Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

HIF1α Protein Stability Is Increased by Acetylation at Lysine 709 *

HIF1α Protein Stability Is Increased by Acetylation at Lysine 709 * THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 42, pp. 35496 –35505, October 12, 2012 © 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. HIF1 Protein Stability Is Increased by Acetylation at □ S Lysine 709 Received for publication, July 12, 2012, and in revised form, August 14, 2012 Published, JBC Papers in Press, August 20, 2012, DOI 10.1074/jbc.M112.400697 Hao Geng, Qiong Liu, Changhui Xue, Larry L. David, Tomasz M. Beer, George V. Thomas, Mu-Shui Dai, and David Z. Qian From the Oregon Health & Science University Knight Cancer Institute, Oregon Health & Science University, Portland, Oregon 97239 Background: HIF1 and p300 are key components of HIF-1 transcription complex. Results: Lysine 709 of HIF1 is acetylated by p300, which increases protein stability and HIF-1 activity. Conclusion: p300 has a novel function in stabilizing HIF1 by Lys-709 acetylation. Significance: New insights in how HIF1 is post-translationally regulated by its cofactor to ensure HIF-1 activity. Lysine acetylation regulates protein stability and function. gen-independent HIF1 to form the core of HIF-1 transcrip- p300 is a component of the HIF-1 transcriptional complex and tional complex (2, 3). The HIF-1 target genes regulate a vast positively regulates the transactivation of HIF-1. Here, we show variety of biological activities that are critical for cell viability, a novel molecular mechanism by which p300 facilitates HIF-1 growth, and differentiation (1). Therefore, the inappropriate activity. p300 increases HIF-1 (HIF1) protein acetylation and accumulation of HIF1 proteins in cancer cells can lead to stability. The regulation can be opposed by HDAC1, but not by inappropriate HIF-1 activities, which underpins malignant HDAC3, and is abrogated by disrupting HIF1-p300 interac- phenotypes including angiogenesis, metabolic reprogramming, tion. Mechanistically, p300 specifically acetylates HIF1 at Lys- metastasis, and therapy resistance (4, 5). 709, which increases the protein stability and decreases polyu- HIF1 protein stability can also be regulated by additional biquitination in both normoxia and hypoxia. Compared with mechanisms that are independent and cross-talk with the oxy- the wild-type protein, a HIF1 K709A mutant protein is more gen/VHL-mediated HIF1 protein degradation pathway. stable, less polyubiquitinated, and less dependent on p300. HSP90 is a key cellular chaperone and is positively associated Overexpression of the HIF1 wild-type or K709A mutant in with HIF1 proteins in both normoxia and hypoxia (6). In nor- cancer cells lacking the endogenous HIF1 shows that the moxia, the HIF1 chaperone function of HSP90 is countered by K709A mutant is transcriptionally more active toward the RACK1 (7). Although significantly stabilized in hypoxia due to HIF-1 reporter and some endogenous target genes. Cancer cells the lack of oxygen/VHL pathway, the HIF1 protein can still be containing the K709A mutant are less sensitive to hypoxia-in- reduced by HSP70-CHIP via polyubiquitination and degrada- duced growth arrest than the cells containing the HIF1 wild- tion (8). In addition, the HIF1 protein stability and HIF-1 tran- type. Taken together, these data demonstrate a novel biological scriptional activity are associated with the protein lysine acety- consequence upon HIF1-p300 interaction, in which HIF1 lation-deacetylation system (9). Multiple members within the can be stabilized by p300 via Lys-709 acetylation. lysine acetylase and deacetylase families have been reported to interact with HIF1 (10–14). The biological consequence is a protein sta- change of HIF-1 activity due to a change in HIF1 Hypoxia-inducible factor 1  (HIF1) is a critical compo- bility in many cases (9, 15, 16). Lysine acetylation changes pro- nent of the HIF-1 transcriptional complex that regulates cellu- tein stability and function (17). Multiple acetylation sites have lar response to hypoxia (1). In non-hypoxic conditions, HIF1 been discovered within the HIF1 protein (11, 14, 18). How- protein is constantly synthesized, but rapidly degraded by oxy- ever, the biological consequence due to these different acetyla- gen. The primary mechanism consists of the post translational tion sites can be very different. Currently, both the lysine acety- hydroxylation of HIF1 at proline residues 402 and 564 by oxy- lase and deacetylase are potential therapeutic targets against gen-dependent prolyl hydroxylases, which is followed by von cancer and other human diseases, in which HIF1 plays a path- Hippel-Lindau (VHL)-mediated polyubiquitination and 26 S ological role. It is important to delineate the functional conse- proteasome-dependent protein degradation (2). In hypoxic quence of HIF1 acetylation at specific lysine locations. conditions, oxygen is not available for the hydroxylation of p300 is a critical component in the HIF-1 transcriptional HIF1 protein, which is stabilized and dimerized with the oxy- complex (19). In the non-hypoxic condition, p300 and HIF1 are not physically associated due to the rapid HIF1 protein * This work was supported by U. S. Public Health Service Grant R01CA149253 degradation and the hydroxylation at asparagine 803 within the from the National Cancer Institute, National Institutes of Health. HIF1 (20). Recently, the acetylation at lysine 674 of HIF1 by □ S This article contains supplemental Figs. S1–S6. PCAF has also been reported to play a role in determining To whom correspondence should be addressed: 3303 SW Bond Ave., CH14R, OHSU, Portland, OR 97239. Tel.: 503-312-5912; Fax: 503-494-6197; E-mail: HIF1-p300 interaction (14). Upon hypoxia, the C-terminal of [email protected]. HIF1 interacts with the CH1 (cysteine/histidine-rich 1) The abbreviations used are: HIF1, hypoxia-inducible factor 1 ; VHL, von domain of p300, which leads to an increase of HIF-1 transcrip- Hippel-Lindau; Ev, empty vector; WCL, whole cell lysate; dm, double mutant; HDAC, histone deacetylase; PCAF, p300/CBP-associated factor. tional activity (21). A small molecule inhibitor, chetomin, has This is an Open Access article under the CC BY license. 35496 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 42 •OCTOBER 12, 2012 HIF1 Lys-709 Acetylation Increases Protein Stability been reported to disrupt the interaction between HIF1 and to validate protein identifications derived from MS/MS sequencing results. p300 and inhibit the HIF-1 target gene expression (22). p300 is In Vitro Acetylation Assay—HIF1 and p300, purified by a multiple domain protein with acetylase activity (23). Despite being closely associated with HIF1 in hypoxia, it is unclear immunoprecipitation, were mixed and incubated at 30 °C in whether p300 has a direct effect on HIF1. In the current study, buffer (25 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, 5 mM DTT, and 0.7 mM acetyl-CoA). Reactions were stopped at indicated time we identified a novel function of p300 in stabilizing HIF1 pro- by adding 6 SDS loading buffer and analyzed by Western teins by lysine acetylation at Lys-709 within HIF1. blots. Cycloheximide HIF1 Half-life—HIF1 and GFP proteins EXPERIMENTAL PROCEDURES were expressed in Hek293T cells. Following exposure to nor- Cell Culture—Human embryonic kidney 293T (Hek293T), mal or hypoxic condition for 5 h, cells were treated with cyclo- 293 (Hek293), osteosarcoma U2OS, Hep3Bc1, and renal cell heximide and harvested at different time points. Western blots carcinoma RCC10 cells were cultured in DMEM with 10% FBS were used to detect HIF1 and GFP proteins, which were quan- and 1% penicillin/streptomycin. Cells were maintained in a tified using LI-COR odyssey Infrared software (version 2.0) as humidified incubator at 37 °C with 5% CO . Hypoxia was described previously (18). induced by exposing cells to 1% oxygen, 5% CO , and 94% nitro- Real-time PCR and Cell Proliferation—The C method gen at 37 °C. was used to calculate mRNA fold change. The trypan blue Vectors—The FLAG-HIF1 wild-type, FLAG-HIF1 proline exclusion method was used to calculate viable cells in the pro- mutant, p2.1 HRE-firefly, pSV40-Renilla were gifts from Dr. liferation experiments as described (18, 24). Gregg Semenza (The Johns Hopkins University). HA-HIF1, Statistical Analysis—Differences between the means of HDAC1, HDAC3, PCAF, and p300 wild-type/catalytic mutant unpaired samples were evaluated by the Student’s t test. vectors were purchased from Addgene. The siRNA against p300 and shRNA against endogenous HIF1 were purchased RESULTS from Sigma. p300 Up-regulates the HIF1 Protein Level—Although p300 Plasmid Construction—Alanine substitutions in HIF1 pro- is known to increase HIF-1 transcriptional activity, its effect on tein were generated by two-step PCR amplification using HIF1 protein is less clear. In the cancer cell line U2OS, we used FLAG-HIF1 wild-type plasmid as a template. The PCR prod- siRNA to knockdown p300 and cultured the cells in either nor- ucts carrying the mutant HIF1 fragments were digested with mal ambient oxygen (21% O ) or hypoxic (1% O ) conditions 2 2 restriction enzymes and inserted into the FLAG empty vector for 6 h to induce HIF1 protein accumulation. We found that used for carrying the wild-type FLAG-HIF1. Each mutation p300 siRNA knockdown (si-p300) significantly decreased the was verified by sequencing. HIF1 in hypoxia (Fig. 1A, U2OS, and supplemental Fig. S1). Immunoprecipitation (IP) and Immunoblot—Cells were This decrease was on the protein level because the mRNA of lysed in prelysis buffer (50 mM Tris-HCl, pH 8.0, 0.1% Nonidet HIF1 was not changed by p300 knockdown (supplemental Fig. P-40, and 150 mM NaCl) in the presence of 1 protease inhib- S1). We also tested the effect of p300 siRNA on HIF1 proteins itor mixture (Thermo), followed by 2 sonication at 15% in a renal cell carcinoma cell line RCC10 (25), in which a loss of amplitude for 10 s. For IP assays, anti-FLAG M2 affinity gel function in VHL renders HIF1 protein stable in normal oxy- (Sigma) was incubated with cell lysates at 4 °C. The precipitates gen condition (Fig. 1A, RCC10). The siRNA knockdown of were extensively washed by prelysis buffer and subjected to p300 significantly decreased HIF1 proteins in both normal Western blotting. The antibodies used in immunoblots and co- and hypoxic conditions (Fig. 1A, RCC10). Next, we measured immunoprecipitations were as follows: HIF1, p300, PCAF, the effect of p300 overexpression on HIF1 protein levels. In ubiquitin, GFP, HDAC3 (Santa Cruz Biotechnology), HDAC1 VHL wild-type U2OS cells, p300 overexpression increased (Millipore), -tubulin and FLAG (Sigma), and acetyl lysine HIF1 proteins in the hypoxic condition (Fig. 1B, U2OS). In (Immuchem). VHL-negative RCC10 cells, the HIF1 protein was significantly Mass Spectrometry—Mass spectrometry was performed by increased by p300 transfection in both normal and hypoxic Proteomics Shared Resource at Oregon Health & Science Uni- conditions (Fig. 1B, RCC10). The effect of p300 on HIF1 versity. Briefly, HIF1 proteins were recovered from SDS- appears to be specific because HIF2 and HIF1 protein levels PAGE gel, reduced with DTT, and digested by trypsin. Each were not changed by p300 (supplemental Fig. S2). Next, we protein digest was analyzed by LC-MS/MS using an Agilent co-overexpressed a FLAG-tagged HIF1 wild-type plasmid 1100 series capillary LC system (Agilent Technologies) and a with either empty vector (Ev) or p300 in Hek293T cells. To VELOS linear ion trap mass spectrometer (Thermo Fisher). ensure the regulation of FLAG-HIF1 by p300 is specific, we Electrospray ionization was performed with an ion max source also included a plasmid encoding the green florescence protein fitted with a 34-gauge metal needle (Thermo Fisher). MS/MS (GFP) that is regulated by the same promoter of FLAG-HIF1 spectra data were searched using SEQUEST against a data- as a control. In Ev cells, hypoxia increased the FLAG-HIF1 base containing human protein sequences from UniProtKB/ protein level, and the overexpressed HIF1 protein was present Swiss-Prot. In-house analysis software was used to calculate in cells cultured in normal oxygen condition (Fig. 1C); presum- discriminant scores and filter out incorrect peptide identifica- ably, this was because that the vector-based FLAG-HIF1 over- tions using sequence-reversed matches to estimate false discov- expression was very robust and overcame the endogenous oxy- ery rates. Scaffold (Proteome Software, Portland, OR) was used gen/VHL-mediated HIF1 degradation. Importantly, p300 OCTOBER 12, 2012• VOLUME 287 • NUMBER 42 JOURNAL OF BIOLOGICAL CHEMISTRY 35497 HIF1 Lys-709 Acetylation Increases Protein Stability reported role of p300, in Ev cells, hypoxia increased the inter- action between FLAG-HIF1 and the endogenous p300 (Fig. 2A). The overexpressed p300 can also interact with FLAG- HIF1 proteins in normoxia, and the interaction was further increased by hypoxia (Fig. 2A). Importantly, the level of FLAG- HIF1 lysine acetylation was increased by hypoxia in the Ev cells and was significantly increased by p300 overexpression in both normal and hypoxic conditions (Fig. 2A). Because both hypoxia and p300 overexpression increased the FLAG-HIF1 protein level (Figs. 1C and 2A), we wanted to know whether the increase of HIF1 acetylation was due to the increase of total HIF1 proteins or the increase of de novo HIF1 acetylation. We performed an in vitro HIF1 acetylation assay by mixing equal amount of IP-purified FLAG-HIF1 proteins with IP- purified p300 proteins in the acetylation assay buffer. After 30 min of incubation at 30 °C, the acetylation level of FLAG- HIF1 was measured by Western blot. As shown in Fig. 2B, the de novo HIF1 acetylation was significantly increased in vitro by incubating with the p300 immunocomplex compared with IgG complex, whereas the total FLAG-HIF1 input remained the same. Next, we repeated the HIF1 and p300 co-overex- pression experiment as in Fig. 2A using a plasmid coding for a FLAG-HIF1 proline double mutant (HIF1-dm) that is resist- ant to the oxygen/VHL-mediated degradation. In Ev cells, the HIF1-dm protein level in WCL was not significantly changed between normal and hypoxic conditions, and hypoxia increased the interaction between HIF1-dm and the endogenous p300 (Fig. 2C) and increased the HIF1-dm protein acetylation in the FIGURE 1. p300 regulates HIF1 proteins. A and B, Western blot analysis of IP samples (Fig. 2C). Co-overexpression of p300 slightly the p300 effect on endogenous HIF1 in U2OS and RCC10 cell lines. Cells increased the HIF1-dm total protein levels in the WCL, were transfected with siRNA control (siC) or si-p300 in A, and empty vector or vector encoding p300 in B. C, Western blot analysis of Hek293T cells co-trans- increased the interaction between HIF1 and p300, and signif- fected with FLAG-HIF1 and p300 or Ev. D, Western blot analysis of Hek293T icantly increased the HIF1-dm protein lysine acetylation (Fig. cells that were transfected with FLAG-HIF1 and treated by 50 nM p300 inhib- itor, chetomin (CTM), or solvent control (V)for6h.In A–D, cells were cultured 2C). These results indicated de novo acetylation of HIF1 by under normal or hypoxic conditions for 6 h before whole cell protein lysates p300. To test whether the HIF1-p300 interaction is required were harvested. In C and D, a vector encoding GFP was used for transfection for the increase of HIF1 protein acetylation, we transfected efficiency controls. The HIF1 protein level (R) was calculated based on the ratio of band intensity of HIF1/tubulin or GFP. All data were representatives FLAG-HIF1 into Hek293T cells and treated cells with chet- of at least three independent experiments. omin. In hypoxia, chetomin treatment significantly reduced the HIF1-p300 interaction, which coincided with the reduction of overexpression further increased the FLAG-HIF1 in both nor- HIF1 acetylation and total HIF1 protein level (Fig. 2D). mal and hypoxic conditions (Fig. 1C). As a control, the co-over- Because HIF1 can also be associated with lysine deacetylases expressed GFP was not changed by either hypoxia or p300 over- (26, 27), we further tested whether the p300-induced acetyla- expression. To further confirm the HIF1 regulation by p300, tion can be countered by deacetylation. We co-overexpressed we transfected the FLAG-HIF1 into Hek293T cells and FLAG-HIF1 with p300 or in combination with either HDAC1 treated cells with chetomin, a chemical inhibitor that disrupts or HDAC3. We found that HDAC1, but not HDAC3, signifi- the HIF1-p300 interaction (22). We found that chetomin cantly reduced the p300-induced HIF1 acetylation, which also treatment decreased the FLAG-HIF1 protein expression in coincided with a reduction of total FLAG-HIF1 protein level both normal and hypoxic conditions (Fig. 1D). These data sug- (Fig. 2E). Recently, HIF1 was reported to be acetylated by a gest that p300 can enhance the HIF1 stability in hypoxia or in p300-interacting protein, PCAF (14). We compared the ability conditions that the oxygen/VHL-mediated HIF1 degradation of p300 and PCAF in terms of HIF1 acetylation. When an is either inactive (RCC10 cell line) or overwhelmed (vector- HA-tagged HIF1 was co-overexpressed with either p300 or based HIF1 overexpression). FLAG-PCAF, we observed that the HIF1-PCAF interaction p300 Acetylates HIF1—One of the primary functions of was not changed by hypoxia, and the interaction was less robust p300 is protein lysine acetylation (23). To understand the molecular mechanism underlying the HIF1 up-regulation by than HIF1-p300 (Fig. 2F). Furthermore, p300 also induced a p300, we transfected the FLAG-HIF1 and p300 in Hek293T more robust HIF1 acetylation than FLAG-PCAF (Fig. 2F). cells, immunoprecipitated the FLAG-HIF1 protein from Taken together, these data suggest that HIF1-p300 interac- whole cell lysates (WCL), and measured the HIF1 lysine acety- tion has two novel biological consequences: the increases of lation and its interaction with p300. In agreement with the HIF1 lysine acetylation and protein stability, which are 35498 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 42 •OCTOBER 12, 2012 HIF1 Lys-709 Acetylation Increases Protein Stability FIGURE 2. p300 acetylates HIF1. A, Hek293T cells were co-transfected with FLAG-HIF1 and p300 or Ev. 48 h after transfection, cells were exposed to either normoxia (n) or hypoxia (h) for 6 h. WCLs were harvested and used for Western blot (IB) or IP with anti-FLAG antibodies and followed by Western blot analysis. Protein acetylation was detected by anti-ace-lysine (Ace-K) antibodies. A GFP vector was also co-transfected as control. B, FLAG-HIF1a and p300 were expressed in Hek293T cells separately and IP purified by anti-FLAG or anti-p300. The immunocomplex containing FLAG-HIF1 was mixed with p300 or IgG immunocom- plex for 0 or 30 min in the in vitro acetylation buffer. Afterward, the acetylated (Ace) HIF1, the total p300, and FLAG-HIF1 protein levels were measured by Western blot. C, similar experiments as described in A were done using a vector encoding for FLAG-HIF1 proline mutant (HIF1-dm) containing proline to alanine mutation at 402 and 564. D, FLAG-HIF1 was overexpressed in Hek293T cells. 24 h later, cells were treated with vehicle (V)or50nM of chetomin (CTM) and exposed to hypoxia for 6 h. Afterward, WCL was used for anti-FLAG IP and Western blots. E, FLAG-HIF1 was co-transfected with Ev and p300 with or without HDAC1 or HDAC3. 48 h after transfection, WCLs were harvested, and used for Western blot (IB) or IP with anti-FLAG antibodies and followed by Western blot analysis. F, a vector encoding for HA-HIF1 was co-overexpressed with Ev, p300, or PCAF in H293T cells. 48 h after transfection, cells were exposed to normal (n) or hypoxia (h) for 6 h, and the HA-HIF1 was IP-purified from WCL and analyzed by Western blot (IB). All data were representative of at least three independent experiments. dependent on HIF1-p300 interaction that can be achieved by that lysine 674 within the FLAG-HIF1 was acetylated in both hypoxia or p300 overexpression. Ev and p300-overexpressed samples (data not shown). Signifi- Lysine 709 of HIF1 Is the Acetylation Target of p300—To cantly, lysine 709 was only acetylated in p300-overexpressed identify the p300 acetylated lysine residue(s) within HIF1,we cells (Fig. 3A and supplemental Fig. S3). Lys-674 has recently co-transfected the full-length wild-type FLAG-HIF1 with been reported as an acetylation target for PCAF (14), and Lys- either p300 or Ev in Hek293T cells. Next, we immunoprecipi- 709 is novel. Based on the FLAG-HIF1 plasmid, we used site- tated and gel-purified FLAG-HIF1 proteins, digested them directed mutagenesis to convert lysine to alanine and generated with trypsin, and analyzed the samples by liquid chromatogra- FLAG-HIF1 mutants containing a single point mutation of phy-tandem mass spectrometry (LC-MS/MS). We observed either K674A or K709A. Then, we overexpressed FLAG- OCTOBER 12, 2012• VOLUME 287 • NUMBER 42 JOURNAL OF BIOLOGICAL CHEMISTRY 35499 HIF1 Lys-709 Acetylation Increases Protein Stability FIGURE 3. HIF1 is acetylated by p300 at lysine 709. A, mass spectrum of HIF1 acetylation (Ace) by p300 at lysine 709. FLAG-HIF1 and p300 were co-overexpressed in Hek293T cells. The FLAG-HIF1 was IP and gel-purified. The gel slice containing FLAG-HIF1 was digested by trypsin and subjected to LC-MS/MS analysis. Mass spectrum of b ions and y ions were shown in red and blue, respectively. Acetylation at the location was marked with an ampersand by additional mass of 42 Da. Image was prepared with Scaffold. B, FLAG-HIF1 WT, FLAG-HIF1 K674A, and FLAG-HIF1 K709A was co-transfected with Ev ()or p300 () in Hek293T cells. 48 h later, WCLs were harvested, IP purified with anti-FLAG antibodies, and analyzed by Western blots. C, FLAG-HIF1 WT and HIF1 K709A were co-transfected with Ev (), p300 WT, or p300 mutant that is defective in acetyltransferase activity. 48 h later, WCL was harvested, IP-purified by anti-FLAG antibodies, and analyzed by Western blots. Data in B and C were representatives of at least three independent experiments. mut, mutant. HIF1-WT, FLAG-HIF1-K674A, or FLAG-HIF1-K709A in  K709A mutant protein observed that the FLAG-HIF1 Hek293T cells with either Ev control or p300. Forty-eight hours expressed at a higher level than FLAG-HIF1-WT in both nor- after transfection, the FLAG-HIF1 wild-type and mutant pro- mal and hypoxic conditions by Western blots coupled with flo- teins were immunopurified from the whole cell lysates. West- rescence densitometry (Fig. 4A). Next, we used cycloheximide ern blots showed that all HIF1 proteins interact with p300 to inhibit protein synthesis and measured the HIF1 WT and similarly (Fig. 3B). In both FLAG-HIF1-WT and FLAG- K709A mutant protein stability in the absence (Ev) or presence HIF1-K674A mutant, the acetylation levels were robustly of p300 overexpression. In normal oxygen condition, the increased by p300 (Fig. 3B). In contrast, the acetylation level of K709A mutant protein was significantly more stable than FLAG-HIF1-K709A mutant was not significantly increased HIF1-WT protein in the Ev cells (#, p  0.01, Fig. 4B). p300 by p300 (Fig. 3B). To further confirm the specific acetylation at overexpression significantly increased the HIF1-WT protein Lys-709 by p300, we co-overexpressed HIF1-WT or Lys-709 stability (a, p  0.01, Fig. 4B) but had no effect on HIF1- mutant with either the wild-type p300 or a p300 mutant that is K709A mutant. A similar trend was observed in the hypoxic defective in the acetyltransferase activity (28). Similar to the condition, in which the HIF1-K709A mutant was more stable results in Fig. 3B, p300 wild-type was able to increase the acety- than the HIF1-WT in Ev cells (##, p  0.01, Fig. 4C), and p300 lation in HIF1-WT, but not in HIF1-K709A mutant proteins overexpression increased the FLAG-HIF1-WT protein stabil- (Fig. 3C). However, the p300 acetylase mutant was unable to ity (aa, p  0.01, Fig. 4C) and had no effect on the K709A increase the acetylation in HIF1-WT and K709A mutant (Fig. mutant (Fig. 4C). 3C). These data suggest that p300 acetylates HIF1 primarily at Lys-709 within the HIF1 has been reported to be a site for Lys-709. polyubiquitination (29). We hypothesize that p300 increases p300 Regulates HIF1 Protein Stability via Lys-709 HIF1-WT protein stability by acetylation at Lys-709, which Acetylation—To investigate the biological significance of Lys- prevents polyubiquitination and protein degradation. To inves- 709 acetylation, we overexpressed equal amounts of FLAG- tigate the relationship between polyubiquitination and acetyla- HIF1-WT or FLAG-HIF1-K709A mutant in Hek293T cells tion at Lys-709, we performed similar co-overexpression exper- with GFP plasmids as transfection controls. Then, we measured iments as in Fig. 4, B and C, and used proteasomal inhibitor the normal and hypoxic FLAG-HIF1 protein levels. We MG132 to prevent FLAG-HIF1 degradation. FLAG-HIF1 35500 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 42 •OCTOBER 12, 2012 HIF1 Lys-709 Acetylation Increases Protein Stability FIGURE 4. p300 regulates HIF1 protein stability via Lys-709. A, FLAG-HIF1 WT or K709A mutant was transfected into the Hek293T cells for 48 h. Then, cells were exposed to normal (N) or hypoxia (H) for 6 h, followed by Western blot analysis. A vector of GFP was co-transfected as the control. The band intensities of FLAG-HIF1 and GFP were quantified by fluorescence densitometry. For each sample, HIF1 signal was divided by the GFP signal, and normalized to WT at normoxia, expressed as mean and S.D. of at least three experiments. *, p 0.05; **, p 0.01, t test. B and C, the WT and K709A plasmids were co-transfected with Ev or p300 in Hek293T cells for 48 h. A vector of GFP was co-transfected as controls. Then cells were exposed to normal (B) or hypoxic mimetic CoCl (C)for6h. Afterward, cells were treated by cycloheximide (CHX) for the indicated time, and WCL was harvested and analyzed by Western blots using anti-FLAG and anti-GFP antibodies. The FLAG-HIF1 WT and mutant protein stability was measured and expressed as in A. Line graphs in B and C were mean and S.D. of three experiments. p  0.01 for all statistical comparisons. WT and mutant proteins were IP-purified, and the level of HIF1 protein can be a target of both polyubiquitination and polyubiquitination and acetylation were measured by Western acetylation. Polyubiquitination at normal and hypoxic condi- blot. In normal oxygen condition, the polyubiquitination level tions cause HIF1 protein degradation. Acetylation by p300 or of HIF1-WT was much higher than HIF1-K709A mutant in the point mutation (Lys3Ala) eliminates the polyubiquitina- Ev cells (Fig. 5A). p300 overexpression reduced polyubiquitina- tion possibility and leads to HIF1 protein stabilization. tion and increased acetylation in HIF1-WT proteins (Fig. 5A). Lys-709 Regulates HIF-1 Reporter and Target Gene Expres- In contrast, p300 did not change the polyubiquitination and sion—We hypothesized that the up-regulation of HIF1 pro- acetylation levels of the HIF1-K709A mutant (Fig. 5A). In tein level due to Lys-709 acetylation can lead to an increase of hypoxic condition, although both wild-type and mutant HIF1 HIF1 transcriptional activity. To directly compare the func- proteins were less polyubiquitinated than in normoxia, the tion of HIF1 WT and K709A mutant in normal and hypoxic HIF1-WT still had a significantly higher level of polyubiquiti- conditions, we transduced Hek293 cells with lentivirus con- nation than the HIF1-K709A mutant (Fig. 5A). p300 only taining shRNA against the 3-UTR of HIF1. As a result, the decreased polyubiquitination and increased acetylation levels transduced Hek293 cell line contained stable shRNA knock- in the HIF1-WT, but not in the HIF1-K709A mutant (Fig. down against the endogenous, but not the exogenous, HIF1 5A). Next, we overexpressed HIF1-WT or K709A mutant in (supplemental Fig. S4). Then, we performed HIF-1 activity the presence of p300 siRNA in U2OS cells. In both normoxia reporter assay using the Dual-Luciferase plasmid system, in and hypoxia, the loss of p300 significantly reduced the which the firefly luciferase reporter gene (p2.1) is under the HIF1-WT protein level, and did not change the HIF1-K709A control of hypoxia and HIF-1 and the Renilla luciferase gene is mutant (Fig. 5B). These data suggest that p300 regulates HIF1 constitutive (30, 31). The HIF1 shRNA effectively inhibited protein stability by Lys-709 acetylation. Lys-709 within the the HIF-1 activity driven by the endogenous HIF1 (supple- OCTOBER 12, 2012• VOLUME 287 • NUMBER 42 JOURNAL OF BIOLOGICAL CHEMISTRY 35501 HIF1 Lys-709 Acetylation Increases Protein Stability mutant activity was not significantly affected (Fig. 6B). This was consistent with the Western blot data in Fig. 5B, in which the protein expression of HIF1-WT, not K709A mutant, was reduced by p300 knockdown. We also compared the HIF-1 target gene expression due to HIF1-WT or the K709A mutant. The Hek293-shHIF1 cell line was transfected with the wild-type or mutant plasmid, and cultured in normal or hypoxic conditions overnight. Similar to Fig. 4A, the Lys-709 mutant protein was more stable than the wild-type (supplemental Fig. S6). The HIF-1 target gene expres- sions were significantly up-regulated by HIF1-WT in hypoxia. For a subset of these genes such as VEGFa, Glut 1, and PDK1, the fold of hypoxia up-regulation was significantly higher by an equal amount of HIF1-K709A plasmid compared with the wild-type (Fig. 6C and supplemental Fig. S6). In contrast, HIF-1 target genes such as hexokinase 2 (HK2) and JMJD1A were not further up-regulated by the K709A mutant (Fig. 6C and supple- mental Fig. S6). Taken together, these results suggest that Lys- 709 is critical for p300 to exert its positive effect on HIF1 protein stability and HIF-1 activity. The elimination of this lysine residue provides a gain of function effect to HIF-1 to phenotypically mimic p300. Finally, to investigate the effect of HIF1-WT and K709A mutant on cancer cell growth, we used shRNA to knockdown the endogenous HIF1 in the liver can- cer cell line Hep3B and subsequently overexpressed either HIF1-WT or K709A mutant. Proliferation studies showed that cells expressing the WT or mutant protein had similar growth patterns in normal oxygen condition (Fig. 6D). Impor- tantly, cells with the K709A mutant grew significantly better than the wild-type in hypoxia (Fig. 6D). DISCUSSION FIGURE 5. p300 regulates HIF1 polyubiquitination and protein stability via Lys-709. A, FLAG-HIF1 WT and K709A mutant were co-transfected with In hypoxic conditions, the increased HIF1-p300 interaction either Ev or p300 in Hek293T cells for 48 h. Afterward, cells were treated with can enhance HIF-1 transcriptional activity. In this study, we proteasomal inhibitor MG132 at normoxia or hypoxia for 4 h, and the WCL identified a novel biological consequence of HIF1-p300 inter- was harvested for anti-FLAG IP and Western blots using anti-ubiquitin, anti- acetylated lysine (Ace-K), anti-p300, or anti-FLAG antibodies. B, U2OS cells action. HIF1 is acetylated at lysine 709 by p300. The conse- were treated with p300 siRNA or control siRNA. 24 h later, cells were co- quence of this acetylation is to allow the hypoxic cells to have a transfected with FLAG-HIF1 WT or K709A mutant and GFP. 24 h after the more sustainable level of HIF1 protein, which can lead to a transfection, cells were exposed to normoxia or hypoxia for 6 h, WCL was harvested for Western blot analysis of FLAG-HIF1 WT, mutant, and GFP. The more sustainable HIF-1 transactivation and HIF-1 target gene FLAG-HIF1 protein level (R) was calculated based on the ratio of band inten- transcription. Our data indicate that p300 can acetylate HIF1 sity of FLAG-HIF1/GFP. All data were representative of three independent experiments. Lys-709 in both normal and hypoxic conditions. However, the acetylation process requires HIF1-p300 interaction, which is mental Fig. S5) and had no effect on HIF-1 activity due to significantly enhanced by hypoxia (26). Therefore, the physio- FLAG-HIF1 overexpression in normal and hypoxic condi- logically relevant Lys-709 acetylation and the biological effect tions (supplemental Fig. S5). In the Hek293 sh-HIF1 cell line, should primarily occur in hypoxia. As suggested in Fig. 6E,in p300 overexpression did not significantly increase the HIF-1 hypoxia, the dehydroxylation at asparagine 803 leads to the reporter activity in normal and hypoxic conditions (Fig. 6A). increase of HIF1-p300 interaction, which shifts Lys-709 to an Consistent with the Western blot results in Fig. 4, the K709A acetylated state and contributes to the HIF1 protein stability mutant had significantly higher activities than the HIF1-WT and HIF-1 transactivation. HIF1 is known to interact directly in both normoxia (*) and hypoxia (**), respectively (Fig. 6A). and/or indirectly with lysine deacetylases (class I–III HDACs) When p300 was co-overexpressed, it significantly increased the (10, 13, 14, 18, 27), including HDAC1, which specifically coun- HIF1-WT activities in both normoxia and hypoxia as ters the Lys-709 acetylation and down-regulates HIF1. Fur- reported. In contrast, p300 did not significantly change the thermore, it has been shown that HDAC1 interacts with HIF1 activity of K709A mutant (Fig. 6A). We also compared the via VHL (27); therefore, it is likely that in normoxia, one or HIF1-WT and K709A mutant activity under the condition multiple HDACs keep the HIF1 (including Lys-709 and Lys- of p300 siRNA. In both normoxia (Fig. 6B, *) and hypoxia 674) in a deacetylated state. This contributes to the inhibition of (Fig. 6B, **), we observed that HIF1-WT activity was signifi- HIF-1 activity via increasing protein degradation and/or cantly reduced by p300 knockdown; in contrast, HIF1-K709A decreasing HIF1-p300 interaction. 35502 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 42 •OCTOBER 12, 2012 HIF1 Lys-709 Acetylation Increases Protein Stability FIGURE 6. Lys-709 regulates HIF-1 transcriptional activity and cancer cell proliferation in hypoxia. A, Hek293 cells with stable shRNA knockdown of endogenous HIF1 (Hek293-shHIF1) were transfected with Ev, p300, FLAG-HIF1 WT, FLAG-HIF1 K709A mutant (mut) as single plasmid or as HIF1 and p300 combined. 24 h later, cells were transfected with hypoxia response element-driven firefly (p2.1) and constitutive Renilla luciferase vectors and cultured in normoxia or hypoxia overnight before Dual-Luciferase measurement. *, p 0.01; **, p 0.05. B, Hek293-shHIF1 cells were treated with p300 siRNA or control siRNA. 24 h later, cells were transfected with Ev, FLAG-HIF1 WT, or K709A mutant. After culturing overnight, cells were further transfected with HRE firefly and constitutive Renilla luciferase vectors and cultured in normoxia or hypoxia overnight before Dual-Luciferase measurement., the difference between si-C and si-p300. *, p  0.05; **, p  0.01. C, Hek293-shHIF1 cells were transfected with equal amount of Ev, FLAG-HIF1 WT, or K709A mutant plasmid. 48 h after transfection, cells were cultured in normoxia (N) or hypoxia (H) overnight, and the HIF-1 target gene expressions were measured by real-time quantitative RT-PCR. *, p 0.01. D, Hep3B-shHIF1 cells were transfected with FLAG-HIF1 WT or K709A mutant. 48 h later, cells were equally divided into 24-well plates and cultured in normoxia (N) or hypoxia (H). The cell proliferation over a 48-h period was measured. *, p  0.01. E, summary of how HIF1 protein stability can be regulated by p300 via Lys-709. ub, ubiquitination. The bar and line graphs in A–D represent mean and S.D. of four experiments; all comparisons were made by t test. Ace, acetylated. We have shown that the Lys-709 acetylation is not the result (29). In this study, the polyubiquitination of HIF1-WT was of hypoxia-induced HIF1 protein stability, but rather, it is one significantly reduced by p300 in normal and hypoxic condi- of the causes for HIF1 to be stable and/or more sustainable in tions. In contrast, the polyubiquitination of HIF1 K709A hypoxic condition. Although the oxygen/VHL pathway pro- mutant was not dramatically affected by p300. We speculate vides a major regulatory mechanism for HIF1 protein stabil- that Lys-709 is an oxygen/VHL-independent polyubiquitina- ity, additional mechanisms do exist to fine-tune the HIF1 pro- tion site. In normoxia, the VHL-dependent mechanism primar- tein stability in both normoxia and hypoxia. For example, ily determines the HIF1 protein stability. In hypoxia or VHL despite being significantly stabilized by hypoxia, HIF1 still loss-of-function conditions, there is a possible competition undergoes polyubiquitination (via HAF and CHIP/HSP70) and between the polyubiquitination (degradation) and acetylation 26 S-dependent proteasomal degradation (8, 32). In the current by p300 (stability) in regulating HIF1 protein. A more detailed study, the post-translational modification at Lys-709 provides a study is required to elucidate the polyubiquitination machinery novel mechanism in regulating HIF1 polyubiquitination and at Lys-709, and the cross-talk or competition between polyu- protein stability in hypoxia. Our data suggest that the acetyla- biquitination and acetylation. tion at Lys-709 by p300 reduces HIF1 polyubiquitination, Although the interaction between HIF1-p300 is function- which serves as a possible mechanism of enhancing the protein ally critical for HIF-1 transactivation, it is unknown whether all stability in hypoxia. Recently, HIF1 Lys-709 has been identi- HIF-1 target gene transactivation depends on the presence of fied as a possible site of polyubiquitination in a proteomic study p300 to the same level. Because p300 can also serve as a chro- OCTOBER 12, 2012• VOLUME 287 • NUMBER 42 JOURNAL OF BIOLOGICAL CHEMISTRY 35503 HIF1 Lys-709 Acetylation Increases Protein Stability -degradative pathway. J. Biol. Chem. 277, hypoxia-inducible factor-1 matin histone acetylase (23, 33), it is possible that the HIF1- 29936–29944 p300 interaction and acetylation occurs at the chromatin level 7. Liu, Y. V., and Semenza, G. L. (2007) RACK1 versus HSP90: competition and is only relevant to HIF-1 target genes that require p300. for HIF-1  degradation versus stabilization. Cell Cycle 6, 656–659 This is a likely interpretation that only a subset of HIF-1 target 8. Luo, W., Zhong, J., Chang, R., Hu, H., Pandey, A., and Semenza, G. L. gene expressions were further increased by p300 or HIF1 Lys- (2010) Hsp70 and CHIP selectively mediate ubiquitination and degrada- 709 mutant overexpression. Therefore, the functional signifi- tion of hypoxia-inducible factor (HIF)-1 but Not HIF-2. J. Biol. Chem. 285, 3651–3663 cance of HIF1-p300 interaction is both gene- and cell 9. Ellis, L., Hammers, H., and Pili, R. (2009) Targeting tumor angiogenesis type-dependent. with histone deacetylase inhibitors. Cancer Lett. 280, 145–153 Multiple sites of the HIF1 protein can be modified by lysine 10. Qian, D. Z., Kachhap, S. K., Collis, S. J., Verheul, H. M., Carducci, M. A., acetylation with different biological consequences. Acetylation Atadja, P., and Pili, R. (2006) Class II histone deacetylases are associated within the oxygen-dependent degradation domain is related to with VHL-independent regulation of hypoxia-inducible factor 1 . Cancer Res. 66, 8814–8821 the VHL-dependent HIF1 degradation (11), whereas acetyla- 11. Jeong, J. W., Bae, M. K., Ahn, M. Y., Kim, S. H., Sohn, T. K., Bae, M. H., Yoo, tion within the N-terminal region is related to VHL-independ- M. A., Song, E. J., Lee, K. J., and Kim, K. W. (2002) Regulation and desta- ent degradation (18). The acetylation at Lys-674 is related to bilization of HIF-1 by ARD1-mediated acetylation. Cell 111, 709–720 HIF1-p300 interaction and HIF-1 transactivation (14). In the 12. Kato, H., Tamamizu-Kato, S., and Shibasaki, F. (2004) Histone deacetylase current study, the acetylation at Lys-709 is related to VHL- 7 associates with hypoxia-inducible factor 1 and increases transcrip- independent HIF1 stability. Furthermore, the acetylation/ tional activity. J. Biol. Chem. 279, 41966–41974 13. Finley, L. W., Carracedo, A., Lee, J., Souza, A., Egia, A., Zhang, J., Teruya- deacetylation enzymes regulating these specific modifications Feldstein, J., Moreira, P. I., Cardoso, S. M., Clish, C. B., Pandolfi, P. P., and can also be different. Our site-directed mutagenesis study indi- Haigis, M. C. (2011) SIRT3 opposes reprogramming of cancer cell metab- cates that p300 primarily acetylates Lys-709 within the HIF1. olism through HIF1 destabilization. Cancer Cell 19, 416–428 In contrast, a p300 associated protein, PCAF, has no effect on 14. Lim, J. H., Lee, Y. M., Chun, Y. S., Chen, J., Kim, J. E., and Park, J. W. (2010) Lys-709 acetylation and is primarily responsible for Lys-674 Sirtuin 1 modulates cellular responses to hypoxia by deacetylating hypox- ia-inducible factor 1. Mol. Cell 38, 864–878 acetylation (14). 15. Laemmle, A., Lechleiter, A., Roh, V., Schwarz, C., Portmann, S., Furer, C., Therapeutically, HIF1 protein stability and HIF-1 tran- Keogh, A., Tschan, M. P., Candinas, D., Vorburger, S. A., and Stroka, D. scriptional activity can be targeted by deacetylase inhibitors (2012) Inhibition of SIRT1 impairs the accumulation and transcriptional targeting class I, II, or III HDACs. Currently, most of the inhib- activity of HIF-1 protein under hypoxic conditions. PLoS One 7, e33433 itors are not specific to an isozyme; therefore, the current study 16. Kim, S. H., Jeong, J. W., Park, J. A., Lee, J. W., Seo, J. H., Jung, B. K., Bae, suggest less than optimal inhibitory results using these deacety- M. K., and Kim, K. W. (2007) Regulation of the HIF-1 stability by histone deacetylases. Oncol. Rep. 17, 647–651 lase inhibitors. For example, the HIF1 protein can be stabi- 17. Sadoul, K., Boyault, C., Pabion, M., and Khochbin, S. (2008) Regulation of lized by deacetylation of N-terminal lysine residues via HDAC4 protein turnover by acetyltransferases and deacetylases. Biochimie 90, (18). In the current study, HIF1 protein can be destabilized by 306–312 deacetylation of Lys-709 via HDAC1. This suggests that a 18. Geng, H., Harvey, C. T., Pittsenbarger, J., Liu, Q., Beer, T. M., Xue, C., and hydroxamic-based HDAC inhibitor such as TSA or SAHA can Qian, D. Z. (2011) HDAC4 protein regulates HIF1 protein lysine acety- cause both stabilization and destabilization activities toward lation and cancer cell response to hypoxia. J. Biol. Chem. 286, 38095–38102 HIF1. The protein level after the treatment is just a net result 19. Arany, Z., Huang, L. E., Eckner, R., Bhattacharya, S., Jiang, C., Goldberg, of these two conflicting mechanisms. Interestingly, this may M. A., Bunn, H. F., and Livingston, D. M. (1996) An essential role for also explain the observation that inhibiting class I HDACs p300/CBP in the cellular response to hypoxia. Proc. Natl. Acad. Sci. U.S.A. (HDAC1–3) does not inhibit HIF1 proteins (10). Therefore, 93, 12969–12973 the current study provides mechanistic rationale of discovering 20. Dann, C. E., 3rd, Bruick, R. K., and Deisenhofer, J. (2002) Structure of factor-inhibiting hypoxia-inducible factor 1: An asparaginyl hydroxylase and developing the HDAC isozyme-specific inhibitors, some of involved in the hypoxic response pathway. Proc. Natl. Acad. Sci. U.S.A. 99, which can potentially be used to either increase or decrease 15351–15356 HIF1 and HIF-1 activities. 21. Kung, A. L., Wang, S., Klco, J. M., Kaelin, W. G., and Livingston, D. M. (2000) Suppression of tumor growth through disruption of hypoxia-in- REFERENCES ducible transcription. Nat. Med. 6, 1335–1340 1. Semenza, G. L. (2012) Hypoxia-inducible factors in physiology and med- 22. Kung, A. L., Zabludoff, S. D., France, D. S., Freedman, S. J., Tanner, E. A., icine. Cell 148, 399–408 Vieira, A., Cornell-Kennon, S., Lee, J., Wang, B., Wang, J., Memmert, K., 2. Ivan, M., Kondo, K., Yang, H., Kim, W., Valiando, J., Ohh, M., Salic, A., Naegeli, H. U., Petersen, F., Eck, M. J., Bair, K. W., Wood, A. W., and Asara, J. M., Lane, W. S., and Kaelin, W. G., Jr. (2001) HIF targeted for Livingston, D. M. (2004) Small molecule blockade of transcriptional co- VHL-mediated destruction by proline hydroxylation: implications for O activation of the hypoxia-inducible factor pathway. Cancer Cell 6, 33–43 sensing. Science 292, 464–468 23. Chan, H. M., and La Thangue, N. B. (2001) p300/CBP proteins: HATs for 3. Yu, F., White, S. B., Zhao, Q., and Lee, F. S. (2001) HIF-1 binding to VHL transcriptional bridges and scaffolds. J. Cell Sci. 114, 2363–2373 is regulated by stimulus-sensitive proline hydroxylation. Proc. Natl. Acad. 24. Geng, H., Rademacher, B. L., Pittsenbarger, J., Huang, C. Y., Harvey, C. T., Sci. U.S.A. 98, 9630–9635 Lafortune, M. C., Myrthue, A., Garzotto, M., Nelson, P. S., Beer, T. M., and 4. Semenza, G. L. (2012) Hypoxia-inducible factors: mediators of cancer Qian, D. Z. (2010) ID1 enhances docetaxel cytotoxicity in prostate cancer progression and targets for cancer therapy. Trends Pharmacol. Sci. 33, cells through inhibition of p21. Cancer Res. 70, 3239–3248 207–214 25. Harten, S. K., Esteban, M. A., and Maxwell, P. H. (2009) Identification of 5. Semenza, G. L. (2004) Intratumoral hypoxia, radiation resistance, and novel VHL regulated genes by transcriptomic analysis of RCC10 renal HIF-1. Cancer Cell 5, 405–406 carcinoma cells. Adv. Enzyme Regul. 49, 43–52 6. Isaacs, J. S., Jung, Y. J., Mimnaugh, E. G., Martinez, A., Cuttitta, F., and 26. Semenza, G. L. (2007) Hypoxia-inducible factor 1 (HIF-1) pathway. Sci. STKE 2007, cm8 Neckers, L. M. (2002) Hsp90 regulates a von Hippel Lindau-independent 35504 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 42 •OCTOBER 12, 2012 HIF1 Lys-709 Acetylation Increases Protein Stability 27. Mahon, P. C., Hirota, K., and Semenza, G. L. (2001) FIH-1: a novel protein quired for O -independent and HSP90 inhibitor-induced degradation of that interacts with HIF-1 and VHL to mediate repression of HIF-1 tran- . Mol. Cell 25, 207–217 HIF-1 scriptional activity. Genes Dev. 15, 2675–2686 31. Zhang, H., Qian, D. Z., Tan, Y. S., Lee, K., Gao, P., and Ren, Y. R. (2008) 28. Chen, L. F., Mu, Y., and Greene, W. C. (2002) Acetylation of RelA at Digoxin and other cardiac glycosides inhibit HIF-1 synthesis and block discrete sites regulates distinct nuclear functions of NF-B. EMBO J. 21, tumor growth. Proc. Natl. Acad. Sci. U.S.A. 105, 19579–19586 6539–6548 32. Koh, M. Y., Darnay, B. G., and Powis, G. (2008) Hypoxia-associated factor, 29. Kim, W., Bennett, E. J., Huttlin, E. L., Guo, A., Li, J., Possemato, A., Sowa, a novel E3-ubiquitin ligase, binds and ubiquitinates hypoxia-inducible fac- M. E., Rad, R., Rush, J., Comb, M. J., Harper, J. W., and Gygi, S. P. (2011) tor 1, leading to its oxygen-independent degradation. Mol. Cell. Biol. 28, Systematic and quantitative assessment of the ubiquitin-modified pro- 7081–7095 teome. Mol. Cell 44, 325–340 33. Luo, W., Hu, H., Chang, R., Zhong, J., Knabel, M., O’Meally, R., Cole, R. N., 30. Liu, Y. V., Baek, J. H., Zhang, H., Diez, R., Cole, R. N., and Semenza, G. L. Pandey, A., and Semenza, G. L. (2011) Pyruvate kinase M2 is a PHD3- (2007) RACK1 competes with HSP90 for binding to HIF-1 and is re- stimulated coactivator for hypoxia-inducible factor 1. Cell 145, 732–744 OCTOBER 12, 2012• VOLUME 287 • NUMBER 42 JOURNAL OF BIOLOGICAL CHEMISTRY 35505 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry American Society for Biochemistry and Molecular Biology

HIF1α Protein Stability Is Increased by Acetylation at Lysine 709 *

Download PDF
10 pages

Loading next page...
 
/lp/american-society-for-biochemistry-and-molecular-biology/hif1-protein-stability-is-increased-by-acetylation-at-lysine-709-0Mf3TrzYiL

References

References for this paper are not available at this time. We will be adding them shortly, thank you for your patience.

Publisher
American Society for Biochemistry and Molecular Biology
Copyright
Copyright © 2012 Elsevier Inc.
ISSN
0021-9258
eISSN
1083-351X
DOI
10.1074/jbc.m112.400697
Publisher site
See Article on Publisher Site

Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 42, pp. 35496 –35505, October 12, 2012 © 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. HIF1 Protein Stability Is Increased by Acetylation at □ S Lysine 709 Received for publication, July 12, 2012, and in revised form, August 14, 2012 Published, JBC Papers in Press, August 20, 2012, DOI 10.1074/jbc.M112.400697 Hao Geng, Qiong Liu, Changhui Xue, Larry L. David, Tomasz M. Beer, George V. Thomas, Mu-Shui Dai, and David Z. Qian From the Oregon Health & Science University Knight Cancer Institute, Oregon Health & Science University, Portland, Oregon 97239 Background: HIF1 and p300 are key components of HIF-1 transcription complex. Results: Lysine 709 of HIF1 is acetylated by p300, which increases protein stability and HIF-1 activity. Conclusion: p300 has a novel function in stabilizing HIF1 by Lys-709 acetylation. Significance: New insights in how HIF1 is post-translationally regulated by its cofactor to ensure HIF-1 activity. Lysine acetylation regulates protein stability and function. gen-independent HIF1 to form the core of HIF-1 transcrip- p300 is a component of the HIF-1 transcriptional complex and tional complex (2, 3). The HIF-1 target genes regulate a vast positively regulates the transactivation of HIF-1. Here, we show variety of biological activities that are critical for cell viability, a novel molecular mechanism by which p300 facilitates HIF-1 growth, and differentiation (1). Therefore, the inappropriate activity. p300 increases HIF-1 (HIF1) protein acetylation and accumulation of HIF1 proteins in cancer cells can lead to stability. The regulation can be opposed by HDAC1, but not by inappropriate HIF-1 activities, which underpins malignant HDAC3, and is abrogated by disrupting HIF1-p300 interac- phenotypes including angiogenesis, metabolic reprogramming, tion. Mechanistically, p300 specifically acetylates HIF1 at Lys- metastasis, and therapy resistance (4, 5). 709, which increases the protein stability and decreases polyu- HIF1 protein stability can also be regulated by additional biquitination in both normoxia and hypoxia. Compared with mechanisms that are independent and cross-talk with the oxy- the wild-type protein, a HIF1 K709A mutant protein is more gen/VHL-mediated HIF1 protein degradation pathway. stable, less polyubiquitinated, and less dependent on p300. HSP90 is a key cellular chaperone and is positively associated Overexpression of the HIF1 wild-type or K709A mutant in with HIF1 proteins in both normoxia and hypoxia (6). In nor- cancer cells lacking the endogenous HIF1 shows that the moxia, the HIF1 chaperone function of HSP90 is countered by K709A mutant is transcriptionally more active toward the RACK1 (7). Although significantly stabilized in hypoxia due to HIF-1 reporter and some endogenous target genes. Cancer cells the lack of oxygen/VHL pathway, the HIF1 protein can still be containing the K709A mutant are less sensitive to hypoxia-in- reduced by HSP70-CHIP via polyubiquitination and degrada- duced growth arrest than the cells containing the HIF1 wild- tion (8). In addition, the HIF1 protein stability and HIF-1 tran- type. Taken together, these data demonstrate a novel biological scriptional activity are associated with the protein lysine acety- consequence upon HIF1-p300 interaction, in which HIF1 lation-deacetylation system (9). Multiple members within the can be stabilized by p300 via Lys-709 acetylation. lysine acetylase and deacetylase families have been reported to interact with HIF1 (10–14). The biological consequence is a protein sta- change of HIF-1 activity due to a change in HIF1 Hypoxia-inducible factor 1  (HIF1) is a critical compo- bility in many cases (9, 15, 16). Lysine acetylation changes pro- nent of the HIF-1 transcriptional complex that regulates cellu- tein stability and function (17). Multiple acetylation sites have lar response to hypoxia (1). In non-hypoxic conditions, HIF1 been discovered within the HIF1 protein (11, 14, 18). How- protein is constantly synthesized, but rapidly degraded by oxy- ever, the biological consequence due to these different acetyla- gen. The primary mechanism consists of the post translational tion sites can be very different. Currently, both the lysine acety- hydroxylation of HIF1 at proline residues 402 and 564 by oxy- lase and deacetylase are potential therapeutic targets against gen-dependent prolyl hydroxylases, which is followed by von cancer and other human diseases, in which HIF1 plays a path- Hippel-Lindau (VHL)-mediated polyubiquitination and 26 S ological role. It is important to delineate the functional conse- proteasome-dependent protein degradation (2). In hypoxic quence of HIF1 acetylation at specific lysine locations. conditions, oxygen is not available for the hydroxylation of p300 is a critical component in the HIF-1 transcriptional HIF1 protein, which is stabilized and dimerized with the oxy- complex (19). In the non-hypoxic condition, p300 and HIF1 are not physically associated due to the rapid HIF1 protein * This work was supported by U. S. Public Health Service Grant R01CA149253 degradation and the hydroxylation at asparagine 803 within the from the National Cancer Institute, National Institutes of Health. HIF1 (20). Recently, the acetylation at lysine 674 of HIF1 by □ S This article contains supplemental Figs. S1–S6. PCAF has also been reported to play a role in determining To whom correspondence should be addressed: 3303 SW Bond Ave., CH14R, OHSU, Portland, OR 97239. Tel.: 503-312-5912; Fax: 503-494-6197; E-mail: HIF1-p300 interaction (14). Upon hypoxia, the C-terminal of [email protected]. HIF1 interacts with the CH1 (cysteine/histidine-rich 1) The abbreviations used are: HIF1, hypoxia-inducible factor 1 ; VHL, von domain of p300, which leads to an increase of HIF-1 transcrip- Hippel-Lindau; Ev, empty vector; WCL, whole cell lysate; dm, double mutant; HDAC, histone deacetylase; PCAF, p300/CBP-associated factor. tional activity (21). A small molecule inhibitor, chetomin, has This is an Open Access article under the CC BY license. 35496 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 42 •OCTOBER 12, 2012 HIF1 Lys-709 Acetylation Increases Protein Stability been reported to disrupt the interaction between HIF1 and to validate protein identifications derived from MS/MS sequencing results. p300 and inhibit the HIF-1 target gene expression (22). p300 is In Vitro Acetylation Assay—HIF1 and p300, purified by a multiple domain protein with acetylase activity (23). Despite being closely associated with HIF1 in hypoxia, it is unclear immunoprecipitation, were mixed and incubated at 30 °C in whether p300 has a direct effect on HIF1. In the current study, buffer (25 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, 5 mM DTT, and 0.7 mM acetyl-CoA). Reactions were stopped at indicated time we identified a novel function of p300 in stabilizing HIF1 pro- by adding 6 SDS loading buffer and analyzed by Western teins by lysine acetylation at Lys-709 within HIF1. blots. Cycloheximide HIF1 Half-life—HIF1 and GFP proteins EXPERIMENTAL PROCEDURES were expressed in Hek293T cells. Following exposure to nor- Cell Culture—Human embryonic kidney 293T (Hek293T), mal or hypoxic condition for 5 h, cells were treated with cyclo- 293 (Hek293), osteosarcoma U2OS, Hep3Bc1, and renal cell heximide and harvested at different time points. Western blots carcinoma RCC10 cells were cultured in DMEM with 10% FBS were used to detect HIF1 and GFP proteins, which were quan- and 1% penicillin/streptomycin. Cells were maintained in a tified using LI-COR odyssey Infrared software (version 2.0) as humidified incubator at 37 °C with 5% CO . Hypoxia was described previously (18). induced by exposing cells to 1% oxygen, 5% CO , and 94% nitro- Real-time PCR and Cell Proliferation—The C method gen at 37 °C. was used to calculate mRNA fold change. The trypan blue Vectors—The FLAG-HIF1 wild-type, FLAG-HIF1 proline exclusion method was used to calculate viable cells in the pro- mutant, p2.1 HRE-firefly, pSV40-Renilla were gifts from Dr. liferation experiments as described (18, 24). Gregg Semenza (The Johns Hopkins University). HA-HIF1, Statistical Analysis—Differences between the means of HDAC1, HDAC3, PCAF, and p300 wild-type/catalytic mutant unpaired samples were evaluated by the Student’s t test. vectors were purchased from Addgene. The siRNA against p300 and shRNA against endogenous HIF1 were purchased RESULTS from Sigma. p300 Up-regulates the HIF1 Protein Level—Although p300 Plasmid Construction—Alanine substitutions in HIF1 pro- is known to increase HIF-1 transcriptional activity, its effect on tein were generated by two-step PCR amplification using HIF1 protein is less clear. In the cancer cell line U2OS, we used FLAG-HIF1 wild-type plasmid as a template. The PCR prod- siRNA to knockdown p300 and cultured the cells in either nor- ucts carrying the mutant HIF1 fragments were digested with mal ambient oxygen (21% O ) or hypoxic (1% O ) conditions 2 2 restriction enzymes and inserted into the FLAG empty vector for 6 h to induce HIF1 protein accumulation. We found that used for carrying the wild-type FLAG-HIF1. Each mutation p300 siRNA knockdown (si-p300) significantly decreased the was verified by sequencing. HIF1 in hypoxia (Fig. 1A, U2OS, and supplemental Fig. S1). Immunoprecipitation (IP) and Immunoblot—Cells were This decrease was on the protein level because the mRNA of lysed in prelysis buffer (50 mM Tris-HCl, pH 8.0, 0.1% Nonidet HIF1 was not changed by p300 knockdown (supplemental Fig. P-40, and 150 mM NaCl) in the presence of 1 protease inhib- S1). We also tested the effect of p300 siRNA on HIF1 proteins itor mixture (Thermo), followed by 2 sonication at 15% in a renal cell carcinoma cell line RCC10 (25), in which a loss of amplitude for 10 s. For IP assays, anti-FLAG M2 affinity gel function in VHL renders HIF1 protein stable in normal oxy- (Sigma) was incubated with cell lysates at 4 °C. The precipitates gen condition (Fig. 1A, RCC10). The siRNA knockdown of were extensively washed by prelysis buffer and subjected to p300 significantly decreased HIF1 proteins in both normal Western blotting. The antibodies used in immunoblots and co- and hypoxic conditions (Fig. 1A, RCC10). Next, we measured immunoprecipitations were as follows: HIF1, p300, PCAF, the effect of p300 overexpression on HIF1 protein levels. In ubiquitin, GFP, HDAC3 (Santa Cruz Biotechnology), HDAC1 VHL wild-type U2OS cells, p300 overexpression increased (Millipore), -tubulin and FLAG (Sigma), and acetyl lysine HIF1 proteins in the hypoxic condition (Fig. 1B, U2OS). In (Immuchem). VHL-negative RCC10 cells, the HIF1 protein was significantly Mass Spectrometry—Mass spectrometry was performed by increased by p300 transfection in both normal and hypoxic Proteomics Shared Resource at Oregon Health & Science Uni- conditions (Fig. 1B, RCC10). The effect of p300 on HIF1 versity. Briefly, HIF1 proteins were recovered from SDS- appears to be specific because HIF2 and HIF1 protein levels PAGE gel, reduced with DTT, and digested by trypsin. Each were not changed by p300 (supplemental Fig. S2). Next, we protein digest was analyzed by LC-MS/MS using an Agilent co-overexpressed a FLAG-tagged HIF1 wild-type plasmid 1100 series capillary LC system (Agilent Technologies) and a with either empty vector (Ev) or p300 in Hek293T cells. To VELOS linear ion trap mass spectrometer (Thermo Fisher). ensure the regulation of FLAG-HIF1 by p300 is specific, we Electrospray ionization was performed with an ion max source also included a plasmid encoding the green florescence protein fitted with a 34-gauge metal needle (Thermo Fisher). MS/MS (GFP) that is regulated by the same promoter of FLAG-HIF1 spectra data were searched using SEQUEST against a data- as a control. In Ev cells, hypoxia increased the FLAG-HIF1 base containing human protein sequences from UniProtKB/ protein level, and the overexpressed HIF1 protein was present Swiss-Prot. In-house analysis software was used to calculate in cells cultured in normal oxygen condition (Fig. 1C); presum- discriminant scores and filter out incorrect peptide identifica- ably, this was because that the vector-based FLAG-HIF1 over- tions using sequence-reversed matches to estimate false discov- expression was very robust and overcame the endogenous oxy- ery rates. Scaffold (Proteome Software, Portland, OR) was used gen/VHL-mediated HIF1 degradation. Importantly, p300 OCTOBER 12, 2012• VOLUME 287 • NUMBER 42 JOURNAL OF BIOLOGICAL CHEMISTRY 35497 HIF1 Lys-709 Acetylation Increases Protein Stability reported role of p300, in Ev cells, hypoxia increased the inter- action between FLAG-HIF1 and the endogenous p300 (Fig. 2A). The overexpressed p300 can also interact with FLAG- HIF1 proteins in normoxia, and the interaction was further increased by hypoxia (Fig. 2A). Importantly, the level of FLAG- HIF1 lysine acetylation was increased by hypoxia in the Ev cells and was significantly increased by p300 overexpression in both normal and hypoxic conditions (Fig. 2A). Because both hypoxia and p300 overexpression increased the FLAG-HIF1 protein level (Figs. 1C and 2A), we wanted to know whether the increase of HIF1 acetylation was due to the increase of total HIF1 proteins or the increase of de novo HIF1 acetylation. We performed an in vitro HIF1 acetylation assay by mixing equal amount of IP-purified FLAG-HIF1 proteins with IP- purified p300 proteins in the acetylation assay buffer. After 30 min of incubation at 30 °C, the acetylation level of FLAG- HIF1 was measured by Western blot. As shown in Fig. 2B, the de novo HIF1 acetylation was significantly increased in vitro by incubating with the p300 immunocomplex compared with IgG complex, whereas the total FLAG-HIF1 input remained the same. Next, we repeated the HIF1 and p300 co-overex- pression experiment as in Fig. 2A using a plasmid coding for a FLAG-HIF1 proline double mutant (HIF1-dm) that is resist- ant to the oxygen/VHL-mediated degradation. In Ev cells, the HIF1-dm protein level in WCL was not significantly changed between normal and hypoxic conditions, and hypoxia increased the interaction between HIF1-dm and the endogenous p300 (Fig. 2C) and increased the HIF1-dm protein acetylation in the FIGURE 1. p300 regulates HIF1 proteins. A and B, Western blot analysis of IP samples (Fig. 2C). Co-overexpression of p300 slightly the p300 effect on endogenous HIF1 in U2OS and RCC10 cell lines. Cells increased the HIF1-dm total protein levels in the WCL, were transfected with siRNA control (siC) or si-p300 in A, and empty vector or vector encoding p300 in B. C, Western blot analysis of Hek293T cells co-trans- increased the interaction between HIF1 and p300, and signif- fected with FLAG-HIF1 and p300 or Ev. D, Western blot analysis of Hek293T icantly increased the HIF1-dm protein lysine acetylation (Fig. cells that were transfected with FLAG-HIF1 and treated by 50 nM p300 inhib- itor, chetomin (CTM), or solvent control (V)for6h.In A–D, cells were cultured 2C). These results indicated de novo acetylation of HIF1 by under normal or hypoxic conditions for 6 h before whole cell protein lysates p300. To test whether the HIF1-p300 interaction is required were harvested. In C and D, a vector encoding GFP was used for transfection for the increase of HIF1 protein acetylation, we transfected efficiency controls. The HIF1 protein level (R) was calculated based on the ratio of band intensity of HIF1/tubulin or GFP. All data were representatives FLAG-HIF1 into Hek293T cells and treated cells with chet- of at least three independent experiments. omin. In hypoxia, chetomin treatment significantly reduced the HIF1-p300 interaction, which coincided with the reduction of overexpression further increased the FLAG-HIF1 in both nor- HIF1 acetylation and total HIF1 protein level (Fig. 2D). mal and hypoxic conditions (Fig. 1C). As a control, the co-over- Because HIF1 can also be associated with lysine deacetylases expressed GFP was not changed by either hypoxia or p300 over- (26, 27), we further tested whether the p300-induced acetyla- expression. To further confirm the HIF1 regulation by p300, tion can be countered by deacetylation. We co-overexpressed we transfected the FLAG-HIF1 into Hek293T cells and FLAG-HIF1 with p300 or in combination with either HDAC1 treated cells with chetomin, a chemical inhibitor that disrupts or HDAC3. We found that HDAC1, but not HDAC3, signifi- the HIF1-p300 interaction (22). We found that chetomin cantly reduced the p300-induced HIF1 acetylation, which also treatment decreased the FLAG-HIF1 protein expression in coincided with a reduction of total FLAG-HIF1 protein level both normal and hypoxic conditions (Fig. 1D). These data sug- (Fig. 2E). Recently, HIF1 was reported to be acetylated by a gest that p300 can enhance the HIF1 stability in hypoxia or in p300-interacting protein, PCAF (14). We compared the ability conditions that the oxygen/VHL-mediated HIF1 degradation of p300 and PCAF in terms of HIF1 acetylation. When an is either inactive (RCC10 cell line) or overwhelmed (vector- HA-tagged HIF1 was co-overexpressed with either p300 or based HIF1 overexpression). FLAG-PCAF, we observed that the HIF1-PCAF interaction p300 Acetylates HIF1—One of the primary functions of was not changed by hypoxia, and the interaction was less robust p300 is protein lysine acetylation (23). To understand the molecular mechanism underlying the HIF1 up-regulation by than HIF1-p300 (Fig. 2F). Furthermore, p300 also induced a p300, we transfected the FLAG-HIF1 and p300 in Hek293T more robust HIF1 acetylation than FLAG-PCAF (Fig. 2F). cells, immunoprecipitated the FLAG-HIF1 protein from Taken together, these data suggest that HIF1-p300 interac- whole cell lysates (WCL), and measured the HIF1 lysine acety- tion has two novel biological consequences: the increases of lation and its interaction with p300. In agreement with the HIF1 lysine acetylation and protein stability, which are 35498 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 42 •OCTOBER 12, 2012 HIF1 Lys-709 Acetylation Increases Protein Stability FIGURE 2. p300 acetylates HIF1. A, Hek293T cells were co-transfected with FLAG-HIF1 and p300 or Ev. 48 h after transfection, cells were exposed to either normoxia (n) or hypoxia (h) for 6 h. WCLs were harvested and used for Western blot (IB) or IP with anti-FLAG antibodies and followed by Western blot analysis. Protein acetylation was detected by anti-ace-lysine (Ace-K) antibodies. A GFP vector was also co-transfected as control. B, FLAG-HIF1a and p300 were expressed in Hek293T cells separately and IP purified by anti-FLAG or anti-p300. The immunocomplex containing FLAG-HIF1 was mixed with p300 or IgG immunocom- plex for 0 or 30 min in the in vitro acetylation buffer. Afterward, the acetylated (Ace) HIF1, the total p300, and FLAG-HIF1 protein levels were measured by Western blot. C, similar experiments as described in A were done using a vector encoding for FLAG-HIF1 proline mutant (HIF1-dm) containing proline to alanine mutation at 402 and 564. D, FLAG-HIF1 was overexpressed in Hek293T cells. 24 h later, cells were treated with vehicle (V)or50nM of chetomin (CTM) and exposed to hypoxia for 6 h. Afterward, WCL was used for anti-FLAG IP and Western blots. E, FLAG-HIF1 was co-transfected with Ev and p300 with or without HDAC1 or HDAC3. 48 h after transfection, WCLs were harvested, and used for Western blot (IB) or IP with anti-FLAG antibodies and followed by Western blot analysis. F, a vector encoding for HA-HIF1 was co-overexpressed with Ev, p300, or PCAF in H293T cells. 48 h after transfection, cells were exposed to normal (n) or hypoxia (h) for 6 h, and the HA-HIF1 was IP-purified from WCL and analyzed by Western blot (IB). All data were representative of at least three independent experiments. dependent on HIF1-p300 interaction that can be achieved by that lysine 674 within the FLAG-HIF1 was acetylated in both hypoxia or p300 overexpression. Ev and p300-overexpressed samples (data not shown). Signifi- Lysine 709 of HIF1 Is the Acetylation Target of p300—To cantly, lysine 709 was only acetylated in p300-overexpressed identify the p300 acetylated lysine residue(s) within HIF1,we cells (Fig. 3A and supplemental Fig. S3). Lys-674 has recently co-transfected the full-length wild-type FLAG-HIF1 with been reported as an acetylation target for PCAF (14), and Lys- either p300 or Ev in Hek293T cells. Next, we immunoprecipi- 709 is novel. Based on the FLAG-HIF1 plasmid, we used site- tated and gel-purified FLAG-HIF1 proteins, digested them directed mutagenesis to convert lysine to alanine and generated with trypsin, and analyzed the samples by liquid chromatogra- FLAG-HIF1 mutants containing a single point mutation of phy-tandem mass spectrometry (LC-MS/MS). We observed either K674A or K709A. Then, we overexpressed FLAG- OCTOBER 12, 2012• VOLUME 287 • NUMBER 42 JOURNAL OF BIOLOGICAL CHEMISTRY 35499 HIF1 Lys-709 Acetylation Increases Protein Stability FIGURE 3. HIF1 is acetylated by p300 at lysine 709. A, mass spectrum of HIF1 acetylation (Ace) by p300 at lysine 709. FLAG-HIF1 and p300 were co-overexpressed in Hek293T cells. The FLAG-HIF1 was IP and gel-purified. The gel slice containing FLAG-HIF1 was digested by trypsin and subjected to LC-MS/MS analysis. Mass spectrum of b ions and y ions were shown in red and blue, respectively. Acetylation at the location was marked with an ampersand by additional mass of 42 Da. Image was prepared with Scaffold. B, FLAG-HIF1 WT, FLAG-HIF1 K674A, and FLAG-HIF1 K709A was co-transfected with Ev ()or p300 () in Hek293T cells. 48 h later, WCLs were harvested, IP purified with anti-FLAG antibodies, and analyzed by Western blots. C, FLAG-HIF1 WT and HIF1 K709A were co-transfected with Ev (), p300 WT, or p300 mutant that is defective in acetyltransferase activity. 48 h later, WCL was harvested, IP-purified by anti-FLAG antibodies, and analyzed by Western blots. Data in B and C were representatives of at least three independent experiments. mut, mutant. HIF1-WT, FLAG-HIF1-K674A, or FLAG-HIF1-K709A in  K709A mutant protein observed that the FLAG-HIF1 Hek293T cells with either Ev control or p300. Forty-eight hours expressed at a higher level than FLAG-HIF1-WT in both nor- after transfection, the FLAG-HIF1 wild-type and mutant pro- mal and hypoxic conditions by Western blots coupled with flo- teins were immunopurified from the whole cell lysates. West- rescence densitometry (Fig. 4A). Next, we used cycloheximide ern blots showed that all HIF1 proteins interact with p300 to inhibit protein synthesis and measured the HIF1 WT and similarly (Fig. 3B). In both FLAG-HIF1-WT and FLAG- K709A mutant protein stability in the absence (Ev) or presence HIF1-K674A mutant, the acetylation levels were robustly of p300 overexpression. In normal oxygen condition, the increased by p300 (Fig. 3B). In contrast, the acetylation level of K709A mutant protein was significantly more stable than FLAG-HIF1-K709A mutant was not significantly increased HIF1-WT protein in the Ev cells (#, p  0.01, Fig. 4B). p300 by p300 (Fig. 3B). To further confirm the specific acetylation at overexpression significantly increased the HIF1-WT protein Lys-709 by p300, we co-overexpressed HIF1-WT or Lys-709 stability (a, p  0.01, Fig. 4B) but had no effect on HIF1- mutant with either the wild-type p300 or a p300 mutant that is K709A mutant. A similar trend was observed in the hypoxic defective in the acetyltransferase activity (28). Similar to the condition, in which the HIF1-K709A mutant was more stable results in Fig. 3B, p300 wild-type was able to increase the acety- than the HIF1-WT in Ev cells (##, p  0.01, Fig. 4C), and p300 lation in HIF1-WT, but not in HIF1-K709A mutant proteins overexpression increased the FLAG-HIF1-WT protein stabil- (Fig. 3C). However, the p300 acetylase mutant was unable to ity (aa, p  0.01, Fig. 4C) and had no effect on the K709A increase the acetylation in HIF1-WT and K709A mutant (Fig. mutant (Fig. 4C). 3C). These data suggest that p300 acetylates HIF1 primarily at Lys-709 within the HIF1 has been reported to be a site for Lys-709. polyubiquitination (29). We hypothesize that p300 increases p300 Regulates HIF1 Protein Stability via Lys-709 HIF1-WT protein stability by acetylation at Lys-709, which Acetylation—To investigate the biological significance of Lys- prevents polyubiquitination and protein degradation. To inves- 709 acetylation, we overexpressed equal amounts of FLAG- tigate the relationship between polyubiquitination and acetyla- HIF1-WT or FLAG-HIF1-K709A mutant in Hek293T cells tion at Lys-709, we performed similar co-overexpression exper- with GFP plasmids as transfection controls. Then, we measured iments as in Fig. 4, B and C, and used proteasomal inhibitor the normal and hypoxic FLAG-HIF1 protein levels. We MG132 to prevent FLAG-HIF1 degradation. FLAG-HIF1 35500 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 42 •OCTOBER 12, 2012 HIF1 Lys-709 Acetylation Increases Protein Stability FIGURE 4. p300 regulates HIF1 protein stability via Lys-709. A, FLAG-HIF1 WT or K709A mutant was transfected into the Hek293T cells for 48 h. Then, cells were exposed to normal (N) or hypoxia (H) for 6 h, followed by Western blot analysis. A vector of GFP was co-transfected as the control. The band intensities of FLAG-HIF1 and GFP were quantified by fluorescence densitometry. For each sample, HIF1 signal was divided by the GFP signal, and normalized to WT at normoxia, expressed as mean and S.D. of at least three experiments. *, p 0.05; **, p 0.01, t test. B and C, the WT and K709A plasmids were co-transfected with Ev or p300 in Hek293T cells for 48 h. A vector of GFP was co-transfected as controls. Then cells were exposed to normal (B) or hypoxic mimetic CoCl (C)for6h. Afterward, cells were treated by cycloheximide (CHX) for the indicated time, and WCL was harvested and analyzed by Western blots using anti-FLAG and anti-GFP antibodies. The FLAG-HIF1 WT and mutant protein stability was measured and expressed as in A. Line graphs in B and C were mean and S.D. of three experiments. p  0.01 for all statistical comparisons. WT and mutant proteins were IP-purified, and the level of HIF1 protein can be a target of both polyubiquitination and polyubiquitination and acetylation were measured by Western acetylation. Polyubiquitination at normal and hypoxic condi- blot. In normal oxygen condition, the polyubiquitination level tions cause HIF1 protein degradation. Acetylation by p300 or of HIF1-WT was much higher than HIF1-K709A mutant in the point mutation (Lys3Ala) eliminates the polyubiquitina- Ev cells (Fig. 5A). p300 overexpression reduced polyubiquitina- tion possibility and leads to HIF1 protein stabilization. tion and increased acetylation in HIF1-WT proteins (Fig. 5A). Lys-709 Regulates HIF-1 Reporter and Target Gene Expres- In contrast, p300 did not change the polyubiquitination and sion—We hypothesized that the up-regulation of HIF1 pro- acetylation levels of the HIF1-K709A mutant (Fig. 5A). In tein level due to Lys-709 acetylation can lead to an increase of hypoxic condition, although both wild-type and mutant HIF1 HIF1 transcriptional activity. To directly compare the func- proteins were less polyubiquitinated than in normoxia, the tion of HIF1 WT and K709A mutant in normal and hypoxic HIF1-WT still had a significantly higher level of polyubiquiti- conditions, we transduced Hek293 cells with lentivirus con- nation than the HIF1-K709A mutant (Fig. 5A). p300 only taining shRNA against the 3-UTR of HIF1. As a result, the decreased polyubiquitination and increased acetylation levels transduced Hek293 cell line contained stable shRNA knock- in the HIF1-WT, but not in the HIF1-K709A mutant (Fig. down against the endogenous, but not the exogenous, HIF1 5A). Next, we overexpressed HIF1-WT or K709A mutant in (supplemental Fig. S4). Then, we performed HIF-1 activity the presence of p300 siRNA in U2OS cells. In both normoxia reporter assay using the Dual-Luciferase plasmid system, in and hypoxia, the loss of p300 significantly reduced the which the firefly luciferase reporter gene (p2.1) is under the HIF1-WT protein level, and did not change the HIF1-K709A control of hypoxia and HIF-1 and the Renilla luciferase gene is mutant (Fig. 5B). These data suggest that p300 regulates HIF1 constitutive (30, 31). The HIF1 shRNA effectively inhibited protein stability by Lys-709 acetylation. Lys-709 within the the HIF-1 activity driven by the endogenous HIF1 (supple- OCTOBER 12, 2012• VOLUME 287 • NUMBER 42 JOURNAL OF BIOLOGICAL CHEMISTRY 35501 HIF1 Lys-709 Acetylation Increases Protein Stability mutant activity was not significantly affected (Fig. 6B). This was consistent with the Western blot data in Fig. 5B, in which the protein expression of HIF1-WT, not K709A mutant, was reduced by p300 knockdown. We also compared the HIF-1 target gene expression due to HIF1-WT or the K709A mutant. The Hek293-shHIF1 cell line was transfected with the wild-type or mutant plasmid, and cultured in normal or hypoxic conditions overnight. Similar to Fig. 4A, the Lys-709 mutant protein was more stable than the wild-type (supplemental Fig. S6). The HIF-1 target gene expres- sions were significantly up-regulated by HIF1-WT in hypoxia. For a subset of these genes such as VEGFa, Glut 1, and PDK1, the fold of hypoxia up-regulation was significantly higher by an equal amount of HIF1-K709A plasmid compared with the wild-type (Fig. 6C and supplemental Fig. S6). In contrast, HIF-1 target genes such as hexokinase 2 (HK2) and JMJD1A were not further up-regulated by the K709A mutant (Fig. 6C and supple- mental Fig. S6). Taken together, these results suggest that Lys- 709 is critical for p300 to exert its positive effect on HIF1 protein stability and HIF-1 activity. The elimination of this lysine residue provides a gain of function effect to HIF-1 to phenotypically mimic p300. Finally, to investigate the effect of HIF1-WT and K709A mutant on cancer cell growth, we used shRNA to knockdown the endogenous HIF1 in the liver can- cer cell line Hep3B and subsequently overexpressed either HIF1-WT or K709A mutant. Proliferation studies showed that cells expressing the WT or mutant protein had similar growth patterns in normal oxygen condition (Fig. 6D). Impor- tantly, cells with the K709A mutant grew significantly better than the wild-type in hypoxia (Fig. 6D). DISCUSSION FIGURE 5. p300 regulates HIF1 polyubiquitination and protein stability via Lys-709. A, FLAG-HIF1 WT and K709A mutant were co-transfected with In hypoxic conditions, the increased HIF1-p300 interaction either Ev or p300 in Hek293T cells for 48 h. Afterward, cells were treated with can enhance HIF-1 transcriptional activity. In this study, we proteasomal inhibitor MG132 at normoxia or hypoxia for 4 h, and the WCL identified a novel biological consequence of HIF1-p300 inter- was harvested for anti-FLAG IP and Western blots using anti-ubiquitin, anti- acetylated lysine (Ace-K), anti-p300, or anti-FLAG antibodies. B, U2OS cells action. HIF1 is acetylated at lysine 709 by p300. The conse- were treated with p300 siRNA or control siRNA. 24 h later, cells were co- quence of this acetylation is to allow the hypoxic cells to have a transfected with FLAG-HIF1 WT or K709A mutant and GFP. 24 h after the more sustainable level of HIF1 protein, which can lead to a transfection, cells were exposed to normoxia or hypoxia for 6 h, WCL was harvested for Western blot analysis of FLAG-HIF1 WT, mutant, and GFP. The more sustainable HIF-1 transactivation and HIF-1 target gene FLAG-HIF1 protein level (R) was calculated based on the ratio of band inten- transcription. Our data indicate that p300 can acetylate HIF1 sity of FLAG-HIF1/GFP. All data were representative of three independent experiments. Lys-709 in both normal and hypoxic conditions. However, the acetylation process requires HIF1-p300 interaction, which is mental Fig. S5) and had no effect on HIF-1 activity due to significantly enhanced by hypoxia (26). Therefore, the physio- FLAG-HIF1 overexpression in normal and hypoxic condi- logically relevant Lys-709 acetylation and the biological effect tions (supplemental Fig. S5). In the Hek293 sh-HIF1 cell line, should primarily occur in hypoxia. As suggested in Fig. 6E,in p300 overexpression did not significantly increase the HIF-1 hypoxia, the dehydroxylation at asparagine 803 leads to the reporter activity in normal and hypoxic conditions (Fig. 6A). increase of HIF1-p300 interaction, which shifts Lys-709 to an Consistent with the Western blot results in Fig. 4, the K709A acetylated state and contributes to the HIF1 protein stability mutant had significantly higher activities than the HIF1-WT and HIF-1 transactivation. HIF1 is known to interact directly in both normoxia (*) and hypoxia (**), respectively (Fig. 6A). and/or indirectly with lysine deacetylases (class I–III HDACs) When p300 was co-overexpressed, it significantly increased the (10, 13, 14, 18, 27), including HDAC1, which specifically coun- HIF1-WT activities in both normoxia and hypoxia as ters the Lys-709 acetylation and down-regulates HIF1. Fur- reported. In contrast, p300 did not significantly change the thermore, it has been shown that HDAC1 interacts with HIF1 activity of K709A mutant (Fig. 6A). We also compared the via VHL (27); therefore, it is likely that in normoxia, one or HIF1-WT and K709A mutant activity under the condition multiple HDACs keep the HIF1 (including Lys-709 and Lys- of p300 siRNA. In both normoxia (Fig. 6B, *) and hypoxia 674) in a deacetylated state. This contributes to the inhibition of (Fig. 6B, **), we observed that HIF1-WT activity was signifi- HIF-1 activity via increasing protein degradation and/or cantly reduced by p300 knockdown; in contrast, HIF1-K709A decreasing HIF1-p300 interaction. 35502 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 42 •OCTOBER 12, 2012 HIF1 Lys-709 Acetylation Increases Protein Stability FIGURE 6. Lys-709 regulates HIF-1 transcriptional activity and cancer cell proliferation in hypoxia. A, Hek293 cells with stable shRNA knockdown of endogenous HIF1 (Hek293-shHIF1) were transfected with Ev, p300, FLAG-HIF1 WT, FLAG-HIF1 K709A mutant (mut) as single plasmid or as HIF1 and p300 combined. 24 h later, cells were transfected with hypoxia response element-driven firefly (p2.1) and constitutive Renilla luciferase vectors and cultured in normoxia or hypoxia overnight before Dual-Luciferase measurement. *, p 0.01; **, p 0.05. B, Hek293-shHIF1 cells were treated with p300 siRNA or control siRNA. 24 h later, cells were transfected with Ev, FLAG-HIF1 WT, or K709A mutant. After culturing overnight, cells were further transfected with HRE firefly and constitutive Renilla luciferase vectors and cultured in normoxia or hypoxia overnight before Dual-Luciferase measurement., the difference between si-C and si-p300. *, p  0.05; **, p  0.01. C, Hek293-shHIF1 cells were transfected with equal amount of Ev, FLAG-HIF1 WT, or K709A mutant plasmid. 48 h after transfection, cells were cultured in normoxia (N) or hypoxia (H) overnight, and the HIF-1 target gene expressions were measured by real-time quantitative RT-PCR. *, p 0.01. D, Hep3B-shHIF1 cells were transfected with FLAG-HIF1 WT or K709A mutant. 48 h later, cells were equally divided into 24-well plates and cultured in normoxia (N) or hypoxia (H). The cell proliferation over a 48-h period was measured. *, p  0.01. E, summary of how HIF1 protein stability can be regulated by p300 via Lys-709. ub, ubiquitination. The bar and line graphs in A–D represent mean and S.D. of four experiments; all comparisons were made by t test. Ace, acetylated. We have shown that the Lys-709 acetylation is not the result (29). In this study, the polyubiquitination of HIF1-WT was of hypoxia-induced HIF1 protein stability, but rather, it is one significantly reduced by p300 in normal and hypoxic condi- of the causes for HIF1 to be stable and/or more sustainable in tions. In contrast, the polyubiquitination of HIF1 K709A hypoxic condition. Although the oxygen/VHL pathway pro- mutant was not dramatically affected by p300. We speculate vides a major regulatory mechanism for HIF1 protein stabil- that Lys-709 is an oxygen/VHL-independent polyubiquitina- ity, additional mechanisms do exist to fine-tune the HIF1 pro- tion site. In normoxia, the VHL-dependent mechanism primar- tein stability in both normoxia and hypoxia. For example, ily determines the HIF1 protein stability. In hypoxia or VHL despite being significantly stabilized by hypoxia, HIF1 still loss-of-function conditions, there is a possible competition undergoes polyubiquitination (via HAF and CHIP/HSP70) and between the polyubiquitination (degradation) and acetylation 26 S-dependent proteasomal degradation (8, 32). In the current by p300 (stability) in regulating HIF1 protein. A more detailed study, the post-translational modification at Lys-709 provides a study is required to elucidate the polyubiquitination machinery novel mechanism in regulating HIF1 polyubiquitination and at Lys-709, and the cross-talk or competition between polyu- protein stability in hypoxia. Our data suggest that the acetyla- biquitination and acetylation. tion at Lys-709 by p300 reduces HIF1 polyubiquitination, Although the interaction between HIF1-p300 is function- which serves as a possible mechanism of enhancing the protein ally critical for HIF-1 transactivation, it is unknown whether all stability in hypoxia. Recently, HIF1 Lys-709 has been identi- HIF-1 target gene transactivation depends on the presence of fied as a possible site of polyubiquitination in a proteomic study p300 to the same level. Because p300 can also serve as a chro- OCTOBER 12, 2012• VOLUME 287 • NUMBER 42 JOURNAL OF BIOLOGICAL CHEMISTRY 35503 HIF1 Lys-709 Acetylation Increases Protein Stability -degradative pathway. J. Biol. Chem. 277, hypoxia-inducible factor-1 matin histone acetylase (23, 33), it is possible that the HIF1- 29936–29944 p300 interaction and acetylation occurs at the chromatin level 7. Liu, Y. V., and Semenza, G. L. (2007) RACK1 versus HSP90: competition and is only relevant to HIF-1 target genes that require p300. for HIF-1  degradation versus stabilization. Cell Cycle 6, 656–659 This is a likely interpretation that only a subset of HIF-1 target 8. Luo, W., Zhong, J., Chang, R., Hu, H., Pandey, A., and Semenza, G. L. gene expressions were further increased by p300 or HIF1 Lys- (2010) Hsp70 and CHIP selectively mediate ubiquitination and degrada- 709 mutant overexpression. Therefore, the functional signifi- tion of hypoxia-inducible factor (HIF)-1 but Not HIF-2. J. Biol. Chem. 285, 3651–3663 cance of HIF1-p300 interaction is both gene- and cell 9. Ellis, L., Hammers, H., and Pili, R. (2009) Targeting tumor angiogenesis type-dependent. with histone deacetylase inhibitors. Cancer Lett. 280, 145–153 Multiple sites of the HIF1 protein can be modified by lysine 10. Qian, D. Z., Kachhap, S. K., Collis, S. J., Verheul, H. M., Carducci, M. A., acetylation with different biological consequences. Acetylation Atadja, P., and Pili, R. (2006) Class II histone deacetylases are associated within the oxygen-dependent degradation domain is related to with VHL-independent regulation of hypoxia-inducible factor 1 . Cancer Res. 66, 8814–8821 the VHL-dependent HIF1 degradation (11), whereas acetyla- 11. Jeong, J. W., Bae, M. K., Ahn, M. Y., Kim, S. H., Sohn, T. K., Bae, M. H., Yoo, tion within the N-terminal region is related to VHL-independ- M. A., Song, E. J., Lee, K. J., and Kim, K. W. (2002) Regulation and desta- ent degradation (18). The acetylation at Lys-674 is related to bilization of HIF-1 by ARD1-mediated acetylation. Cell 111, 709–720 HIF1-p300 interaction and HIF-1 transactivation (14). In the 12. Kato, H., Tamamizu-Kato, S., and Shibasaki, F. (2004) Histone deacetylase current study, the acetylation at Lys-709 is related to VHL- 7 associates with hypoxia-inducible factor 1 and increases transcrip- independent HIF1 stability. Furthermore, the acetylation/ tional activity. J. Biol. Chem. 279, 41966–41974 13. Finley, L. W., Carracedo, A., Lee, J., Souza, A., Egia, A., Zhang, J., Teruya- deacetylation enzymes regulating these specific modifications Feldstein, J., Moreira, P. I., Cardoso, S. M., Clish, C. B., Pandolfi, P. P., and can also be different. Our site-directed mutagenesis study indi- Haigis, M. C. (2011) SIRT3 opposes reprogramming of cancer cell metab- cates that p300 primarily acetylates Lys-709 within the HIF1. olism through HIF1 destabilization. Cancer Cell 19, 416–428 In contrast, a p300 associated protein, PCAF, has no effect on 14. Lim, J. H., Lee, Y. M., Chun, Y. S., Chen, J., Kim, J. E., and Park, J. W. (2010) Lys-709 acetylation and is primarily responsible for Lys-674 Sirtuin 1 modulates cellular responses to hypoxia by deacetylating hypox- ia-inducible factor 1. Mol. Cell 38, 864–878 acetylation (14). 15. Laemmle, A., Lechleiter, A., Roh, V., Schwarz, C., Portmann, S., Furer, C., Therapeutically, HIF1 protein stability and HIF-1 tran- Keogh, A., Tschan, M. P., Candinas, D., Vorburger, S. A., and Stroka, D. scriptional activity can be targeted by deacetylase inhibitors (2012) Inhibition of SIRT1 impairs the accumulation and transcriptional targeting class I, II, or III HDACs. Currently, most of the inhib- activity of HIF-1 protein under hypoxic conditions. PLoS One 7, e33433 itors are not specific to an isozyme; therefore, the current study 16. Kim, S. H., Jeong, J. W., Park, J. A., Lee, J. W., Seo, J. H., Jung, B. K., Bae, suggest less than optimal inhibitory results using these deacety- M. K., and Kim, K. W. (2007) Regulation of the HIF-1 stability by histone deacetylases. Oncol. Rep. 17, 647–651 lase inhibitors. For example, the HIF1 protein can be stabi- 17. Sadoul, K., Boyault, C., Pabion, M., and Khochbin, S. (2008) Regulation of lized by deacetylation of N-terminal lysine residues via HDAC4 protein turnover by acetyltransferases and deacetylases. Biochimie 90, (18). In the current study, HIF1 protein can be destabilized by 306–312 deacetylation of Lys-709 via HDAC1. This suggests that a 18. Geng, H., Harvey, C. T., Pittsenbarger, J., Liu, Q., Beer, T. M., Xue, C., and hydroxamic-based HDAC inhibitor such as TSA or SAHA can Qian, D. Z. (2011) HDAC4 protein regulates HIF1 protein lysine acety- cause both stabilization and destabilization activities toward lation and cancer cell response to hypoxia. J. Biol. Chem. 286, 38095–38102 HIF1. The protein level after the treatment is just a net result 19. Arany, Z., Huang, L. E., Eckner, R., Bhattacharya, S., Jiang, C., Goldberg, of these two conflicting mechanisms. Interestingly, this may M. A., Bunn, H. F., and Livingston, D. M. (1996) An essential role for also explain the observation that inhibiting class I HDACs p300/CBP in the cellular response to hypoxia. Proc. Natl. Acad. Sci. U.S.A. (HDAC1–3) does not inhibit HIF1 proteins (10). Therefore, 93, 12969–12973 the current study provides mechanistic rationale of discovering 20. Dann, C. E., 3rd, Bruick, R. K., and Deisenhofer, J. (2002) Structure of factor-inhibiting hypoxia-inducible factor 1: An asparaginyl hydroxylase and developing the HDAC isozyme-specific inhibitors, some of involved in the hypoxic response pathway. Proc. Natl. Acad. Sci. U.S.A. 99, which can potentially be used to either increase or decrease 15351–15356 HIF1 and HIF-1 activities. 21. Kung, A. L., Wang, S., Klco, J. M., Kaelin, W. G., and Livingston, D. M. (2000) Suppression of tumor growth through disruption of hypoxia-in- REFERENCES ducible transcription. Nat. Med. 6, 1335–1340 1. Semenza, G. L. (2012) Hypoxia-inducible factors in physiology and med- 22. Kung, A. L., Zabludoff, S. D., France, D. S., Freedman, S. J., Tanner, E. A., icine. Cell 148, 399–408 Vieira, A., Cornell-Kennon, S., Lee, J., Wang, B., Wang, J., Memmert, K., 2. Ivan, M., Kondo, K., Yang, H., Kim, W., Valiando, J., Ohh, M., Salic, A., Naegeli, H. U., Petersen, F., Eck, M. J., Bair, K. W., Wood, A. W., and Asara, J. M., Lane, W. S., and Kaelin, W. G., Jr. (2001) HIF targeted for Livingston, D. M. (2004) Small molecule blockade of transcriptional co- VHL-mediated destruction by proline hydroxylation: implications for O activation of the hypoxia-inducible factor pathway. Cancer Cell 6, 33–43 sensing. Science 292, 464–468 23. Chan, H. M., and La Thangue, N. B. (2001) p300/CBP proteins: HATs for 3. Yu, F., White, S. B., Zhao, Q., and Lee, F. S. (2001) HIF-1 binding to VHL transcriptional bridges and scaffolds. J. Cell Sci. 114, 2363–2373 is regulated by stimulus-sensitive proline hydroxylation. Proc. Natl. Acad. 24. Geng, H., Rademacher, B. L., Pittsenbarger, J., Huang, C. Y., Harvey, C. T., Sci. U.S.A. 98, 9630–9635 Lafortune, M. C., Myrthue, A., Garzotto, M., Nelson, P. S., Beer, T. M., and 4. Semenza, G. L. (2012) Hypoxia-inducible factors: mediators of cancer Qian, D. Z. (2010) ID1 enhances docetaxel cytotoxicity in prostate cancer progression and targets for cancer therapy. Trends Pharmacol. Sci. 33, cells through inhibition of p21. Cancer Res. 70, 3239–3248 207–214 25. Harten, S. K., Esteban, M. A., and Maxwell, P. H. (2009) Identification of 5. Semenza, G. L. (2004) Intratumoral hypoxia, radiation resistance, and novel VHL regulated genes by transcriptomic analysis of RCC10 renal HIF-1. Cancer Cell 5, 405–406 carcinoma cells. Adv. Enzyme Regul. 49, 43–52 6. Isaacs, J. S., Jung, Y. J., Mimnaugh, E. G., Martinez, A., Cuttitta, F., and 26. Semenza, G. L. (2007) Hypoxia-inducible factor 1 (HIF-1) pathway. Sci. STKE 2007, cm8 Neckers, L. M. (2002) Hsp90 regulates a von Hippel Lindau-independent 35504 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 42 •OCTOBER 12, 2012 HIF1 Lys-709 Acetylation Increases Protein Stability 27. Mahon, P. C., Hirota, K., and Semenza, G. L. (2001) FIH-1: a novel protein quired for O -independent and HSP90 inhibitor-induced degradation of that interacts with HIF-1 and VHL to mediate repression of HIF-1 tran- . Mol. Cell 25, 207–217 HIF-1 scriptional activity. Genes Dev. 15, 2675–2686 31. Zhang, H., Qian, D. Z., Tan, Y. S., Lee, K., Gao, P., and Ren, Y. R. (2008) 28. Chen, L. F., Mu, Y., and Greene, W. C. (2002) Acetylation of RelA at Digoxin and other cardiac glycosides inhibit HIF-1 synthesis and block discrete sites regulates distinct nuclear functions of NF-B. EMBO J. 21, tumor growth. Proc. Natl. Acad. Sci. U.S.A. 105, 19579–19586 6539–6548 32. Koh, M. Y., Darnay, B. G., and Powis, G. (2008) Hypoxia-associated factor, 29. Kim, W., Bennett, E. J., Huttlin, E. L., Guo, A., Li, J., Possemato, A., Sowa, a novel E3-ubiquitin ligase, binds and ubiquitinates hypoxia-inducible fac- M. E., Rad, R., Rush, J., Comb, M. J., Harper, J. W., and Gygi, S. P. (2011) tor 1, leading to its oxygen-independent degradation. Mol. Cell. Biol. 28, Systematic and quantitative assessment of the ubiquitin-modified pro- 7081–7095 teome. Mol. Cell 44, 325–340 33. Luo, W., Hu, H., Chang, R., Zhong, J., Knabel, M., O’Meally, R., Cole, R. N., 30. Liu, Y. V., Baek, J. H., Zhang, H., Diez, R., Cole, R. N., and Semenza, G. L. Pandey, A., and Semenza, G. L. (2011) Pyruvate kinase M2 is a PHD3- (2007) RACK1 competes with HSP90 for binding to HIF-1 and is re- stimulated coactivator for hypoxia-inducible factor 1. Cell 145, 732–744 OCTOBER 12, 2012• VOLUME 287 • NUMBER 42 JOURNAL OF BIOLOGICAL CHEMISTRY 35505

Journal

Journal of Biological Chemistry – American Society for Biochemistry and Molecular Biology

Published: Oct 12, 2012

Keywords: Hypoxia; Hypoxia-inducible Factor (HIF); p300; Post-translational Modification; Protein Stability; Acetylation

There are no references for this article.