Glycogen Synthase Kinase 3 Phosphorylates Hypoxia-Inducible ...

MOLECULAR AND CELLULAR BIOLOGY, May 2007, p. 3253–3265 0270-7306/07/$08.00⫹0 doi:10.1128/MCB.00015-07 Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Vol. 27, No. 9

Glycogen Synthase Kinase 3 Phosphorylates Hypoxia-Inducible Factor 1␣ and Mediates Its Destabilization in a VHL-Independent Manner䌤 Daniela Flu ¨gel,1 Agnes Go ¨rlach,2 Carine Michiels,3 and Thomas Kietzmann1* Fachbereich Chemie, Abteilung Biochemie, Universita ¨t Kaiserslautern, D-67663 Kaiserslautern, Germany1; Experimental Pediatric Cardiology, Department of Pediatric Cardiology and Congenital Heart Disease, German Heart Center Munich at the Technical University Munich, Lazarettstrasse 36, 80636 Munich, Germany2; and Laboratory of Biochemistry and Cellular Biology, University of Namur, 61 rue de Bruxelles, 5000 Namur, Belgium3 Received 3 January 2007/Returned for modification 12 February 2007/Accepted 13 February 2007

Hypoxia-inducible transcription factor 1␣ (HIF-1␣) is a key player in the response to hypoxia. Additionally, HIF-1␣ responds to growth factors and hormones which can act via protein kinase B (Akt). However, HIF-1␣ is not a direct substrate for this kinase. Therefore, we investigated whether the protein kinase B target glycogen synthase kinase 3 (GSK-3) may have an impact on HIF-1␣. We found that the inhibition or depletion of GSK-3 induced HIF-1␣ whereas the overexpression of GSK-3␤ reduced HIF-1␣. These effects were mediated via three amino acid residues in the oxygen-dependent degradation domain of HIF-1␣. In addition, mutation analyses and experiments with von Hippel-Lindau (VHL)-defective cells indicated that GSK-3 mediates HIF-1␣ degradation in a VHL-independent manner. In line with these observations, the inhibition of the proteasome reversed the GSK-3 effects, indicating that GSK-3 may target HIF-1␣ to the proteasome by phosphorylation. Thus, the direct regulation of HIF-1␣ stability by GSK-3 may influence physiological processes or pathophysiological situations such as metabolic diseases or tumors. (39). The hydroxylation within the ODD allows for the binding of the von Hippel-Lindau (VHL) tumor suppressor protein (pVHL), a component of an E3 ubiquitin ligase complex that targets HIF-␣ subunits for degradation by the ubiquitin-proteasome pathway (64). The N hydroxylation by the factorinhibiting HIF (42) inhibits the recruitment of the coactivator CBP/p300, thus resulting in a loss of the transactivation potential (39). In addition to proline and asparagine hydroxylation, HIF-1␣ protein levels and transcriptional activity have been shown to be regulated by various growth factors and hormones, including insulin, via the phosphatidylinositol 3-kinase and protein kinase B (PKB, also known as Akt) pathway (24, 36, 44, 62, 70–72). Therefore, it appeared that the enhancement of HIF-1␣ was not due to direct phosphorylation by PKB rather than the action of another PKB target, such as mammalian target of rapamycin (mTOR) or glycogen synthase kinase 3 (GSK-3). Indeed, some investigations pointed to a role for mTOR (1, 40, 65) in the regulation of HIF-1␣ protein levels, and others showed the involvement of GSK-3 (10, 46, 59). GSK-3 exists in two isoforms (␣ and ␤), which can be phosphorylated by PKB at Ser-21 and Ser-9, respectively, thus leading to an inhibition of GSK-3 activity. Although first identified as a negative regulator of glycogen synthesis (15), GSK-3 is now known to be involved also in the regulation of transcription factors such as the cyclic AMP-responsive element binding (CREB) protein (12), Foxo forkhead transcription factors (37), AP-1 (5, 47), Myc (52), and NF-␬B (4, 25). While the action of mTOR on HIF-␣ seems to occur via a translational process, GSK-3 appears to exert its effect on HIF-1␣ in such a way that the activation of GSK-3 down-regulates HIF-1␣ protein levels (46). However, the underlying mechanisms and the exact localization of GSK-3 phosphorylation sites have remained unknown.

Increasing evidence has been provided that most, if not all, cells are able to adapt to changes in O2 tension by regulating the level of gene expression. Transcriptional control of gene expression is brought about by the function of several transcription factors. Under hypoxic conditions, the transcription factor hypoxia-inducible factor 1 (HIF-1) (7, 53–55, 67) appears to play a critical role in the transcriptional induction of many genes, including those encoding erythropoietin and glycolytic enzymes but also those for plasminogen activator inhibitor 1 (PAI-1) and vascular endothelial growth factor (43, 50, 66). HIF-1 is composed of an inducible subunit (HIF-1␣) and a constitutively expressed subunit (HIF-1␤, also known as ARNT), and both are members of the basic helix-loop-helixPAS protein family. Two other HIF-␣ subunits, HIF-2␣ (EPAS/HLF/HRF/MOP2) (14, 17, 26, 66) and HIF-3␣ (22, 33), have been cloned from human, mouse, and rat, and together with two other ARNT isoforms (ARNT2 and ARNT3/ BMAL-1/MOP3), they give rise to the existence of several HIF dimers composed of different HIF-␣ subunits and ARNT isoforms (56, 66). The mechanisms by which hypoxia stimulates the activation of HIF-1 are not understood to the last detail. A major pathway appears to be the posttranslational modification of HIF-1␣ via the oxygen-dependent hydroxylation (29, 30) of two proline residues (P402 and P564) within the O2-dependent degradation domain (ODD), which overlaps with an N-terminal transactivation domain (TAD-N), and that of an asparagine residue (N803) within the C-terminal transactivation domain (TAD-C) * Corresponding author. Mailing address: Fachbereich Chemie, Abteilung Biochemie, Universita¨t Kaiserslautern, Erwin Schro ¨dinger Str. Geb 54, D-67663 Kaiserslautern, Germany. Phone: 49-631-2054953. Fax: 49-631-2053419. E-mail: [email protected]. 䌤 Published ahead of print on 26 February 2007. 3253

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Among the various HIF-1 target genes (68), the PAI-1 gene appears to be of special importance due to the finding that enhanced levels of PAI-1 are associated with various types of tumors and indicate a poor prognosis (13). Although several cell types secrete PAI-1, the liver appears to be a major source of PAI-1. Interestingly, the liver acinus exhibits a periportalto-perivenous oxygen gradient (31). This O2 gradient is generated due to the blood flow and the metabolism of the cells inside the acinus and is considered to be responsible for the zonation of metabolic processes, e.g., gluconeogenesis in the periportal area and glycolysis in the perivenous area. The periportal region is normoxic, with conditions similar to 16% O2. By contrast, the perivenous area is mildly hypoxic, with conditions similar to 8% O2 (31). Thus, it was the aim of the present study to identify the GSK-3 target sites within the HIF-1␣ molecule and to investigate their functional relevance in the regulation of HIF-1␣ protein stability and the expression of the HIF-1 target PAI-1 gene in liver-derived cells under periportal and perivenous O2 conditions. MATERIALS AND METHODS All biochemicals and enzymes were of analytical grade and were obtained from commercial suppliers. Cell culture. HepG2 cells were cultured in a normoxic atmosphere of 16% O2, 79% N2, and 5% CO2 (by volume) in minimal essential medium supplemented with 10% fetal calf serum for 24 h. At 24 h, the medium was changed and the culture was continued under normoxic conditions of 16% O2 or hypoxic conditions of 8% O2 (with 87% N2 and 5% CO2 [by volume]). The pVHL-defective renal cell carcinoma (RCC4) cells and RCC4 cells with reintroduced pVHL (RCC4/VHL cells) were cultured in Dulbecco’s modified Eagle’s medium with 4.5 g of glucose/liter, supplemented with 2 mM glutamine, 100 U of penicillin/ml, 100 ␮g of streptomycin/ml, 1 mM sodium pyruvate, and 10% fetal calf serum. Plasmid constructs. The pGL3-hPAI-796 plasmid, containing the human PAI-1 gene promoter 5⬘-flanking region from ⫺796 to ⫹13, as well as the pGL3-hPAI-796-M2 construct with a mutation in the hypoxia-responsive element (HRE), have been described previously (34). The constructs for pG5E1B-LUC (38) and pSG424 (49) and those for Gal4HIF-1␣-TAD-N and Gal4-HIF-1␣-TAD-C were described previously (9, 42, 64). The various constructs for full-length HIF-1␣, Gal4-HIF-1␣-TAD-N, and pGEX-HIF1␣-TADN in which the proline 564, serine 551, threonine 555, or serine 589 was mutated to alanine were constructed by using the QuickChange mutagenesis kit (Promega, Heidelberg, Germany). The constructs for the respective GSK-3 expression vectors (pcDNA3-HAGSK-3␤ and pcDNA3-HA-GSK-3␤S9A) were a kind gift from Jim Woodgett and were described previously (61). The expression vector for the constitutive active protein kinase B (myrPKB) was described previously (34). RNA preparation and Northern blot analysis. The isolation of total RNA and Northern blot analysis were performed as described previously (35). Digoxigeninlabeled antisense RNAs served as hybridization probes; they were generated by in vitro transcription from pBS-PAI1 by using T3 polymerase and from pBSActin by using T7 polymerase. Blots were quantified with a video densitometer (Biotech Fischer, Reiskirchen, Germany). Western blot analysis, HIF-1␣ protein half-life studies, and HIF-1␣ ubiquitylation assays. Western blot analysis was carried out as described previously (28). In brief, medium and lysates from HepG2 cells were collected and 100 ␮g of protein was loaded onto a 10% or 7.5% sodium dodecyl sulfate (SDS)polyacrylamide gel. After electrophoresis and electroblotting onto a methanolactivated polyvinylidene difluoride membrane, proteins were detected with a monoclonal antibody against human PAI-1 (1:100; American Diagnostics, Pfungstadt, Germany), a monoclonal antibody against human HIF-1␣ (1:2,000; BD Biosciences, Heidelberg, Germany), a monoclonal antibody against Flag M2 (1:1,000; Sigma), a monoclonal antibody against hemagglutinin (HA; 1:500; Santa Cruz, California), or a monoclonal antibody against the Myc or V5 tag (1:10,000; Invitrogen, Karlsruhe, Germany) or a polyclonal antibody against phospho-GSK-3␣/␤ (1:2,000; Cell Signaling, Frankfurt, Germany), a polyclonal antibody against total GSK-3␣/␤ (1:2,000; Cell Signaling), or a polyclonal antibody against the Golgi membrane (1:10,000; Biosciences, Go ¨ttingen, Germany). The secondary antibody was either an anti-mouse or an anti-rabbit immunoglob-

MOL. CELL. BIOL. ulin G conjugated to horseradish peroxidase (1:5,000; Sigma). The ECL system (Amersham, Freiburg, Germany) was used for detection. For half-life studies, HepG2 cells were transfected with plasmids expressing V5 epitope-tagged versions of HIF-1␣ along with a plasmid expressing HAtagged GSK-3␤. After 24 h, cycloheximide (100 ␮g/ml; Sigma) was added to the medium, and at indicated time points thereafter, cells were removed and protein abundance was measured by immunoblot analysis. For ubiquitylation assays, the cells were treated with the proteasome inhibitor MG132 (10 ␮M) 24 h after transfection. Six hours later, cells were washed with 1⫻ phosphate-buffered saline and lysed in a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton, 2 mM EDTA, 2 mM EGTA, 10 mM Na2PO7, and protease inhibitors. After scraping, lysates were incubated with continuous shaking at 4°C for 20 min and then they were centrifuged at 8,000 ⫻ g at 4°C for 15 min. To recover anti-V5 and anti-HA immunoprecipitates, 150 ␮g of protein was incubated with 2 ␮g of antibody at 4°C for 1 h before Sepharose beads (30 ␮l per reaction mixture) were added for 12 h. Thereafter, the beads were washed five times with lysis buffer and recovered pellets were dissolved in 2⫻ Laemmli buffer, loaded onto 7.5% SDS gels, and immunoblotted with antibodies against ubiquitin and the V5 or HA epitope. Cell transfection and luciferase assay. About 4 ⫻ 105 HepG2 cells per 60mm-diameter dish were transfected as described previously (36). In brief, cells were transfected with 2.5 ␮g of pGL3-hPAI-796 or pGL3-hPAI-796-M2. Transfection efficiency was controlled for by cotransfecting the cells with 0.25 ␮g of a Renilla luciferase expression vector (pRLSV40; Promega). The detection of luciferase activity was performed with a luciferase assay kit (Berthold, Pforzheim, Germany). To investigate HIF-1␣ transactivation, cells were cotransfected with 2 ␮g of reporter construct pG5-E1B-Luc and 500 ng of the Gal4-HIF-1␣TAD-N, Gal4-HIF-1␣-TAD-C, or respective mutant construct (41). For Western blot experiments, cells were transfected with 5 ␮g of the respective GSK-3 expression vectors or the control vector. After 5 h, the medium was changed and cells were cultured for 16 h under normoxic or hypoxic conditions. Cell transfection with siRNA. About 4 ⫻ 105 HepG2 cells per 60-mm-diameter dish were transfected with the Oligofectamine reagent (Invitrogen). Before transfection, cells were washed with serum-free medium and subsequently transfected with small interfering RNA (siRNA) targeting GSK-3␤ (final concentration, from 100 nM) in 1.65 ml of serum-free medium. After transfection, cells were cultured for 4 h under normoxia before the addition of 825 ␮l of medium containing 3⫻ fetal calf serum, and cell culture was continued for the next 72 h under normoxia or hypoxia. Then cells were further cultured for 24 h under normoxia and hypoxia. Purification of the GST-TAD-N fusion proteins. Escherichia coli BL21(DE3) cells transformed with pGEX-GST-TADN or pGEX-5X1 were grown at 37°C in Luria-Bertani media containing 100 ␮g of ampicillin/ml and induced with 0.1 mM isopropyl-␤-D-thiogalactoside (IPTG) for 4 h. Then cells were harvested, and glutathione S-transferase (GST)-TAD-N and GST proteins were prepared essentially as described previously (41). The integrity and yield of purified GST proteins were assessed by SDS-polyacrylamide gel electrophoresis followed by Coomassie blue staining. In vitro HIF phosphorylation assay. The purified wild-type or mutant GSTHIF-TAD-N fusion proteins (20 ␮g) were incubated in kinase buffer [0.2 M MOPS (morpholinepropanesulfonic acid; pH 7.4), 0.5 M EDTA, 0.1 M Mg(CH3COO)2] in the presence of active GSK-3␤ (50 mU; Cell Signaling) and 1 ␮Ci/␮l [␥-32P]ATP (Amersham). After 30 min of incubation at 30°C, samples were loaded onto a 10% SDS gel, and after electrophoresis and blotting onto a polyvinylidene difluoride membrane, phosphorylated proteins were visualized by phosphorimaging. Statistical analysis. Each experiment was performed at least three times, and representative data are shown. Data in bar graphs are given as mean values ⫾ standard errors of the means (SEM). Statistical differences were calculated by using the Student t test, with error probabilities of P of ⬍0.05 considered to be significant.

RESULTS Inhibition of GSK-3 by insulin, LiCl, and GSK-3␤ siRNA enhances HIF-1␣ levels. To investigate whether GSK-3 can modulate HIF-1␣ levels, we treated HepG2 cells with insulin and LiCl, both known inhibitors of GSK-3, under normoxia and hypoxia. We found that hypoxia induced an increase in HIF-1␣ protein levels of about 11-fold. Treatment with insulin enhanced

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HIF-1␣ protein levels by about eightfold under normoxia and by about 12-fold under hypoxia. In line with these results, LiCl enhanced HIF-1␣ levels by about fivefold under normoxia while under hypoxia HIF-1␣ levels were again induced to increase by about 11-fold (Fig. 1A and B). In contrast to other kinases which are activated upon phosphorylation, GSK-3␣ and GSK-3␤ become inactivated upon phosphorylation at Ser-21 and Ser-9, respectively. To prove that LiCl and insulin inactivated GSK-3, we investigated the phosphorylation of GSK-3␣ and GSK-3␤ with an antibody specifically recognizing Ser-21- and Ser-9-phosphorylated GSK-3. Both agents, LiCl and insulin, were found to effectively inhibit GSK-3, as indicated by the enhanced levels of phosphorylated GSK-3␣ and GSK-3␤. To investigate the involvement of GSK-3 in the regulation of HIF-1␣ in more detail, we aimed to specifically deplete cells of GSK-3␤ by using siRNA. After using an unspecific control siRNA, we found that hypoxia enhanced HIF-1␣ levels by about 14-fold. By contrast, the use of GSK-3␤ siRNA induced an increase in HIF-1␣ protein levels of about 16-fold under normoxia. Interestingly, the GSK-3␤ siRNA did not additively enhance HIF-1␣ levels under hypoxia (Fig. 1C and D). Thus, the inhibition of GSK-3␤ activity elicits the induction of HIF-1␣ preferentially under normoxia. Concomitantly, the depletion of GSK-3␤ induced the levels of the HIF-1 target PAI-1. Hypoxia enhanced PAI-1 levels by about sixfold. GSK-3␤ siRNA enhanced PAI-1 levels under both normoxia and hypoxia by about sixfold (Fig. 1E and F). Similar results could also be obtained with GSK-3␣ (data not shown). GSK-3␤ down-regulates PAI-1 promoter activity via HIF-1 at the HRE. To specify whether the GSK-3-mediated regula-

FIG. 1. Regulation of HIF-1␣ and the HIF-1 target PAI-1 gene by the inhibition of GSK-3. (A) HepG2 cells were cultured under normoxia (16% O2) for 48 h and then stimulated with either LiCl or insulin and further cultured for 4 h under normoxic or hypoxic (8% O2) conditions. HIF-1␣ protein levels were measured by Western blot analysis. The HIF-1␣ protein levels under normoxic conditions (16% O2) were set at 1. (B, D, and F) Representative Western blots. One hundred micrograms of total protein from HepG2 cells was analyzed with antibodies against HIF-1␣, phospho-GSK-3␣/␤ (p-GSK-3␣/␤), and GSK-3␣/␤. PAI-1 levels in 100 ␮g of protein from the culture medium were determined and analyzed with an antibody against human PAI-1. (C) HepG2 cells were transfected with siRNA against GSK-3␤ and cultured for 72 h under normoxia. Then cells were further cultured under normoxia (16% O2) or hypoxia (8% O2) for the next 24 h. The HIF-1␣ and PAI-1 protein levels were measured by Western blot analysis, and protein levels under normoxic conditions (16% O2) were set at 1. Values are means ⫾ SEM of results from at least three independent experiments. Statistics were calculated by using the Student t test for paired values. *, significant difference between results with 16% O2 and those with 8% O2; **, significant difference between results with 8% O2 and those with 8% O2 and a stimulus or siRNA; ***, significant difference between results with 16% O2 and those with 16% O2 and a stimulus or siRNA; P ⱕ 0.05. (E) HepG2 cells were transfected with either the PAI-1 promoter construct (pGL3-hPAI-796 Luc; PAI-766) or the HRE mutant (pGL3-hPAI-796HREm Luc; PAI766HREm) construct and an empty control vector or an expression vector carrying GSK-3␤. Then cells were cultured under normoxia (16% O2; control) or exposed to hypoxia (8% O2) or to insulin for the next 24 h. In each experiment, the percentage of Luc activity relative to the activity in the pGL3PAI-766 and pGL3PAI-766M2 controls, which was set at 100%, was determined.

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FIG. 2. GSK-3 downregulates HIF-1␣ via the TAD-N. (A) HepG2 cells were cotransfected with expression vectors for GSK-3␤, Gal4-HIF1␣-TAD-N-C, Gal4-HIF-1␣-TAD-N, and Gal4-HIF-1␣-TAD-C and the p5GE1B-Luc gene construct. After transfection, cells were cultured with fresh culture medium for the next 48 h under normoxia (16% O2). In each experiment, the percentage of Luc activity relative to that in the Gal4-HIF-1␣-TAD-N-C, Gal4-HIF-1␣-TAD-N, or Gal4-HIF-1␣-TAD-C control, which was set at 100%, was determined. The values represent

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tion of PAI-1 occurs via HIF-1 binding at the HRE in the PAI-1 promoter, we cotransfected HepG2 cells with a GSK-3␤ expression vector along with a wild-type PAI-1 promoter Luc gene or a PAI-1 promoter construct mutated at the HRE. In line with findings of previous studies, we found that hypoxia and insulin enhanced the Luc activity of the wild-type promoter by about threefold. By contrast, the overexpression of GSK-3␤ decreased Luc activity by about 70% (Fig. 1E). However, when the PAI-1 promoter construct with the mutated HRE was used, neither hypoxia nor insulin nor GSK-3␤ affected Luc activity. Thus, GSK-3␤ may regulate PAI-1 expression via the modulation of HIF-1␣ levels. GSK-3␤ affects HIF-1␣ protein stability via the N-terminal transactivation domain. HIF-1␣ activity is regulated mainly on the level of protein stability and cofactor recruitment. This regulation is achieved by the integrated action of proline hydroxylases and an asparagine hydroxylase (6). To find out whether the effects of GSK-3␤ on HIF-1␣ affect protein stability, we transfected cells with the GSK-3␤ expression vector together with constructs expressing fusion proteins from the Gal4 DNA binding domain and the respective HIF-1␣ TAD along with a Luc construct containing five Gal4 binding sites in front of the adenovirus E1B promoter. By using a Gal4 construct expressing a fusion protein with the entire HIF-1␣ region from the N-terminal to the C-terminal end (Gal4-HIF-1␣-TAD-N-C), we found that GSK-3␤ reduced Luc activity by about 60%. Likewise, GSK-3␤ reduced Luc activity by 80% when a Gal4 fusion construct containing only the TAD-N was used. By contrast, GSK-3␤ did not affect Luc activity when a Gal4 construct containing only the TAD-C was used (Fig. 2A and B). In addition, this pattern of regulation could be detected on the level of the fusion proteins (Fig. 2A and B). Since PKB inactivates GSK-3␤ by phosphorylation at serine 9, we investigated whether cotransfection with myrPKB/Akt could increase the activity of the Gal4-HIF-1␣-TAD-N and whether this effect would be lost when cells were cotransfected with a mutant of GSK-3␤, such as GSK-3␤ with serine 9 replaced with alanine (GSK-3␤ S9A), which cannot be phosphorylated by PKB/Akt. We found that myrPKB/Akt increased Gal4-HIF-1␣-TAD-N activity and protein levels. However, this effect was antagonized when the PKB/Akt-neutral mutant GSK-3␤ S9A was used (Fig. 2C and D). These data suggest that the PKB/Akt pathway may involve GSK-3 to regulate HIF-1␣ at the TAD-N. Mutation of the GSK-3 phosphorylation sites within HIF-1␣ enhances HIF-1␣ protein levels. In order to identify the putative GSK-3 phosphorylation sites within HIF-1␣, we searched for the presence of a GSK-3 consensus site. We found that the

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regions around S551, T555, and S589 would match the consensus site, and in line with the above-mentioned findings, these residues are located within the TAD-N. To test their functional role, we again performed cotransfection assays with appropriate constructs for wild-type Gal4-HIF-1␣ fusion proteins or fusion proteins in which the respective GSK-3 sites had been mutated, along with the GSK-3␤ expression vector. The wild-type HIF-1␣-TAD-N construct was down-regulated by GSK-3␤ by about 70%. The conversion of serine 551 into an alanine enhanced Luc activity and protein levels by about 150%. The overexpression of GSK-3␤ did not affect Luc activity and protein levels. Further, the replacement of threonine 555 with valine displayed the same effect as the S551A mutation. When the third GSK-3 site at Ser-589 was mutated, Luc activity and protein levels were increased about 250% compared to the levels in the control. As in the case of the other mutants, the overexpression of GSK-3␤ did not reduce this enhanced Luc activity. Further, when a construct in which all GSK-3 sites were mutated was used, the enhancement of Luc activity and protein levels was about 300% and the overexpression of GSK-3␤ did not mediate a reduction of Luc activity and fusion protein levels (Fig. 3A and B). Thus, GSK-3 appears to contribute to the stabilization of the HIF-1␣ protein. To investigate this idea in more detail, we generated expression vectors for full-length Myc- or V5-tagged HIF-1␣ in which the respective GSK-3 sites were mutated either alone or in combination. When cells were cotransfected with wild-type Myc-tagged HIF-1␣ and a GSK-3␤ expression vector, the HIF-1␣ protein levels were reduced by about 70%. These data are in line with our analyses from the Gal4-luciferase assay. When GSK-3␤ was overexpressed along with HIF-1␣ variants containing either the S551A, T555V, S551A/T555V, or S551A/ T555V/S589A mutations, the HIF-1␣ protein levels were enhanced and GSK-3␤ no longer down-regulated HIF-1␣ (Fig. 3C and D). Thus, these data suggest that GSK-3 regulates HIF-1␣ protein stability. The regulation of HIF-1␣ by GSK-3 is independent of the hydroxylation sites. In order to find out whether the GSK-3mediated regulation of HIF-1␣ is linked to the pVHL-dependent protein destabilization, we coexpressed GSK-3␤ with the Gal4-HIF-1␣ constructs. As shown in Fig. 4, GSK-3␤ decreased Luc activity by about 70% when the Gal4-HIF-1␣TAD-N construct was used. When a Gal4-HIF-1␣-TAD-N construct in which the proline 564 was converted into alanine to prevent hydroxylation was used, Luc activity increased about 300% compared to that of the control. However, cotransfection with the GSK-3␤ expression vector and the P564A mutant

means ⫾ SEM of results from three independent experiments. Numbers below the upper left panel represent amino acid positions. Statistics were calculated by using the Student t test for paired values. *, significant difference between results for the control and those for GSK-3␤-transfected cells (P ⱕ 0.05); bHLH, basic helix-loop-helix; ⫺, control. (B) Representative Western blot. One hundred micrograms of total protein from the transfected HepG2 cells was analyzed by Western blotting with antibodies against Flag M2 and the Golgi membrane (GM). (C) HepG2 cells were cotransfected with expression vectors for GSK-3 S9A and Gal4-HIF-1␣-TAD-N and the p5GE1B-Luc gene construct and cultured as described for panel A. In each experiment, the percentage of Luc activity relative to that in the Gal4-HIF-1␣-TAD-N control, which was set at 100%, was determined. Values represent means ⫾ SEM of results from three independent experiments. *, significant difference between results for the control and those for myrPKB and/or GSK-3 S9A (P ⱕ 0.05). (D) Representative Western blot analyzed as described for panel B.

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FIG. 4. The destabilization of HIF-1␣ by GSK-3 is independent of the hydroxylation site proline 564. (A) HepG2 cells were cotransfected with expression vectors for GSK-3␤, wild-type Gal4-HIF-1␣-TAD-N (TADN), or the Gal4-HIF-1␣-TAD-N P564A (P564A), Gal4-HIF-1␣-TAD-N P564A/S551A (P/S), or Gal4-HIF-1␣-TAD-N P564A/T555V/S589A (P/T/S/) mutant and the p5GE1B-Luc gene construct. After transfection, cells were cultured with fresh culture medium for the next 48 h under normoxia (16% O2). In each experiment, the percentage of Luc activity relative to that in the wild-type Gal4-HIF-1␣-TAD-N control, which was set at 100%, was determined. The values represent means ⫾ SEM of results from three independent experiments. Numbers below the upper left panel represent amino acid positions. Statistics were calculated by using the Student t test for paired values. *, significant difference between results for the respective control and those for GSK-3␤-transfected cells; **, significant difference between results for the TAD-N wild type and those for TAD-N mutants; P ⱕ 0.05. bHLH, basic helix-loop-helix; ⫺, control. (B) Representative Western blot. One hundred micrograms of total protein from the transfected HepG2 cells was analyzed by Western blotting with antibodies against Flag M2 and the HA tag.

reduced Luc activity by about 50%, suggesting that the GSK-3␤ effect is independent of P564 (Fig. 4). Interestingly, when a Gal4-HIF-1␣-TAD-N double mutant containing the S551A mutation, which prevents GSK-3 phosphorylation, together with P564A (Gal4-HIF-1␣ P564A/S551A)

was used, GSK-3␤ no longer down-regulated Luc activity or protein levels enhanced by the P564A/S551A double mutant (Fig. 4). Further, the GSK-3␤ effect was not seen when a Gal4-HIF-1␣-TAD-N variant containing mutations of the GSK-3 phosphorylation sites T555 and S589, together with

FIG. 3. Mutation of the GSK-3 target sites abolishes the destabilization of HIF-1␣. (A) HepG2 cells were cotransfected with expression vectors for GSK-3␤, wild-type Gal4-HIF-1␣-TAD-N (TADN), or the Gal4-HIF-1␣-TAD-N S551A, Gal4-HIF-1␣-TAD-N T555V, Gal4-HIF-1␣-TAD-N S589A, or Gal4-HIF-1␣-TAD-N S551S/T555V/S589A (S/T/S) mutant and the p5GE1B-Luc gene construct. After transfection, cells were cultured with fresh culture medium for the next 48 h under normoxia (16% O2). In each experiment, the percentage of Luc activity relative to that in the wild-type Gal4-HIF-1␣-TAD-N control, which was set at 100%, was determined. The values represent means ⫾ SEM of results from three independent experiments. Statistics were calculated by using the Student t test for paired values. *, significant difference between results for the control and those for GSK-3␤-transfected cells; **, significant difference between results for wild-type Gal4-HIF-1␣-TAD-N and those for Gal4-HIF-1␣-TAD-N mutants; P ⱕ 0.05. bHLH, basic helix-loop-helix. (B) Representative Western blot. One hundred micrograms of total protein from the transfected HepG2 cells was analyzed by Western blotting with antibodies against Flag M2 and GSK-3␣/␤. (C) HepG2 cells were cotransfected with expression vectors for GSK-3␤, Myc- or V5-tagged full-length wild-type human HIF-1␣ (WT), or the human HIF-1␣ S551A, human HIF-1␣ T555V, human HIF-1␣ S589, human HIF-1␣ S551A/T555V (S/T), or human HIF-1␣ S551A/T555V/S589A (S/T/S) mutant. After transfection, cells were cultured with fresh culture medium for the next 24 h. The HIF-1␣ protein levels were measured by Western blotting, and the wild-type HIF-1␣ levels in the controls were set at 100%. Values are means ⫾ SEM of results from at least three independent experiments. Statistics were calculated by using the Student t test for paired values. *, significant difference between results for the wild type in control and GSK-3␤-transfected cells; **, significant difference between results for the mutants and those for the wild type in GSK-3␤-transfected cells; ***, significant difference between results for the wild type and those for the mutants; P ⱕ 0.05. ⫺, control. (D) Representative Western blot. One hundred micrograms of total protein from the transfected HepG2 cells was analyzed by Western blotting with antibodies against the Myc, HA, and V5 tags and the Golgi membrane (GM).

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FIG. 5. GSK-3-mediated destabilization of HIF-1␣ is independent of the hydroxylation sites within HIF-1␣ and of pVHL. (A) HepG2 cells were cotransfected with expression vectors for GSK-3␤, full-length V5-tagged wild-type human HIF-1␣ (WT), or the human HIF-1␣ P402A/ P564A/N803A (P/P/N), human HIF-1␣ P402A/P564A/N803A/S551A/T555V (P/P/N/S/T), or human HIF-1␣ P402A/P564A/N803A/S551A/T555V/ S589A (P/P/N/S/T/S) mutant. After transfection, the cells were cultured with fresh culture medium for the next 24 h. The HIF-1␣ protein levels were measured by Western blotting, and the wild-type HIF-1␣ levels in the controls were set at 100%. Values are means ⫾ SEM of results from at least three independent experiments. Statistics were calculated by using the Student t test for paired values. *, significant difference between results for the control and those for GSK-3␤-transfected cells; **, significant difference between results for the mutants and those for the wild type

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P564A, was used. Again, these phenomena indicated that the pVHL binding sites and GSK-3 phosphorylation sites contribute not only to protein stability but also to the transactivation of HIF-1␣. Next, we aimed to investigate the importance of the GSK-3 sites alone and in combination with the hydroxylation sites on the level of the full-length HIF-1␣ protein. To do this, we used expression vectors for Myc- or V5-tagged HIF-1␣ with mutations in the hydroxylation and GSK-3 sites. The use of a hydroxylation-resistant HIF-1␣ variant with the mutations P402A, P564A, and N803A (PPN mutant) did not prevent the down-regulation of the protein by GSK-3␤, although HIF-1␣ levels were threefold higher under control conditions (Fig. 5A and B). However, the additional introduction of S551A and T555V mutations into the PPN mutant slightly enhanced HIF-1␣ protein levels under control conditions and abolished downregulation by GSK-3␤. In addition, the mutation of the third GSK-3 site in this mutant further enhanced HIF-1␣ levels by about sixfold under control conditions. Again, the overexpression of GSK-3␤ did not show any effect on this mutant (Fig. 5A and B). In order to gain further support that GSK-3 affects HIF-1␣ protein stability, we determined the half-lives of HIF-1␣ and the respective GSK-3 site mutation variants in response to GSK-3␤. To do this, we cotransfected HepG2 cells with GSK-3␤ expression vectors and vectors expressing full-length wild-type V5-tagged HIF-1␣ or the respective variants in which either the hydroxylation sites alone or both the hydroxylation sites and the GSK-3 sites were mutated. After the blocking of protein synthesis with cycloheximide, the HIF-1␣ protein levels were determined by Western analysis with the antibody against the V5 tag. Our data show that GSK-3␤ reduced the half-life of HIF-1␣ even when the hydroxylation sites at P402, P564, and N803 had been mutated. By contrast, when GSK-3␤ was overexpressed along with the HIF-1␣ variant additionally containing the S551A, T555V, and S589A mutations (the PPNSTS mutant), the HIF-1␣ protein half-life was no longer decreased by GSK-3␤ (Fig. 5C). These findings suggested that GSK-3 may down-regulate HIF-1␣ in a pVHL-independent manner. To further underline this idea, we transfected pVHL-defective RCC4 cells and RCC4 cells in which VHL was reintroduced with the GSK-3␤ expression vector. The RCC4 cells display a constitutive level of HIF-1␣ since it is not degraded via the pVHL-mediated

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pathway. The transfection of RCC4 cells with GSK-3␤ revealed a reduction in HIF-1␣ protein levels of about 50% (Fig. 5D and E). A reduction in HIF-1␣ protein levels upon the expression of GSK-3␤ in the RCC4/VHL cells was also seen. Together, these data show that GSK-3 can down-regulate HIF-1␣ in a pVHL-independent manner. The downregulation of HIF-1␣ by GSK-3␤ involves ubiquitylation and the proteasome. Since our data suggested that GSK-3 destabilizes HIF-1␣ in a VHL-independent manner, we were interested to test whether this destabilization involves ubiquitylation and the proteasome. To find out whether GSK-3␤ may promote the ubiquitylation of HIF-1␣, we performed coimmunoprecipitation analyses with proteins from HepG2 cells transfected with vectors for V5-tagged wild-type HIF-1␣, the PPN mutant, or the HIF-1␣ PPNSTS mutant together with HA-GSK-3␤ expression vectors. After the immunoprecipitation of wild-type HIF-1␣ proteins with the V5 tag antibody, these proteins were found to be strongly ubiquitylated, and this result was further enhanced in the presence of GSK-3␤. Although ubiquitylation was greatly reduced when the PPN mutant was used, GSK-3␤ still promoted ubiquitylation. By contrast, ubiquitylation was nearly abolished and was no longer promoted by GSK-3␤ when the HIF-1␣ PPNSTS mutant was used. In addition, the detection of precipitates with the HA antibody revealed that wild-type HIF-1␣ and the HIF-1␣ PPN mutant interacted with GSK-3␤ whereas this interaction was greatly reduced with the HIF-1␣ PPNSTS mutant. In line with this finding, precipitation with the HA antibody also showed the interaction of GSK-3␤ with HIF-1␣ and HIF-1␣ PPN but almost no interaction with the HIF-1␣ PPNSTS mutant (Fig. 5F). Thus, these data further indicated that GSK-3␤ promotes the ubiquitylation and proteasomal degradation of HIF-1␣ in a VHL-independent manner. To investigate the involvement of the proteasome, we transfected HepG2 cells with the V5-tagged HIF-1␣ wild-type, the HIF-1␣ PPN mutant, or the HIF-1␣ PPNSTS mutant together with the GSK-3␤ expression vector and treated these cells with the proteasome inhibitor MG132 under normoxia and hypoxia. We found that hypoxia enhanced the levels of HIF-1␣ and that MG132 mimicked hypoxia even under normoxia. The overexpression of GSK-3␤ decreased HIF-1␣ levels by about 70% under hypoxia. This decrease was completely abolished by the addition of MG132 (Fig. 6). After transfection with the PPN mutant, which was stable

in GSK-3␤-transfected cells; ***, significant difference between results for the mutants and those for the wild type; P ⱕ 0.05. bHLH, basic helix-loop-helix. (B and E) Representative Western blots. One hundred micrograms of total protein from the cells was analyzed by Western blotting with antibodies against HIF-1␣, pVHL, actin, and the V5 and HA tags. ⫹, present; ⫺, absent. (C) Determination of HIF-1␣ protein half-life. HepG2 cells were cotransfected with vectors for HA epitope-tagged GSK-3␤ or either V5-tagged full-length wild-type HIF-1␣ or the respective HIF-1␣ mutant human HIF-1␣ P402A/P564A/N803A or human HIF-1␣ P402A/P564A/N803A/S551A/T555V/S589A. After the inhibition of protein synthesis with cycloheximide (CHX; 100 ␮g/ml), the HIF-1␣ protein levels were determined by Western analysis with an antibody against the V5 tag. (D) The VHL-defective RCC4 cells (⫺) and the RCC4 cells with reintroduced VHL (⫹) were transfected with GSK-3␤ expression vectors or an empty control vector. After transfection, the cells were cultured with fresh culture medium for the next 24 h. The HIF-1␣ levels were measured by Western blotting, and the wild-type HIF-1␣ levels in the RCC4 cells were set at 100%. Values are means ⫾ SEM of results from at least three independent experiments. Statistics were calculated by using the Student t test for paired values. *, significant difference between results for the control and those for GSK-3␤-transfected cells; **, significant difference between results for RCC4/VHL cells and those for RCC4 cells; ***, significant difference between results for RCC4/VHL cells and RCC4 cells transfected with GSK-3␤; P ⱕ 0.05. (F) Immunoblot (IB) analysis of anti-V5 and anti-HA immunoprecipitates (IP) and whole-cell extracts (WCE) of HepG2 cells treated with the proteasomal inhibitor MG132 after transfection with the indicated plasmids. GM, Golgi membrane.

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FIG. 6. GSK-3 mediates the destabilization of HIF-1␣ in a VHL-independent manner via the proteasome. HepG2 cells were cotransfected with expression vectors for GSK-3␤, full-length V5-tagged wild-type human HIF-1␣, or the human HIF-1␣ P402A/P564A/N803A or human HIF-1␣ P402A/P564A/N803A/S551A/T555V/S589A mutant. After transfection, cells were cultured for the next 24 h under normoxia (16% O2) and then they were treated with MG132 and further cultured for 4 h under normoxia or hypoxia (8% O2). (A) The HIF-1␣ protein levels were measured by Western blotting, and the levels of wild-type human HIF-1␣ and the human HIF-1␣ P402A/P564A/N803A and human HIF-1␣ P402A/P564A/ N803A/S551A/T555V/S589A mutants under hypoxia were set at 100%. Values are means ⫾ SEM of results from at least three independent experiments. Statistics were calculated by using the Student t test for paired values. *, significant difference between results with 16% O2 and those with 8% O2; **, significant difference between results for the respective control and those for GSK-3␤-transfected cells; ***, significant difference between results for the respective control and those for MG132-treated cells; P ⱕ 0.05. bHLH, basic helix-loop-helix. (B) Representative Western blot. One hundred micrograms of total protein from the transfected HepG2 cells was analyzed by Western blotting with antibodies against human HIF-1␣, the V5 tag, the HA tag, and the Golgi membrane (GM). WT, wild type; P/P/N, PPN mutant; P/P/N ⫹ S/T/S, PPNSTS mutant. Numbers represent oxygen concentrations.

under normoxic and hypoxic conditions, MG132 further enhanced HIF-1␣ levels by about 200%. The overexpression of GSK-3␤ reduced HIF-1␣ PPN levels by about 70 to 80%. Again, the addition of MG132 reversed this effect so that the PPN levels were in the range of those of the controls (Fig. 6). When we transfected cells with the HIF-1␣ PPNSTS construct, which contains mutations in the hydroxylation sites and the GSK-3 phosphorylation sites, neither hypoxia nor MG132 affected the levels of this mutant protein. The overexpression of GSK-3␤ along with this mutant had also no effect, again confirming the role of these phosphorylation sites in protein stabilization (Fig. 6). Together, these data show that GSK-3␤ induces the destabilization of HIF-1␣ via the proteasome in a VHL-independent manner. All three sites contribute to the phosphorylation of the HIF1␣-TAD-N by GSK-3␤. Our data indicate that the HIF-1␣TAD-N is a direct target of GSK-3, and we investigated this possibility by performing phosphorylation assays. When the

wild-type GST-HIF-1␣-TAD-N protein was used as a substrate, it was found that GSK-3␤ phosphorylated the TAD-N whereas the GST protein alone was not phosphorylated (Fig. 7). Next, we used the GST-HIF-1␣-TAD-N proteins in which each single GSK-3 site was mutated. Interestingly, we found that GSK-3␤ was still able to phosphorylate all these mutants. However, when all three sites were mutated together, GSK-3␤ no longer phosphorylated the HIF-1␣-TAD-N (Fig. 7). These data show that the TAD-N of HIF-1␣ is a direct target of GSK-3, but it appears that all three sites act in a cooperative manner. DISCUSSION The present study has shown that GSK-3 negatively regulates HIF-1␣ by phosphorylating the TAD-N and promoting proteasomal degradation independent of prolyl hydroxylation and VHL binding. Our findings link recent reports showing that hypoxia and

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FIG. 7. HIF-1␣ is phosphorylated by GSK-3. (A) GST-HIF-1␣TAD-N wild-type (WT) or mutant (listed by mutations) fusion proteins were prepared from E. coli, and 20 ␮g of these fusion proteins was incubated with 50 mU of active GSK-3␤ and 10 ␮Ci of [␥-32P]ATP for 30 min at 30°C. (B) Afterwards, the phosphorylated proteins were separated from unbound radioactivity by electrophoresis on a 10% SDS gel. Radioactive proteins were visualized by phosphorimaging.

nonhypoxic stimuli such as insulin-like growth factors 1 and 2 (8, 16, 19, 70), insulin (36, 65), thrombin (20), nitric oxide (51), and heregulin (40) can stimulate HIF-1␣ via the phosphatidylinositol 3-kinase/PKB pathway. Although PKB/Akt appears to induce HIF-1␣ stabilization (24, 44, 71, 72), translation (60, 63), and coactivator recruitment (32), HIF-1␣ is not a direct target of PKB/Akt. Instead, the PKB/Akt-dependent HIF-1␣ activation appears to involve the PKB/Akt target HDM2 (3, 58), mTOR (65), or GSK-3␤ (46). The latter two have been implicated in both the activation of HIF-1␣ translational mechanisms and the enhancement of HIF-1␣ protein stability. GSK-3 appears to have a more important role since it was found to be activated by hypoxia (10, 46) and growth factors (57). Although our findings do not rule out the involvement of GSK-3 in the regulation of HIF-1␣ translation, they are in line with previous findings showing that GSK-3␤ phosphorylates the ODD of HIF-1␣ (59). However, the previous study did not define the exact location of the proposed GSK-3 phosphorylation sites. In continuation of our previous findings, we show here that the HIF-1␣ ODD contains three sites which are

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subject to phosphorylation by GSK-3, including S551, T555, and S589. The GSK-3 phosphorylation site is in general defined as S/TXXXS/T, where X represents any amino acid residue, and serine or threonine residues situated N terminally next to a proline residue tend to be the residues phosphorylated. A neighboring proline residue was found only with S589 within HIF-1␣, thus indicating that this site may have a stronger effect on HIF-1␣ stability. This was indeed the case, since the conversion of S589 into alanine led to about 2.5-fold-higher Luc activities and Gal4-HIF-TAD-N levels than the S551A or T555V mutation. It has been shown that the affinity of GSK-3 for its substrate is enhanced if the substrate is already primed at a ⫹4 serine or threonine residue. This priming can be done by other kinases, such as casein kinases 1a and 2, protein kinase A, dual-specificity tyrosine phosphorylation-regulated protein kinase (69), and GSK-3 itself (18). However, priming is not necessarily a prerequisite for GSK-3-catalyzed phosphorylation since the axin-adenomatous polyposis complex does not require priming for phosphorylation (48). The presence of a GSK-3 phosphorylation site at S551 may imply that T555 within HIF-1␣, which may also be a GSK-3 site, may be a target site for a priming kinase. In this case, the mutation of this site should abolish the phosphorylation of HIF-1␣ by GSK-3. However, this appeared not to be the case. Instead, only when all three GSK-3 sites were mutated was the phosphorylation of HIF-1␣ completely abolished, indicating cooperation between all three sites. This result is also underlined by the finding that Luc activity was highest when TAD-N constructs with all GSK-3 site mutations present at the same time were used. The importance of GSK-3 for the regulation of HIF-1␣ was further demonstrated by the use of insulin and LiCl, which phosphorylate GSK-3 and thus inhibit the activation of this kinase. Both reagents induced an accumulation of HIF-1␣ under normoxia, whereas the effect under hypoxia was less prominent but still significant. In line with this observation, the specific depletion of GSK-3␤ by siRNA dramatically increased HIF-1␣ protein levels. Importantly, the GSK-3-dependent modulation of HIF-1␣ levels also had functional consequences since the siRNA-mediated depletion of GSK-3 induced the expression of the HIF-1␣ target PAI-1 gene. By contrast, the overexpression of GSK-3 down-regulated PAI-1 promoter activity, and this effect could be completely eliminated by the mutation of the HRE within the promoter, thus again indicating the specificity of the effects observed. The present study shows that GSK-3 exerts its effects within the N-terminal transactivation domain. These effects first indicated that the GSK-3-mediated down-regulation of HIF-1␣ might act in conjunction with the prolyl residues necessary for VHL binding. However, our data clearly show that this is not the case. The mutation of the critical proline residues in the Gal4-TAD-N or the full-length HIF-1␣ construct failed to abolish down-regulation by GSK-3. By contrast, the enhanced stability of HIF-1␣ obtained after the mutation of P402 and P564 was further increased when the GSK-3 site mutations were introduced either alone or in combination. The GSK-3-mediated degradation of HIF-1␣ implies a scenario similar to that of ␤-catenin, which upon phosphorylation by GSK-3 is ubiquitylated and degraded in the proteasome. The results of this study showing that GSK-3␤ interacts with

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HIF-1␣ and promotes its ubiquitylation, together with the finding that the use of the proteasome inhibitor MG132 counteracts the GSK-3-mediated degradation of HIF-1␣, support this notion. In line with these observations, the degradation induced by the overexpression of ubiquitin together with GSK-3 could also be inhibited by MG132. Together, our data support a model in which the activation of Akt/PKB by either hypoxia or various stimuli, including insulin, may lead to an inhibition of GSK-3 activity, which then cannot phosphorylate HIF-1␣ and target it for proteasomal degradation. Thus, the HIF-1␣ system displays enormous plasticity since the degradation of HIF-1␣ can be induced by hydroxylation and phosphorylation events either alone or in combination. Our findings suggest that GSK-3 is a novel master regulator of HIF-1␣ stability independent from the established hydroxylase-VHL pathway. This system may constitute a novel reserve mechanism of a cell for adapting its function in response to various physiological and nonphysiological signals. Although initially recognized for its function in the inhibition of glycogen synthesis through the phosphorylation of glycogen synthase (11), GSK-3 regulates a wide range of cellular functions, including metabolism, gene expression, cell attachment, and cell integrity functions (2, 21). GSK-3 is also involved in a number of disease processes, like cardiac hypertrophy (23) and Alzheimer’s disease (27, 45). Since GSK-3 is a central molecule not only for the Akt/PKB pathway but also for the Wnt pathway, which are both frequently activated in cancer cells, GSK-3 may be important for the regulation of HIF in cancer. Together, our findings that GSK-3 phosphorylates HIF-1␣ and initiates its degradation in a pVHL-independent manner provide evidence that GSK-3 operates like a tumor suppressor to inhibit cellular transformation.

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10.

11. 12. 13.

14.

15. 16. 17.

18. 19.

20.

21. 22.

ACKNOWLEDGMENT This study was supported by a grant from the Fonds der Chemischen Industrie to T.K. and by grants from the sixth European framework program (EUROXY) and Fondation Leducq to A.G. C.M. is senior research associate of FNRS (National Funds for Scientific Research, Brussels). There are no conflicts of interest with this study.

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