Sprouty2 Protein Regulates Hypoxia-inducible Factor-α (HIFα) Protein Levels and Transcription of HIFα-responsive Genes.

crossmark THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 291, NO. 32, pp. 16787–16801, August 5, 2016 © 2016 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

Sprouty2 Protein Regulates Hypoxia-inducible Factor-␣ (HIF␣) Protein Levels and Transcription of HIF␣-responsive Genes*□ S

Received for publication, January 8, 2016, and in revised form, May 27, 2016 Published, JBC Papers in Press, June 8, 2016, DOI 10.1074/jbc.M116.714139

Kristin C. Hicks‡ and Tarun B. Patel§1 From the ‡Department of Molecular Pharmacology and Therapeutics, Loyola University Chicago, Maywood, Illinois 60153, and the § Albany College of Pharmacy and Health Sciences, Albany, New York 12208 The ␣-subunits of hypoxia-inducible factors (HIF1␣ and HIF2␣) promote transcription of genes that regulate glycolysis and cell survival and growth. Sprouty2 (Spry2) is a modulator of receptor tyrosine kinase signaling and inhibits cell proliferation by a number of different mechanisms. Because of the seemingly opposite actions of HIF␣ subunits and Spry2 on cellular processes, we investigated whether Spry2 regulates the levels of HIF1␣ and HIF2␣ proteins. In cell lines from different types of tumors in which the decreased protein levels of Spry2 have been associated with poor prognosis, silencing of Spry2 elevated HIF1␣ protein levels. Increases in HIF1␣ and HIF2␣ protein levels due to silencing of Spry2 also up-regulated HIF␣ target genes. Using HIF1␣ as a prototype, we show that Spry2 decreases HIF1␣ stability and enhances the ubiquitylation of HIF1␣ by a von Hippel-Lindau protein (pVHL)-dependent mechanism. Spry2 also exists in a complex with HIF1␣. Because Spry2 can also associate with pVHL, using a mutant form of Spry2 (3P/3A-Spry2) that binds HIF1␣, but not pVHL, we show that WT-Spry2, but not the 3P/3A-Spry2 decreases HIF1␣ protein levels. In accordance, expression of WT-Spry2, but not 3P/3A-Spry2 results in a decrease in HIF1␣-sensitive glucose uptake. Together our data suggest that Spry2 acts as a scaffold to bring more pVHL/associated E3 ligase in proximity of HIF1␣ and increase its ubiquitylation and degradation. This represents a novel action for Spry2 in modulating biological processes regulated by HIF␣ subunits.

The four Sprouty proteins (Spry1 to Spry4), which are products of different genes, regulate downstream signaling from certain receptor tyrosine kinases and therefore play a major role in development (1–5). Because cell proliferation processes in normal development and tumor growth overlap, some of the Spry2 proteins, such as Spry1, Spry2, and Spry4, also regulate tumor growth (6 –16). Among the four isoforms, Spry2 is ubiq-

* This work was supported by a Schmitt fellowship (to K. H.) and Veterans Affairs Merit Review Award 5IBX002355 (to T. B. P.). The authors declare that they have no conflicts of interest with the contents of this article. □ S This article contains supplemental Tables S1 and S2. 1 To whom correspondence should be addressed: Albany College of Pharmacy and Health Sciences, O’Brien Bldg, Ste. 104G, 106 New Scotland Ave., Albany, NY 12208. E-mail: [email protected]. 2 The abbreviations used are: Spry, Sprouty; HIF, hypoxia-inducible factor; PHD, prolyl hydroxylase domain protein; pVHL, von Hippel-Lindau protein; CHX, cycloheximide; Co-IP, co-immunoprecipitation; IP, immunoprecipitated; ICC, immunocytochemistry; PLA, proximity ligation assay; PFK, phosphofructose kinase; EPO, erythropoietin; WCL, whole cell lysate; qRT,

AUGUST 5, 2016 • VOLUME 291 • NUMBER 32

uitously expressed and well studied in cancer. In cancers of the liver, lung, breast, and prostate, Spry2 levels are decreased (6 –11, 17–19). The decrease in Spry2 levels in these cancers has been correlated to poor patient prognosis and shorter survival of patients implicating Spry2 as a tumor suppressor. Therefore, a number of studies have investigated mechanisms that regulate the expression of Spry2, as well as how Spry2 modulates signaling via receptor tyrosine kinases (20 –25). At the posttranslational level, Spry2 has been shown to be ubiquitylated and targeted for proteosomal degradation by c-Cbl (26, 27), Siah2 (28), Nedd4-1 (29), and pVHL with its associated E3 ligase (30). Interestingly, in some patient-derived hepatocellular carcinomas, when Spry2 levels were decreased, the levels of Nedd4-1 were elevated (8). It is now well established that as tumors proliferate rapidly prior to angiogenesis, the oxygen from the nearby vasculature cannot diffuse throughout the entire tumor resulting in the formation of a hypoxic environment (31). Cells adapt to the hypoxic environment by altering a variety of cellular processes that are heralded by an increase in the levels of the transcription factors, hypoxia-inducible factors (HIFs) (32–36). HIFs are heterodimers composed of one of three ␣-subunits (HIF1␣, HIF2␣, and HIF3␣) and a ␤-subunit (HIF1␤). The role of HIF3␣ in hypoxic gene regulation is not well understood, but a splice variant of HIF3␣ may function as an inhibitor of gene transcription during hypoxia (37, 38). In normoxia, HIF1␣ and HIF2␣ are degraded by a well characterized proteosomal mechanism (39, 40). Prolyl hydroxylase domain proteins (PHDs) bind to the HIF␣ subunits and hydroxylate two proline residues (41, 42). These hydroxy-prolyl residues serve as the docking site for pVHL and its associated E3 ubiquitin ligase resulting in the ubiquitylation and subsequent proteosomal degradation of HIF␣ subunits (43– 46). Because the hydroxylation reaction requires oxygen, in hypoxia the activities of the PHDs are attenuated, and therefore, the hydroxylation, ubiquitylation, and degradation of HIF␣ subunits are inhibited. This permits the accumulation of HIF␣ protein levels and consequently enhances their ability to regulate transcription of genes, such as those that regulate proliferation, angiogenesis, drug metabolism, and glycolysis to promote tumor survival and growth (47– 49).

quantitative RT; GOI, gene of interest; ANOVA, analysis of variance; MEM, minimum Eagle’s medium.

JOURNAL OF BIOLOGICAL CHEMISTRY

16787

Sprouty2 Regulates Hypoxia-inducible Factor-␣ Because of the anti-proliferative and tumor suppressor actions of Spry2, we investigated whether Spry2 alters the levels of HIF␣ subunits and/or their ability to alter transcription of their target genes. Herein, we report the novel ability of endogenous Spry2 to promote the degradation of HIF1␣ and HIF2␣ resulting in a decrease in the expression of the genes that the two HIFs regulate. With HIF1␣ as the prototype of the two HIF␣ proteins (HIF1␣ and HIF2␣), we demonstrate that Spry2 decreases HIF1␣ protein stability and enhances the ubiquitylation of HIF1␣. Using different approaches, we also show that Spry2 exists in a complex with HIF1␣. Because we previously reported that Spry2 is capable of binding to pVHL (30), we hypothesized that Spry2, by binding with HIF1␣, increases the amount of pVHL in the vicinity of HIF1␣ and promotes its degradation through a pVHL-dependent mechanism. Indeed, our studies show that when pVHL is silenced, Spry2 cannot alter HIF1␣ protein levels. Additionally, a mutant form of Spry2 that does not bind to pVHL also does not decrease HIF1␣ protein levels. In agreement, wild-type (WT) Spry2 inhibits HIF1␣sensitive glucose uptake, whereas the mutant form of Spry2, which does not associate with pVHL, does not alter glucose uptake.

Results Endogenous Spry2 Regulates HIF1␣ and HIF2␣ Protein Levels—Because previous studies have suggested that Spry2 plays a critical role in the regulation of hepatocellular, prostate, breast, and lung carcinomas, we utilized HuH7 cells that are derived from a human hepatocellular carcinoma (6 –11, 17–19). In these cells, the siRNA-mediated silencing of endogenous Spry2 resulted in a 2.5-fold elevation of endogenous HIF1␣ protein levels (Fig. 1A). As established by numerous studies, HIF1␣ levels are not detectable in normoxia. However, once the cells are exposed to hypoxia, HIF1␣ protein levels are elevated (Fig. 1A). It is also important to note that as reported in our earlier report (30), Spry2 levels are also elevated by hypoxia (Fig. 1A). The increase in HIF1␣ protein levels upon silencing of endogenous Spry2 is not accompanied by a change in HIF1␣ mRNA levels (Fig. 1B). Like HIF1␣, endogenous HIF2␣ protein levels in HuH7 cells were also increased by hypoxia and further elevated by 2-fold upon silencing of Spry2 (Fig. 1C) without any changes in HIF2␣ mRNA levels (Fig. 1D). A second Spry2 siRNA targeting a different sequence in the mRNA also elevated HIF1␣ and HIF2␣ protein levels in HuH7 cells (Fig. 2A). Notably, Spry2 is modified by phosphorylation and migrates as multiple bands (50), which depending on length of exposure of the blots can be observed as a single band or a doublet (cf. Figs. 1, A and B, and 2A). Together, these findings suggest that Spry2 alters HIF1␣ and HIF2␣ protein levels post-transcriptionally. The generality of our findings is exemplified by the observations that silencing of Spry2 with two different siRNAs results in an elevation of HIF1␣ protein levels in the following panel of cell lines derived from tumors in which Spry2 plays a crucial role: breast cancer-derived cell lines (MCF-7 and MDA-MB231); lung cancer cell line (A549); and another hepatocellular carcinoma cell line (Hep3B) (Fig. 2B). Different cell types express different amounts of Spry2 and different amounts of post-translationally modified Spry2. As mentioned above,

16788 JOURNAL OF BIOLOGICAL CHEMISTRY

FIGURE 1. Silencing of Spry2 increases HIF1␣ and HIF2␣ protein amounts without altering their mRNA levels. HuH7 cells transfected with control (Cntrl) siRNA or siRNA targeting Spry2 were cultured in normoxia (N) (21% O2) or hypoxia (H) (3% O2) for 8 h (A and B) or 24 h (C and D). Representative Western blots probed for HIF1␣ (A), HIF2␣ (images from same blot) (C), Spry2, and actin (loading control) are shown. Graphs in lower panels show mean ⫾ S.E. of densitometric analysis of HIF1␣ (A) or HIF2␣ (B) normalized to actin from three (A) or four (C) independent experiments. Transcript levels of HIF1␣ (B), HIF2␣ (D), and SPRY2 were monitored using qRT-PCR, and relative levels were calculated as described under “Experimental Procedures.” Graphs are mean ⫾ S.E. from three independent experiments. Statistical significance was assessed using unpaired Student’s t tests (A and C); n.s., not significant.

Spry2 migrates as a doublet representing its phosphorylated and dephosphorylated forms (29, 50), and thus, it would appear that in A549 cells a larger amount of Spry2 is phosphorylated, whereas in Hep3B cells a larger amount of dephosphorylated Spry2 is present (Fig. 2B). Likewise, HIF1␣ is also phosphorylated and migrates as a doublet (51–53), and depending upon the cell type, the relative amounts of phosphorylated and dephosphorylated HIF1␣ as well as the total amount of HIF1␣ vary. In contrast to the findings with Spry2 silencing, the ectopic expression of Spry2 in HeLa cells, which do not express detectable protein levels of endogenous Spry2, decreased HIF1␣ protein levels (Fig. 2C). Moreover, expression of the other isoforms of Spry (Spry1, Spry3, and Spry4) also decreased HIF1␣ protein levels (data not shown). These findings suggest that all isoforms of Spry proteins regulate HIF1␣ protein levels. However, to elucidate the mechanisms and impact of the regulation of HIF␣ protein levels, herein we have focused on Spry2 as a prototypic member of this family. Spry2 Regulates the Ability of HIF1␣ and HIF2␣ to Modulate the Transcription of Their Target Genes—HIF1␣ and HIF2␣ form heterodimers with HIF1␤ and act as transcription factors to regulate a variety of genes, including those that regulate glycolysis (54 – 60). To determine whether the ability of HIF1␣ and HIF2␣ to function as transcription factors was altered upon silencing of endogenous Spry2, mRNA levels of HIF1␣- and HIF2␣-regulated genes were monitored using quantitative RTVOLUME 291 • NUMBER 32 • AUGUST 5, 2016

Sprouty2 Regulates Hypoxia-inducible Factor-␣

FIGURE 2. Two siRNAs against Spry2 enhance HIF1␣ protein levels in a variety of cell lines, and ectopic expression of Spry2 decreases HIF1␣ protein levels. A, HuH7 cells transfected with control (Cntrl) siRNA or a different siRNA targeting Spry2 were cultured in hypoxia (H) (3% O2). Western blots probed for HIF1␣, HIF2␣, Spry2, and actin (loading control) are shown. B, MCF7, MDA-MB-231, A549, and Hep3B cells transfected with control (⫺) or one of two different Spry2 siRNAs (1 and 2) were cultured in hypoxia (3% O2). A representative blot probed for HIF1␣, Spry2, and actin and captured with the Bio-Rad ChemiDoc XRS⫹ instrument is shown. Graph shows mean ⫾ S.E. of densitometric analysis of HIF1␣ normalized to actin from three independent experiments. C, control or HA-Spry2-expressing HeLa cells were cultured in normoxia (N) (21% O2) or hypoxia (H) (3% O2). Representative blot probed for HIF1␣, HA-Spry2, and ERK1/2 (loading) is shown. Graph below shows mean ⫾ S.E. of densitometric analysis of HIF1␣ normalized to actin from four independent experiments. Statistical significance was assessed by unpaired Student’s t test (B and C).

PCR. As examples of HIF1␣-regulated genes, we monitored the mRNA levels of the glucose transporter GLUT1 and the glycolytic enzyme phosphoglycerate kinase 1 (PGK1). Consistent with the changes in protein levels of HIF1␣, Spry2 silencing resulted in a significant elevation of GLUT1 and PGK1 mRNA levels (Fig. 3, A and B). The silencing of HIF1␣, but not HIF2␣, abolished the ability of siRNA against Spry2 to elevate GLUT1 and PGK1 mRNA levels demonstrating that silencing of Spry2 mediated its effects on GLUT1 and PGK1 via HIF1␣ and not HIF2␣ (Fig. 3, A and B). As a second approach to monitor the ability of Spry2 to modulate HIF1␣-elicited transcription, we transfected HuH7 cells with a luciferase reporter plasmid composed of the phosphofructose kinase (PFK) promoter fused to a luciferase reporter gene (PFK-Luc) and a Renilla luciferase plasmid as a control for AUGUST 5, 2016 • VOLUME 291 • NUMBER 32

transfection efficiency. Transcription of the PFK gene and the PFK promoter-Luc construct is regulated by HIF1␣ but not by HIF2␣ (58). In these cells, the ability of HIF1␣ to regulate transcription, as measured by luciferase activity, was elevated 2.5fold by hypoxia (Fig. 3C). Moreover, silencing of Spry2 further elevated luciferase activity by 63% (Fig. 3C). Silencing of HIF1␣, but not HIF2␣, abolished the ability of Spry2 targeting siRNA to enhance luciferase activity (Fig. 3C) demonstrating that silencing of Spry2 enhances the transcription of PFK-Luc via changes in HIF1␣ levels. These data (Fig. 3, A–C) also show that, as reported by others (54, 55, 58, 59), the expressions of GLUT1, PGK1, and PFK-Luc are regulated by HIF1␣ and not HIF2␣. Recently, erythropoietin (EPO) was identified as an HIF2␣responsive gene (61, 62). Therefore, to determine whether the enhanced HIF2␣ levels observed upon silencing of Spry2 (Fig. JOURNAL OF BIOLOGICAL CHEMISTRY

16789


FIGURE 3. Spry2 silencing enhances the transcript levels of HIF1␣- and HIF2␣-regulated genes and activity of the PFK-Luc reporter. HuH7 cells were transfected with control siRNA or siRNAs against Spry2, Spry2 ⫹ HIF1␣, or Spry2 ⫹ HIF2␣ and cultured in hypoxia (3% O2). Transcript levels of GLUT-1 (A), PGK1 (B), EPO (D), SPRY2 (E), HIF1␣ (F), and HIF2␣ (G) were monitored using qRT-PCR, and relative levels were calculated as described under “Experimental Procedures.” C, HuH7 cells were transfected with the same siRNAs as described for A, B, and D along with pGL2-Pfkfb3/⫺3566 and pRG-TK (as control) and cultured in normoxia (21% O2) or hypoxia (3% O2) for 24 h. Luciferase assays were performed as described under “Experimental Procedures.” Data shown are the mean ⫾ S.E. from four (B and D–G), three (A), or at least three (C) independent experiments. Statistical significance was assessed by one-way ANOVA with Tukey’s multiple comparisons (A, B, and D–G) or two-way ANOVA with Sidak’s multiple comparisons (C).

1B) also altered the transcription of the HIF2␣-responsive EPO gene, we monitored EPO mRNA levels in HuH7 cells. Akin to the findings with GLUT1 and PGK1 for HIF1␣, the silencing of Spry2 elevated EPO mRNA levels, and the silencing of HIF2␣, but not HIF1␣, obliterated the ability of the siRNA against Spry2 to elevate EPO mRNA levels (Fig. 3D). The efficient silencing of the SPRY2, HIF1␣, and HIF2␣ expression by the different siRNAs for the qRT-PCR data is shown in Fig. 3, E–G. Overall, the data presented thus far demonstrate that Spry2 decreases HIF1␣ and HIF2␣ protein amounts without altering the mRNA levels of HIF1␣ or HIF2␣, and when Spry2 expression is silenced, the elevation in their protein levels increases the transcription of their respective target genes. Hence, endogenous Spry2 regulates the endogenous HIF␣ protein content and the transcription of their target genes.


Spry2 Regulates the Stability and Ubiquitylation of HIF1␣— The lack of any changes in mRNA levels of HIF1␣ or HIF2␣ when expression of Spry2 is silenced in the face of a 2–2.5-fold increase in the levels of the two proteins (Fig. 1) would suggest that Spry2 regulates HIF1␣ and HIF2␣ post-translationally. The post-translational regulation of HIF1␣ and HIF2␣ involves very similar mechanisms consisting of hydroxylation of two prolyl residues that serve as the binding site for von HippelLindau protein (pVHL) and associated E3 ligase (39, 40). Therefore, using HIF1␣ as a prototypic member, we first examined whether Spry2 altered the stability of the HIF1␣ protein. Essentially, HuH7 cells transfected with control siRNA or siRNA against Spry2 were treated with the protein translation inhibitor cycloheximide, and the levels of HIF1␣ were monitored at different times. As shown in Fig. 4A, silencing of Spry2 increased the half-life of HIF1␣ from 4.3 to 7.1 min. The major VOLUME 291 • NUMBER 32 • AUGUST 5, 2016


FIGURE 4. Spry2 decreases the stability of HIF1␣ by enhancing the ubiquitylation of HIF1␣. A, HuH7 cells transfected with control (Cntrl) siRNA or siRNA targeting Spry2 were cultured in hypoxia (3% O2) for 8 h. Cycloheximide (CHX) (200 ␮M) was added, and cells were lysed at the indicated times. Representative Western blots probed for HIF1␣, Spry2, actin, and ERK1/2 are shown. Graph shows mean ⫾ S.E. of densitometric analysis of HIF1␣ normalized to the average of actin and ERK1/2 from five independent experiments. Half-lives are listed in legend. Statistical significance was assessed by unpaired Student’s t test. B, HEK293T cells were transfected with constructs of HIF1␣ and FLAG-tagged ubiquitin (Ub) along with either control (Cntrl) or Spry2 siRNAs. Cells were cultured in hypoxia (3% O2) for 16 h, and MG132 (25 ␮M) was added in during the last 4 h of incubation. All FLAG-tagged ubiquitylated proteins were IP, and the amount of ubiquitylated HIF1␣ was monitored by Western blotting analyses; immunoprecipitation with nonspecific mouse IgG served as control. WCL were analyzed for HIF1␣, Spry2, FLAG-tagged ubiquitin, and actin (loading). Graph shows mean ⫾ S.E. of densitometric analysis of ubiquitylated HIF1␣ divided by total immunoprecipitated FLAG-tagged ubiquitylated proteins from three independent experiments. Statistical significance was assessed by unpaired Student’s t test.

degradation pathway for HIF1␣ involves ubiquitylation followed by proteosomal degradation (63). Therefore, we directly determined whether Spry2 altered the ubiquitylation of HIF1␣. For this purpose, we utilized HEK293T cells because the transfection efficiency of HuH7 cells with FLAG-ubiquitin was very low. HEK293T cells were transfected with FLAG-ubiquitin and either control siRNA or siRNA against Spry2. The whole cell lysate (WCL) shows efficient silencing of Spry2 and equal expression of FLAG-ubiquitin (Fig. 4B, left panel). As mentioned earlier, HIF1␣ appears as a single or double band depending upon its phosphorylation state (see Fig. 2B). In HEK293T, cells we only observe a single band; whether this is the phosphorylated or dephosphorylated HIF1␣ is difficult to determine. Immunoprecipitation of HEK293T cell lysates under denaturing conditions with nonspecific IgG or antiFLAG antibody showed that the anti-FLAG antibody immunoprecipitates similar amounts of total FLAG-tagged ubiquitylated proteins from cells treated with control siRNA and siRNA against Spry2 (Fig. 4B, right panel). Probing the immunoprecipitates for HIF1␣ assessed the amount of ubiquitylated HIF1␣ in each sample. When Spry2 was silenced, the amount of ubiquitylated HIF1␣ was ⬃50% lower than that in control siRNA-transfected cells (Fig. 4B, right panel and bar graph at bottom). These data (Fig. 4) suggest that endogenous Spry2 decreases HIF1␣ stability by increasing the ubiquitylation of HIF1␣ and targeting it for proteosomal degradation. Spry2 Exists in a Complex with HIF1␣ and Regulates HIF1␣ through pVHL-dependent Mechanism—To identify the mechanism by which Spry2 regulates HIF1␣ ubiquitylation and degradation, we determined whether endogenous Spry2 and HIF1␣ exist in the same complex. In hypoxic HuH7 cells treated AUGUST 5, 2016 • VOLUME 291 • NUMBER 32

with the proteosomal inhibitor MG132, immunoprecipitates of Spry2 contained endogenous HIF1␣ (Fig. 5A). Additionally, by immunocytochemistry, we also observed the co-localization of Spry2 and HIF1␣, and this co-localization was markedly decreased upon silencing of either HIF1␣ or Spry2 (Fig. 5B). The co-localization of Spry2 and HIF1␣ within intact cells was further confirmed using the proximity ligation assay (PLA) that permits detection of interacting proteins within intact cells (64). As shown in Fig. 5C, the PLA approach also showed that Spry2 and HIF1␣ are in close proximity, and the PLA signal is markedly diminished when HIF1␣ is silenced. Interestingly, the data in Fig. 5, B and C, show that Spry2 and HIF1␣ interact in the nucleus. Although the localization of Spry2 in cytoplasm and membrane ruffles has been previously reported (65, 66), this is the first demonstration of Spry2 being present in the nucleus. Overall, using three different approaches, the data in Fig. 5 demonstrate that endogenous Spry2 and HIF1␣ exist in a complex. Previous work from our laboratory has shown that Spry2 is also hydroxylated by PHDs and can bind to pVHL (30). With this in mind and the data demonstrating the existence of endogenous Spry2 in a complex with HIF1␣ (Fig. 5), we postulated that Spry2 by associating with HIF1␣ brings more pVHL in proximity of HIF1␣, and therefore, it enhances the degradation of HIF1␣. To investigate the involvement of pVHL in the ability of Spry2 to regulate HIF1␣, HuH7 cells were transfected with two different siRNAs targeting pVHL and the effect of Spry2 silencing on HIF1␣ protein was monitored. Indeed, as shown previously (Fig. 1A), silencing of Spry2 resulted in a significant increase in HIF1␣ protein levels without altering pVHL protein levels (Fig. 6A). However, once pVHL is silenced JOURNAL OF BIOLOGICAL CHEMISTRY

16791


FIGURE 5. Spry2 exists in a complex with HIF1␣. A, HuH7 cells incubated in hypoxia (3% O2) for 8 h were treated with MG132 (25 ␮M) during the last 4 h of the incubation. Spry2 was IP, and the co-immunoprecipitation of HIF1␣ was monitored; nonspecific rabbit IgG was used in control immunoprecipitations. A representative blot from three independent experiments is shown. WCL were analyzed for HIF1␣ and Spry2. B, HuH7 cells transfected with indicated siRNAs (20 nM each) were incubated in hypoxia for 8 h and treated with MG132 (10 ␮M) for the last 2 h of incubation. Immunocytochemistry was performed as described under “Experimental Procedures” to stain for Spry2 (green) and HIF1␣ (red). Images shown are ⫻126 magnification. White bar (bottom left) is the scale for 20 ␮m. C, HuH7 cells were incubated in hypoxia (3% O2) for 8 h. A PLA reaction was performed as described under “Experimental Procedures” to look at Spry2 and HIF1␣ interaction. Images shown are ⫻63 magnification, and the inset (top left) is three times greater. White bar (bottom right) is scale for 100 ␮m. Controls included PLA secondary antibodies (secondary only) alone and complete PLA assay without HIF1␣ antibody (omit one primary). Graph shows mean ⫾ variance of the quantified puncta divided by the number of cells in the field with the background from the control with 2° antibody only (no 1° antibody) subtracted and normalized to control siRNA-transfected cells for two independent experiments in which 10 –15 fields were analyzed for each condition.

with either of the two pVHL targeting siRNAs, the silencing of Spry2 no longer affected HIF1␣ protein levels (Fig. 6A). These data suggest that Spry2 regulates HIF1␣ in a pVHLdependent mechanism. To further investigate the contribution of pVHL in the ability of Spry2 to regulate HIF1␣, we utilized a mutant form of Spry2 that cannot bind pVHL. Previously, using proteomic analyses we showed that Pro-18, Pro-144, and Pro-160 on Spry2 are hydroxylated (30). The substitution of these three Pro residues to Ala abolished the ability of Spry2 to bind pVHL (30). Here, we have designated this 3Pro 3 3Ala substituted form of Spry2 as “3P/3A-Spry2.” To determine whether the 3P/3A-Spry2 could alter HIF1␣ protein levels, HEK293T cells were transfected with empty vector, wild-type (WT)-Spry2, or 3P/3A-Spry2, and HIF1␣ levels were monitored. As expected and shown for HeLa cells (Fig. 2C), the expression of WT-Spry2 resulted in a 50% reduction in HIF1␣ protein levels compared with empty vector transfected cells (Fig. 6B). Intriguingly, the expression of 3P/3A-Spry2,


which cannot bind pVHL, had no significant effect on HIF1␣ protein levels compared with vector-transfected cells (Fig. 6B). Similar results were observed in HeLa cells stably expressing no Spry2, WT-Spry2, or 3P/3A-Spry2 (Fig. 6C). Once again, HeLa cells expressing WT-Spry2 had a 50% reduction in HIF1␣ protein levels, although the expression of 3P/3A-Spry2 had no significant effect on HIF1␣ levels when compared with cells not expressing Spry2 (Fig. 6C). These data provide further evidence that Spry2 regulates HIF1␣ in a pVHL-dependent manner because when Spry2 cannot bind pVHL, it cannot alter HIF1␣ protein levels. To determine whether the ubiquitylation of HIF1␣ was altered when WT-Spry2 or 3P/3A-Spry2 were overexpressed in HEK293T cells, we transfected these two forms of Spry2 together with FLAG-ubiquitin and HIF1␣ and monitored HIF1␣ ubiquitylation after immunoprecipitating HIF1␣. As shown in Fig. 6D, ubiquitylation of HIF1␣ in cells expressing WT-Spry2 was approximately twice as high as that in cells expressing 3P/3A-Spry2. VOLUME 291 • NUMBER 32 • AUGUST 5, 2016


FIGURE 6. Spry2 enhances the degradation of HIF1␣ in a pVHL-dependent manner. A, HuH7 were transfected with control (Cntrl) or Spry2 siRNAs along with control (⫺) or one of the two pVHL siRNAs (1 or 4) and cultured in hypoxia (3% O2) for 8 h. Representative Western blots probed for HIF1␣, Spry2, pVHL, and actin (loading) are shown. Graph below shows mean ⫾ S.E. of densitometric analysis of HIF1␣ normalized to actin from four independent experiments. B, HEK293T cells transfected with empty vector or vector constructs to express WT-Spry2 or 3P/3A-Spry2 were cultured in hypoxia (3% O2) for 16 h. Representative Western blots probed for HIF1␣, HA-Spry2, actin, and ERK1/2 are shown. Graph below shows mean ⫾ S.E. of densitometric analysis of HIF1␣ normalized to the average of actin and ERK1/2 from four independent experiments. C, control and HA-Spry2 (either WT-Spry2 or 3P/3A-Spry2) expressing HeLa cells were cultured in hypoxia for 24 h. Representative Western blots probed for HIF1␣, HA-Spry2, and tubulin (loading control) are shown. Graph below shows mean ⫾ S.E. of densitometric analysis of HIF1␣ normalized to tubulin from four independent experiments. D, HEK293T cells were transfected with constructs of HIF1␣ and FLAG-tagged ubiquitin along with constructs of WT-Spry2 or 3P/3A-Spry2. Cells were cultured in hypoxia (3% O2) for 16 h, and MG132 (10 ␮M) was added in during the last 2 h of incubation. HIF1␣ was IP, and the amount of ubiquitylated HIF1␣ was monitored by Western blotting analyses; immunoprecipitation with nonspecific mouse IgG served as control. WCL were analyzed for HIF1␣, Spry2, FLAG-tagged ubiquitin and tubulin (loading). Western blot images were captured using the Bio-Rad ChemiDoc XRS⫹ instrument. Graph shows mean ⫾ S.E. of densitometric analysis of ubiquitylated HIF1␣ divided by total immunoprecipitated HIF1␣ from three independent experiments. E, HuH7 cells transfected with control or Spry2 siRNAs were incubated in hypoxia (3% O2) for 8 h. pVHL was IP, and the co-immunoprecipitation of HIF1␣ was monitored. Western blot images were captured using the Bio-Rad ChemiDoc XRS⫹ instrument. Graph below shows mean ⫾ S.E. of densitometric analysis of HIF1␣ normalized to pVHL immunoprecipitated from four independent experiments. Statistical significance was assessed by unpaired Student’s t test (A and C–E) and one-way ANOVA (B) with Tukey’s multiple comparisons; n.s., not significant.

We reasoned that if Spry2 was bringing more pVHL in proximity to HIF1␣, then in immunoprecipitates of pVHL the amount of HIF1␣ co-immunoprecipitating should be diminished when Spry2 is silenced by siRNA. Indeed, when Spry2 was silenced in HuH7 cells, immunoprecipitates of pVHL contained ⬃50% less HIF1␣ (Fig. 6E). Notably, the protein levels of PHD1, PHD2, PHD3, and pVHL were not significantly altered by the expression of WT-Spry2 or 3P/3A-Spry2 compared with empty vector-transfected cells (Fig. 7A). 3P/3A-Spry2 Can Associate with HIF1␣ and Inhibit the Phosphorylation of AKT—To address the possibility that the mutation of the three proline residues in 3P/3A-Spry2 abrogated its ability to associate with HIF1␣ and therefore abolished its abilAUGUST 5, 2016 • VOLUME 291 • NUMBER 32

ity to alter HIF1␣ protein levels, we investigated whether the 3P/3A-Spry2 interacted with HIF1␣. HEK293T cells were transfected with HIF1␣ and either WT-Spry2 or 3P/3A-Spry2 and placed in hypoxia. The WCL indicates that equal amounts of the two Spry2 constructs were transfected into the cells (Fig. 7B). Because both WT-Spry2 and 3P/3A-Spry2 co-immunoprecipitated with HIF1␣ (Fig. 7B), the substitution of the three prolines on Spry2 with alanines does not alter the ability of Spry2 to associate with HIF1␣. Spry2 has been shown to inhibit the downstream signaling processes such as phosphorylation and activation of AKT (22, 67, 68). Therefore, we also determined whether the 3P/3ASpry2 retained its ability to modulate downstream signaling JOURNAL OF BIOLOGICAL CHEMISTRY

16793


FIGURE 7. 3P/3A-Spry2 interacts with HIF1␣ and inhibits phosphorylation of AKT to a similar extent as WT-Spry2. A, HEK293T lysates from Fig. 6B were used to monitor the levels of pVHL, PHD1, PHD2, PHD3, Spry2, actin, and ERK1/2 by Western blotting analyses. A representative blot is shown, and the graph shows mean ⫾ S.E. of densitometric analysis of protein of interest normalized to the average of actin and ERK1/2 from four independent experiments. B, HEK293T cells transfected with vector constructs to express HIF1␣ and either WT-Spry2 or 3P/3A-Spry2 were cultured in hypoxia (3% O2) for 16 h. HIF1␣ was IP, and the co-immunoprecipitation of Spry2 was monitored; immunoprecipitation with nonspecific IgG was used a control. Western blot images were captured on the Bio-Rad ChemiDoc XRS⫹ instrument. A representative blot from three independent experiments is shown. WCLs were probed for HIF1␣, HA-Spry2, and actin (loading). C, HEK293T lysates from Fig. 6B were used to probe for phospho-AKT Ser-473, and HA-Spry2 and actin (loading) are shown. Graph below shows mean ⫾ S.E. of densitometric analysis of pAKT normalized to actin from four independent experiments. Statistical significance was assessed by a one-way ANOVA with Tukey’s multiple comparisons.

such as phosphorylation of AKT. Using the HEK293T cell lysate from experiments described in Fig. 6B, the levels of phosphorylation of AKT (pAKT) on Ser-473 were monitored. As expected from our previous findings (69), the expression of WT-Spry2 reduced pAKT content by more than 50%. Likewise, 3P/3A-Spry2 also reduced pAKT levels to a similar extent (Fig. 7C). Thus, 3P/3A-Spry2 retains its ability to inhibit the phosphorylation of AKT. Taken together, these data indicate that 3P/3A-Spry2 only lacks the ability to bind to pVHL (30) and to alter HIF1␣ levels (Fig. 6, B and C) but retains its other functions such as binding to HIF1␣ and inhibiting AKT phosphorylation (Fig. 7, B and C). WT-Spry2, but Not 3P/3A-Spry2, Regulates HIF1␣-sensitive Glucose Uptake—Because 3P/3A-Spry2 does not alter HIF1␣ protein levels, we reasoned that a biological function that is regulated by HIF1␣ would be susceptible to modulation by WT-Spry2 but not 3P/3A-Spry2. In this context, it is well established that HIF1␣ regulates the expression of certain glucose transporters, such as GLUT1 and GLUT3, and the increased expression of these glucose transporters enhances glucose


FIGURE 8. WT-Spry2, but not 3P/3A-Spry2, reduces HIF1␣-sensitive 2-deoxy-D-[3H]glucose uptake. A, HeLa cells transfected with control (Cntrl) (closed circle) or HIF1␣-specific siRNA (closed square) were cultured in hypoxia for 24 h. 2-Deoxy-D-[3H]glucose (100 ␮M) uptake was monitored over the time course shown following the protocol described under “Experimental Procedures.” Samples treated in an identical manner but with excess unlabeled 2-deoxy-D-glucose (20 mM) were used a control to demonstrate specificity (open circle and square). Cells were lysed, and the amount of radioactivity was measured in a scintillation counter, and a protein determination was performed on the lysates. Data are presented as picomoles of 2-deoxy-D-[3H]glucose uptake per ␮g of protein. HeLa cells (B) and HuH7 cells (C) were transfected with empty vector or vector constructs to express WT-Spry2 or 3P/3A-Spry2 along with control (Cntrl) or HIF1␣-specific siRNA. 2-Deoxy-D-[3H]glucose uptake over 10 min was monitored. HIF1␣-sensitive uptake was calculated by subtracting the 2-deoxy-D-[3H]glucose uptake from HIF1␣ siRNA-transfected cells from the uptake in control siRNA-transfected cells. Graph shows mean ⫾ S.E. of picomoles of HIF1␣-sensitive 2-deoxy-D-[3H]glucose per ␮g of protein from three independent experiments. Statistical significance was assessed by one-way ANOVA with Dunnett’s multiple comparisons (B) or unpaired student t test (C), n.s., not significant.

uptake into cells (59, 70). Therefore, in HuH7 and HeLa cells, we monitored HIF1␣-sensitive glucose uptake after transfecting either WT- or 3P/3A-Spry2. As shown in Fig. 8A, 2-deoxyVOLUME 291 • NUMBER 32 • AUGUST 5, 2016

Sprouty2 Regulates Hypoxia-inducible Factor-␣ D-[

3

H]glucose uptake in HeLa cells exposed to hypoxia was linear over time, and silencing of HIF1␣ decreases 2-deoxy-D[3H]glucose uptake by 31%. Because HIF1␣ does not regulate all isoforms of glucose transporters, silencing of HIF1␣ only diminishes that portion of the glucose transport that is HIF1␣dependent; in the case of HeLa cells, this is 30% of the total glucose uptake in hypoxia. Here, we refer to this as the HIF1␣sensitive glucose uptake. Moreover, the addition of unlabeled 2-deoxy-D-glucose markedly inhibited 2-deoxy-D-[3H]glucose uptake in the presence and absence of HIF1␣ demonstrating the specificity of glucose uptake via glucose transporters (Fig. 8A). For the experimental data presented in Fig. 8, B and C, by monitoring 2-deoxy-D-[3H]glucose uptake with or without HIF1␣ silencing, we determined the amount of HIF1␣-sensitive glucose uptake in cells transfected with vector, WT-Spry2, or 3P/3A-Spry2. In HeLa cells, the HIF1␣-sensitive portion of glucose uptake was significantly inhibited (61%) by WT-Spry2, but it was not affected by 3P/3A-Spry2 (Fig. 8B). Similarly, in HuH7 cells, WT-Spry2 reduced HIF1␣-sensitive 2-deoxy-D-[3H]glucose uptake by 73%, whereas 3P/3A-Spry2 did not affect it (Fig. 8C). Additionally, in HuH7 cells, total glucose uptake was elevated by ⬃3.5-fold upon exposure to hypoxia, and interestingly, the transfection of WT-Spry2 or 3P/3A Spry2 did not alter glucose uptake in cells incubated in normoxia in which HIF1␣ protein levels would be much lower (data not shown). These findings are consistent with the observations that WT-Spry2, but not 3P/3A-Spry2, regulates HIF1␣ protein content in cells (Fig. 6, B and C).

Discussion Sprouty proteins, especially Spry2, working via a number of different mechanisms, have been shown to modulate signaling downstream of receptor tyrosine kinases to inhibit cell proliferation and migration (20, 21, 71, 72). Consistent with these findings, several studies have shown that Spry2 levels are decreased in different forms of cancer, including those of the breast, liver, prostate, and lung, suggesting a potential role for Spry2 as a tumor suppressor (6 –11, 17–19). Because decreased protein levels of Spry2 in hepatocellular carcinomas have been associated with poor patient survival and prognosis, it has been suggested that Spry2 levels could be utilized as a prognostic marker (73). However, the role of HIF␣ subunits, in particular HIF1␣, is now well established in promoting tumor survival, growth, and metastasis. Specifically, HIF1␣ by augmenting the transcription of genes encoding for glucose transporters, GLUT1 and GLUT3, as well as several glycolytic enzymes, provides tumors with a growth advantage in hypoxia (54 – 60). Hence, although Spry2 may have tumor suppressor functions, HIF␣ subunits, especially HIF1␣, act as tumor promoters (47– 49). In this study, using HIF1␣ as a prototype of HIF␣ subunits, we have uncovered a novel mechanism for regulation of HIF␣ protein levels by Spry2. Our findings, for the first time, demonstrate that endogenous Spry2 regulates HIF1␣ protein levels in a variety of different cell types derived from breast (MCF-7 and MDA-MB-231), liver (HuH7 and Hep3B), and lung (A549) cancer in which Spry2 (Fig. 2B) plays a critical role. Additionally, silencing of Spry2 in HEK293T (Fig. 4B) or its ectopic expresAUGUST 5, 2016 • VOLUME 291 • NUMBER 32

sion in these cells and HeLa cells regulates HIF1␣ protein levels (Figs. 2C and 6, B and C). These observations establish the generality of our findings. The silencing of endogenous Spry2 elevates HIF1␣ and HIF2␣ protein levels (Fig. 1, A and C) and augments their ability to regulate their respective target genes GLUT1, PGK1, and PFK for HIF1␣ and EPO for HIF2␣ (Fig. 3, A–D). Consistently, the overexpression of Spry2 (Figs. 2C and 6B) or other Spry isoforms, Spry1, Spry3, and Spry4 (data not shown), decrease HIF1␣ levels demonstrating the ability of different Spry isoforms to modulate HIF1␣ levels. Hence, the findings described in this study are also applicable to the other Spry isoforms. It is well established that pVHL and associated E3 ligase degrades HIF1␣ and HIF2␣ under normoxic conditions via a process that involves their hydroxylation by PHDs (41, 42). In hypoxia, the lack of free molecular oxygen reduces, but does not completely inhibit, activities of the PHDs (74, 75). This results in a reduction of pVHL/associated E3 ligase binding to HIF␣ subunits, and therefore, a decrease in their ubiquitylation and degradation in hypoxia (39, 40). Herein, we demonstrate that Spry2 decreases the amount of HIF1␣ by increasing its ubiquitylation and degradation even in hypoxic conditions suggesting that Spry2 enhances the proteosomal degradation of HIF1␣ (Fig. 4). This process requires pVHL because silencing of pVHL abrogated the ability of Spry2 to regulate HIF1␣ (Fig. 6A). Furthermore, for the reasons described below, our data suggest that Spry2, which we have previously shown to bind pVHL (30), exists in a complex with HIF1␣ and thereby brings more pVHL in the proximity of HIF1␣ to permit its ubiquitylation and degradation. First, using three different approaches, we demonstrate that Spry2 and HIF1␣ exist in a complex (Fig. 5). Second, the silencing of pVHL expression abrogates the increase in HIF1␣ levels when Spry2 is silenced, and the expression of WT-Spry2, but not 3P/3A-Spry2 that does not bind pVHL (30), decreases HIF1␣ levels (Fig. 6, A–C). Notably, 3P/3A-Spry2 is as effective as WT-Spry2 in attenuating AKT phosphorylation (Fig. 7C) and associating with HIF1␣ (Fig. 7B). Thus, 3P/3A-Spry2 does not lose all of its biological functions. Third, ubiquitylation of HIF1␣ when WT-Spry2 is overexpressed is enhanced 2-fold as compared to when 3P/3A-Spry2 is overexpressed (Fig. 6D), and immunoprecipitates of pVHL from HuH7 cells in which Spry2 has been silenced contain ⬃50% less HIF1␣ (Fig. 6E). All of this evidence strongly supports the notion that Spry2, by interacting with both pVHL and HIF1␣, brings more pVHL in proximity of HIF1␣ and thereby enhances HIF1␣ ubiquitylation and degradation. The latter is supported by our data that show silencing of Spry2 diminishes the ubiquitylation of HIF1␣ (Fig. 4B). Consistent with our proposal that Spry2 acts as a scaffold to bring more pVHL/associated E3 ligase in proximity of HIF1␣, unlike WT-Spry2, the 3P/3A-Spry2 that does not bind pVHL also does not inhibit HIF1␣-sensitive glucose transport in HeLa and HuH7 cells (Fig. 8). The paradigm we propose fits well with the function of Spry2 as a scaffolding protein, which by interacting with other proteins regulates different signaling pathways and biological functions (20 –25). One of the interesting and novel observations by immunocytochemistry and PLA (Fig. 5, B and C) from our studies is the JOURNAL OF BIOLOGICAL CHEMISTRY

16795

Sprouty2 Regulates Hypoxia-inducible Factor-␣ nuclear co-localization of Spry2 with HIF1␣. Although we and others have shown cytoplasmic and membrane localization of Spry2 (65, 66), to date its nuclear localization has not been reported. Notably, Spry2 does not have a nuclear localization sequence, and how it enters the nucleus is not known. However, as clearly shown here, it can interact with nuclear proteins such as HIF1␣ and perhaps other nuclear proteins not yet identified. This adds a novel dimension by which Spry2 can regulate biological processes. Although increasing HIF stability by inhibiting pVHL has been shown previously (76 –78), only one report, aside from this study, has shown that HIF1␣ degradation by pVHL can be enhanced in hypoxia. Specifically, that report showed that SSAT2 binding to pVHL stabilized the interactions between pVHL and elongin C and enhanced the ability of pVHL to degrade HIF1␣ (79). Likewise, it has been shown that recruitment of PHDs in the proximity of HIF1␣ can enhance its degradation in hypoxia. Osteosarcoma-amplified 9 (OS-9) and mitogen-activated protein kinase organizer 1 (MORG1) by associating with PHDs and bringing them in proximity to HIF1␣ enhance the hydroxylation and therefore the degradation of HIF1␣ in hypoxia (80, 81). Furthermore, by a mechanism similar to what we have shown here, HIF1␣ degradation is enhanced by p53 interacting with HIF1␣ and bringing the E3 ubiquitin ligase MDM2 in proximity of HIF1␣ (82). Altogether, these previous reports support the concept that a protein can enhance the degradation of another protein by bringing it into proximity to an enzyme that causes its degradation. However, our findings reported here for the first time demonstrate that Spry2 by scaffolding HIF1␣ and pVHL can augment the ubiquitylation of HIF1␣ and decrease its stability. Reports in the literature suggest that AKT and ERK can regulate HIF1␣ translation, protein levels, and activity (83– 88). The role of AKT regulation of HIF1␣ appears to be cell typespecific and not critical for HIF1␣ stabilization in hypoxia (89). Nevertheless, the ability of Spry2 to inhibit ERK and AKT activation could provide a possible mechanism by which Spry2 regulates HIF1␣ protein levels. However, we have shown that both WT-Spry2 and 3P/3A-Spry2 inhibit the phosphorylation of AKT (Fig. 7C), but only WT-Spry2 decreases HIF1␣ protein levels suggesting AKT activity has no role in the ability of Spry2 to regulate HIF1␣ (Fig. 6, B and C). Likewise, under the same conditions as those in Fig. 1, silencing of Spry2 in HuH7 cells did not alter ERK1/2 phosphorylation status suggesting that ERK1/2 do not contribute to the elevation of HIF1␣ protein levels (data not shown). One study by Liu et al. (84) demonstrated that microRNA-21 (miR-21) elevated HIF1␣ levels due to modest increases in AKT and ERK1/2 activation. However, miR-21 also decreases the expression of Spry2 (90). Hence, in light of our findings, one could propose that in the studies of Liu et al. (84) miR21-mediated decrease in Spry2 levels, which was not monitored in that study, contributes to the elevation in HIF1␣ protein levels. Our findings that silencing of endogenous Spry2 elevates the transcription of HIF1␣-responsive genes (Fig. 3, A–C) and that Spry2 also inhibits HIF1␣-sensitive glucose uptake (Fig. 8) implicate Spry2 as a regulator of glycolysis. In this context, Wang et al. (91) showed that expression of a dominant negative


Spry2 with AKT resulted in increased membrane levels of GLUT1 and GLUT4 as well as an increase in the glycolysis master regulator pyruvate kinase M2 (PKM2). However, that study did not delineate the mechanism by which Spry2 up-regulated PKM2. Given our findings in this report and the fact that PKM2 transcription and expression are elevated by HIF1␣ (92), it is tempting to speculate that the up-regulation of PKM2, GLUT1, and GLUT4 by dominant negative Spry2 is due to an elevation of HIF1␣ levels because dominant negative Spry2 would ablate the ability of endogenous Spry2 to decrease HIF1␣ levels. In summary, we showed that Spry2 acts as a scaffold that complexes with HIF1␣ and pVHL elevating the local concentrations of pVHL to ubiquitylate HIF1␣ in hypoxia and target it for degradation. The regulation of HIF1␣ levels by Spry2 also translates into regulation of HIF1␣-responsive genes and glucose uptake. Thus, Spry2 also inhibits the expression of glycolytic genes and HIF1␣-sensitive glucose uptake. Because glucose uptake and glycolysis are critical in providing survival advantage and promotion of cancer, our data suggest Spry2, in part, may exert its “tumor suppressor” actions by regulating HIF1␣ protein levels and gene transcription. As a corollary, when Spry2 is decreased or lost in certain forms of cancer, the transcription of HIF1␣-regulated genes would be elevated and further promote the progression of the cancer. Overall, these findings reveal a novel mode of action for Spry2 in regulating cellular signaling and biological functions such as glucose uptake.

Experimental Procedures Chemicals and Reagents—Cycloheximide and N-ethylmaleimide were purchased from Sigma. MG132 was purchased from Selleck Chem (Houston, TX). All primers, probes, and siRNAs were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA). The sequences of the various siRNAs used are described in supplemental Table 1A. Antibodies used for different applications and their dilutions are listed in supplemental Table 1B. DNA Constructs—The cloning of human Spry2 cDNA into the pHM6-HA-vector is described elsewhere (93). pHM6-HA3P/3A-Spry2 was generated using site-directed mutagenesis of the wild-type pHM6-HA-Spry2 to mutate prolines 18, 144, and 160 (30). The cDNA of HIF1␣ was purchased from Origene and subcloned into pcDNA3 vector using HindIII and NotI restriction sites. pRG-TK was kindly provided by Dr. Neil Clipstone, Loyola University Chicago. pGL2-Pfkfb3/⫺3566 was kindly provided by Ramon Bartrons, University of Barcelona. pCMV3⫻-FLAG-ubiquitin was kindly provided by Dr. Adriano Marchese, Medical College of Wisconsin. The cloning of FLAG-Spry2 was described previously (29). FLAG-Spry1, FLAG-Spry3, and FLAG-Spry4 were generous gifts from Dr. Graeme Guy, Institute of Molecular and Cell Biology, Singapore. All constructs were verified by sequencing. Cell Culture—HuH7 and Hep3B cells were kindly provided by Dr. Basabi Rana, University of Illinois at Chicago. HuH7 cells were cultured in DMEM/nutrient mixture F-12 50:50 supplemented with HEPES (10 mM), 10% FBS, penicillin (100 units/ ml), and streptomycin (100 ␮g/ml). Hep3B cells were cultured VOLUME 291 • NUMBER 32 • AUGUST 5, 2016

Sprouty2 Regulates Hypoxia-inducible Factor-␣ in MEM supplemented with HEPES (10 mM), sodium pyruvate (1 mM), MEM non-essential amino acid solution (1⫻), 10% FBS, penicillin (100 units/ml), and streptomycin (100 ␮g/ml). HEK293T cells were kindly provided by Dr. Jody Martin, Loyola University, Chicago. HeLa cells were kindly gifted by the late Dr. Jill Lahti, St. Jude Children’s Research Hospital, Memphis, TN. HEK293T and HeLa cells were cultured in high glucose containing DMEM supplemented with 10% FBS, penicillin (100 units/ml), and streptomycin (100 ␮g/ml). MDA-MB-231 and MCF7 cells were kindly provided by Dr. Ajay Rana, University of Illinois at Chicago or Dr. Paula McKeown-Longo, Albany Medical College. MDA-MB-231 cells were cultured in RMPI 1640 medium or DMEM supplemented with 10% FBS, penicillin (100 units/ml), and streptomycin (100 ␮g/ml). MCF7 cells were cultured in MEM supplemented with 0.01 mg/ml human recombinant insulin, 10% FBS, penicillin (100 units/ml), and streptomycin (100 ␮g/ml) or DMEM as described previously. A549 cells were kindly provided by Dr. Maurizio Bocchetta, Loyola University Chicago, or Dr. Paula McKeown-Longo, Albany Medical College, and were cultured in F-12K supplemented with 10% FBS, penicillin (100 units/ml), and streptomycin (100 ␮g/ml). Cells were maintained under normoxic conditions (21% O2) in a water-jacket CO2 incubator (Thermo Fisher Scientific, Waltham, MA) at 37 °C, 5% CO2. Cells were maintained under hypoxic conditions (3% O2) in a Coy Hypoxic Chamber (Grass Lake, MI) at 37 °C, 5% CO2. Media used for hypoxic experiments were pre-equilibrated under hypoxia for 16 h. Silencing of Endogenous Spry2—Cells (150,000 –250,000/ 35-mm dish) were plated, and the next day they were transfected using Trans-IT TKO (Mirus Bio LLC, Madison, WI) transfection reagent with 20 nM 27-mer (control) mutant Spry2 siRNA containing three ribonucleotide substitutions of the Spry2 siRNA or Spry2 siRNA duplex following the manufacturer’s protocol. The cells were placed in hypoxia the next day, and the media were changed to pre-equilibrated hypoxic media. Cell lysates were collected after either 8 h (for HIF1␣ studies) or 24 h (for HIF2␣ studies). Isolating RNA, cDNA Synthesis, and qRT-PCR—HuH7 cells were plated (250,000/35-mm dish or 80,000/12-well plate) and transfected the following day with control (40 nM), Spry2 (20 nM ⫹20 nM control (for HIF1/2␣)), Spry2 (20 nM) ⫹ HIF1␣ (20 nM), or Spry2 (20 nM) ⫹ HIF2␣ (20 nM) siRNAs. After 16 or 24 h in hypoxia, the cells were lysed in TRIzol (Life Technologies, Inc.), and RNA was extracted according to the manufacturer’s protocol with the addition of GlycoBlue (Life Technologies, Inc.) to aid in precipitation of RNA. At least duplicate samples were collected for each condition. The RNA was re-precipitated with sodium acetate and ethanol to remove impurities. All RNA used had a 260:280 ratio above 1.8 and a 260:230 ratio above 1.9. Total RNA (1 ␮g or 500 ng) was reverse-transcribed into cDNA using the SuperScript威 VILO cDNA synthesis kit (Life Technologies, Inc.) following the manufacturer’s protocol. The cDNA (10 ng) was used for qRT-PCR using either FastStart Universal Probe Master Mix with Rox (Roche Applied Science) and primer probe mixes or FastStart Universal SYBR Master Mix with Rox (Roche Applied Science) with primers. All primers and probes with their calculated efficiencies are listed in AUGUST 5, 2016 • VOLUME 291 • NUMBER 32

supplemental Table 2, A and B. The cycle threshold (CT) was calculated using a single threshold. Three housekeeping genes were used: 18S ribosomal RNA (RN18S1), hypoxanthine-guanine phosphoribosyltransferase (HPRT), and ribosomal protein, large, P0 (RPLP0). The average was taken for the CT values of the control siRNA-transfected cells for all housekeeping genes and genes of interest (GOI). The fold change of each housekeeping gene and GOI was calculated by taking the efficiency (E) of the gene to the power of the difference of the experimental CT and the average CT of control CT (fold change ⫽ E∧(CTexp ⫺ average(CTcontrol)). The geometric mean was taken of three housekeeping genes. To calculate the relative mRNA amount of each GOI, the fold change of the GOI was divided by the geometric mean of the housekeeping genes (relative mRNA amount ⫽ fold change(GOI)/geomean(fold change(RN18S1, HPRT, RPLP0)). Luciferase Assays—HuH7 cells were plated (50,000/24-well plate), and the following day were transfected using Trans-IT TKO (Mirus Bio LLC, Madison, WI) with control siRNA or siRNAs against Spry2, Spry2 ⫹ HIF1␣, or Spry2 ⫹ HIF2␣ (as described above under “Isolating RNA, cDNA Synthesis, and qRT-PCR”) and 1 ␮g of pGL2-Pfkfb3/⫺3566 plus 10 ng of pRG-TK and incubated in normoxia or hypoxia (3% O2) for 24 h. The cells were lysed in Passive Lysis Buffer (Promega, Madison, WI). Luciferase assays were performed using the Promega Dual-Luciferase kit and read on a PHERAStar FS plate reader (BMG Labtech, Cary, NC). Stability of HIF1␣—HuH7 cells (250,000/35-mm dish) were plated and the following day transfected with 20 nM each of control or Spry2 siRNAs as stated above. The cells were placed in hypoxia, and 8 h later cycloheximide (200 ␮M) was added. The cells were lysed after 0, 5, or 10 min in Laemmli sample buffer. HIF1␣, Spry2, ERK1/2, and actin levels were monitored using Western blotting. Ubiquitylation Assays—HEK293T cells (300,000/60-mm dish) were plated and the following day transfected with 1.25 ␮g of pJX40-FLAG-ubiquitin and 625 ng of pcDNA3-HIF1␣ along with either control or Spry2 siRNAs or WT-Spry2 or 3P/3ASpry2. The following day the cells were placed in hypoxia for 12 h, and 25 ␮M MG132 was added for an additional 4 h for silencing of Spry2 experiments or 2 h for overexpression experiments. The cells were lysed in denaturing lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% SDS, 1% Nonidet P-40, and 0.5% sodium deoxycholate with the following protease inhibitors: 50 mM NaF, 5 mM ␤-glycerol phosphate, 1 mM sodium orthovanadate, 150 nM aprotinin, 1.5 mM pepstatin A, 25 ␮M MG132, 4.7 mM leupeptin, 6.6 mM benzamidine, 8.3 mM sodium pyrophosphate, 5 mM N-ethylmaleimide, 0.1 mM phenylmethylsulfonyl fluoride, pH 7.4). The samples were sonicated twice for 10 s on ice. Lysate (100 ␮g) was diluted 1:10 with lysis buffer without SDS and incubated with 3 ␮g of antiFLAG-M2, anti-HIF1␣, or mouse IgG for 1 h. Protein G beads (Roche Applied Science) (20 ␮l slurry) were incubated with the lysate for 2 h. The beads were washed three times with lysis buffer with 0.1% SDS. Heating in Laemmli sample buffer eluted the bound proteins, which were then separated by SDS-PAGE, and HIF1␣ and FLAG-ubiquitin were detected by Western blotting analysis. JOURNAL OF BIOLOGICAL CHEMISTRY

16797

Sprouty2 Regulates Hypoxia-inducible Factor-␣ Co-immunoprecipitation (Co-IP) of Endogenous Spry2 and HIF1␣—HuH7 cells (800,000/60-mm dish) were plated and the following day exposed to hypoxia for 8 h with the proteosomal inhibitor MG132 (25 ␮M) treatment during the last 4 h. Cells were washed twice with ice-cold PBS, lysed in lysis buffer (as described above except with 1% Triton X-100 instead of SDS, Nonidet P-40, and sodium deoxycholate), and rotated for 30 min at 4 °C. Lysates were centrifuged at 15,000 ⫻ g for 15 min, and 500 ␮g of protein was rotated with 3 ␮g of anti-N-terminal Spry2 antibody overnight at 4 °C. The next day the lysates were incubated rotating with 30 ␮l of a slurry of protein G beads (Roche Applied Science) for 2 h at 4 °C. The beads were washed three times in lysis buffer and then heated to 95 °C for 10 min in Laemmli buffer to collect bound proteins, which were analyzed for HIF1␣ and Spry2 by Western blotting. ICC and PLA—HuH7 cells were plated in an 8-well chamber slide and the following day transfected with 20 nM each of control (for Spry2) or Spry2 siRNAs (ICC only) or control (for HIF1␣) or HIF1␣ siRNAs (ICC and PLA) as stated above. The next day cells were placed in hypoxia for 8 h and then fixed with 3.7% formaldehyde in PIPES, pH 6.8, for 10 min at room temperature, washed, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with 10% normal goat serum for 1 h at room temperature (ICC) or the PLA kit (Sigma) blocking solution for 30 min at 37 °C (PLA). Rabbit Spry2 (Rockland, Limerick, PA) (1:400 dilution) and mouse HIF1␣ (BD Biosciences) (1:50 dilution) were incubated overnight 4 °C. For ICC, the secondary antibodies of goat anti-rabbit conjugated with Alexa Fluor 488 or goat anti-mouse conjugated to Alexa Fluor 594 (ThermoFisher, Grand Island, NY) (1:500 dilution) were incubated on the slide for 1 h. For PLA (Sigma), the manufacturer’s protocol was followed. Confocal images were obtained with a TCS SP5 laser scanning confocal microscope (Leica, Exton, PA) equipped with DMI6000 inverted microscope with blue diode (405 nm), argon (458/476/488/496/514 nm), diode pumped solid state (561 nm), and HeNe (633 nm) lasers and a ⫻63 HCX PL APO ␭blue (numerical aperture, 1.4 oil immersion) objective lens. The software used to capture the images was Leica Application Suite Advanced Fluorescence. For ICC, five fields were taken for each condition. For PLA, 10 –15 fields were taken of each condition, and PLA puncta were quantified using ImageJ64 software (National Institutes of Health) using the “analyze particles” function. The ⫻3 zoomed insets were generated using “Zoom in images and stacks” macro by Gilles Carpentier. Co-IP of HIF1␣ with pVHL—HuH7 cells (650,000/60-mm dish) were plated and the next day transfected with 20 nM control or Spry2 siRNAs as stated above. The following day, the cells were exposed to hypoxia for 8 h with proteosomal inhibitor MG132 (10 ␮M) treatment during the last 4 h. Immunoprecipitation of pVHL was performed using the buffer described under “Ubiquitylation Assays” except the buffer contained 0.1% SDS instead of 1% SDS. The samples were centrifuged at 15,000 rpm for 15 min, and 250 ␮g of protein was rotated with 3 ␮g of anti-pVHL (BD Biosciences)-bound bead slurry (15 ␮l/IP) (Roche Applied Science) overnight at 4 °C. The next day the beads were washed three times with the buffer diluted 1:1 with


PBS. The bound proteins were eluted in Laemmli buffer and analyzed using Western blotting for HIF1␣ and pVHL. Co-IP of WT-Spry2 and 3P/3A Spry2 with HIF1␣—HEK293T cells (500,000/60-mm dish) were plated, and the next day the cells were transfected with 750 ng of pHM6-vector, pHM6WT-HA-Spry2, or pHM6 –3P/3A-HA-Spry2 along with 750 ng of pcDNA3-HIF1␣ with TransIT-2020 (Mirus Bio LLC, Madison, WI) following the manufacturer’s protocol. The next day the cells were incubated in hypoxia for 16 h. Immunoprecipitation protocol was modified from the two-step lysis method in Ref. 94. Protein G bead slurry (Roche Applied Science) was incubated overnight at 4 °C with 2 ␮g of antibody (anti-HIF1␣, Novus Biologicals, Littleton, CO) or mouse IgG (Sigma) in PBS with 0.5% CHAPS and 5% BSA. The next day the cells were lysed in 500 ␮l of Triton X-100 hypotonic buffer (94) with the addition of protease and phosphatase inhibitors stated above. The samples were sonicated twice at 15% power for 10 s. NaCl was added to each tube to a final concentration of 420 mM, and the samples were incubated on ice for 1 h. The samples were sonicated again as stated previously and centrifuged at 10,000 ⫻ g for 10 min. The supernatant (150 ␮g) was incubated with antibody-bound beads (either mouse IgG or HIF1␣) for 2 h. The samples were washed three times in the hypotonic buffer with 150 mM NaCl. The final wash was removed, and the bound proteins were eluted off the beads by heating in Laemmli sample buffer. pVHL Silencing—HuH7 cells were plated (250,000/35-mm dish) and transfected with 20 nM each of control or Spry2 siRNAs along with 40 nM each of control or one of two pVHL siRNAs (pVHL siRNA1 or siRNA4). The next day the cells were incubated in hypoxia for 8 h. The cells were lysed in Laemmli sample buffer, and the levels of HIF1␣, Spry2, actin, and pVHL were monitored using Western blotting. Expression of Wild-type (WT) Spry2 and Spry2 P18A/P144A/ P160A (3P/3A) Mutant Spry2—HEK293T cells were plated (250,000/35-mm dish) and transfected the next day using TransIT-2020 transfection reagent (Mirus Bio LLC, Madison WI) with 250 ng of pHM6 vector, pHM6-WT-HA-Spry2, or pHM6 –3P/3A-HA-Spry2 following the manufacturer’s protocol. After 24 h, the cells were placed in hypoxia for 16 h. The cells were lysed in Laemmli sample buffer and analyzed for HIF1␣, HA-Spry2, actin, ERK1/2, pAKT Ser-473, PHD1, PHD2, PHD3, and pVHL levels using Western blotting. Glucose Uptake Assays—HeLa or HuH7 cells (40,000/well of 24-well plate) were plated and the following day transfected with 20 nM each of control or HIF1␣ siRNAs and 200 ng each of either pHM6-vector, pHM6-WT-HA-Spry2, or pHM6 –3P/ 3A-HA-Spry2 using TransIT-HeLaMONSTER (Mirus) (HeLa) or TransIT-X2 (Mirus) (HuH7). Quadruplicate sets of wells for each condition were transfected. The next day the cells were placed in the hypoxic chamber, and the media were changed to pre-equilibrated hypoxic media. After 24 h, the cells were washed with Krebs-Ringer HEPES Buffer (KRH) (modified from Cold Spring Harbor Protocols) and then incubated in KRH with 100 ␮M 2-deoxy-D-[3H]glucose (0.5 ␮Ci/well) for 10 min. One set of duplicate wells was incubated with an additional 20 mM 2-deoxy-D-glucose to compete with 2-deoxy-D[3H]glucose and determine specific glucose uptake. The cells VOLUME 291 • NUMBER 32 • AUGUST 5, 2016

Sprouty2 Regulates Hypoxia-inducible Factor-␣ were then washed three times in ice-cold KRH and lysed in 0.5 M NaOH with 0.1% Triton X-100. An aliquot (10 ␮l) of the lysate from each sample was used for protein determination. Another aliquot of the lysate (140 ␮l) was transferred to a scintillation vial. Each well was washed once with 300 ␮l of KRH that was also transferred to the scintillation vials for counting after mixing with 5 ml of scintillation fluid. The vials were counted on a Beckman Coulter scintillation counter. Data were calculated to represent picomoles of 2-deoxy-D-[3H]glucose uptake/␮g of total protein. To determine the HIF1␣-specific glucose uptake, the average of the HIF1␣ siRNA-transfected samples was subtracted from the control siRNA-transfected samples. Statistical Analysis—All data are represented as means ⫾ S.E. of the mean. Data were analyzed as indicated in the figure legends with two-tailed unpaired t tests, one-way analysis of variance (ANOVA), or two-way ANOVA; p ⬍ 0.05 was considered significant, n.s. indicates not significant. All statistical tests were calculated using Prism 6 (GraphPad, La Jolla, CA). Author Contributions—K. C. H. planned and designed the experiments, collected and interpreted data, and helped write the manuscript. T. B. P. planned and designed the experiments, interpreted data, helped write the manuscript. Acknowledgment—We thank Dr. Xianlong Gao, Dept. of Cell and Molecular Physiology, Stritch School of Medicine, Loyola University at Chicago, for helpful discussions and suggestions during the progress of this work. References 1. Zhang, S., Lin, Y., Itäranta, P., Yagi, A., and Vainio, S. (2001) Expression of Sprouty genes 1, 2 and 4 during mouse organogenesis. Mech. Dev. 109, 367–370 2. Chi, L., Zhang, S., Lin, Y., Prunskaite-Hyyryläinen, R., Vuolteenaho, R., Itäranta, P., and Vainio, S. (2004) Sprouty proteins regulate ureteric branching by coordinating reciprocal epithelial Wnt11, mesenchymal Gdnf and stromal Fgf7 signalling during kidney development. Development 131, 3345–3356 3. Perl, A. K., Hokuto, I., Impagnatiello, M. A., Christofori, G., and Whitsett, J. A. (2003) Temporal effects of Sprouty on lung morphogenesis. Dev. Biol. 258, 154 –168 4. Lee, S. H., Schloss, D. J., Jarvis, L., Krasnow, M. A., and Swain, J. L. (2001) Inhibition of angiogenesis by a mouse sprouty protein. J. Biol. Chem. 276, 4128 – 4133 5. Tefft, J. D., Lee, M., Smith, S., Leinwand, M., Zhao, J., Bringas, P., Jr., Crowe, D. L., and Warburton, D. (1999) Conserved function of mSpry-2, a murine homolog of Drosophila sprouty, which negatively modulates respiratory organogenesis. Curr. Biol. 9, 219 –222 6. Lo, T. L., Yusoff, P., Fong, C. W., Guo, K., McCaw, B. J., Phillips, W. A., Yang, H., Wong, E. S., Leong, H. F., Zeng, Q., Putti, T. C., and Guy, G. R. (2004) The ras/mitogen-activated protein kinase pathway inhibitor and likely tumor suppressor proteins, sprouty 1 and sprouty 2 are deregulated in breast cancer. Cancer Res. 64, 6127– 6136 7. Fong, C. W., Chua, M. S., McKie, A. B., Ling, S. H., Mason, V., Li, R., Yusoff, P., Lo, T. L., Leung, H. Y., So, S. K., and Guy, G. R. (2006) Sprouty 2, an inhibitor of mitogen-activated protein kinase signaling, is down-regulated in hepatocellular carcinoma. Cancer Res. 66, 2048 –2058 8. Lee, S. A., Ladu, S., Evert, M., Dombrowski, F., De Murtas, V., Chen, X., and Calvisi, D. F. (2010) Synergistic role of Sprouty2 inactivation and c-Met up-regulation in mouse and human hepatocarcinogenesis. Hepatology 52, 506 –517


9. Lee, S. A., Ho, C., Roy, R., Kosinski, C., Patil, M. A., Tward, A. D., Fridlyand, J., and Chen, X. (2008) Integration of genomic analysis and in vivo transfection to identify sprouty 2 as a candidate tumor suppressor in liver cancer. Hepatology 47, 1200 –1210 10. Fritzsche, S., Kenzelmann, M., Hoffmann, M. J., Müller, M., Engers, R., Gröne, H. J., and Schulz, W. A. (2006) Concomitant down-regulation of SPRY1 and SPRY2 in prostate carcinoma. Endocr. Relat. Cancer 13, 839 – 849 11. Sutterlüty, H., Mayer, C. E., Setinek, U., Attems, J., Ovtcharov, S., Mikula, M., Mikulits, W., Micksche, M., and Berger, W. (2007) Down-regulation of Sprouty2 in non-small cell lung cancer contributes to tumor malignancy via extracellular signal-regulated kinase pathway-dependent and -independent mechanisms. Mol. Cancer Res. 5, 509 –520 12. Liu, X., Lan, Y., Zhang, D., Wang, K., Wang, Y., and Hua, Z. C. (2014) SPRY1 promotes the degradation of uPAR and inhibits uPAR-mediated cell adhesion and proliferation. Am. J. Cancer Res. 4, 683– 697 13. Taniguchi, K., Ishizaki, T., Ayada, T., Sugiyama, Y., Wakabayashi, Y., Sekiya, T., Nakagawa, R., and Yoshimura, A. (2009) Sprouty4 deficiency potentiates Ras-independent angiogenic signals and tumor growth. Cancer Sci. 100, 1648 –1654 14. Ma, Y., Yu, S., Zhao, W., Lu, Z., and Chen, J. (2010) miR-27a regulates the growth, colony formation and migration of pancreatic cancer cells by targeting Sprouty2. Cancer Lett. 298, 150 –158 15. Macià, A., Gallel, P., Vaquero, M., Gou-Fabregas, M., Santacana, M., Maliszewska, A., Robledo, M., Gardiner, J. R., Basson, M. A., Matias-Guiu, X., and Encinas, M. (2012) Sprouty1 is a candidate tumor-suppressor gene in medullary thyroid carcinoma. Oncogene 31, 3961–3972 16. Sabatel, C., Cornet, A. M., Tabruyn, S. P., Malvaux, L., Castermans, K., Martial, J. A., and Struman, I. (2010) Sprouty1, a new target of the angiostatic agent 16K prolactin, negatively regulates angiogenesis. Mol. Cancer 9, 231 17. Sirivatanauksorn, Y., Sirivatanauksorn, V., Srisawat, C., Khongmanee, A., and Tongkham, C. (2012) Differential expression of sprouty genes in hepatocellular carcinoma. J. Surg. Oncol. 105, 273–276 18. Faratian, D., Sims, A. H., Mullen, P., Kay, C., Um, I., Langdon, S. P., and Harrison, D. J. (2011) Sprouty 2 is an independent prognostic factor in breast cancer and may be useful in stratifying patients for trastuzumab therapy. PLoS ONE 6, e23772 19. McKie, A. B., Douglas, D. A., Olijslagers, S., Graham, J., Omar, M. M., Heer, R., Gnanapragasam, V. J., Robson, C. N., and Leung, H. Y. (2005) Epigenetic inactivation of the human sprouty2 (hSPRY2) homologue in prostate cancer. Oncogene 24, 2166 –2174 20. Yusoff, P., Lao, D. H., Ong, S. H., Wong, E. S., Lim, J., Lo, T. L., Leong, H. F., Fong, C. W., and Guy, G. R. (2002) Sprouty2 inhibits the Ras/MAP kinase pathway by inhibiting the activation of Raf. J. Biol. Chem. 277, 3195–3201 21. Impagnatiello, M. A., Weitzer, S., Gannon, G., Compagni, A., Cotten, M., and Christofori, G. (2001) Mammalian sprouty-1 and -2 are membraneanchored phosphoprotein inhibitors of growth factor signaling in endothelial cells. J. Cell Biol. 152, 1087–1098 22. Gross, I., Bassit, B., Benezra, M., and Licht, J. D. (2001) Mammalian sprouty proteins inhibit cell growth and differentiation by preventing ras activation. J. Biol. Chem. 276, 46460 – 46468 23. Wong, E. S., Lim, J., Low, B. C., Chen, Q., and Guy, G. R. (2001) Evidence for direct interaction between Sprouty and Cbl. J. Biol. Chem. 276, 5866 –5875 24. Egan, J. E., Hall, A. B., Yatsula, B. A., and Bar-Sagi, D. (2002) The bimodal regulation of epidermal growth factor signaling by human Sprouty proteins. Proc. Natl. Acad. Sci. U.S.A. 99, 6041– 6046 25. Lao, D. H., Yusoff, P., Chandramouli, S., Philp, R. J., Fong, C. W., Jackson, R. A., Saw, T. Y., Yu, C. Y., and Guy, G. R. (2007) Direct binding of PP2A to Sprouty2 and phosphorylation changes are a prerequisite for ERK inhibition downstream of fibroblast growth factor receptor stimulation. J. Biol. Chem. 282, 9117–9126 26. Fong, C. W., Leong, H. F., Wong, E. S., Lim, J., Yusoff, P., and Guy, G. R. (2003) Tyrosine phosphorylation of Sprouty2 enhances its interaction with c-Cbl and is crucial for its function. J. Biol. Chem. 278, 33456 –33464 27. Hall, A. B., Jura, N., DaSilva, J., Jang, Y. J., Gong, D., and Bar-Sagi, D. (2003) hSpry2 is targeted to the ubiquitin-dependent proteasome pathway by c-Cbl. Curr. Biol. 13, 308 –314


16799

Sprouty2 Regulates Hypoxia-inducible Factor-␣ 28. Nadeau, R. J., Toher, J. L., Yang, X., Kovalenko, D., and Friesel, R. (2007) Regulation of Sprouty2 stability by mammalian Seven-in-Absentia homolog 2. J. Cell Biochem. 100, 151–160 29. Edwin, F., Anderson, K., and Patel, T. B. (2010) HECT domain-containing E3 ubiquitin ligase Nedd4 interacts with and ubiquitinates Sprouty2. J. Biol. Chem. 285, 255–264 30. Anderson, K., Nordquist, K. A., Gao, X., Hicks, K. C., Zhai, B., Gygi, S. P., and Patel, T. B. (2011) Regulation of cellular levels of Sprouty2 protein by prolyl hydroxylase domain and von Hippel-Lindau proteins. J. Biol. Chem. 286, 42027– 42036 31. Bertout, J. A., Patel, S. A., and Simon, M. C. (2008) The impact of O2 availability on human cancer. Nat. Rev. Cancer 8, 967–975 32. Semenza, G. L., Nejfelt, M. K., Chi, S. M., and Antonarakis, S. E. (1991) Hypoxia-inducible nuclear factors bind to an enhancer element located 3⬘ to the human erythropoietin gene. Proc. Natl. Acad. Sci. U.S.A. 88, 5680 –5684 33. Semenza, G. L., and Wang, G. L. (1992) A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation. Mol. Cell. Biol. 12, 5447–5454 34. Wang, G. L., and Semenza, G. L. (1993) Characterization of hypoxiainducible factor 1 and regulation of DNA binding activity by hypoxia. J. Biol. Chem. 268, 21513–21518 35. Wang, G. L., and Semenza, G. L. (1995) Purification and characterization of hypoxia-inducible factor 1. J. Biol. Chem. 270, 1230 –1237 36. Wang, G. L., Jiang, B. H., Rue, E. A., and Semenza, G. L. (1995) Hypoxiainducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc. Natl. Acad. Sci. U.S.A. 92, 5510 –5514 37. Gu, Y. Z., Moran, S. M., Hogenesch, J. B., Wartman, L., and Bradfield, C. A. (1998) Molecular characterization and chromosomal localization of a third ␣-class hypoxia inducible factor subunit, HIF3␣. Gene Expr. 7, 205–213 38. Makino, Y., Cao, R., Svensson, K., Bertilsson, G., Asman, M., Tanaka, H., Cao, Y., Berkenstam, A., and Poellinger, L. (2001) Inhibitory PAS domain protein is a negative regulator of hypoxia-inducible gene expression. Nature 414, 550 –554 39. Ziello, J. E., Jovin, I. S., and Huang, Y. (2007) Hypoxia-inducible factor (HIF)-1 regulatory pathway and its potential for therapeutic intervention in malignancy and ischemia. Yale J. Biol. Med. 80, 51– 60 40. Semenza, G. L. (2003) Targeting HIF-1 for cancer therapy. Nat. Rev. Cancer 3, 721–732 41. Jaakkola, P., Mole, D. R., Tian, Y. M., Wilson, M. I., Gielbert, J., Gaskell, S. J., von Kriegsheim, A., Hebestreit, H. F., Mukherji, M., Schofield, C. J., Maxwell, P. H., Pugh, C. W., and Ratcliffe, P. J. (2001) Targeting of HIF-␣ to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science 292, 468 – 472 42. Ivan, M., Kondo, K., Yang, H., Kim, W., Valiando, J., Ohh, M., Salic, A., Asara, J. M., Lane, W. S., and Kaelin, W. G., Jr. (2001) HIF␣ targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing. Science 292, 464 – 468 43. Cockman, M. E., Masson, N., Mole, D. R., Jaakkola, P., Chang, G. W., Clifford, S. C., Maher, E. R., Pugh, C. W., Ratcliffe, P. J., and Maxwell, P. H. (2000) Hypoxia-inducible factor-␣ binding and ubiquitylation by the von Hippel-Lindau tumor suppressor protein. J. Biol. Chem. 275, 25733–25741 44. Kamura, T., Sato, S., Iwai, K., Czyzyk-Krzeska, M., Conaway, R. C., and Conaway, J. W. (2000) Activation of HIF1␣ ubiquitination by a reconstituted von Hippel-Lindau (VHL) tumor suppressor complex. Proc. Natl. Acad. Sci. U.S.A. 97, 10430 –10435 45. Maxwell, P. H., Wiesener, M. S., Chang, G. W., Clifford, S. C., Vaux, E. C., Cockman, M. E., Wykoff, C. C., Pugh, C. W., Maher, E. R., and Ratcliffe, P. J. (1999) The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature 399, 271–275 46. Tanimoto, K., Makino, Y., Pereira, T., and Poellinger, L. (2000) Mechanism of regulation of the hypoxia-inducible factor-1 ␣ by the von HippelLindau tumor suppressor protein. EMBO J. 19, 4298 – 4309 47. Maxwell, P. H. (2005) The HIF pathway in cancer. Semin. Cell Dev. Biol. 16, 523–530


48. Semenza, G. L. (2010) HIF-1: upstream and downstream of cancer metabolism. Curr. Opin. Genet. Dev. 20, 51–56 49. Majmundar, A. J., Wong, W. J., and Simon, M. C. (2010) Hypoxia-inducible factors and the response to hypoxic stress. Mol. Cell 40, 294 –309 50. DaSilva, J., Xu, L., Kim, H. J., Miller, W. T., and Bar-Sagi, D. (2006) Regulation of sprouty stability by Mnk1-dependent phosphorylation. Mol. Cell. Biol. 26, 1898 –1907 51. Kwon, S. J., Song, J. J., and Lee, Y. J. (2005) Signal pathway of hypoxiainducible factor-1␣ phosphorylation and its interaction with von HippelLindau tumor suppressor protein during ischemia in MiaPaCa-2 pancreatic cancer cells. Clin. Cancer Res. 11, 7607–7613 52. Richard, D. E., Berra, E., Gothié, E., Roux, D., and Pouysségur, J. (1999) p42/p44 mitogen-activated protein kinases phosphorylate hypoxia-inducible factor 1␣ (HIF-1␣) and enhance the transcriptional activity of HIF-1. J. Biol. Chem. 274, 32631–32637 53. Minet, E., Michel, G., Mottet, D., Raes, M., and Michiels, C. (2001) Transduction pathways involved in hypoxia-inducible factor-1 phosphorylation and activation. Free Radic. Biol. Med. 31, 847– 855 54. Semenza, G. L., Roth, P. H., Fang, H. M., and Wang, G. L. (1994) Transcriptional regulation of genes encoding glycolytic enzymes by hypoxiainducible factor 1. J. Biol. Chem. 269, 23757–23763 55. Ebert, B. L., Firth, J. D., and Ratcliffe, P. J. (1995) Hypoxia and mitochondrial inhibitors regulate expression of glucose transporter-1 via distinct cis-acting sequences. J. Biol. Chem. 270, 29083–29089 56. Maxwell, P. H., Dachs, G. U., Gleadle, J. M., Nicholls, L. G., Harris, A. L., Stratford, I. J., Hankinson, O., Pugh, C. W., and Ratcliffe, P. J. (1997) Hypoxia-inducible factor-1 modulates gene expression in solid tumors and influences both angiogenesis and tumor growth. Proc. Natl. Acad. Sci. U.S.A. 94, 8104 – 8109 57. Kim, J. W., Tchernyshyov, I., Semenza, G. L., and Dang, C. V. (2006) HIF-1-mediated expression of pyruvate dehydrogenase kinase: a metabolic switch required for cellular adaptation to hypoxia. Cell Metab. 3, 177–185 58. Obach, M., Navarro-Sabaté, A., Caro, J., Kong, X., Duran, J., Gómez, M., Perales, J. C., Ventura, F., Rosa, J. L., and Bartrons, R. (2004) 6-Phosphofructo-2-kinase (pfkfb3) gene promoter contains hypoxia-inducible factor-1 binding sites necessary for transactivation in response to hypoxia. J. Biol. Chem. 279, 53562–53570 59. Chen, C., Pore, N., Behrooz, A., Ismail-Beigi, F., and Maity, A. (2001) Regulation of glut1 mRNA by hypoxia-inducible factor-1. Interaction between H-ras and hypoxia. J. Biol. Chem. 276, 9519 –9525 60. Firth, J. D., Ebert, B. L., and Ratcliffe, P. J. (1995) Hypoxic regulation of lactate dehydrogenase A. Interaction between hypoxia-inducible factor 1 and cAMP response elements. J. Biol. Chem. 270, 21021–21027 61. Mastrogiannaki, M., Matak, P., Mathieu, J. R., Delga, S., Mayeux, P., Vaulont, S., and Peyssonnaux, C. (2012) Hepatic HIF-2 down-regulates hepcidin expression in mice through epo-mediated increase in erythropoiesis. Haematologica 97, 827– 834 62. Rankin, E. B., Biju, M. P., Liu, Q., Unger, T. L., Rha, J., Johnson, R. S., Simon, M. C., Keith, B., and Haase, V. H. (2007) Hypoxia-inducible factor-2 (HIF-2) regulates hepatic erythropoietin in vivo. J. Clin. Invest. 117, 1068 –1077 63. Salceda, S., and Caro, J. (1997) Hypoxia-inducible factor 1␣ (HIF-1␣) protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions. Its stabilization by hypoxia depends on redox-induced changes. J. Biol. Chem. 272, 22642–22647 64. Söderberg, O., Gullberg, M., Jarvius, M., Ridderstråle, K., Leuchowius, K. J., Jarvius, J., Wester, K., Hydbring, P., Bahram, F., Larsson, L. G., and Landegren, U. (2006) Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat. Methods 3, 995–1000 65. Lim, J., Wong, E. S., Ong, S. H., Yusoff, P., Low, B. C., and Guy, G. R. (2000) Sprouty proteins are targeted to membrane ruffles upon growth factor receptor tyrosine kinase activation. Identification of a novel translocation domain. J. Biol. Chem. 275, 32837–32845 66. Hanafusa, H., Torii, S., Yasunaga, T., and Nishida, E. (2002) Sprouty1 and Sprouty2 provide a control mechanism for the Ras/MAPK signalling pathway. Nat. Cell Biol. 4, 850 – 858 67. Gao, M., Patel, R., Ahmad, I., Fleming, J., Edwards, J., McCracken, S.,

VOLUME 291 • NUMBER 32 • AUGUST 5, 2016


68.

69. 70.

71.

72.

73.

74.

75.

76.

77.

78.

79.

80.

81.

Sahadevan, K., Seywright, M., Norman, J., Sansom, O., and Leung, H. Y. (2012) SPRY2 loss enhances ErbB trafficking and PI3K/AKT signalling to drive human and mouse prostate carcinogenesis. EMBO Mol. Med. 4, 776 –790 Edwin, F., Singh, R., Endersby, R., Baker, S. J., and Patel, T. B. (2006) The tumor suppressor PTEN is necessary for human Sprouty 2-mediated inhibition of cell proliferation. J. Biol. Chem. 281, 4816 – 4822 Edwin, F., and Patel, T. B. (2008) A novel role of Sprouty 2 in regulating cellular apoptosis. J. Biol. Chem. 283, 3181–3190 Baumann, M. U., Zamudio, S., and Illsley, N. P. (2007) Hypoxic upregulation of glucose transporters in BeWo choriocarcinoma cells is mediated by hypoxia-inducible factor-1. Am. J. Physiol. Cell Physiol. 293, C477–C485 Ozaki, K., Kadomoto, R., Asato, K., Tanimura, S., Itoh, N., and Kohno, M. (2001) ERK pathway positively regulates the expression of Sprouty genes. Biochem. Biophys. Res. Commun. 285, 1084 –1088 Ozaki, K., Miyazaki, S., Tanimura, S., and Kohno, M. (2005) Efficient suppression of FGF-2-induced ERK activation by the cooperative interaction among mammalian Sprouty isoforms. J. Cell Sci. 118, 5861–5871 Song, K., Gao, Q., Zhou, J., Qiu, S. J., Huang, X. W., Wang, X. Y., and Fan, J. (2012) Prognostic significance and clinical relevance of Sprouty 2 protein expression in human hepatocellular carcinoma. Hepatobiliary Pancreat. Dis. Int. 11, 177–184 Pan, Y., Mansfield, K. D., Bertozzi, C. C., Rudenko, V., Chan, D. A., Giaccia, A. J., and Simon, M. C. (2007) Multiple factors affecting cellular redox status and energy metabolism modulate hypoxia-inducible factor prolyl hydroxylase activity in vivo and in vitro. Mol. Cell. Biol. 27, 912–925 Hirsilä, M., Koivunen, P., Günzler, V., Kivirikko, K. I., and Myllyharju, J. (2003) Characterization of the human prolyl 4-hydroxylases that modify the hypoxia-inducible factor. J. Biol. Chem. 278, 30772–30780 Bemis, L., Chan, D. A., Finkielstein, C. V., Qi, L., Sutphin, P. D., Chen, X., Stenmark, K., Giaccia, A. J., and Zundel, W. (2004) Distinct aerobic and hypoxic mechanisms of HIF-␣ regulation by CSN5. Genes Dev. 18, 739 –744 Kim, B. Y., Kim, H., Cho, E. J., and Youn, H. D. (2008) Nur77 upregulates HIF-␣ by inhibiting pVHL-mediated degradation. Exp. Mol. Med. 40, 71– 83 Jung, J. E., Kim, H. S., Lee, C. S., Shin, Y. J., Kim, Y. N., Kang, G. H., Kim, T. Y., Juhnn, Y. S., Kim, S. J., Park, J. W., Ye, S. K., and Chung, M. H. (2008) STAT3 inhibits the degradation of HIF-1␣ by pVHL-mediated ubiquitination. Exp. Mol. Med. 40, 479 – 485 Baek, J. H., Liu, Y. V., McDonald, K. R., Wesley, J. B., Zhang, H., and Semenza, G. L. (2007) Spermidine/spermine N(1)-acetyltransferase-1 binds to hypoxia-inducible factor-1␣ (HIF-1␣) and RACK1 and promotes ubiquitination and degradation of HIF-1␣. J. Biol. Chem. 282, 33358 –33366 Baek, J. H., Mahon, P. C., Oh, J., Kelly, B., Krishnamachary, B., Pearson, M., Chan, D. A., Giaccia, A. J., and Semenza, G. L. (2005) OS-9 interacts with hypoxia-inducible factor 1␣ and prolyl hydroxylases to promote oxygendependent degradation of HIF-1␣. Mol. Cell 17, 503–512 Hopfer, U., Hopfer, H., Jablonski, K., Stahl, R. A., and Wolf, G. (2006) The


82.

83.

84.

85.

86.

87.

88. 89.

90.

91.

92.

93.

94.

novel WD-repeat protein Morg1 acts as a molecular scaffold for hypoxiainducible factor prolyl hydroxylase 3 (PHD3). J. Biol. Chem. 281, 8645– 8655 Ravi, R., Mookerjee, B., Bhujwalla, Z. M., Sutter, C. H., Artemov, D., Zeng, Q., Dillehay, L. E., Madan, A., Semenza, G. L., and Bedi, A. (2000) Regulation of tumor angiogenesis by p53-induced degradation of hypoxia-inducible factor 1␣. Genes Dev. 14, 34 – 44 Majumder, P. K., Febbo, P. G., Bikoff, R., Berger, R., Xue, Q., McMahon, L. M., Manola, J., Brugarolas, J., McDonnell, T. J., Golub, T. R., Loda, M., Lane, H. A., and Sellers, W. R. (2004) mTOR inhibition reverses Akt-dependent prostate intraepithelial neoplasia through regulation of apoptotic and HIF-1-dependent pathways. Nat. Med. 10, 594 – 601 Liu, L. Z., Li, C., Chen, Q., Jing, Y., Carpenter, R., Jiang, Y., Kung, H. F., Lai, L., and Jiang, B. H. (2011) MiR-21 induced angiogenesis through AKT and ERK activation and HIF-1␣ expression. PLoS ONE 6, e19139 Jiang, B. H., Jiang, G., Zheng, J. Z., Lu, Z., Hunter, T., and Vogt, P. K. (2001) Phosphatidylinositol 3-kinase signaling controls levels of hypoxia-inducible factor 1. Cell Growth Differ. 12, 363–369 Zhong, H., Chiles, K., Feldser, D., Laughner, E., Hanrahan, C., Georgescu, M. M., Simons, J. W., and Semenza, G. L. (2000) Modulation of hypoxiainducible factor 1␣ expression by the epidermal growth factor/phosphatidylinositol 3-kinase/PTEN/AKT/FRAP pathway in human prostate cancer cells: implications for tumor angiogenesis and therapeutics. Cancer Res. 60, 1541–1545 Minet, E., Arnould, T., Michel, G., Roland, I., Mottet, D., Raes, M., Remacle, J., and Michiels, C. (2000) ERK activation upon hypoxia: involvement in HIF-1 activation. FEBS Lett. 468, 53–58 Yee Koh, M., Spivak-Kroizman, T. R., and Powis, G. (2008) HIF-1 regulation: not so easy come, easy go. Trends Biochem. Sci. 33, 526 –534 Arsham, A. M., Plas, D. R., Thompson, C. B., and Simon, M. C. (2002) Phosphatidylinositol 3-kinase/Akt signaling is neither required for hypoxic stabilization of HIF-1 ␣ nor sufficient for HIF-1-dependent target gene transcription. J. Biol. Chem. 277, 15162–15170 Sayed, D., Rane, S., Lypowy, J., He, M., Chen, I. Y., Vashistha, H., Yan, L., Malhotra, A., Vatner, D., and Abdellatif, M. (2008) MicroRNA-21 targets Sprouty2 and promotes cellular outgrowths. Mol. Biol. Cell 19, 3272–3282 Wang, C., Delogu, S., Ho, C., Lee, S. A., Gui, B., Jiang, L., Ladu, S., Cigliano, A., Dombrowski, F., Evert, M., Calvisi, D. F., and Chen, X. (2012) Inactivation of Spry2 accelerates AKT-driven hepatocarcinogenesis via activation of MAPK and PKM2 pathways. J. Hepatol. 57, 577–583 Luo, W., Hu, H., Chang, R., Zhong, J., Knabel, M., O’Meally, R., Cole, R. N., Pandey, A., and Semenza, G. L. (2011) Pyruvate kinase M2 is a PHD3stimulated coactivator for hypoxia-inducible factor 1. Cell 145, 732–744 Yigzaw, Y., Cartin, L., Pierre, S., Scholich, K., and Patel, T. B. (2001) The C terminus of sprouty is important for modulation of cellular migration and proliferation. J. Biol. Chem. 276, 22742–22747 Klenova, E., Chernukhin, I., Inoue, T., Shamsuddin, S., and Norton, J. (2002) Immunoprecipitation techniques for the analysis of transcription factor complexes. Methods 26, 254 –259


16801

Sprouty2 protein regulates hypoxia-inducible factor-α (HIFα) protein levels and transcription of HIFα-responsive genes.

Hypoxia inducible factors regulate the transcription of the sprouty2 gene and expression of the sprouty2 protein.

Werner syndrome protein positively regulates XRCC4-like factor transcription.

The tyrosine phosphatase SHP-1 regulates hypoxia inducible factor-1α (HIF-1α) protein levels in endothelial cells under hypoxia.

20-Hydroxyecdysone is required for, and negatively regulates, transcription of Drosophila pupal cuticle protein genes.

Identification of a STOP1-like protein in Eucalyptus that regulates transcription of Al tolerance genes.

Evolutionary Dynamics of Floral Homeotic Transcription Factor Protein-Protein Interactions.

KDM4A regulates HIF-1 levels through H3K9me3.

Homeodomain Protein Scr Regulates the Transcription of Genes Involved in Juvenile Hormone Biosynthesis in the Silkworm.

Sumoylation of Hes6 Regulates Protein Degradation and Hes1-Mediated Transcription.

A TRIP230-retinoblastoma protein complex regulates hypoxia-inducible factor-1α-mediated transcription and cancer cell invasion.

Transcription Factor Functional Protein-Protein Interactions in Plant Defense Responses.

The prolyl isomerase Pin1 regulates hypoxia-inducible transcription factor (HIF) activity.

Heat shock transcription factor HSF1 regulates the expression of the Huntingtin-interacting protein HYPK.

Bacterial nucleoid-associated protein uncouples transcription levels from transcription timing.

Hypoxia regulates alternative splicing of HIF and non-HIF target genes.

Hypoxia inducible factor (HIF) as a model for studying inhibition of protein-protein interactions.

Specificity protein 1 transcription factor regulates human ARTS promoter activity through multiple binding sites.

The cAMP-responsive element binding protein (CREB) transcription factor regulates furin expression during human trophoblast syncytialization.

SUMOylation of GPS2 protein regulates its transcription-suppressing function.

Myocardin-related Transcription Factor Regulates Nox4 Protein Expression: LINKING CYTOSKELETAL ORGANIZATION TO REDOX STATE.

Sestrin 2 protein regulates platelet-derived growth factor receptor β (Pdgfrβ) expression by modulating proteasomal and Nrf2 transcription factor functions.

Activation of protein kinase-C differentially regulates insulin-like growth factor-I and basic fibroblast growth factor messenger RNA levels.

Manidipine regulates the transcription of cytokine genes.