A Variant of Nuclear Localization Signal of Bipartite-Type Is Required for the Nuclear Translocation of Hypoxia Inducible Factors (1A,2A and 3A)

A variant of nuclear localization signal of bipartite-type is required for the nuclear translocation of hypoxia inducible factors (1a,2a and 3a)

Jin Cai Luo1 and Masabumi Shibuya*,1

1Department of Genetics, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan

Hypoxia inducible factors (HIF1, 2 and 3), consisting of (HRE) (Semenza, 1998). HIF1a is unique to HIF1 a and b subunits, play an essential role in various whereas HIF1b is identical to the aryl hydrocarbon responses to hypoxia. Nuclear entry of a subunits is a receptor nuclear translocator (Arnt), that also di- necessary step for the formation of DNA-binding merizes with the aryl hydrocarbon receptor (Wang et complex with b subunit, which is constitutively localized al., 1995). Recently, two HIF1a-related proteins have in the nucleus. We show here that the nuclear been found: HIF2a and HIF3a. The former, which is accumulation of HIF2a induced by hypoxia is mediated also named EPAS1, MOP2, HLF and HRF (Ema et through a novel variant of bipartite-type nuclear al., 1997; Hogenesch et al., 1998 and references localization signal (NLS) in the C-terminus of the therein), like HIF1a, forms a heterodimeric complex protein, which has an unusual length of spacer sequence with HIF1b and activate VEGF gene transcription between two adjacent basic domains. We further show after exposure to hypoxia. The latter, which is isolated that when the ubiquitin-proteasome system was de®cient from a mouse EST cDNA clone (Gu et al., 1998), has or inhibited, HIF2a accumulated in the nucleus even a N-terminal domain quite similar to HIF1a and under normoxia, also mediated through the bipartite HIF2a (57% and 53% identity, respectively). NLS. These ®ndings indicate that the protein stability is At the protein level, HIF1b is constitutively critical for the nuclear localization of HIF2a and expressed in the nucleus and not signi®cantly aected hypoxia is not a necessary factor for the process. by oxygen concentration. In contrast, HIFa subunits Importantly, the NLS of HIF2a is also conserved in are abundant under hypoxia but rapidly degraded the other HIF family members, HIF1a and HIF3a. under normoxia. The ubiquitin-proteasome system Mutational analyses proved that the NLS mediating the plays a pivotal role in the degradation of HIF a nuclear localization of HIF1a is indeed bipartite-, but subunits, which is negatively regulated by the tumor not monopartite-type as thought before. Our results suppressor von hippel-lindau (VHL) protein (Kaelin suggest that the newly identi®ed NLS is crucial for the and Maher, 1998; Maxwell et al., 1999; Cockman et functional regulation of HIF family. Oncogene (2001) al., 2000; Ohh et al., 2000; Tanimoto et al., 2000). In 20, 1435 ± 1444. fact, VHL has been shown to be a putative ubiquitin ligase that speci®cally targets HIFa subunits (includ- Keywords: nuclear localization signal; hypoxia indu- ing HIF2a) for ubiquitination (Stebbins et al., 1999; cible factor; ubiquitin-proteasome system; von hippel- Cockman et al., 2000). Thus, the HIF complex lindau tumor suppressor; protein stability formation and transcription-activating function mainly depend on the regulation of the protein stability and nuclear entry of HIFa subunits. Progress Introduction has been made in understanding the protein stability and the transcriptional activation of the HIFa In response to hypoxia, a set of genes encoding subunits during hypoxia, but the mechanism under- erythropoietin, vascular endothelial growth factor lying its nuclear import is poorly understood (Conrad (VEGF), several glycolytic enzymes are adaptively et al., 1999; Ema et al., 1999). activated in the regulation of erythropoiesis, angiogen- Proteins larger than 45 kDa require NLS as the esis and energy metabolism. HIF1, a heterodimeric targeting signal, which is de®ned as the sequences complex composed of the two basic helix ± loop ± helix sucient and necessary for nuclear localization of their (bHLH) Per ± Arnt ± Sim (PAS) subunits HIF1a and respective proteins (Jans and Hubner, 1996; Dingwall HIF1b, has been shown to activate these diverse genes and Laskey, 1991). The NLS generally consists of two under hypoxic conditions, through binding a common classes. One is monopartite-type of basic amino acids enhancer domain termed hypoxia responsive element (aa) represented by the SV40 large T antigen NLS (PKKKRKV), and the other is bipartite-type represented by the nucleoplasmin NLS (KRPAATKKAG- QAKKKK), which is characterized by two sets of *Correspondence: M Shibuya Received 18 October 2000; revised 26 December 2000; accepted 4 adjacent basic residues separated by a spacer of January 2001 approximately 10 aa (Dingwall and Laskey, 1991; A variant of bipartite NLS for nuclear import of HIFs JC Luo and M Shibuya 1436 Burglin et al., 1987). The two sets of basic residues of bipartite-type NLS are essentially required for sucient nuclear localization, while the spacer is mutant-tolerant in sequence. A recent study has proposed that the nuclear import of human HIF1a induced by hypoxia is mediated through a monopartite-type NLS (Kallio et al., 1998), although it has not been carefully characterized. However, the regulation of intracellular localization of HIF2a and HIF3a is hardly known. Here, we report the identi®cation of a novel variant of bipartite-type NLS in the C-terminus of human HIF2a, which is necessary for the nuclear translocation of the protein induced by hypoxic condition or by the inhibition of ubiquitin-proteasome system under normoxia. Impor- tantly, this NLS is well conserved in other HIF family members, HIF1a and HIF3a.

Results

Nuclear accumulation of HIF2a induced by hypoxia Using three cell lines, Hela, HepG2 and PC12, we found that HIF2a protein levels in the whole cell extracts were signi®cantly induced by hypoxia (data not shown), consistent with previous observations (Wiesener et al., 1998). We further found that similar to HIF1a, HIF2a induced by hypoxia was predomi- nantly localized to the nucleus (Figure 1a). Immuno- ¯uorescent analysis of HA-HIF2a-expressed cells with an antibody for the HA tag con®rmed this ®nding (Figure 1b). To investigate the localization of HIF2a easily, we generated pEGFP-HIF2a, which was tagged with EGFP at the N-terminus of HIF2a. The chimeric constructs were introduced into COS-7 cells, and their localization was examined. The ¯uorescence of EGFP alone uniformly distributed between the cytoplasm and nucleus under both normoxia and hypoxia. In contrast, the ¯uorescence of EGFP-HIF2a exclusively accumulated in the nucleus after exposure to hypoxia (Figure Figure 1 Hypoxia-induced nuclear accumulation of HIF2a.(a) 2). In addition, treatment of the cells with Dipyridyl to Immunoblot of representative nuclear (N) and cytoplasmic (C) mimic hypoxic condition also induced the nuclear fractions of Hela, HepG2 and PC12 cells treated with or without accumulation of EGFP-HIF2a. Furthermore, using a 2% hypoxia for 12 h. Protein samples (100 mg per lane) were construct tagged with EGFP at the C-terminus of loaded for the HIF1a and HIF2a blot, 25 mg of protein per lane was used for a-Tubulin and Oct-1 blots. (b) Immuno¯uorescence HIF2a, we obtained similar results (data not shown). of pCMV-HA-HIF2a-transfected Hela cells with HA epitope These results show that fusion with EGFP did not antibody. After transfection, cells were incubated for 12 h under aect the regulation of intracellular localization of normoxia or hypoxia. Cells were processed for immuno¯uores- HIF2a by hypoxia. cence with HA antibody (aHA) and for the visualization of the nuclei with PI staining (PI)

Two candidate regions involved in the regulation of nuclear localization of HIF2a (EGFP-HIF2a/689 ± 870) displayed a constitutive nu- To identify the region required for the nuclear clear localization under both normoxia and hypoxia. localization of HIF2a, we generated a series of C- Sequence analyses revealed two regions bearing NLS- terminal and internal deletion mutants of HIF2a fused like basic motifs (Figure 3a): the N-terminal one is with EGFP (Figure 3b) and the localization of these located in the bHLH domain, and the C-terminal one fusion proteins was examined. As shown in Figure 3c, lies between the N-terminal transactivation domain two fusion proteins of relatively minimal N-terminal (NAD) and C-terminal transactivation domain (CAD) region (EGFP-HIF2a/1 ± 50) and C-terminal region (Ema et al., 1999). To con®rm the presence of NLS

Oncogene A variant of bipartite NLS for nuclear import of HIFs JC Luo and M Shibuya 1437

Figure 2 Regulation of EGFP-HIF2a under hypoxic conditions. Hela cells were transfected with pEGFP or pEGFP-HIF2a and treated with 2% hypoxia or normoxia for 12 h, or normoxia in the presence of 100 mm of Dipyridyl (DP) to mimic hypoxic condition for 6 h before ®xation. The localization of the expressed proteins was examined by ¯uorescent microscopy. The nuclei were visualized with PI staining (PI) motifs in these regions, we introduced several point et al., 1998; Kallio et al., 1998). In fact, inclusion of the mutations into some basic residues. As shown in PAS domain to the minimal N-terminal region of Figure 3c, all the four mutants of HIF2a/1 ± 50/ HIF2a (EGFP-HIF2a/1 ± 333) did suppress the nuclear R14A, HIF2a/1 ± 50/R14G/R15G, HIF2a/689 ± 870/ localization of the fusion protein (Figure 3c). K709T and HIF2a/689 ± 870/K738T lost their abilities In contrast, the mutations of either K709T or K738T to accumulate in the nucleus. This result con®rmed partly impaired the nuclear accumulation of HIF2a that both terminal regions of HIF2a contain NLS induced by hypoxia. Double mutation of K709T/ motifs. K738T abolished hypoxia-induced nuclear accumulation of HIF2a. These results indicate that both basic motifs within the C-terminal are essential for the The NLS motif in the C-terminus of HIF2a is required for nuclear localization of HIF2a under hypoxia. Interest- the nuclear localization of the protein under both hypoxia ingly, the two basic motifs were also required for the and normoxia nuclear import of EGFP-fused HIF2a under normoxia Since two regions containing potential NLS were since mutations of K709T and K738T led to the identi®ed, we then examined their roles in the retention of fusion proteins in the cytoplasm under regulation of nuclear localization of HIF2a in the normoxia (Figure 4). context of full length molecule. Introduction of point Taken together, the NLS motif in the C-terminus is mutation (R14A) or double mutations (R14G/R15G) functional and required for the nuclear localization of did not obviously aect the intracellular localization of HIF2a under both hypoxia and normoxia. HIF2a under both normoxia and hypoxia (Figure 4), indicating that the N-terminal NLS motif of HIF2a The NLS motif in the C-terminus of HIF2a is a novel does not function in the context of the full-length variant of bipartite-type protein. This may be due to the presence of adjacent PAS domain, which has been shown to mask the NLS To examine the ability of the NLS motif in the C- in human aryl hydrocarbon receptor and HIF1a (Ikuta terminus of HIF2a to target a large heterologous

Oncogene A variant of bipartite NLS for nuclear import of HIFs JC Luo and M Shibuya 1438

Figure 3 Mapping of the regions involved in the regulation of localization of HIF2a.(a) Schematic representation of the localization of potential NLSs in human HIF2a. Bold letters Figure 4 The NLS in the C-terminus of HIF2a is required for indicate the basic amino acid residues within the NLS-like motifs. the nuclear translocation of full length HIF2a. Point mutations (b) Deletion and/or mutation constructs of EGFP-HIF2a. The within the NLS motifs of the N-terminal and C-terminal region mutations were either single (Arg to Ala) or double (Arg to Gly) was introduced into the residues as shown. After transfection, amino acid exchange at residues 14 and 15, and a single or double COS-7 cells were treated with (+) or without (7) 100 mmof amino acid exchanges (Lys to Thr) at residues 709 and 738. (c) Dipyridyl (DP) for 6 h before ®xation Representative examples of the subcellular distributions of EGFP- HIF2a fusion proteins presented in (b). After transfection, COS-7 cells were treated with (+) or without (7) 100 mm of Dipyridyl exclusively localized to the cytoplasm (data not shown). (DP) for 6 h before ®xation and the localization of green In contrast, fusion of fragments containing HIF2a aa ¯uorescence in the transfected cells were observed. The ¯uorescence patterns are indicated as: N, nucleus; C, cytoplasm; N/C, 708 ± 742 to the tag (EGFP-b-Gal-HIF2a/708 ± 742) led both the nucleus and cytoplasm to a complete nuclear localization (Figure 5), indicating that this fragment contains strong NLS(s). To further characterize the two basic motifs in the fragment of protein to the nucleus, we fused the fragments HIF2a, we introduced single mutations of either containing aa 708 ± 742 or its mutants of key basic K709T or K738T, and double mutation of K709T/ residues to EGFP-b-Gal (Figure 5). EGFP-b-Gal tag K738T into EGFP-b-Gal-HIF2a/708 ± 742, and found (about 190 kDa) is too big to diuse into the nucleus that these mutations impaired and abolished the passively, ensuring identi®cation of a functional NLS nuclear localization, respectively (Figure 5). which actively targets the fused EGFP-b-Gal to the The above observations showed that the two basic nucleus (Bear et al., 1999). EGFP-b-Gal tag alone motifs in the C-terminus of HIF2a are interdepen-

Oncogene A variant of bipartite NLS for nuclear import of HIFs JC Luo and M Shibuya 1439 in the whole cell extracts under normoxia (data not shown). We then found that the inhibitor-induced HIF2a accumulated remarkably in the nucleus (Figure 6a). Since some proteasome inhibitors might be toxic to the integrity of nuclear structure (Chilov et al., 1999), we thus chose a VHL-de®cient cell line (786-0) (Maxwell et al., 1999) to avoid the inhibitor in¯uences. As described in `Introduction', VHL has been shown to be a putative ubiquitin ligase which speci®cally targets HIFa subunits (including HIF2a) for ubiquitination Figure 5 The NLS in the C-terminus of HIF2a actively targets and degradation (Stebbins et al., 1999; Cockman et al., the fused EGFP-b-Gal to the nucleus. Subcellular distributions of chimeric proteins of EGFP-b-Gal-tagged NLS motif of HIF2a or 2000). In Caki cells, whose VHL is intact, only a low various mutants were examined. The construct of HIF2a/705 ± level of HIF2a protein was detected from the nuclear 742/NP 10 aa was created by the replacement of the spacer fraction under normoxia, but a high level of HIF2a sequence between 716 and 732 aa with that of X. laevis protein was induced by hypoxia, just like those in other nucleoplasmin NLS. The mutated basic residues are indicated cells (Figure 1a). Interestingly, a signi®cant accumula- with outline letters. The ¯uorescence patterns are indicated as: N, nucleus; C, cytoplasm. N/C, both the nucleus and cytoplasm tion of HIF2a protein was observed in the nuclear fraction of 780-6 cells both under normoxia and hypoxia (Figure 6b), indicating a constitutive nuclear accumulation of HIF2a under VHL-minus condition. dently required for full nuclear localization of the These observations suggest that HIF2a stabilized by chimeric protein, and suggest that the two basic motifs the loss of VHL protein or the inhibition of VHL- may constitute a bipartite-type NLS. However, the ubiquitin-proteasome system is able to translocate to critical residues in the basic motifs are dierent from the nucleus under normoxia. To clarify in more detail the consensus sequences of the typical bipartite type the relationship between the stability as well as NLS of X. laevis nucleoplasmin (see Introduction). In accumulation of HIF2a in the nucleus and VHL addition, the sequence separating the two basic regions protein, VHL-de®cient 780-6 cells were transfected in the C-terminal NLS-like motif of HIF2a (27 aa) is with Myc-tagged wild type (VHLwt) or mutant type even longer than the spacer of the typical bipartite-type (VHLmt) VHL plasmids, and HIF2a protein expres- NLS of X. laevis nucleoplasmin (10 aa). Therefore, we sion in the whole cell extracts was examined. We found next examined the possible length requirement of the that the introduction of wild type VHL, but not Spacer for ecient NLS activity. Internal deletion of mutant type, dose-dependently decreased the protein the amino acid residues between 716 and 732 to level of HIF2a in 786-0 cells. This down-regulation of generate a spacer of 10 aa length did not apparently HIF2a by the wild type VHL was suppressed by the aect their nuclear targeting function for the fusion treatment of proteasome inhibitor N-cbz-LLL (Figure protein. We then replaced the spacer of HIF2a with 6b). This observation con®rmed that VHL is involved that of nucleoplasmin with the same length and in the degradation of HIF2a. We then investigated the obtained similar results (Figure 5). These results expression of HIF2a protein in the nuclei of 786-0 cells indicate that a spacer of 10 aa length is sucient for after the introduction of VHL. Figure 6c showed that full HIF2a NLS function. However, further deletion of introduction of wild type VHL signi®cantly decreased the amino acid residues between 713 and 733 to the nuclear accumulation of HIF2a protein, which was generate a spacer of 6 aa impaired the NLS activity. restored by the treatment of N-cbz-LLL. This result Taken together, these results indicate that the two suggests that accumulation of HIF2a in the nucleus in basic motifs in the C-terminus of HIF2a constitute a 786-0 cells is due to the defect of VHL-ubiquitin- novel NLS of bipartite type. proteasome system. Finally, we studied whether new protein synthesis is required for the nuclear accumulation of HIF2a induced by proteasome inhibitors. As Stabilized HIF2a can be imported to the nucleus under shown in Figure 6d, protein synthesis inhibitor normoxia, mediated through the bipartite NLS Cycloheximide signi®cantly decreased the amount of It is thought that the nuclear import of HIF1a is HIF2a accumulated in the nucleus induced by dependent on hypoxia (Semenza, 1998). Since the proteasome inhibitor N-cbz-LLL, strongly suggesting nuclear translocation of HIFa subunits involves at that the turnover of HIF2a is quite rapid under least two steps: the stabilization and nuclear import, it normoxia and the major part of HIF2a accumulated is interesting to investigate the eect of protein stability in the nucleus is the newly synthesized HIF2a. This is on the nuclear localization of HIFa subunits. To consistent with the observation that the half-life of approach this, we ®rst examined the eect of treatment HIF proteins under normoxia is very short (Wang et with the three ubiquitin-proteasome inhibitors, N-cbz- al., 1995). However, another possibility that a newly LLL (Sigma), Lactacystin (Calbiochem) and N-(2- synthesized protein(s) other than HIF2a is required for mercaptopropiony)-glycine (NMPG) (Fluka). We ob- the nuclear translocation of HIF2a in the presence of served that these inhibitors signi®cantly induced HIF2a proteasome inhibitor cannot be ruled out.

Oncogene A variant of bipartite NLS for nuclear import of HIFs JC Luo and M Shibuya 1440

Figure 6 Stabilized HIF2a was able to translocate to the nucleus under normoxia. (a) Immunoblot analysis of representative nuclear (N) and cytoplasmic (C) fractions of several cells with anti-HIF 2a antibody. Left panel: treatment of Hela cells with ubiquitin-proteosome inhibitor: 40 mM N-CBZ-LLL-AL (NCLA), 60 mM Lactacystin or 2 mM NMPG for 8 h. Hela cells were left nontreated (Non) or treated with 100 mm DP (PC) for 8 h were used as a control. Right panel: comparison of HIF2a expression in Caki and 786-0 cells in response to DP. Cells were treated with (Hy) or without (Nr) 100 mmDPfor8h.(b) Immunoblot analysis of whole cell lysates from pVHLwt- or pVHLmt-transfected 786-0 cells with anti-HIF2a antibody. 786-0 cells were transfected with pCMV-Myc-VHLwt or pCMV-Myc-VHLmt with indicated amounts (mg). After 12 h culture under normoxia, cells were further incubated under normoxia in the presence or absence of 40 mM N-CBZ-LLL-AL (NCLA) for 8 h. (c) Immunoblot analysis of representative nuclear (N) and cytoplasmic (C) fractions of pVHLwt- or pVHLmt-transfected 786-0 cells with anti-HIF2a antibody. 786-0 cells were transfected with pCMV-Myc-VHLwt or pCMV-Myc-VHLmt with indicated amounts (mg). After 12 h culture, cells were further incubated under normoxia in the presence or absence of 40 mM N-CBZ-LLL-AL (NCLA) for 8 h. Caki cells were used as control. (d) Immunoblot analysis of representative nuclear fractions of Caki cells with anti-HIF2a antibody. Caki cells were treated with 100 mg/ml Cycloheximide (Sigma), 40 mM N-CBZ-LLL-AL (NCLA), or with both of them for indicated hours before harvesting. Immunoblot analysis of Ran protein is used as control as we found that Ran is a relatively abundant and stable nuclear protein. The intensity of Western blot band was measured as described in Materials and methods

Taken together, our results suggest that protein terminal basic motifs of HIF1a is a monopartite type or stability is critical for the nuclear import of HIF2a for bipartite type NLS, we used the same protocol as which hypoxia is not a necessary factor. described above, i.e. fusing the fragments spanning To determine whether the nuclear localization of HIF1a aa 716 ± 757 or its mutants of key basic residues HIF2a resulting from the loss of VHL is mediated with EGFP-b-Gal tag and examining their intracellular through the C-terminal NLS, we expressed HA-tagged localization. As shown in Figure 8c fusion protein HIF2a or HIF2a mutants of C-terminal NLS in 786-0 containing HIF1a aa 716 ± 757 accumulated in the cells and examined the intracellular localization by nucleus. Interestingly, introduction of point mutation Western blot and immunostaining analyses. As shown of either K719T or K753T almost abolished the nuclear in Figure 7, HA-tagged HIF2a, but not HA-tagged localization. Unexpectedly, internal deletion of the HIF2a/K738T, HIF2a/K709T, HIF2a/705 ± 742/D713 ± amino acid residues (aa 724 ± 750) between the two 733 or HIF2a/K738T/K709T, exclusively accumulated basic motifs led to a similar result. These ®ndings in the nucleus under normoxia, proved that the nuclear indicate that each set of the two basic motifs and the localization of HIF2a resultant from the loss of VHL is intervening sequences are necessary for the nuclear mediated through the C-terminal NLS. localization. To extend these ®ndings, we constructed a series of mutants of full length HIF1a protein and obtained similar results (Figure 8d). These data Bipartite NLS of HIF2a is conserved in hypoxia inducible provided evidence that the two basic motifs in the C- factor family terminus of HIF1a also constitute a bipartite type, but Sequence comparisons revealed that the newly identi®ed not a monopartite type, or two separate monopartite bipartite NLS of human HIF2a is highly conserved in type NLSs. Interestingly, the spacer of HIF1a NLS those among vertebrate animals (Figure 8a), suggesting (31 aa) is a little longer than that of HIF2a (Figure 8b). its biological importance in the function of HIF2a. Since the key basic residues are conserved in HIF3a Interestingly, the key basic residues in the NLS of (Figure 8b), it is reasonable to predict that the bipartite- HIF2a are also conserved in the C-terminal basic motifs type NLS is also required for the nuclear localization of of HIF family members, HIF1a and HIF3a, although HIF3a. In summary, these observations strongly suggest HIF1a was thought to contain a monopartite-type NLS that the novel bipartite-type NLS is required for the (Kallio et al., 1998). To determine whether the C- nuclear localization of all HIF a family members.

Oncogene A variant of bipartite NLS for nuclear import of HIFs JC Luo and M Shibuya 1441

Figure 7 Stabilized HIF2a can be imported to the nucleus in 786-0 cells under normoxia, mediated through the bipartite-type NLS in the C-terminus. 786-0 cells were transfected with HA-tagged HIF2a or various mutants of the NLS in the C-terminus and incubated for 24 h under normoxia. (a) Immunoblot analysis of representative nuclear (N) and cytoplasmic (C) fractions of transfected cells with HA antibody. Fifty mg of protein per lane was used. (b) Immuno¯uorescence analysis of transfected cells. Transfected cells were ®xed and stained with HA antibody as described in Materials and methods

Discussion The nuclear translocation of HIFa subunits is a process consisting of at least two steps: stabilization Considering the fact that HIF1b is constitutively and nuclear import. Although the nuclear import of localized to the nucleus (Eguchi et al., 1997), the HIF1a has been suggested to be dependent on hypoxia, mechanism underlying the nuclear translocations of the tight link between the nuclear imports and hypoxia HIFa subunits has become one of the central themes has not been established. In this article, we present in the study of the regulation of HIF activities. In evidence that hypoxia is not a necessary factor for the this article, we demonstrated a variant of bipartite- nuclear localization of HIF1a, instead, the stability of type NLS in the C-terminus of HIF2a. The NLS of protein is critical. First, treatment of ubiquitin- HIF2a was functional, not only necessary for the proteasome inhibitors remarkably led to the nuclear nuclear import of HIF2a under both normoxia and accumulation of HIF2a protein under normoxia hypoxia, but also sucient for targeting a hetero- (Figure 6a). Our preliminary data have shown that logous cytoplasmic protein to the nucleus (Figures 3- the treatment of Hela cells with one of the inhibitors 5). This NLS shares some common features of known N-cbz-LLL, up-regulated the level of transcription of bipartite type NLSs. For instance, two sets of VEGF gene, a major target of HIF2a (Wakiya and adjacent basic motif are essential for the full nuclear Shibuya, unpublished data). Second, a signi®cant localization function of the NLS, and the spacer is accumulation of HIF2a protein was found in the important but not absolute and resistant to the nucleus of VHL-de®cient 786-0 cells under normoxia, sequence replacement or limited deletion (Figure 5). which was partly abrogated by the expression of VHL However, it is a new member because of two reasons. gene wild type (Figure 6b,c). Third, under normoxia First, there is the dierence between the critical basic both EGFP-fused HIF1a and HIF2a are partly residues of HIF2a NLS and the consensus sequences localized to the nucleus. GFP fusion has been shown of other bipartite ones. Second, the spacer between by our (Figure 2) and others' studies (Kallio et al., the basic motifs is unusually longer (27 aa) than that 1998) to partly stabilize HIFa subunits. of the other bipartite NLSs (approximately 10 aa) VHL, as a component of ubiquitin ligase complex (Figure 8b). (Stebbins, 1999), has been shown to speci®cally target

Oncogene A variant of bipartite NLS for nuclear import of HIFs JC Luo and M Shibuya 1442

Figure 8 The bipartite-type NLS in the C-terminus of human HIF2a is conserved both in those derived from a variety of vertebrate animals and in HIF1a and HIF3a.(a) The bipartite-type NLS in the C-terminus of human HIF2a is conserved in those derived from mouse, rat, bovine and avian. Their amino acid sequences are available in GenBank under the accession numbers of 3914285, 8953577, 5019614 and 6644408, respectively. (b) Sequence comparisons of the bipartite-type NLS in the C-terminus of human HIF2a with the corresponding regions of HIF1a and HIF3a and the consensus sequences of the NLS of X. laevis nucleoplasmin, IL-5 (18) and RB (8). (c) The NLS in the C-terminus of HIF1a is a bipartite, but not monopartite type. Subcellular distributions of chimeric proteins of EGFP-b-Gal-tagged NLS motif of HIF1a or various mutants were examined. Bold letters indicate the basic amino acid residues in the NLS motifs. The mutated basic residues are indicated with outline letters. The ¯uorescence patterns are indicated as: N, nucleus; C, cytoplasm. N/C, both the nucleus and cytoplasm. (d) The bipartite-type NLS in the C-terminus is also required for the nuclear localization of full length HIF1a. Point mutations or deletion mutation within the NLS motifs in the C-terminus of HIF1a were introduced into full length HIF1a as shown. After transfection, COS-7 cells were treated with (+) or without (7) 100 mm of Dipyridyl (DP) for 6 h before ®xation

HIF a proteins (including HIF2a) for ubiquitination lysine (K) are completely conserved and their im- and degradation (Cockman et al., 2000; Ohh et al., portance in the nuclear localization of HIF1a and 2000). These observations established a model for the HIF2a has been con®rmed. Mutational analyses nuclear translocation of HIF2a. Under normoxia, proved that the NLS mediating the nuclear localization HIF2a is unstable, which is stabilized by three of HIF1a is indeed bipartite-, but not monopartite-type conditions, i.e. (1) exposure of cells to hypoxia; (2) as thought before (Kallio et al., 1998). Interestingly, the loss of VHL; and (3) the inhibition of VHL- the spacer between the basic motifs in the NLS of ubiquitin-proteasome system. The stabilized HIF2a is HIF1a is longer (33 aa) than that of of HIF2a (27 aa) imported to the nucleus mediated by the newly (Figure 8b) and probably is the longest one in the identi®ed bipartite NLS in the C-terminus. Until bipartite-type NLSs known. In addition, the mutation now, however, it is unknown whether the protein of critical basic residue in either of the basic domains stabilization only increases the level of HIF2a protein of HIF1a bipartite NLS led to the complete cytoplas- or can unmask the bipartite NLS and thus stimulates mic retention of the protein, and in contrast, the the nuclear import of HIF2a as well. mutation of that in both of the basic domains of Since several studies have demonstrated that the HIF2a bipartite NLS led to the same eect. This inhibition of ubiquitin-proteasome pathways also led to ®nding indicates that each basic motif of HIF2a NLS the accumulation and transcriptional activation of only partly targets the protein to the nucleus and both endogenous HIF1a, it is quite possible that the protein basic motifs together can completely target HIF2a to stability is also important for the nuclear localization the nucleus. In contrast, each basic motif of HIF1a of HIF1a (Salceda and Caro, 1997; Huang et al., NLS is absolutely required for the nuclear localization 1998). of the protein. The NLS of HIF2a is strikingly conserved in the It is known that VHL mutations cause the other HIF family members, HIF1a and HIF3a. In the development of haemangioblastoma with numerous three NLSs, two basic residues of arginine (R) and blood vessels (Kaelin and Maher, 1998). Further

Oncogene A variant of bipartite NLS for nuclear import of HIFs JC Luo and M Shibuya 1443 studies revealed that although there is plenty of oxygen To construct GFP-NLS-b-Gal fusion proteins, the GST-GFP available, cells lacking VHL continue to produce HIF cassette vector was prepared as follows. BglII site was added proteins and express VEGF, which is normally to the initiation codon of b-galactosidase (Clontech), expressed in a hypoxia-dependent pattern (Maxwell et subsequently a BglII ± PstI fragment having full coding region al., 1999; Iliopoulos et al., 1996). The concept, that the of b-galactosidase gene was digested and ligated to XhoI± PstI opened pEGFP-C1 (linked XhoI and BglII sites with an stabilized HIF protein is able to localize to the nucleus in frame XhoI±BglII adaptor containing NLS sequence). and activate the target genes under normoxia, may Each construct was characterized by restriction and sequen- provide a reasonable explanation for the development cing analyses. of multiple vascular tumors of VHL syndrome. Transient transfection, preparation of whole cell extracts and subcellular fractions Materials and methods Cells were grown on 90 mm plates overnight to 50% con¯uency and transfected according to Eectene protocol Cell culture and reagents (Qiagen). A typical transfection was performed by using 2 mg 786-0, Caki, COS-7, Hela and HepG2 cells were grown in plasmid with 60 ml Eectene and incubating for 48 h before DMEM containing 10% fetal bovine serum (FBS), and PC12 harvest. Whole cell extracts were prepared as described cells in DMEM with 10% calf serum (CS) and 5% horse (Kallio et al., 1998). Fractionation of nuclear and cytoplasm fractions was performed as described elsewhere (Andrews and serum, under nromoxia (95% air and 5% CO2) or hypoxia (93% N2, 2% oxygen and 5% CO2) in an incubator (Tapei/ Faller, 1991). The protein concentrations in extracts were Espec, Osaka, Japan). In some experiments, cells were determined with a protein assay (Bio-Rad). cultured in the presence of 100 mm of Dipyridyl (Sigma) to mimic hypoxic condition (Kallio et al., 1998). Monoclonal Western blot analysis antibodies (mAbs) for the myc epitope and for the HA tag were obtained from Invitrogen and Boehringer Mannheim, Western blot analysis was performed as described (Luo et al., respectively. A mAb for HIF1a and a rabbit polyclonal 1998) with some modi®cations, by using 100 mg extracts if antibody for HIF2a were from Novus Biologicals. Goat there is no special description and analysing with SDS/7.5% polyclonal antibody for Ran was from Santa Cruz. PAGE. Mouse monoclonal antibodies (mAb) speci®c for Oct- 1 (Oncogene Research Products) and for a-tubulin (Sigma) were used on immunoblots as markers for nuclear and Plasmids cytoplasmic fractions, respectively. The intensity of Western The human HIF2a cDNA as well as full-length (1 ± 213 blot band was measured with a commercial method Quantity residues, wt) and truncated (1 ± 115 residues, mt) VHL cDNA One (Bio-Rad). were isolated from human umbilical vein endothelial cells (HUVEC) mRNA with a RT ± PCR protocol (Luo et al., Detection of transiently expressed proteins by green fluorecence + 1998) and inserted into pBluescript II/SK (Stratagene). The protein and immunofluorescence coding region of HIF2a from the full-length HIF2a cDNA was ampli®ed by PCR and cloned into the enhanced green Cells grown on coverslips were washed with PBS once and ¯uorescent protein (EGFP) expression vectors (Clontech), ®xed with 3% paraformaldehyde in PBS. For immuno¯uor- pEGFP-C1 or pEGFP-N1 to generate pEGFP-HIF2a escence staining, cells were permeabilized with 0.5% Triton constructs, which were tagged with EGFP at the N or C X-100 in PBS, blocked with 5% skim-milk and then terminus. The same fragment was inserted into EcoRI site of incubated with 10 mg/ml anti-HA mAb. A ¯uorescein- pCMV-HA (Clontech) in-frame with the upstream of HA conjugated goat anti-mouse antibody (ICN/Cappel) was used epitope sequences to generate CMV-HA-HIF2a construct. as the secondary antibody and DNA in the nuclei of cells was pCMV-Myc/VHLs were constructed by ligating full length or detected with Propidium Iodide (PI) (Wako, Osaka, Japan). truncated VHL cDNA into pCMV-Myc (Invitrogen) with the Finally, the ¯uorescence patterns were observed and photo- downstream of Myc epitope sequences. pEGFP-HIF1a was graphed by using a laser scanning cofocal imaging system created by cutting the HIF-1 coding region from pEBB/ (Bio-Rad). HIF1a (Novus Biologicals) as a NcoI±KpnI fragment and ligating this into BglII ± KpnI opened pEGFP-C1 (linked BglII and NcoI sites with an in frame BglII ± NcoI adaptor). Acknowledgments Deletion constructs of various length HIF2a fused with We wish to thank Dr Tetsuya Nosaka and Dr Yoshiharu EGFP were generated by taking advantage of conveniently Amasaki for their helpful discussions. This work was placed restriction sites in the construct EGFP-HIF2a. Point supported by a Grant-in-Aid for Special Project Research mutation mutants of HIF2a and HIF1a NLS were generated on Cancer-Bioscience 04253204 from the Ministry of by an oligonucleotide-based site-directed mutagenesis method Education (to M Shibuya) and a fellowship from the with Pfu DNA polymerase (Stratagene) (Imai, et al., 1991). Japan Society for the Promotion of Science (to JC Luo).

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