Received 22 January 2003; revised 15 May 2003; accepted 5 June 2003 growth. DUSP5 as a p53-target K Ueda et al 5587 Results and discussion To examine further whether p53BS1 in fact has p53- dependent transcriptional activity, we carried out To identify additional p53-target genes, we applied a reporter assays. A 331-bp genomic DNA fragment cDNA-microarray technique to screen human cDNAs including the promoter and p53BS1 was cloned into for p53-inducible transcripts, as previously described pGL3-basic vector, and this construct (pGL3-p53BS1) (McGrory et al., 1988; Ono et al., 2000). Transcription was cotransfected into SaOS-2 cells along with wt-p53 of the gene that encodes dual-specificity phosphatase 5 or mt-p53 expression vectors. As shown in Figure 2c, (DUSP5) was induced to a remarkable degree by the luciferase activity was strikingly enhanced, up to nearly introduction of exogenous p53 into p53-deficient cells. 50-fold, by co-transfection with wt-p53 expression The induction was confirmed on Northern blots, which vector, but not with mt-p53. Moreover, point mutations indicated that the DUSP5 transcript was induced by in the p53BS1 sequence (mt1) completely blocked this overexpression of p53 using Ad-p53 (Figure 1a) and it enhancement, suggesting that p53-dependent activity was 2.5 kb long. We also examined whether endogenous was specific to the sequence of p53BS1. These results, p53 could activate the transcription of DUSP5 in colon- taken together, suggested that DUSP5 is a bona fide cancer cell lines HCT116 (p53 þ / þ ) and HCT116 target of p53. (p53À/À) in response to DNA damage. As shown in To explore the role of DUSP5 among the physiolo- Figure 1b, the expression of DUSP5 mRNA increased gical functions of p53, we constructed a plasmid remarkably in HCT116 (p53 þ / þ ) cells, but not in designed to express DUSP5 protein containing HA-tag HCT116 (p53À/À) cells, even though both cell lines had at the N-terminus (N-HA-DUSP). N-HA-DUSP pro- suffered genotoxic stress through adriamycin treatment teins were ectopically and stably expressed in A549 and or UV radiation. These results clearly suggested that H1299 lung-cancer cells, and in HCT116 p53 þ / þ or DUSP5 was likely to be a strong candidate as a direct p53À/À colon-cancer cells, by transfection of the N- transcriptional target of p53. HA-DUSP plasmid (Figure 3a). As shown in Figure 3b, Therefore we evaluated binding of the p53 protein to immunostaining experiments indicated that DUSP5 was the transcriptional regulatory region of this gene by localized in the nuclei of HCT116 (p53 þ / þ ) cells; performing ChIP assays. We identified several candidate similar results were obtained with A549, H1299, and sequences as p53-binding targets in the promoter region HCT116 (p53À/À) cells (data not shown). and in intron 1, and then tested each of them for Using the same plasmid, we investigated a possible interaction with endogenous p53 protein. One potential role of DUSP5 in cell growth by means of a colony- p53-binding sequence (p53BS1), located 1.2 kb upstream formation assay. We transfected pcDNA3.1( þ )/ of the transcription starting point (Figure 2a), was DUSP5, pcDNA3.1(À)/DUSP5, or pcDNA3.1( þ )/ into reproducibly present in the chromatin DNAs that were each of three cancer-cell lines (H1299, HCT116 p53 þ / immunoprecipitated in endogenous p53-protein com- þ , HCT116 p53À/À). The cells were cultured in the plexes, whereas none of the other fragments including presence of G418, and the number of colonies was p53BS2 was amplified by PCR using the same chroma- counted 2 weeks later. As shown in Figure 4a, enforced tin DNAs. We interpreted this result to mean that expression of DUSP5 significantly reduced the number endogenous p53 protein had interacted with only of colonies in all the three cancer cell lines compared p53BS1 in response to adriamycin-induced DNA with the same lines using pcDNA3.1(À)/DUSP5 or damage (Figure 2b). pcDNA3.1( þ )/ vector, suggesting a growth-suppressive Figure 1 (a) Induction of DUSP5 and b-actin mRNAs in the human glioblastoma cell line U373MG by overexpressed p53, and (b) expression of DUSP5 mRNA by endogenous p53 after cytotoxic damage, as shown by Northern blotting. Expression of the b-actin gene served as a quantity control. DUSP5 mRNA was induced in cells infected by Ad-p53, but not in cells infected by Ad-LacZ (data not shown). DUSP5 and p21waf1 mRNAs increased with time following adriamycin or UV treatment in HCT116 p53 þ / þ cells, but not in the HCT116 cell line lacking wild-type p53 Oncogene DUSP5 as a p53-target K Ueda et al 5588 Figure 2 (a) Genomic structure of DUSP5. A potential p53-binding site is present in the promoter region, between nucleotide positions À1179 and À1159, where þ 1 represents the translation-initiation site. (below) Sequence of the putative p53BS1-binding site compared to the consensus sequence. R, purine; Y, pyrimidine; W, A, or T. E1–E4 represent exons. (b) ChIP assay of a genomic fragment, nucleotide positions À1359 to À1028 containing the p53BS1 sequence, in damaged HCT116 p53 þ / þ or HCT116 p53À/À cells.