RESEARCH ARTICLE

ERK1/2 phosphorylates HIF-2α and regulates its activityby controlling its CRM1-dependent nuclear shuttlingIoanna-Maria Gkotinakou1, Christina Befani1, George Simos1,2 and Panagiotis Liakos1,*

ABSTRACTHypoxia-inducible factor 2 (HIF-2) is a principal component of thecellular response to oxygendeprivation (hypoxia). Its inducible subunit,HIF-2α (also known as EPAS1), is controlled by oxygen-dependent aswell as oxygen-independent mechanisms, such as phosphorylation.We showhere that HIF-2α is phosphorylated under hypoxia (1%O2) byextracellular signal-regulated protein kinases 1 and 2 (ERK1/2; alsoknown as MAPK3 and MAPK1, respectively) at serine residue 672,as identified by in vitro phosphorylation assays. Mutation of this siteto an alanine residue or inhibition of the ERK1/2 pathway decreasesHIF-2 transcriptional activity and causes HIF-2α to mislocalize to thecytoplasm without changing its protein expression levels. Localization,reporter gene and immunoprecipitation experiments further showthat HIF-2α associates with the exportin chromosomal maintenance 1(CRM1, also known as XPO1) in a phosphorylation-sensitive mannerand identify two critical leucine residues as part of an atypicalCRM1-dependent nuclear export signal (NES) neighboring serine672. Inhibition of CRM1 or mutation of these residues restores nuclearaccumulation and activity of HIF-2α lacking the ERK1/2-mediatedmodification. In summary, we reveal a novel regulatory mechanism ofHIF-2, involving ERK1/2-dependent phosphorylation of HIF-2α, whichcontrols its nucleocytoplasmic shuttling and the HIF-2 transcriptionalactivity.

This article has an associated First Person interview with the firstauthor of the paper.

KEY WORDS: HIF-2, ERK1/2, Phosphorylation, CRM1,Nuclear export signal, Nuclear transport

INTRODUCTIONHuman tissues are exposed to reduced oxygen concentration(hypoxia) during physiological processes like development anddifferentiation, but also in pathological situations like ischemia,inflammation and cancer (Semenza, 2011). In order to adapt to thehypoxic environment, cells upregulate the expression of genesinvolved in processes such as metabolic reprogramming,erythropoiesis, cell proliferation, invasion and metastasis, andangiogenesis (Rankin and Giaccia, 2016). This cellular response tohypoxia is orchestrated by hypoxia-inducible factors (HIFs). HIFsare a small family of heterodimeric transcriptional activatorscomposed of a hypoxia-inducible α-subunit (HIF-α) and a stably

expressed β subunit [aryl hydrocarbon receptor nuclear translocator(ARNT), also known as HIF-1β]. After heterodimerization, HIFsbind to E-box-like hypoxia response elements (HREs) within thepromoter or enhancer regions of target genes that contain the coresequence 5′-[A/G]CGTG-3′ (Kaelin and Ratcliffe, 2008).

HIF-1α is ubiquitously expressed and triggers a wide range ofadaptive changes with a more-prominent role in the regulation ofgenes coding for metabolic enzymes (Majmundar et al., 2010). Theexpression of the second, less-studied isoform, HIF-2α (encoded bythe EPAS1 gene, a paralog of HIF1A), is restricted to specific celltypes. HIF-2 is a transcriptional activator (Koh and Powis, 2012)and a key mediator of the cellular adaptation to oxygen deprivation(hypoxia), playing important roles in physiological processes, suchas erythropoiesis and vascularization (Keith et al., 2011) and inpathological conditions including the progression of solid tumorsand metastasis (Patel and Simon, 2008; Zhao et al., 2015). HIF-2,thus, constitutes a validated target of anti-cancer therapy (Meteloet al., 2015; Chen et al., 2016).

HIF-2α is regulated by oxygen in a similar fashion to HIF-1α. Inthe presence of oxygen, HIF-2α is modified by HIF-specific prolyl-4-hydroxylases (PHDs), leading to proteasomal degradationmediated in part by the Von Hippel Lindau tumor suppressorprotein (VHL) (Ohh et al., 2000). HIF-2α is also hydroxylated atan asparagine residue by factor inhibiting HIF (FIH, also knownas HIF1AN), which inhibits HIF-2α interaction with thetranscriptional co-activators CREB-binding protein (CBP, alsoknown as CREBBP) and p300 (EP300) (collectively termed CBP/p300) (Kaelin and Ratcliffe, 2008). Under hypoxia, PHDs and FIH,which use oxygen as a substrate, become inactive. As a result,HIF-2α hydroxylation is inhibited, leading to HIF-2α stabilization,transport into the nucleus, dimerization with ARNT, DNA binding,co-activator recruitment and formation of an active transcriptionalcomplex. Growth factors, hormones or elevated oncogenicsignaling in cancer cells can induce HIF-α independently ofoxygen levels, both in terms of expression levels and activity (Déryet al., 2005; Koh and Powis, 2012). Those oxygen-independentmechanisms involve the extensive post-translational modificationof HIF-α subunits and their interaction with different proteins.The most well-studied post-translational modification of HIF-αsubunits that affects their function other than hydroxylation,is phosphorylation. Modification of HIF-α subunits byphosphorylation can affect their stability, subcellular localizationand transactivation potential. There are a lot of kinases known tomodify HIF-1α and the effect of phosphorylation on HIF-1αfunction has been extensively studied (Kietzmann et al., 2016).However, our knowledge concerning HIF-2α regulation byphosphorylation is more limited. Open questions include whetherthe phosphorylation-dependent mechanisms that regulate HIF-1αactivity also apply to HIF-2α or whether there are isoform-specificmodifications mediated by different protein kinases. It has beenreported that HIF-2α is phosphorylated at T324 by protein kinaseReceived 20 September 2018; Accepted 31 January 2019

1Laboratory of Biochemistry, Faculty of Medicine, University of Thessaly, 41500,Biopolis, Larissa, Greece. 2Gerald Bronfman Department of Oncology, Faculty ofMedicine, McGill University, Montreal, Canada, H4A 3T2.

*Author for correspondence (pliakos@med.uth.gr)

G.S., 0000-0001-5453-3185; P.L., 0000-0002-3800-5056

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D1 (PKD1, also known as PRKD1) and, as a result, its interactionwith the transcription factor specificity protein 1 (SP1) is inhibited,thus promoting the expression of nijmegen breakage syndromeprotein 1 (NBS1) (To et al., 2006). HIF-2α is also phosphorylatedby casein kinase 2 (CK2) at T844, which results in increased HIF-2transcriptional activity, possibly by lowering the affinity of HIF-2αfor FIH (Gradin et al., 2002). We have recently identified tworesidues of HIF-2α, S383 and T528 that are targeted by caseinkinase 1δ (CK1δ, also known as CSNK1D). These modificationslead to efficient HIF-2α nuclear accumulation and full HIF-2transcriptional activity (Pangou et al., 2016).Cellular stress conditions, such as reactive species, mitogens and

hypoxia, are known to activate the extracellular signal-regulatedprotein kinase (ERK) pathway, which controls many principalprocesses of cell metabolism, motility and proliferation. Theproteins ERK1 and ERK2 (ERK1/2, also known as MAPK3and MAPK1, respectively) are known to be critical for thetransactivation of many genes and mediates their effects primarilythrough the phosphorylation of downstream transcription factors.The Ras-MEK-ERK1/2 pathway has been implicated both in theregulation of HIF-1 protein synthesis (Fukuda et al., 2002) and inthe potentiation of its transcriptional activity (Bárdos and Ashcroft,2004; Brahimi-Horn et al., 2005). Our lab previously addressed therole of ERK1/2 in the hypoxic response and has shown that HIF-1αis controlled by direct ERK1/2-dependent phosphorylation(Mylonis et al., 2008; Mylonis et al., 2006). In the case of HIF-2α, older work by others has suggested that it is not a direct ERK1/2target and that ERK1/2 mediates its positive effects on HIF-2indirectly, possibly by modifying other proteins critical for HIF-2transactivation functions (Conrad et al., 1999). To clarify this issue,we analyzed further the effect of ERK1/2 on HIF-2α expression andtranscriptional activity in cancer cell lines under hypoxia (1% O2).

Our findings suggest a novel mechanism of HIF-2α regulationinvolving ERK1/2-dependent phosphorylation of S672 andinhibition of CRM1-mediated nuclear export.

RESULTSERK1/2 enhances HIF-2α target gene expression byfacilitating its nuclear accumulationTo investigate the involvement of ERK1/2 in the regulation of HIF-2α, Huh7 cells were treated with the MEK inhibitor U0126 (5 μM)under either 21% or 1% O2. This treatment drastically reduced themRNA expression levels of EPO and PAI-1 (also known asSERPINE1) (Fig. 1A,B), without affecting HIF-2α protein levels(Fig. 1C). These two genes are specific targets of HIF-2, aspreviously shown in the literature (Befani et al., 2013; Rankin et al.,2007, and Pawlus et al., 2012; Hu et al., 2007; Sato et al., 2004;Befani and Liakos, 2017, respectively) and confirmed in our model(Fig. S1). These data suggest that inhibition of ERK1/2 activationresults in the inhibition of HIF-2α transcriptional activity at a stepsubsequent to its hypoxia-induced stabilization. To identify thisstep, we performed subcellular fractionation (Fig. 1D). As expected,HIF-2α stabilized by hypoxia was mainly located in the nuclearfraction. However, when ERK1/2 activation was inhibited byU0126, HIF-2α was mostly recovered in the cytoplasmic fraction.These data suggest that activation of ERK1/2 stimulates HIF-2activity under hypoxia by enhancing its nuclear accumulation.

ERK 1/2 phosphorylates HIF-2α on S672In order to further analyze the observed HIF-2 regulation by ERK1/2,we tested whether ERK1/2 can directly modify HIF-2α in vitro. Full-length HIF-2α and its smaller fragments (corresponding to aminoacids 1–679, 1–640, 1–620, 1–560, 1–542, 640–870 and 680–870)were expressed in bacteria as GST fusion proteins (Fig. 2A), purified

Fig. 1. Inhibition of ERK1/2 activation decreasesHIF-2 target gene mRNA levels and mislocalizesHIF-2α to the cytoplasm. (A) Determination ofEPO mRNA levels by quantitative real-time PCR inHuh7 cells incubated and treated for 16 h with thespecific MEK inhibitor U0126 (5 μM) under hypoxia(1% O2). Results are shown as the fold increase inrelation to the value of corresponding normoxicconditions and represent the mean (±s.e.m.) ofthree independent experiments performed induplicate (**P<0.01). (B) Determination of PAI-1mRNA levels by quantitative real-time PCR in Huh7cells incubated and treated as in A. Results areshown as in A (*P<0.05). (C) Western blot analysisof Huh7 total cell extracts treated as in A. Actin isused as a loading control. (D) Immunoblotting ofsubcellular fraction lysates derived from HeLa cells,incubated and treated as in A using the indicatedantibodies. Actin serves as a cytoplasmic marker,while ARNT serves as a nuclear marker.

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and used as substrates in radioactive kinase assays using recombinantERK2 (Fig. 2B). Full-length HIF-2α could indeed be directlyphosphorylated by ERK2. HIF-2α fragments 1–542, 1–560 and680–870 were very poor substrates for ERK2 compared to full-length HIF-2α. Fragment 1–620, 1–640, 1–679 and 640–870exhibited significant phosphorylation levels compared to full-lengthHIF-2α, with the 1–679 fragment being an equally good ERK2substrate as the full-length form. Taking these results into account, weconclude that HIF-2α is predominantly phosphorylated by ERK2 inan area between residues 620–680, a region that is part of theinhibitory domain.To identify the residues that are modified by ERK2, we examined

the amino acid sequence of the 620–680 fragment for SP or TPdipeptides, which fit, at least partly, the ERK1/2 consensus sitePx(S/T)P. S672 and T626 are both found in SP or TP dipeptides(Fig. 3A). Furthermore, sequence analysis revealed the presence ofthese two residues in a conserved position in all known vertebrateHIF-2α homologs (Fig. 3A). To test the possibility that one of theseresidues is the target of ERK2, each one was converted into analanine residue by site-directed mutagenesis (Fig. 3A). The two

mutant proteins, namely HIF-2αS672A (HIF-2α with an alanineresidue at position 672) and HIF-2αT626A (HIF-2αwith an alanineresidue at position 626), were expressed as GST fusions in bacteria,purified and used as substrates for in vitro phosphorylation assayswith recombinant ERK2 (Fig. 3B) or Huh7 total cell extracts(Fig. 3C). Phosphorylation of HIF-2αS672A and HIF-2αT626A byERK2 was reduced by ∼80% and ∼20%, respectively, relative towild-type HIF-2α (Fig. 3B), showing that ERK2 preferentiallytargets S672. When recombinant ERK2 was replaced by totalsoluble protein extract of Huh7 cells as a kinase source (Fig. 3C),mutation of S672 caused an ∼65% reduction in the HIF-2αphosphorylation level, while mutation of T626 had no significanteffect. These data identify S672 of human HIF-2α as thepredominant ERK2 in vitro phosphorylation site. To confirm thisfinding for endogenous ERK1/2, Huh7 cells were treated for 16 hwith U0126 under hypoxia before preparation of the extracts as akinase source. As also shown in Fig. 3C, the phosphorylation ofwild-type HIF-2α with extracts from U0126-treated cells wasreduced to 40% and was similar to the phosphorylation of theHIF-2αS672A mutant seen with extracts from untreated cells.

Fig. 2. ERK2 phosphorylates HIF-2α invitro between amino acids 620 and 679.(A) Schematic representation of full-lengthHIF-2α and its fragments cloned, expressedand purified as GST fusion proteins andphosphorylated by recombinant ERK2. Anapproximate estimation of the relativephosphorylation level (based on the resultsof three independent experimentsperformed as in B), is shown. (B) Theindicated GST-tagged HIF-2α fragmentswere subjected to in vitro phosphorylationby 100 units of recombinant ERK2 and[γ-32P] ATP, and analyzed by SDS-PAGEfollowed by Coomassie Blue staining (upperpanel) and autoradiography (lowerpanel,32P). Dots indicate the positions of therecombinant GST-tagged HIF-2α proteins.Numbers on the left indicate the positions ofmolecular mass markers (kDa).

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Furthermore, treatment with U0126 did not influence thephosphorylation of the HIF-2αS672A mutant while it reducedphosphorylation of the HIF-2αT626A mutant by 40%. Takentogether, our data suggest that phosphorylation of HIF-2α on S672represents a major modification mediated by ERK1/2 in Huh7 cellextracts.

Phosphorylation of S672byERK1/2 enhances transcriptionalactivity and nuclear accumulation of HIF-2α by blocking itsCRM1-dependent nuclear exportTo further study the effect of S672 phosphorylation on HIF-2αfunction, we constructed HIF-2αS672D (HIF-2α with an aspartateresidue at position 672) as a phospho-mimetic mutant since thenegative charge of aspartate residue could potentially mimic thecharge and effects of phosphorylated serine. Wild-type HIF-2α and

all its mutant forms were sub-cloned into the pFLAG-CMV2mammalian expression vector and introduced as Flag-taggedproteins into Huh7 and HeLa cells grown under 21% O2 (i.e. inthe absence of endogenous HIF-2α) by transient transfection.Western blotting analysis showed that the protein expression levelsof all mutant forms were similar to those of the wild-type HIF-2αform (Fig. S3). As the data shown in Fig. 1D suggests that activatedERK1/2 positively affects HIF-2 activity by increasing theaccumulation of HIF-2α inside the nucleus, localization of wild-type Flag–HIF-2α and its mutant forms was monitored byimmunofluorescence microscopy in both HeLa (Fig. 4A) andHuh7 (Fig. S4) cells. Wild-type Flag–HIF-2α was detectedexclusively inside the nucleus in ∼80% of the transfected cellsand displayed both nuclear and cytoplasmic localization in only20% of the cells (Fig. 4A,B). When cells were treated with U0126,

Fig. 3. S672 of HIF-2α is an in vitro ERK2 phosphorylation site. (A) Schematic representation of full-length HIF-2α showing the position of the sequence(amino acids 621–676) targeted in vitro by ERK1/2 and alignment of corresponding amino acid sequences from vertebrate HIF-2α homologs. T626and S672, potential sites for phosphorylation by ERK1/2, are shown in green, and their neighboring conserved hydrophobic residues are shown in blue. Alanineresidues introduced by mutation are depicted in red. (B) GST–HIF-2α (1 μg) or its indicated point mutant forms were subjected to in vitro phosphorylationby 100 units of recombinant ERK2 and [γ-32P] ATP and analyzed by SDS-PAGE followed by Coomassie Blue staining (upper panel) and autoradiography (lowerpanel, 32P). Dots indicate the positions of the recombinant GST-tagged HIF-2α proteins. Only the relevant parts of the gels are shown. The numbers under eachlane represent relative phosphorylation levels measured as described in the Materials and Methods, and are means of three independent experiments.(C) Same as in B, but the GST fusion proteins were subjected to in vitro phosphorylation by 1 μg of Huh7 total protein extract prepared from cells treated with 5 μMU0126 for 16 h or left untreated as indicated.

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Fig. 4. See next page for legend.

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wild-type Flag–HIF-2α was exclusively nuclear in only 35% of thetransfected cell population, showing that inhibition of ERK1/2activation impairs HIF-2α accumulation inside the nucleus(Fig. 4A,B). Localization of the phospho-deficient HIF-2αS672Amutant form was exclusively nuclear in only ∼30% of thetransfected cell population, again suggesting that lack ofmodification by ERK1/2 impairs HIF-2α nuclear accumulation.On the other hand, localization of the phospho-mimetic mutant HIF-2αS672D form was predominantly nuclear in ∼90% of thetransfected cell population mimicking the distribution of the wild-type Flag–HIF-2α and, furthermore, this distribution did not changeafter treatment with U0126 (Fig. 4A,B).Increased cytoplasmic localization of mutant HIF-2αS672A

protein or wild-type HIF-2α protein in the presence of U0126 couldbe either due to diminished nuclear import or increased nuclearexport. To clarify which one of these processes is affected, cellswere treated with leptomycin B (LMB), a potent inhibitor of CRM1,the major exportin in human cells. Exposure to LMB did notsignificantly affect the localization of wild-type HIF-2α or thephospho-mimetic HIF-2αS672D mutant form, which remainedaccumulated inside the nucleus (Fig. 4A,B). However, LMBtreatment resulted in an exclusive nuclear localization for thephospho-deficient HIF-2αS672Amutant form in almost 90% of thecells (compared to 30% in the absence of LMB) (Fig. 4A,B). Thesedata suggest that HIF-2α can shuttle between the nucleus and thecytoplasm and that lack of phosphorylation by ERK1/2 enhancesCRM1-mediated HIF-2α nuclear export, leading to its cytoplasmicmislocalization.To investigate the functional significance of the phosphorylation-

dependent subcellular distribution of HIF-2α, HIF-2 transcriptional

activity was determined via a luciferase reporter gene assay in Huh7cells expressing Flag-tagged wild-type, phospho-deficient SA andphospho-mimetic SD mutant forms of HIF-2α under 21% O2 (i.e. inthe absence of endogenous HIF-2α) and in the absence or presence ofU0126 and/or LMB (Fig. 4C). In the absence of any treatment, thephosphorylation-deficient HIF-2αS672A mutant form demonstratedan ∼50% decrease in its HRE-dependent transcriptional activitycompared with that of wild-type HIF-2α (Fig. 4C, compare columns5 and 9), in agreement with the increased cytoplasmicmislocalizationof HIF-2αS672A in most of the cells. By contrast, the activity of thephospho-mimetic HIF-2αS672D mutant form was similar to that ofwild-type HIF-2α (Fig. 4C, compare columns 5 and 12), suggestingthat wild-type HIF-2α is subjected to substantial phosphorylation atS672 inside Huh7 cells. In the presence of U0126, the wild-type HIF-2α form exhibited a significantly lower activity compared towhat wasseen upon no treatment (Fig. 4C, compare columns 5 and 6), again inagreement with the localization data and reproducing the behavior ofthe endogenous, hypoxically induced, HIF-2α (Fig. 1A,B). Neitherthe low transcriptional activity of the phospho-deficient HIF-2αS672A mutant form nor the normal (compared to wild-type)activity of the phospho-mimetic HIF-2αS672D form weresignificantly affected by U0126 (Fig. 4C, compare columns 9 and12 to 10 and 13, respectively). Overall, these data suggest thatphosphorylation of HIF-2α by ERK1/2 predominantly involves S672and stimulates HIF-2 transcriptional activity in human cancer cells.

Treatment of the cells with LMB did not significantly affect thetranscriptional activity of wild-type HIF-2α or its phospho-mimeticHIF-2αS672D mutant form, but it restored the activity of thephospho-deficient HIF-2αS672A mutant to levels comparable tothe wild-type form activity (Fig. 4C, compare columns 5, 9 and 12to 8, 11 and 14, respectively), suggesting that the predominantreason for partial HIF-2α inactivation upon inhibition of theERK1/2 pathway or a mutation abolishing ERK-phosphorylation isenhanced HIF-2α nuclear export mediated by the target of LMB, theexportin CRM1.

To confirm the involvement of CRM1, wild-type Flag–HIF-2αand its mutant forms were expressed in HeLa cells grown under 21%O2 and subsequently subjected to immunoprecipitation with an anti-Flag antibody. Analysis of the immunoprecipitates revealed a stronginteraction of endogenous CRM1 with the phosphorylation-deficient HIF-2αS672A mutant (Fig. 4D, lane 2) whereas CRM1was almost non-detectable in wild-type or phospho-mimetic HIF-2αS672D immunoprecipitates (Fig. 4D, lanes 1 and 3), showingthat CRM1 can only bind to HIF-2α when it lacks its ERK1/2-mediated phosphorylation on S672. In addition, we tested apreviously described mutant form of HIF-2α that lacks the CK1δphosphorylation sites S383 and T528 and is, as a result, alsomislocalized in the cytoplasm (Pangou et al., 2016). However, thismutant did not interact with CRM1 (Fig. 4D, lane 4), suggestingthat lack of phosphorylation by CK1δ leads to cytoplasmicmislocalization through a mechanism distinct from the oneinvolving interaction of HIF-2αS672A with CRM1. To furtherverify these results with endogenousHIF-2α, HeLa cells grown underhypoxia (1% O2) were treated with U0126 in the presence or absenceof LMB and subjected to immunoprecipitation with an anti-HIF-2αantibody. Analysis of the immunoprecipitates showed that CRM1strongly associated with endogenous wild-type HIF-2α aftertreatment with U0126 (Fig. 4E, lane 4). In contrast, when theERK1/2 pathway was active, the interaction of HIF-2α with CRM1was hardly detectable (Fig. 4E, lane 2). As expected, treating the cellswith LMB abolished the interaction between CRM1 and HIF-2αirrespective of the ERK1/2 activation status (Fig. 4E, lanes 3 and 5).

Fig. 4. Mutation of S672 into alanine or inhibition of ERK1/2 activationenhances CRM1-dependent nuclear export of HIF-2α and decreases itstranscriptional activity. (A) Transfected HeLa cells, incubated under 21%O2,expressing wild-type Flag–HIF-2α (wt), its phosphorylation-deficient mutantFlag–HIF-2αS672A (S→A) or its phospho-mimetic mutant Flag–HIF-2αS672D(S→D), were fixed at 24 h post transfection and subjected toimmunofluorescence microscopy with anti-Flag mouse monoclonal antibody(green). Nuclei were stained with DAPI (magenta). In some cases, at 8 h post-transfection, cells were treated with either the specific MEK inhibitor U0126(5 μM) or at 20 h post-transfection with the CRM1 inhibitor LMB (10 ng/ml) (asindicated). Scale bars: 10 μm. (B) Quantification of the results from theexperiment described in A. Transfected cells showing exclusive nuclearstaining under each condition were counted, and their percentage wascalculated relative to the total number of transfected cells. At least 100–150transfected cells were examined in each case. Data plotted are the mean ofthree independent experiments (± s.e.m.) (****P<0.0001). (C) HIF-2transcriptional activity was determined 24 h after transfection of Huh7 cells,incubated under 21% O2, with the pGL3-5HRE-VEGF reporter plasmid, theplasmid pCI-Renilla and the pFLAG-CMV2 plasmids carrying the indicatedinserts in the presence or absence of either U0126 (5 μM) or LMB (10 ng/ml) asdescribed in A. Values show the fold increase of relative luciferase units (fireflyover Renilla activity) in relation to the values obtained from cells expressingpFLAG-CMV2 alone and represent the mean of three independentexperiments performed in triplicate (±s.e.m.) (**P<0.01; ****P<0.0001).(D) HeLa cells grown under 21% O2 were transfected as in A or additionallywith mutant Flag–HIF-2αS383A/T528A (S383→A/T528→A) which lacks theCK1δ phosphorylation sites (Pangou et al., 2016). At 24 h post transfection,cells were lysed and subjected to immunoprecipitation with anti-Flag antibody.Total cell extracts (input) and precipitated proteins (I.P.) were analyzed bywestern blotting using anti-CRM1 and anti-HIF-2α antibodies as indicated.(E) HeLa cells were treated with U0126 (5 μM), LMB (10 ng/ml) and incubatedfor 16 h under hypoxia (1% O2). Then, cells were lysed and subjected toimmunoprecipitation with anti-HIF-2α antibody. Total cell extracts (input) andprecipitated proteins (IP) were analyzed by western blotting using anti-CRM1,anti-phospho-serine (P-Ser) and anti-HIF-2α antibodies as indicated. Westernblots are representative from three independent experiments.

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Importantly and in support of all our previous data, associationwith CRM1 was inversely correlated with the ERK-dependentphosphorylation status of HIF-2α as shown by phospho-serinedetection in the HIF-2α immunoprecipitates (Fig. 4E, compare lanes2 and 4). These results further corroborate that ERK1/2-mediatedphosphorylation of HIF-2α inhibits its interaction with CRM1.

Identification of leucine residues required for associationwith CRM1 and nuclear export of phospho-deficient HIF-2αGiven the association of CRM1 with HIF-2α that is notphosphorylated at S672, we examined its amino acid sequence forthe presence of a leucine-rich nuclear export signal (NES), knownto be the best-characterized binding site of CRM1 (Hutten andKehlenbach, 2007). Although we could not detect a sequencefollowing exactly the typical consensus NES motif (Φx2–3Φx2–3ΦxΦ; Φ=leucine, isoleucine, valine, phenylalanine or methionine;x, any amino acid), we noticed a number of conserved hydrophobicresidues neighboring the ERK1/2 phosphorylation site (Fig. 3A),which, despite their atypical spacing and composition, couldpotentially function as a leucine-rich NES. Among these, twoleucine residues at positions 662 and 667 were conserved in allknown vertebrate HIF-2α homologs (Fig. 3A). To test theinvolvement of these residues in the phosphorylation-dependentnucleocytoplasmic trafficking of HIF-2α, two additional HIF-2αmutant variants were constructed, namely, HIF-2αL662A/L667A(L662 and L667 converted into alanine residues) and HIF-2αL662A/L667A/S672A (L662, L667 and S672 converted toalanine residues). These were expressed as Flag-tagged proteins inHuh7 or HeLa cells grown under 21% O2 and their subcellulardistribution was analyzed by immunofluorescence microscopy andcompared to that of wild-type HIF-2α and the phospho-deficientmutant HIF-2αS672A (Fig. 5A). In the absence of U0126, bothmutant HIF-2αL662A/L667A and HIF-2αL662A/L667A/S672Aforms were exclusively nuclear in most cells, behaving like wild-type Flag-HIF-2α and unlike HIF-2αS672A, which, as also shownabove, was distributed in most cells in both nucleus and cytoplasm.Therefore, mutation of the two leucine residues inhibited nuclearexport of the phospho-deficient mutant HIF-2αS672A, suggestingthat they are indeed part of a phosphorylation-sensitive NES. In ananalogous manner, when cells were treated with U0126, unlike thewild-type HIF-2α protein, which remained inside the nucleus inonly ∼35% of the transfected cell population (as also shown beforein Fig. 4A,B), the HIF-2αL662A/L667A mutant protein retained itsexclusive nuclear localization in most of the transfected cells(Fig. 5A,B), showing again that the two leucine residues arerequired for efficient nuclear export of HIF-2α lacking ERK1/2-mediated phosphorylation. These data suggest that the hydrophobicsequence bordering the ERK1/2 phosphorylation site behavesindeed as a functional NES, but only when the neighboringphospho-acceptor site remains unmodified.As mutation of the neighboring leucine residues could efficiently

re-establish the nuclear localization of the phospho-deficient HIF-2αS672A mutant form, we examined whether it could also restoreits transcriptional activity. Indeed, while the transcriptional activityof the phospho-deficient HIF-2αS672Amutant was 50% lower thanthat of the wild-type and phospho-mimetic HIF-2αS672D forms(Fig. 5C, columns 2, 3, 4), the activity of the phospho-deficientand NES-lacking HIF-2αL662A/L667A/S672A mutant form wascomparable to that of the wild-type and HIF-2αS672D forms(Fig. 5C, columns 2, 6). Finally, abolishing the NES alone in theHIF-2αL662A/L667A form retained, as expected, transcriptionalactivity of equal levels to wild-type HIF-2α (Fig. 5C, columns 2, 4,

5). These data indicate that the predominant reason for partialinactivation of HIF-2α lacking the ERK1/2 phospho-acceptor site isthe extensive nuclear exclusion of HIF-2α due to an exposedleucine-containing NES.

To investigate whether this leucine-containing NES is indeedresponsible for the association of HIF-2α with CRM1, HeLa cellsgrown under 21% O2 and expressing Flag-tagged wild-type HIF-2αor phospho-deficient HIF-2αS672A or NES-deficient HIF-2αL662A/L667A or the double NES-deficient and phospho-deficient HIF-2αL662A/L667A/S672A mutant forms were treatedor not with U0126 and subjected to immunoprecipitation with ananti-Flag antibody. The results show that inhibition of ERK1/2activation or/and mutation of the Leu residues greatly affected boththe phospho-serine content of HIF-2α and/or its association withCRM1, respectively (Fig. 6A,B). More specifically, impairing HIF-2α phosphorylation by ERK1/2 either by treatment with U0126(Fig. 6A, compare lanes 4 and 6 to lanes 3 and 5; Fig. 6B, comparelane 4 to lane 3) or by mutation (Fig. 6B, compare lanes 1, 2 and 5 tolane 3) greatly reduced the phospho-serine content of HIF-2α,confirming that ERK1/2-mediated phosphorylation represents amajor HIF-2α post-translational modification. The fact that U0126did not exert an effect on the phospho-serine levels of HIF-2αS672A(Fig. 6B; compare lane 1 to 2 and 5 to 6) indicates that S672 is apredominant ERK1/2 phosphorylation site. More importantly,although CRM1 associated with HIF-2α when its phosphorylationwas inhibited by either U0126 (Fig. 4A, lane 4) or the S672Amutation (Fig. 4B, lanes 1 and 2), CRM1 did not associate with theNES-deficient mutant (Fig. 6A, lane 5; Fig. 6B, lane 3) even underthe phosphorylation-inhibiting conditions (Fig. 6A, lane 6; Fig. 6B,lanes 4–6), confirming that the two leucine residues at positions 662and 667 are indeed required for binding of unmodified HIF-2α toCRM1.

DISCUSSIONAlthough, it has been long known that HIF-2 activity is affected byERK1/2 (Conrad et al., 1999), the regulatory mechanism remainedunclear. In this study, we have revealed a novel O2-independentregulatory step of HIF-2α activity involving ERK1/2-mediatedphosphorylation of a distinct residue of HIF-2α, serine at position672. Our data suggest that this phosphorylation of HIF-2α byERK1/2 controls its CRM1-dependent nuclear shuttling bymaskinga functional atypical NES in HIF-2α (Fig. 6B). The identified site ofERK1/2 modification, S672, as well as the NES are localized inthe inhibitory domain (ID) of HIF2α, which is located betweenthe N-terminal activation domain (N-TAD) and the C-terminaltransactivation domain (C-TAD). The ID is not well characterizedbut it has been reported to be involved in the transactivation of HIFsbecause its deletion increases the transcriptional activity of both theN-TAD and C-TAD (Jiang et al., 1997; Pugh et al., 1997; O’Rourkeet al., 1999). Our data showing that the ID contains both an ERK1/2phosphorylation site and a CRM1-dependent NES, could explainthese older data, as removal of the ID would remove the NES and,therefore, increase the nuclear accumulation and, subsequently, thetranscriptional activity of HIF-2α irrespective of the activationstatus of the ERK1/2 pathway (i.e. elicit similar behavior to ourHIF-2αL662A/L667A/S672A mutant carrying mutations in boththe NES and the ERK1/2 site).

The HIF-2α serine residue (S672) targeted by ERK1/2 isconserved in the same position in all vertebrate homologs ofHIF-2α. The fact that this particular site (S672) represents a validphospho-acceptor site is further confirmed by a recent phospho-proteomics analysis in breast tumor samples, which connects

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somatic mutations to signaling events (Mertins et al., 2016). This,together with our data, showing that both mutation of S672 intoalanine and inhibition of ΕRΚ1/2 pathway by U0126 impaired

HIF-2α transcriptional activity in human cells, strongly suggeststhat HIF-2α S672 constitutes a major in vitro and in vivo ERK1/2phosphorylation target, important for HIF-2 function. Our data have

Fig. 5. See next page for legend.

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further shown that phosphorylation of S672 by ERK1/2 is requiredto maintain HIF-2α inside the nucleus by inhibiting its associationwith CRM1. It is well known that regulation of subcellularlocalization of transcription factors adds an additional layer ofcomplexity in the regulation of their transcriptional activity,enabling their fine tuning in order to access their target genespromoters or enhancers as well as modulating their interaction withco-activators and/or co-repressors depending on the needs of thecell. Inhibition of nuclear export by phosphorylation is indeed anefficient way to ensure nuclear accumulation and full activation ofshuttling transcription factors as has been shown previously inseveral cases (Ikuta et al., 2004; Lee and Bai, 2002; Zhang andXiong, 2001).Although the nuclear import mechanism of HIF-2α is well studied

(Luo and Shibuya, 2001), its ability to shuttle (i.e. move in as well asout of the nucleus), was unknown as its nuclear export had not beeninvestigated. HIF-2α far exceeds the generally accepted nuclear porediffusional cut-off size of 40 kDa (Gorlich and Kutay, 1999) andwould thus also need specific shuttling signals such as an NES inaddition to an NLS. Our analysis of the HIF-2α amino acid sequenceadjacent to ERK1/2 phosphorylation site revealed a number ofhydrophobic residues that are nearly universally conserved acrossvertebrates (Fig. 3A). The spacing of the conserved hydrophobicresidues in the FLGAAPLGPPV sequence deviates from theproposed wide CRM1-dependent hydrophobic NES consensus(Φx2–3Φx2–3ΦxΦ; Φ=leucine, isoleucine, valine, phenylalanine andmethionine; x=any amino acid). Nevertheless, the L662A/L667Amutation apparently inactivated this potential and atypical NESsequence, as it increased the accumulation of non-phosphorylatedHIF-2α in the nucleus and stimulated its transcriptional activity.Furthermore, the same mutation abolished the physical interactionof non-phosphorylated HIF-2α with CRM1. All these indicate thatthe FLGAAPLGPPV sequence neighboring the S672 ERK1/2phosphorylation site in HIF-2α indeed represents an atypical NESsequence which mediates CRM1 association and nuclear exportof phospho-deficient HIF-2α. It is noteworthy that thenucleocytoplasmic transport of HIF-1α has also been reported to beregulated by ERK1/2 via direct phosphorylation of HIF-1α andinhibition of CRM1-dependent nuclear export (Mylonis et al., 2008).It appears that the regulation of both HIF-1 and HIF-2 activity byERK1/2 involves the nuclear export machinery and in an analogousmanner, despite the fact that both the ERK1/2 and the NES sites on

HIF-1α are distinct from those identified here on HIF-2α (Fig. S5) asthey are located in a region of very low sequence similarity betweenHIF-1α and HIF-2α. These regulatorymechanisms, although similar,must have, therefore, evolved independently and after the duplicationof the primordial HIF-α gene and the appearance of the two isoformsin vertebrates (Rytkonen and Storz, 2011), in a kind of convergentmolecular evolution. This highlights the functional importance ofcontrolling the nucleocytoplasmic distribution of both HIF-αisoforms in response to the activation level of ERK1/2 and theirupstream effectors in theMAPK pathway. However, regulation of thenucleocytoplasmic distribution of HIF-2α appears to be morecomplex than HIF-1α, as ERK1/2 is the second kinase involved inthis process.We have previously shown that phosphorylation of HIF-2α by CK1δ at S383 and T528 also affects nucleocytoplasmicdistribution of HIF-2α by inhibiting its CRM1-dependent nuclearexport (Pangou et al., 2016). However, no NES-like sequence couldbe detected close to these CK1δ sites. Moreover, our present datashow that HIF-2α lacking the ERK1/2 sites (HIF-2αS672A) is able tointeract with CRM1 while the HIF-2α form lacking the CK1δ sites(HIF-2αS383A/T528A) is unable to form a tight complexwith CRM1. These data suggest that phosphorylation by ERK1/2directly controls the interaction of HIF-2α with CRM1 by masking aneighboringNESwhile modification by CK1δ indirectly affects HIF-2α nuclear export, probably by facilitating binding of HIF-2α toimmobile nuclear or chromatin components, which results in nuclearretention. Lack of the CK1δmodification could thus shift the balancemore towards nuclear export despite the weak interaction of CRM1with ERK1/2-modified HIF-2α. Similarly, lack of the ERK1/2-mediated phosphorylation would result in a stronger interaction withCRM1 and more efficient nuclear export despite the CK1δ-mediatedmodification. Alternatively, it is also possible that phosphorylation byCK1δ somehow facilitates or permits modification of HIF-2α byERK1/2 and, therefore, also indirectly inhibits the activity of NES-like sequence identified in this work. In any case, fine-tuning controlof HIF-2α activity by nucleocytoplasmic transport apparentlyinvolves at least two kinases (Fig. 6C) and, therefore, can beconsidered an important and complex mechanism worthy of furtherinvestigation.

ERK1/2 family members are evolutionarily conserved serine/threonine kinase members involved in many cellular biologicalprocesses including cell proliferation, cell cycle regulation,differentiation and inflammation (Kyriakis and Avruch, 2012). Thedysregulation of ERK1/2 is implicated in a variety of human diseasesas well as cancers (Kim and Choi, 2015; Yang and Huang, 2015). Inthe literature, ERK1/2 have been reported to target severaltranscription factors (Monje et al., 2005; Roskoski, 2012), includingHIF-1 (Mylonis et al., 2008; Mylonis et al., 2006). Our findingsadding HIF-2α to this list contribute to the understanding of themolecular mechanisms underlying the interaction between thehypoxia response and ERK1/2 signaling. Overall, it appears that, inorder to adapt to hypoxia, cells with activated ERK1/2 (e.g.proliferating or transformed cells) require a full HIF-mediatedtranscriptional response, which is ensured by shifting the balance infavor of nuclear import (through inhibiting association with CRM1)and maximizing the concentration of HIF-α inside the nucleus. Uponinactivation of ERK1/2 (e.g. in quiescent or somatic cells), successfuladaptation to hypoxia may require a weaker transcriptional activationof HIF-target genes or, alternatively, a cytoplasmic non-genomic HIF-α function made possible by shifting the balance in favor of nuclearexport (by allowing association of HIF-α with CRM1). In the case ofHIF-1α, such a cytoplasmic function has indeed been recentlyreported: upon inhibition of its ERK1/2-mediated phosphorylation

Fig. 5. Disruption of a potential NES restores nuclear accumulation andtranscriptional activity of HIF-2α lacking the ERK1/2 phosphorylationsite. (A) Transfected HeLa cells grown under in 21%O2 expressing one of wild-type Flag–HIF-2α (wt), or the phospho-deficient Flag–HIF-2αS672A (S→A),NES-deficient mutant Flag–HIF-2αL662A/L667A (L→A) or the doublephospho- and NES-deficient Flag–HIF-2αL662A/L667A/S672A (S→A/L→A)mutant were fixed at 24 h post-transfection and subjected toimmunofluorescence microscopy with anti-Flag mouse monoclonal antibody(green). Nuclei were stained with DAPI (magenta). In some cases, at 8 h post-transfection, cells were treated with (5 μM) U0126 (as indicated). Scale bars:10 μm. (B) Quantification of the results from the experiment described inA. Transfected cells showing exclusive nuclear staining under each conditionwere counted, and their percentage was calculated relative to the total numberof transfected cells. At least 100–150 transfected cells were examined in eachcase. Data plotted are the mean of three independent experiments (±s.e.m.)(***P<0.001). (C) HIF-2 transcriptional activity was determined 24 h aftertransfection for Huh7 cells grown under 21% O2 with the pGL3-5HRE-VEGFreporter plasmid, plasmid pCI-Renilla and the pFLAG-CMV2 plasmids carryingthe indicated inserts as described in A. Values show the fold increase ofrelative luciferase units (firefly over Renilla activity) in relation to the valuesobtained from cells expressing pFLAG-CMV2 alone and represent the mean ofthree independent experiments performed in triplicate (±s.e.m.) (**P<0.01).

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and translocation to the cytoplasm, HIF-1α interacts with proteins ofthe outer mitochondrial membrane forming an anti-apoptotic complex(Mylonis et al., 2017). This leaves open the possibility that the

regulation of the nucleocytoplasmic distribution of HIF-2α by ERK1/2 may not only serve the control of HIF-2-target gene expression butalso a cytoplasmic function of HIF-2α such as the one involving the

Fig. 6. HIF-2α nuclear export is mediated by a CRM1-interacting NES sequence that can be masked by ERK1/2-catalyzed phosphorylation of S672.(A) HeLa cells were transfected with wild-type Flag–HIF-2α (wt) or the NES-deficient Flag–HIF-2αL662A/L667A (L→A) mutants, and at 8 h post transfection weretreated or not with U0126 (5 μM) under 21% O2. At 24 h post transfection, cells were lysed and subjected to immunoprecipitation with anti-Flag antibody.Total cell extracts (input) and immunoprecipitated proteins (I.P.) were analyzed by western blotting using anti-CRM1, anti-HIF-2α and anti-phospho-serine (p-Ser)antibodies as indicated. (B) HeLa cells were transfected with the phospho-deficient flag-HIF-2αS672A (S→A), the NES-deficient flag-HIF-2αL662A/L667A (L→A)or double NES-deficient and phospho-deficient Flag–HIF-2αL662A/L667A/S672A (L→A, S→A) mutants and, at 8 h post transfection, treated or not withU0126 (5 μM) under 21% O2. At 24 h post-transfection, cells were lysed and subjected to immunoprecipitation with anti-Flag antibody. Total cell extracts (input)and precipitated proteins (I.P.) were analyzed by western blotting using anti-CRM1, anti-HIF-2α and anti-phospho-serine (p-Ser) antibodies as indicated.(C) A proposedmodel for regulation of HIF-2α nucleocytoplasmic trafficking by phosphorylation. Under hypoxia, HIF-2α is stabilized and enters the nucleus. In theabsence of phosphorylation, binding of CRM1 to the NES neighboring S672 returns HIF-2α to the cytoplasm and diminishes its activity. When ERK1/2phosphorylates S672, the NES is masked, interaction with CRM1 is inhibited and HIF-2α nuclear export is blocked promoting its nuclear accumulation andmaximizing its activity. CK1δ-dependent phosphorylation (at S383/T528) can also inhibit nuclear export of HIF-2α (Pangou et al., 2016) but through amechanismnot involving the CRM1–NES interaction (this work). Nuclear HIF-2α interacts with ARNT to form an active HIF-2 heterodimer, which can bind to HRE sequencesin the promoter/enhancer regions of its target genes (such as EPO and PAI-1) and stimulate their transcription.

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interaction of HIF-2α with RNA-binding protein RBM4 and proteintranslation factors, which mediates an O2-regulated switch in theprotein synthesis machinery (Uniacke et al., 2012).In summary, our work suggests an additional O2-independent

HIF-2 regulatory mechanism that involves control of HIF-2αnucleocytoplasmic trafficking, responds to MAPK pathwayactivation, and may thus connect HIF-2 activity to cancer cellproliferation under hypoxia. It involves a leucine-containing atypicalhydrophobic CRM1-dependent NES which is ‘masked’ when S672of HIF-2α is phosphorylated by ERK1/2. Taking into account theimportant role of HIF-2 in hepatocellular carcinoma (HCC) and renalcell carcinoma (RCC) (Nath and Szabo, 2012; Luo et al., 2016) manycompounds that directly interfere with HIF-2 function or targetdownstream hypoxia-related pathways have been tested inexperimental trials as anti-cancer agents but have proven ineffective(Zhao et al., 2015). The novel regulatory communication betweenERK1/2 and HIF-2 described in this work provides an alternativemeans to control HIF-2 activity in disease that needs to be furtherinvestigated as a potential target of therapeutic intervention.

MATERIALS AND METHODSPlasmid constructionscDNAs encoding various fragments of HIF-2α were amplified by PCR andcloned as BamHI fragments into pGEX-4T-1 bacterial expression vector (GEHealthcare), yielding pGEX-HIF-2α full-length (1–870) and pGEX-HIF-2αfragments 1–542, 1–560, 1–620, 1–640, 1–679, 640–870, 680–870. Singlepoint HIF-2α mutants S672A, S672D, T626A were constructed using, as atemplate, pBS-SK(+)-HIF-2α containing the full-length cDNA of HIF-2α,with the QuikChange II site-directed mutagenesis kit (Agilent Technologies).These HIF-2α mutant forms were then subcloned as BamHI fragments intothe mammalian expression vector pFlag-CMV2 and into the bacterialexpression vector pGEX-4T-1. HIF-2α point mutants L662A/L667A andL662A/L667A/S672A were constructed using, as a template, pFlag-HIF-2αor pFlag-HIF-2α (S672A) with the QuikChange II site-directed mutagenesiskit. The primer sequences used are available upon request. The DNAsequence of the point mutants was confirmed by sequencing performed byCemia SA (Larissa, Greece). pFlag-HIF-2α (S383A/T528A) was previouslydescribed (Pangou et al., 2016). Reporter plasmids pGL3-5xHRE-VEGFcontaining five copies of the HRE from the VEGF gene promoter, andpCI-Renilla were previously described (Mylonis et al., 2006).

Cell culture, transfection and luciferase assaysHuh7 and HeLa cells (supplied by ATCC and checked for mycoplasma)were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Biosera)containing 10% fetal bovine serum (FBS; Biochrom, Berlin, Deutschland)and 100 U/ml penicillin–streptomycin (Biosera) in normal atmospheric aircontaining 5%CO2 (denoted as 21%O2) in a standard humidified incubator.Transient transfections were carried out by using TurboFect™ transfectionreagent (Thermo Fisher Scientific) according to the manufacturer’sinstructions. When required, 4 h after transfection, cells were treated withthe MEK inhibitor U0126 (5 μM; Cell Signaling Danvers) for 16 h and with10 ng/ml leptomycin B (LMB; Sigma-Aldrich) for 4 h or with DMSO(Applichem, Darmstadt, Deutschland) as a solvent control. For hypoxictreatment, cells were exposed for 16 h to a gas mixture containing 1% O2,94% N2 and 5% CO2 (denoted as 1% O2) in an IN VIVO2 200 hypoxiaworkstation (Baker Ruskinn, Sanford, ME). These conditions were chosenas closest to physiological hypoxia since they resulted in completestabilization of HIF-2α and full activation of HIF-2 target genes (i.e.efficient cellular response to hypoxia) compared to the incomplete HIF-2αstabilization and partial or no activation of HIF-2 target genes (i.e.inefficient response to hypoxia) observed when cells were exposed to a gasmixture containing 5% O2, 90% N2 and 5% CO2 (denoted as 5% O2) (Fig.S2). Luciferase assays were performed in cells transiently co-transfectedwith equal amounts of plasmids expressing different forms of HIF-2α (or theempty parental vector pFlag-CMV2 as control), the firefly luciferasereporter plasmid pGL3-5HRE-VEGF and the Renilla luciferase expressing

plasmid PCI Renilla. At 24 h post transfection, cells were washed with PBSand lysed, and luciferase activity was determined by using the DualLuciferase Reporter Assay system (Promega) with a luminometer (TD20/20,Turner Designs).

siRNA-mediated silencingHuh7 cells were incubated in serum-free DMEM for 4 hwith siRNAs targetingHIF-1α (10 nM; SI02664053) or HIF-2α (10 nM; SI02663038) (Qiagen) inthe presence of LipofectamineTM 2000 (Invitrogen, Thermo Fisher Scientific)as described by themanufacturer. AllStars siRNA (1027280, Qiagen) was usedas negative control. At 24 h post transfection, cells were subjected to hypoxiaand, 16 h later they were harvested, and cell lysates were prepared for real-timequantitative PCR or for western blot analysis.

RNA extraction and quantitative PCRTotal RNA from Huh7 cells was isolated using the Nucleozol reagent(Macherey-Nagel, Duren, Deutschland) and cDNAwas synthesized with theHigh Capacity Reverse Transcription kit (Applied Biosystems, ThermoFisher Scientific). Real-time quantitative PCR was performed with SYBRGreen qPCR SuperMix Universal (Invitrogen, Thermo Fisher Scientific) in aLightCycler 96 (Roche Life Science, Basel, Switzerland). The mRNAsencoding EPO, PAI-1 and 18S were amplified using primers available uponrequest. Each sample was assayed in duplicate for both target and internalcontrol. Relative quantitative gene expression was calculated using the ΔΔCTmethod and is presented as a fold increase in relation to the respective controls.

SDS-PAGE, western blotting and subcellular fractionationProteins were resolved by 10% SDS-PAGE, and analyzed by CoomassieBlue or western immunoblotting using the antibodies: rabbit polyclonalantibody against HIF-2α (ΝΒ100-122, 1:750 dilution; Novus Europe),affinity-purified rabbit polyclonal antibody against HIF-1α (Lyberopoulouet al., 2007), mouse monoclonal antibody against Flag (F4042, 1:10,000dilution; Sigma-Aldrich), mouse monoclonal antibody against β-actin(3700, 1:5000 dilution; Cell Signaling), and mouse monoclonal antibodiesagainst CRM1 (611832, 1:1000 dilution), phospho-serine (612547, 1:1000dilution) and ARNT (611079, 1:500 dilution) from BD Biosciences.Subcellular fractionations were performed with HeLa cells as previouslydescribed (Andrews and Faller, 1991). Western blot images were takenusing Uvitec Cambridge chemiluminescence imaging system with the helpof Alliance Software (ver. 16.06) (Uvitec Cambridge, Cambridge, UK).

ImmunoprecipitationImmunoprecipitation experiments were performed as previously described(Mylonis et al., 2006). HeLa cells incubated under hypoxia in the presenceor absence of U0126 and/or LMB or transfected with pFlag-HIF-2α or itspoint mutants, werewashed with cold PBS and lysed (30 min, 4°C) in buffercontaining 25 mMHEPES pH 7.5, 150 mMNaCl, 1% Triton X-100, 2 mMMgCl2, 2 mM phenylmethylsulfonyl fluoride, 4 mM β-glycerolphosphateand 2 mM Na3VO4. Immunoprecipitation from total cell lysates ofendogenous HIF-2α or Flag-tagged proteins was performed using rabbitpolyclonal antibody against HIF-2α (ΝΒ100-122; Novus Europe) andprotein A–Sepharose beads or anti-Flag M2 Affinity Gel (Sigma-Aldrich),respectively. Precipitates were eluted with glycine 0.2 M, pH 2 and analyzedthrough SDS-PAGE and western blotting.

ImmunofluorescenceFor immunofluorescence experiments, Huh7 or HeLa cells were grown oncoverslips and transiently transfected with pCMV2-FLAG vectorexpressing wild-type HIF-2α and its mutant forms. In some cases, cells at4 h post transfection were treated with U0126 for 16 h or treated with LMBfor 4 h prior to observation. Coverslips were incubated for 1 h at roomtemperature with anti-Flag mAb (Sigma-Aldrich), washed three times withPBS, and incubated for 1 h at room temperature with Cy3-conjugated anti-mouse secondary antibody (Jackson ImmunoResearch Europe Ltd, Ely,Cambridgeshire, UK). Analysis by immunofluorescence was carried out aspreviously described (Mylonis et al., 2008). Images were taken on a ZeissAxio Imager.Z2 microscope equipped with an AxioCamMRm or AxioCamIcC5 sensor and a 40× objective or 100× oil-immersion lens.

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Protein purification and phosphorylation assaysWild-type and mutant forms of GST-HIF-2α and its GST fragments 1–542,1–560, 1–620, 1–640, 1–679, 640–820, 680–820 were expressed inEscherichia coli and purified as previously reported for GST–HIF-1α(Chachami et al., 2005). Phosphorylation reactions were carried out aspreviously described (Mylonis et al., 2006) at 30°C for 60 min with 1 μg ofthe appropriate substrate in a total volume of 25 μl in a buffer containing25 mM Tris-HCl pH 7.5, 10 mM MgCl2, 50 mM NaCl, 50 µM ATPsupplied with 0.5 μCi [γ-32P] ATP (6000 Ci/mmol). When required, 1 μgof total Huh7 protein extracts or 5 units of active recombinant ERK2(New England Biolabs #P6080) were added to the mix. The reaction wasstopped by adding SDS sample buffer and heating at 95°C for 5 min.Samples were analyzed by SDS-PAGE followed by Coomassie Bluestaining and autoradiography. To quantify the relative phosphorylationlevels of the different substrates, bands corresponding to the substrates wereexcised from the gels and their radioactivity was measured throughscintillation counting.

Statistical analysisStatistical differences between two groups of data were assessed using theunpaired t-test in the GraphPad Prism version 5.04 software; P<0.05 wasconsidered to be significant (*P<0.05; **P<0.01; ***P<0.001).

AcknowledgementsWe would like to thank Assist. Prof. Ilias Mylonis (University of Thessaly, Larissa,Greece) and Dr Evanthia Pangou (IGBMC, Illkirch, France), for their help withradiolabeling experiments in the preliminary stages of this project.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: P.L.; Methodology: I.-M.G., C.B.; Validation: P.L.; Formalanalysis: I.-M.G., C.B.; Investigation: I.-M.G., C.B.; Writing - original draft: I.-M.G.,C.B., P.L.; Writing - review & editing: G.S., P.L.; Supervision: G.S., P.L.; Projectadministration: P.L.; Funding acquisition: G.S., P.L.

FundingI.-M.G. was a recipient of a scholarship from the State Scholarships Foundation (IKY).

Supplementary informationSupplementary information available online athttp://jcs.biologists.org/lookup/doi/10.1242/jcs.225698.supplemental

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