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Vol. 20, Issue 17, 3878-3887, September 1, 2009
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/Sima in Oxygen-dependent Nuclear ExportInstituto Leloir and FCEyN, Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas, 1405 Buenos Aires, Argentina
Submitted January 13, 2009;
Revised June 17, 2009;
Accepted June 29, 2009
Monitoring Editor: Marianne Bronner-Fraser
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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homologue, Sima, is localized mainly in the cytoplasm in normoxia and accumulates in the nucleus upon hypoxic exposure. We have characterized the mechanism governing Sima oxygen-dependent subcellular localization and found that Sima shuttles continuously between the nucleus and the cytoplasm. We have previously shown that nuclear import depends on an atypical bipartite nuclear localization signal mapping next to the C-terminus of the protein. We show here that nuclear export is mediated in part by a CRM1-dependent nuclear export signal localized in the oxygen-dependent degradation domain (ODDD). CRM1-dependent nuclear export requires both oxygen-dependent hydroxylation of a specific prolyl residue (Pro850) in the ODDD, and the activity of the von Hippel Lindau tumor suppressor factor. At high oxygen tension rapid nuclear export of Sima occurs, whereas in hypoxia, Sima nuclear export is largely inhibited. HIF/Sima nucleo-cytoplasmic localization is the result of a dynamic equilibrium between nuclear import and nuclear export, and nuclear export is modulated by oxygen tension. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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/β heterodimers of basic-helix-loop-helix-PAS (bHLH-PAS) proteins in which the β-subunit is constitutive and the -subunit is regulated by oxygen levels (Wang et al., 1995
). Oxygen-dependent regulation of the -subunit is exerted through different mechanisms, including control of protein half-life (Maxwell et al., 1999), recruitment of transcriptional coactivators (Sang et al., 2002), and regulation of subcellular localization (Kallio et al., 1998; Tanimoto et al., 2000). In normoxia, -subunits undergo polyubiquitination and rapid degradation at the 26S proteasome, and in hypoxia proteolysis is largely prevented (Ivan et al., 2001; Jaakkola et al., 2001). The molecular basis of this regulation is now well understood; in normoxia two key prolyl residues that reside in the so-called oxygen-dependent degradation domain (ODDD) are hydroxylated in a reaction catalyzed by 2-oxoglutarate and iron (Fe2+)-dependent dioxygenases that utilize molecular oxygen as a cosubstrate (Ivan et al., 2001; Jaakkola et al., 2001; Masson et al., 2001; Yu et al., 2001). Prolyl hydroxylation enables interaction between the HIF ODDD and the von Hippel Lindau (VHL) tumor suppressor protein, which is the substrate recognition subunit of the VCB (VHL; Cullin2; Elongin B-C) E3 ubiquitin ligase complex (Maxwell et al., 1999; Ohh et al., 2000). Thus, at low oxygen levels hydroxylation is prevented, ubiquitination does not occur, and hence HIF is stabilized. Therefore, the enzymes that catalyze prolyl hydroxylation, termed PHD1, PHD2. and PHD3, are considered bona fide molecular oxygen sensors that ultimately control the half-life of HIF subunits (Bruick and McKnight, 2001; Epstein et al., 2001).
Recruitment of HIF transcriptional coactivators is another oxygen-regulated step of HIF activity. It is governed by a second oxygen-sensing system that involves hydroxylation of a specific asparagine residue located in the transactivation domain, next to HIF C-terminal end (Lando et al., 2002b). This hydroxylation reaction is catalyzed by another member of the 2-oxoglutarate and iron-dependent family of dioxygenases, called factor-inhibiting HIF (FIH; Lando et al., 2002a). This regulatory mechanism is also well characterized: FIH-mediated hydroxylation of the asparagine residue prevents physical interaction with the transcriptional coactivator P-300, which was previously shown to be required for maximal HIF transcriptional activity. Thus, in hypoxia asparaginyl hydroxylation is prevented and interaction with P-300 can take place, enabling full transcriptional activation of the system.
Regulation of HIF oxygen-dependent subcellular localization is less well understood. Studies in cell culture have shown that HIF is mostly cytoplasmic in normoxia and accumulates in the nucleus in hypoxia and that hypoxia-dependent nuclear import accounts for this differential localization (Kallio et al., 1998; Chilov et al., 1999). Although structure-function studies have led to the identification of a relevant nuclear localization signal (NLS) and other protein domains required for this regulation (Kallio et al., 1998; Luo and Shibuya, 2001), the molecular mechanism governing HIF subcellular localization is so far unclear.
We and others have demonstrated that an oxygen-inducible transcriptional response occurs in Drosophila (Lavista-Llanos et al., 2002; Douglas and Haddad, 2003; Gorr et al., 2004; Centanin et al., 2005; Dekanty et al., 2005), and that Similar (Sima; Nambu et al., 1996) and Tango (Tgo; Sonnenfeld et al., 1997) bHLH-PAS proteins are the HIF- and HIF-β homologues, respectively (Lavista-Llanos et al., 2002; Gorr et al., 2004; Romero et al., 2007). Although Tango is expressed constitutively regardless of oxygen levels, Sima protein is rapidly degraded in normoxia and stabilized in hypoxia; stability of the protein depends on a central protein domain encompassing amino acids 692-863, functionally homologous to HIF ODDD (Lavista-Llanos et al., 2002). More recently, we have identified Drosophila mutants for a HIF prolyl hydroxylase homologue that we have named Fatiga, which exhibited strong constitutive upregulation of both Sima protein and HRE reporter gene expression (Centanin et al., 2005, 2008). Thus the whole basic machinery controlling the transcriptional response to hypoxia is apparently conserved between Drosophila and mammalian cells.
By overexpressing Sima in transgenic embryos under control of an engrailed-gal4 driver, we overrode the rapid rate of protein degradation and found that Sima was mostly cytoplasmic in normoxia and nuclear in hypoxia (Lavista-Llanos et al., 2002). This phenomenon is not an all-or-none response but rather, Sima localization depends on oxygen levels in a graded manner and interestingly, it is influenced by developmental parameters (Dekanty et al., 2005). More recently, we have found that Sima nuclear import depends on an atypical bipartite NLS, localized next to the C-terminal end of the protein and that nuclear export is mediated in part by two conserved nuclear export signals (NESs) mapping at the bHLH domain of the protein (Romero et al., 2008).
In this study we have further explored the mechanism of oxygen-dependent regulation of HIF/Sima subcellular localization. We show that Sima localization emerges from a dynamic equilibrium between nuclear import and nuclear export and that oxygen levels can modify the ratio between these two processes. A cryptic CRM1-dependent NES occurs in the ODDD, contributing to the regulation of Sima nuclear export. Consistent with the occurrence of this NES in the ODDD, prolyl hydroxylation and VHL activity are absolutely required for CRM1-dependent nuclear export of Sima and moreover, graded activation of the prolyl hydroxylase/VHL pathway modulates the rate of Sima nuclear export. This regulatory mechanism has not been so far reported in mammalian HIF proteins.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Sima constructs for transgenic lines generation were all engineered in a pNB40 plasmid and then subcloned into the pCaSpeR-UAS vector as EcoRI-NotI fragments. EGFP-ODDD1-3 chimeras were generated by in-frame cloning of sequences amplified by PCR into XbaI-NotI sites of the pMT/Bip/V5-His-EGFP plasmid (Roth et al., 2003). The EGFP-ODDD fusion was generated by cloning in-frame the EGFP sequence in a SacII site upstream of the sequence encoding Sima 665-1507 (Bacon et al., 1998) in a pcDNA3.1 vector (Invitrogen, Carlsbad, CA). The plasmid encoding the EGFP-Sima 665-1507 fusion was used as a template to amplify the EGFP-ODDD sequence (Sima amino acids 665-871) using the primers: 5'-GCTTCGCCGCGGTCTAGATGGTGAGCAAGGGCGAGGAG-3'and 5'-GCTTCGTCTAGACCACATGAGATCCGTCTCCGTTAGC-3' that included XbaI restriction sites on their 5' ends and was subcloned in an XbaI site of a pMT/Bip/V5-His vector. Mutated EGFP-ODDD was generated by mutagenic divergent PCR in the original pMT/Bip/V5-His-EGFP-ODDD plasmid, with the QuikChange Site-directed Mutagenesis Kit (Stratagene, La Jolla, CA) by using the primers 5'-CTCGGGCAGCGCGCAGGTGCCCATGGCGCCAACCAACATGTC-3' and 5'-GACATGTTGGTTGGCGCCATGGGCACCTGCGCGCTGCCCGAG-3'. Underlined codons represent the mutations introduced in the sequence.
S2 Cell Transfections and Analysis of Subcellular Localization of EGFP Fusion Constructs
All expression constructs were cloned in a pMT/Bip/V5-His plasmid (Invitrogen). S2 cells were maintained at 25°C in Schneider Drosophila medium (Sigma, St. Louis, MO), with 10% fetal bovine serum (Invitrogen), 50 U/ml penicillin, and 50 µg/ml streptomycin in 25- or 75-cm2 T-flasks (Greiner, Monroe, NC). Transfections (5 µg of total DNA) were performed with a calcium phosphate transfection kit (Invitrogen), and after 24 h expression was induced by addition of 0.4 mM CuSO4 for 5–16 h. Cells were then observed at the confocal microscope (Carl Zeiss, Thornwood, NY; LSM5, Pascal). For hypoxic treatments, cells were exposed to hypoxia (3% O2) during 20 h in a Forma Scientific 3131 incubator (Marietta, OH). Leptomycin B (Sigma-Aldrich) was applied for 2 h, and results were immediately analyzed in the confocal microscope; cell nuclei were visualized by Hoechst 33342 staining. Quantitative analysis of subcellular localization was performed in the confocal microscope by measuring intensity of EGFP fluorescence per µm2 in the nucleus (N) and cytoplasm (C) and assessing the N–C ratio.
Fly Stocks and Analysis of Sima Localization In Vivo
The transgenic lines UAS-embargoed, UAS-VHL, UAS-simaP850A, and other UAS-sima–mutagenized variants were generated by P-element germline transformation using standard procedures. The following fly strains were also used: UAS-sima and UAS-nGFP-LacZ (Lavista-Llanos et al., 2002), embargoed3 (Collier et al., 2000), fatiga1 (Centanin et al., 2005), engrailed-Gal4 and Df(2R)en-A deficiency (Bloomington Stock Center). The UAS-VHL double-stranded RNA line (no. 32163) was obtained from the Vienna Drosophila RNAi Centre (VDRC).
Analysis of Sima subcellular localization in vivo was performed in transgenic embryos overexpressing the Sima wild-type protein or altered variants of Sima under control of an en-Gal4 driver. To obtain synchronized embryos, egg-laying agar plates were replaced every 3 h, and embryos were grown at 25°C in normoxia until the desired stage and then transferred to hypoxia for 4 h. Hypoxic treatments were applied in a Forma Scientific 3131 incubator at 25°C; embryos were then recovered, fixed, and immunostained with anti-Sima and/or anti-βgal antibodies (Bacon et al., 1998; Lavista-Llanos et al., 2002). Observations were performed in an Olympus BX60 microscope (Melville, NY) or in a Carl Zeiss LSM5 Pascal confocal microscope. To analyze the effect of embargoed, fatiga, or VHL loss-of-function, recombinant chromosomes carrying UAS-sima or en-gal4 elements and the mutation of interest were generated by meiotic recombination. Similarly, for emb or VHL gain-of-function experiments UAS-sima/UAS-VHL or UAS-emb recombinant chromosomes were generated and used for crosses with the en-gal4 driver. Quantitative analyses of subcellular localization were carried out as previously described (Dekanty et al., 2005). Briefly, each embryo was classified into one of three categories of Sima subcellular localization: cytoplasmic, where more than 90% of the cells in the engrailed-expressing stripes show cytoplasmic localization; nuclear, where more than 90% of the cells in the engrailed-expressing stripes have nuclear localization; and ubiquitous, where <90% of the cells have cytoplasmatic or nuclear localization. The percentage of embryos assigned to each subcellular localization category was calculated, and category distributions in the different genotypes were compared with a 
2 test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). We therefore, sought to investigate whether Sima steady-state subcellular localization depends on CRM1 in living embryos, by analyzing en-Gal4/UAS-sima individuals carrying a homozygous loss-of-function mutation in the Drosophila CRM1 homologous gene embargoed (Collier et al., 2000; Roth et al., 2003). Sima subcellular localization in these mutants was compared with that in wild-type control embryos. As we reported previously (Dekanty et al., 2005), in wild-type embryos Sima was predominantly cytoplasmic in normoxia and gradually more nuclear as oxygen concentration decreased (Figure 1, A and B). In embargoed mutant embryos (emb3/emb3), Sima localization was clearly more nuclear than in wild-type controls (Figure 1B), supporting the notion that Sima steady-state subcellular localization depends at least in part on active nuclear export mediated by the nuclear export receptor CRM1. To get direct evidence that a nuclear export defect in emb mutants accounts for the shift in Sima subcellular localization, we measured nuclear export directly in a reoxygenation assay as described before (Romero et al., 2008). Embryos expressing transgenic Sima protein were exposed to 1% O2 for 4 h (Sima is fully nuclear in this condition) and then transferred to normoxia and fixed at different times after reoxygenation for Sima subcellular localization analysis. As depicted in Figure 1C, Sima nuclear export is affected in the emb mutant embryos, supporting the notion that increased Sima steady-state nuclear localization in these mutants is due to slower nuclear export that partially depends on Embargoed.
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Next, we sought to investigate whether the Emb-dependent shift in Sima subcellular localization can modify Sima transcriptional activity, and for this purpose, we analyzed induction of a Sima-inducible LacZ reporter (Lavista-Llanos et al., 2002) in emb mutants and in embryos overexpressing Emb, in comparison with wild-type controls. As shown in Figure 1F, β-galactosidase activity derived from a Sima-inducible LacZ reporter was significantly increased in emb mutant embryos, and reduced in embryos overexpressing emb. These results provide direct functional evidence that alteration of Sima subcellular localization impinges on its transcriptional activity.
Occurrence of a Functional NES in the Sima Oxygen-dependent Degradation Domain
Because deletion of the entire ODDD provokes constitutive nuclear localization of Sima in normoxia, resulting in increased transcriptional activity of the protein (Lavista-Llanos et al., 2002), we explored whether CRM1-dependent NESs occur within this domain. Close inspection of the entire amino acid sequence of the ODDD did not reveal the presence of a canonical NES consensus (L-x(2,3)-[LIVFM]-x(2,3)-L-x[LI] (Bogerd et al., 1996) within this domain. However, because CRM1-dependent NESs often diverge from this canonical consensus, we tested the ability of the ODDD (amino acids 665-871) to promote nuclear export of an EGFP reporter in Drosophila S2 cultured cells. Analysis of subcellular localization of EGFP-ODDD revealed an ubiquitous (slightly nuclear) localization of the chimera, with an N–C fluorescence ratio of 1.4 (Figure 2A). To answer if the steady-state subcellular localization of the fusion protein is influenced by CRM1-dependent nuclear export, we investigated whether subcellular localization is sensitive to LMB. LMB provoked a significant increase of nuclear localization, shifting the N–C fluorescence ratio to 1.9 (Figure 2A). As expected, subcellular localization of an EGFP-NES positive control construct, bearing a NES derived from PKI (Roth et al., 2003), also became more nuclear upon addition of LMB (Figure 2A), and an EGFP construct lacking an export signal (negative control), was insensitive to LMB (Figure 2A). The above results strongly suggest that the Sima ODDD includes a functional CRM1-dependent NES.
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), we decided to analyze this stretch of hydrophobic amino acids as a candidate NES. We generated a mutated version of the complete EGFP-ODDD chimera by replacing Leu689 and Leu694 by alanines (Figure 2B). Subcellular localization of the mutated EGFP-ODDD in transfected S2 cells was clearly more nuclear than the wild-type version (Figure 2C), suggesting that the two leucine residues are part of a functional NES. When the cells transfected with mutated EGFP-ODDD were exposed to LMB, the construct became even more nuclear (Figure 2C), suggesting that the functionality of the ODDD NES was reduced but not completely abrogated by the mutations. To investigate whether this novel NES plays a role in the regulation of subcellular localization of the Sima full-length protein, we replaced Leu689 and Leu694 by alanines (SimaL689A/L694A), generated a transgenic (fly) line expressing this Sima variant, and analyzed the rate of nuclear export upon reoxygenation of the transgene embryos as above. Nuclear export of SimaL689A/L694A was clearly impaired in comparison with wild-type Sima (Figure 2D), confirming that the sequence under study is a functional NES that contributes to the overall subcellular localization of Sima.
Prolyl Hydroxylation and VHL Mediate Sima Nuclear Export
The occurrence of a CRM1-dependent NES in the ODDD prompted us to investigate whether oxygen-dependent hydroxylation of a prolyl residue within this domain is required for Sima nuclear export. We began by performing a quantitative analysis of Sima subcellular localization in embryos carrying a loss-of-function mutation in the fatiga (fga) gene, which encodes the Drosophila HIF prolyl-4-hydroxylase (Centanin et al., 2005). As shown in Figure 3A, embryos homozygous for fga1, the strongest of the fga alleles (Centanin et al., 2005), exhibited a major increase in Sima nuclear localization throughout embryogenesis at different oxygen tensions, indicating that reduced prolyl hydroxylase activity can shift the Sima import/export balance toward nuclear localization.
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; Centanin et al., 2008), and analyzed subcellular localization in UAS transgenic embryos. After replacing this proline by an alanine (SimaP850A) Sima became totally nuclear throughout embryogenesis, regardless of oxygen tension (Figure 3B). Taken together, these results indicate that prolyl hydroxylation is likely required for Sima nuclear export in normoxia.
In mammalian VHL-deficient cell lines high levels of stabilized HIF protein occur, and strong oxygen-independent expression of a battery of HIF target genes was demonstrated (Maxwell et al., 1999), suggesting that VHL loss-of-function leads to HIF nuclear accumulation (Bonicalzi et al., 2001). To explore the role of Drosophila VHL (Adryan et al., 2000) in Sima subcellular localization, we began by analyzing VHL loss-of-function in embryos expressing VHL double-stranded RNA (VHL RNAi/VHLi) (Dietzl et al., 2007) in UAS transgenic lines. Heat-shock driven expression of VHLi provoked 93% reduction of VHL mRNA expression (data not shown). When VHLi was expressed under an en-Gal4 driver, Sima was remarkably more nuclear than in wild-type controls (Figure 4A), suggesting that VHL is required for cytoplasmic localization in normoxia. Consistent with this, embryos homozygous for the Df(2R)en-A chromosomal deficiency, which includes the VHL gene, exhibited Sima more nuclear than in wild-type siblings (Supplemental Figure S2A). To answer if this effect was indeed due to reduced VHL levels, and not to other genes mapping in the deficiency, we performed a specific rescue by overexpressing the VHL transgenic protein in Df(2R)en-A homozygous embryos. Normal Sima subcellular distribution was largely restored upon reexpressing VHL (Supplemental Figure S2B), confirming that VHL loss-of-function accounts for Sima nuclear localization in Df(2R)en-A homozygous embryos. To get direct evidence for a role of VHL in Sima nuclear export, we subjected the embryos expressing VHLi to a reoxygenation assay as above. We found that Sima nuclear export was dramatically impaired (Figure 4B), supporting the notion that VHL is required in this process. Similar results were obtained upon reoxygenation of embryos homozygous for the Df(2R)en-A deficiency (Supplemental Figure S2C), further supporting a role for VHL in nuclear export.
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CRM1-dependent Nuclear Export of Sima Requires P850 Hydroxylation and VHL Function
As shown above, overexpression of CRM1/Embargoed can increase the rate of Sima nuclear export (Figure 1D,E). On the basis of this observation, we explored if hydroxylation of Sima is required for this effect, by studying if subcellular localization of SimaP850A is modified upon overexpression of the Emb protein. As depicted in Figure 5A, SimaP850A subcellular localization was not modified upon overexpression of Emb: The protein remained totally nuclear, indicating that Proline 850 is critically required for CRM1-dependent nuclear export of Sima.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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/Sima is cytoplasmic in normoxia and imported into the nucleus upon hypoxic exposure, Sima localization depends on a dynamic equilibrium between nuclear import and nuclear export in which both processes occur simultaneously (Figure 6). This model is supported by the fact that genetic or pharmacological impairment of either, nuclear import or nuclear export (Romero et al., 2008 and this study) led to an alteration of Sima subcellular localization at different oxygen concentrations. Furthermore, our data suggest that oxygen levels, acting in a dose-dependent manner, can modify the ratio between nuclear import and nuclear export, thereby setting the steady-state subcellular localization of Sima. In this context, the predominant cytoplasmic localization of Sima observed in normoxia is likely due to rapid nuclear export of the protein that overcomes the rate of nuclear import.
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, and its Drosophila homologue Sima, nuclear export depends at least in part, on the nuclear export receptor CRM1 (Mylonis et al., 2006; Romero et al., 2008). We have identified a functional NES within the Sima ODDD that contributes to regulated nuclear export of the protein, and hydroxylation of Pro850, the prolyl residue involved in Sima normoxic proteasomal degradation (Arquier et al., 2006; Centanin et al., 2008), is absolutely required for cytoplasmic localization of Sima. Consistent with this, Fatiga, the enzyme responsible for hydroxylation of this prolyl residue (Centanin et al., 2005) is necessary for cytoplasmic localization as well. Thus, our results support the notion that subcellular localization of Sima depends on graded levels of prolyl hydroxylation that can fine tune the ratio between nuclear import and nuclear export in an oxygen-dependent manner, so that high hydroxylation levels favor a cytoplasmic localization, and low hydroxylation levels shift the equilibrium toward Sima nuclear localization (Figure 6). It remains to be determined the molecular mechanism that accounts for oxygen-mediated regulation of nuclear export. It is tempting to speculate that the NES within the Sima ODDD becomes accessible to the export machinery only upon hydroxylation of Pro850.
VHL was shown to be another critical determinant of Sima subcellular localization. Paralleling the requirement of prolyl-hydroxylation for Sima localization in the cytoplasm, high levels of VHL push the equilibrium toward cytoplasmic localization of the protein; conversely, reduced levels of VHL shift the balance in favor of nuclear localization. Thus, our data have shown that, in order for VHL to control Sima subcellular localization, hydroxylation of the Pro850 residue is required. We therefore conclude that the cellular machinery previously reported to be involved in ubiquitination (Maxwell et al., 1999; Ivan et al., 2001; Jaakkola et al., 2001) of HIF- subunits is also required for cytoplasmic localization of the protein in normoxia.
Sima Nuclear Export Is an Oxygen-regulated Step
Having established that VHL, acting on hydroxylated Pro850 can affect Sima subcellular localization, leading to modification of the import/export ratio of the protein, the next step was to define which of the two processes, nuclear import or nuclear export, is under regulation of the prolyl hydroxylation/VHL pathway. By overexpressing in vivo the Drosophila CRM1 nuclear export receptor homologue, Embargoed, we have been able to increase the rate of Sima nuclear export, thereby enhancing Sima cytoplasmic localization. This strategy led us to conclude that the VHL/hydroxylation pathway is absolutely necessary for Sima Embargoed-dependent nuclear export (Figure 6), because either VHL loss-of-function or mutagenesis of Proline 850 provoked increased nuclear localization of Sima, and, in either case, this localization could not be altered by overexpression of CRM1/Embargoed.
Thus, our data suggest that Sima nuclear export is oxygen-regulated, but do not rule out the possibility that additional mechanisms controlling Sima/HIF localization, as for example, oxygen-dependent cytoplasmic retention, might occur in parallel. Nuclear import of another transcription factor of the bHLH-PAS family, the aryl hydrocarbon receptor (Ahr), was shown to be elicited by xenobiotic compounds that can bind to its PAS domain, provoking a conformational change that lifts cytoplasmic retention mediated by the heat-shock protein 90 (Hsp90) chaperone complex (Kazlauskas et al., 2001). Interestingly, Hsp90 was shown to bind to HIF PAS domain (Katschinski et al., 2002), and thus it is possible that the Hsp-90 complex can mediate HIF cytoplasmic retention and then lifts such retention upon a hypoxia-elicited conformational change. Thus, the possibility that HIF/Sima is regulated through oxygen-dependent cytoplasmic retention still needs to be investigated.
Compartmentalization of Prolyl Hydroxylation and the Dual Function of VHL
Given that VHL acting on hydroxyprolyl residues is required for Sima nuclear export, hydroxyprolyl Sima molecules are predicted to be present in the nucleus. Several lines of evidence suggest that HIF/Sima prolyl hydroxylation occurs at least in part in the nuclear compartment. It was reported that the Drosophila HIF-prolyl-hydroxylase/Fatiga is localized in the nucleus in imaginal discs (Frei and Edgar, 2004), and consistent with this, in mammalian cells in culture, prolyl hydroxylase activity occurs in the nuclear compartment (Groulx and Lee, 2002). Subcellular localization of the three mammalian HIF-prolyl hydroxylase isoforms was directly assessed. Although this issue is still matter of some controversy, it is clear that at least one of the PHD isoforms is localized in the nucleus (Metzen et al., 2003).
Nuclear export of p53 is mediated by CRM1 and was shown to be regulated by Mdm2-dependent ubiquitination (Boyd et al., 2000; Geyer et al., 2000). It was demonstrated that Mdm2 can catalyze either p53 mono or polyubiquitination, apparently depending on Mdm2 intracellular levels. Interestingly, monoubiquitination promotes p53 nuclear export and polyubiquitination leads to proteasomal degradation (Li et al., 2003). Evidence from mammalian HIF suggests that this might indeed be the case. Groulx and Lee (2002), working in adenovirus-transfected mammalian cells, showed that HIF ubiquitination can occur in the nuclear compartment and proposed that HIF-ubiquitinated species are exported to the cytoplasm previous to degradation at the 26S proteasome. Whether mono- or polyubiquitination of Sima are required for nuclear export remains to be investigated (Figure 6).
The Interplay between HIF-/Sima protein Degradation and Subcellular Localization
Why is it necessary that Sima undergoes rapid nuclear export in normoxia, whereas in parallel, the protein is actively degraded at the 26S proteasome? Regulation of transcription factor activity typically occurs at several different levels through more than one mechanism that operate simultaneously to ensure that regulation is efficiently achieved. Sima regulation does not seem to be an exception to this rule. Degradation and nuclear export of Sima appear to take place simultaneously in an oxygen dose-dependent manner, thereby synergizing in the reduction of Sima nuclear concentration and preventing hypoxia-inducible transcription. According to this model (Figure 6), both degradation and nuclear export are inhibited in hypoxic conditions, provoking an increase of Sima protein levels in the nuclear compartment, where transcription takes place. We therefore propose that Sima regulation involves a dual mechanism (i.e., proteasomal degradation and nuclear export) that relies on a single cellular machinery (i.e., the VHL/prolyl-hydroxylation system) and that this dual mechanism contributes to secure adaptive changes in gene expression to cope with variations of environmental oxygen tension.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: Pablo Wappner (pwappner{at}leloir.org.ar)
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, cytoplasmic localization of Sima; 
, nuclear localization; and 
, ubiquitous localization. Sima subcellular localization in wild-type embryos (WT) was progressively more nuclear as embryogenesis proceeds and gradually more nuclear as oxygen levels decrease (
