Regulation of STRA13 by the von Hippel-Lindau Tumor Suppressor Protein, Hypoxia, and the UBC9/Ubiquitin Proteasome Degradation Pathway*

Alla V. IvanovaDagger §, Sergey V. Ivanov, Alla Danilkovitch-MiagkovaDagger , and Michael I. LermanDagger

From the Dagger  Laboratory of Immunobiology, NCI-Frederick Cancer Research Development Center, Frederick, Maryland 21702 and the  Intramural Research Support Program, SAIC Frederick, National Cancer Institute-Frederick Cancer Research Development Center, Laboratory of Immunobiology, Frederick, Maryland 21702

Received for publication, November 21, 2000, and in revised form, January 19, 2001

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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In this study, we focus on different modes of regulation of STRA13, a human ortholog of the mouse basic helix-loop-helix transcriptional factor, previously identified by us as a new von Hippel-Lindau tumor suppressor gene (VHL) target. The gene was overexpressed in VHL-deficient cell lines and tumors, specifically clear cell renal carcinomas and hemangioblastomas. Introduction of wild type VHL transgene into clear cell renal carcinoma restored low level expression of STRA13. Overexpression was also detected in many common malignancies with an intact VHL gene, suggesting the existence of another, VHL-independent pathway of STRA13 regulation. Similar to many other von Hippel-Lindau tumor-suppressor protein (pVHL) targets, the expression of STRA13 on the mRNA level was hypoxia-sensitive, indicating oxygen-dependent regulation of the gene, presumably through the pVHL/hypoxia-inducible factor 1 (HIF-1) pathway. The yeast two-hybrid screening revealed interaction of the STRA13 protein with the human ubiquitin-conjugating enzyme (UBC9) protein, the specificity of which was confirmed in mammalian cells. By adding the proteasome inhibitor acetyl-leucinyl-leucinyl-norleucinal, we demonstrated that the 26 S proteasome pathway regulates the stability of pSTRA13. Co-expression of STRA13 and UBC9 led to an increase of the pSTRA13 ubiquitination and subsequent degradation. These data established that UBC9/STRA13 association in cells is of physiological importance, presenting direct proof of UBC9 involvement in the ubiquitin-dependent degradation of pSTRA13. Hypoxia treatment of mammalian cells transiently expressing STRA13 protein showed that stability of pSTRA13 is not affected by hypoxia or VHL. Thus, STRA13, a new pVHL target, is regulated in cells on multiple levels. We propose that STRA13 may play a critical role in carcinogenesis, since it is a potent transcriptional regulator, abundant in a variety of common tumors.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Functional inactivation of the von Hippel-Lindau tumor-suppressor protein (pVHL)1 causes a hereditary cancer syndrome characterized by the predisposition to develop tumors of kidney, central nervous system, retina, pancreas, and adrenal gland. Recent studies highlighted pVHL as a key regulator of cellular responses to oxygen deprivation. pVHL regulates activity of the hypoxia-inducible factor 1 (HIF-1), consisting of an HIF-1alpha /HIF-1beta heterodimer. Under normoxic conditions, the alpha  subunit interacts with the pVHL-elongin B-elongin C complex and is rapidly degraded (1). Hypoxia or pVHL deficiency stabilizes HIF-1alpha , which results in activation of a set of genes including vascular endothelial growth factor (VEGF) (2, 3), glucose transporter GLUT-1 (3), glycolytic enzymes (reviewed in Ref. 4), transforming growth factor-alpha (5), and transforming growth factor-beta 1 (6). We have recently expanded the list of hypoxia-sensitive pVHL targets. Using the RNA differential display technique, we identified and characterized two novel hypoxia-responsive VHL targets, carbonic anhydrases 9 and 12 (7). Here we describe a third gene down-regulated by pVHL, STRA13 (DEC1)2 (8), the human ortholog of the mouse Stra13 (stimulated with retinoic acid) gene (9).

STRA13 belongs to the basic helix-loop-helix (bHLH) family of transcription factors that play important roles in the regulation of cell proliferation, differentiation, and apoptosis (reviewed in Ref. 10). The amino acid sequence of STRA13 shows the highest homology in the bHLH domain with Drosophila Hairy (H), Enhancer of Split (E(Spl)), and mouse Hes1 proteins (9). Outside of the bHLH domain, STRA13 contains three putative alpha -helices, which have no obvious similarity with any known proteins. Although mouse Stra13, unlike many other bHLH proteins (10), does not bind E- or N-boxes, it exhibits DNA binding properties and transcriptional repression activity in the chloramphenicol acetyltransferase assay, which is mediated through the alpha -helices (9). The in vitro interaction between Stra13 and either TBP or TFIIB, the subunits of RNA polymerase II complex, supports the idea that Stra13 may repress transcription by interacting with the basal transcription machinery (9). Stra13 expression is regulated through a negative autoregulatory mechanism that is brought about by its interaction with the co-repressor histone deacetylase (11). Little is known about Stra13 function, although its ability to repress mesodermal and endodermal differentiation, promoting, instead, neuronal differentiation, strongly suggests that Stra13 may play a critical role during mouse development (9). Its expression in kidney tubules at early stages (9) is of special interest, since these structures are known to be involved in carcinogenesis due to mutations of the VHL gene (12, 13).

Our findings that STRA13 is a pVHL target (7) and that it is overexpressed in many common malignancies (this paper) prompted us to propose STRA13 association with carcinogenesis and stimulated further research into its expression regulation in cells. We have focused on STRA13 control on mRNA and protein levels. On the mRNA level, STRA13 expression is regulated by pVHL with involvement of both beta -domain and elongin C-binding domains. We found that the gene is hypoxia-inducible, suggesting involvement of HIF-1 in its regulation. Our data imply that STRA13 may function as another important mediator of cellular responses to hypoxia in the pVHL/HIF-1 pathway. We further showed that STRA13 is also tightly controlled on the protein level. Using the yeast two-hybrid assay, we found that the ubiquitin-conjugating enzyme UBC9 specifically interacts with pSTRA13. We demonstrated also that pSTRA13 accumulates upon proteasome inhibitor treatment, producing multiubiquitinated forms. Co-expression of UBC9 and pSTRA13 increased ubiquitination of STRA13 and shortened its half-life. Hypoxia experiment showed that regulation of STRA13 on the protein level does not depend on hypoxia and VHL. Localization of the protein in the nucleus and its ability to homodimerize conform to its proposed role as a transcriptional regulator. Overall, our data suggest that STRA13 is a cancer-associated protein regulated by pVHL and hypoxia on the mRNA level and by the UBC9/ubiquitin proteasome degradation pathway on the protein level.

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ABSTRACT
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Cell Lines and Tumor Samples-- HEK 293 cells were routinely maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Cell lines transfected with VHL constructs are described in Ref. 7. 28 mRNA samples were isolated from cancer cell lines by the Developmental Therapeutics Program (NCI-Frederick Cancer Research Development Center). The human glioblastoma cell line U87 was purchased from ATCC (Manassas, VA). Human brain tumor samples were kindly provided by Marsha Merrill (NINDS, National Institutes of Health, Department of Microbiology and Molecular Genetics).

Northern Blot Analysis-- mRNA isolation from cell lines and tumors was done using commercially available kits (Invitrogen, Carlsbad, California). Electrophoresis in formaldehyde gels and Northern blot analysis were performed as described (7). Quantification of Northern hybridization signals was done with a Cyclone Storage Phosphor System (Packard) or using a VE-1000 Video Camera System (Dage-MTI, Inc., Michigan City, IN) and NIH Image, version 1.6.1, software.

Hypoxia Experiment-- Flasks with cells in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum were exposed to a constant flow of 1% O2, 5% CO2, and 94% N2 (Airgas; Mid-Atlantic, Baltimore, MD) in a humidified modular incubator chamber (Bellco Glass, Vineland, NJ) at 37 °C. After treatment, the cells were immediately lysed, and mRNA samples were isolated with the FastTrack Kit (Invitrogen).

Co-immunoprecipitation and Western Blot Analysis-- HEK 293 cells were transfected using Superfect (Qiagen) according to the manufacturer's recommendations. 48 h after transfection, the cells were lysed in radioimmune precipitation buffer (150 mM NaCl, 50 mM Hepes (pH 7.4), 10 mM NaF, 1 mM EDTA, 2 mM orthovanadate, 10% glycerol, 1% Triton, 1% deoxychelate, 0.5% SDS, protease inhibitors), the lysates were sheared by ultrasound and precleared by centrifugation at 13,000 rpm for 15 min at 4 °C. Cell lysates containing equal amounts of total protein were incubated for 2 h at 4 °C with 50 µl of 30% protein G-Sepharose beads (Stratagene) and either anti-FLAG monoclonal (Upstate Biotechnology, Inc., Lake Placid, NY) or anti-FLAG goat polyclonal antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The beads were washed extensively with lysis buffer, and bound proteins were fractionated by 8-16% SDS-PAGE, transferred to a nitrocellulose membrane, and incubated with the respective antibodies (anti-hemagglutinin (HA) or anti-ubiquitin (Santa Cruz Biotechnology). FLAG- or HA-fused proteins were visualized using the ECL kit (DURA; Pierce).

Yeast Two-hybrid Screen-- The entire open reading frame of STRA13 (411 amino acids residues) was cloned in frame with GAL4 DNA-binding domain in the pAS2-1 expression vector (CLONTECH). The STRA13-pAS2-1 plasmid was then transformed using the standard LiAC method into CG1945 yeast strain containing stable constructs of the reporter genes LacZ and His3. In the next step, the cDNA fetal kidney Matchmaker library (CLONTECH) was transformed into -trp cells containing STRA13-pAS2-1. Out of 5.5 × 106 independent transformants, 360 yeast colonies were selected on AA synthetic medium lacking leucine, tryptophan, and histidine and containing 5 mM 3-aminotriazol. 186 out of those 360 successfully passed secondary screening for high beta -galactosidase activity by the filter assay according to the manufacturer.

Immunostaining of Cells-- HEK 293 cells were transiently transfected with either FLAG-fused STRA13 constructs or HA-fused UBC9 construct, and after 24 h of incubation they were cultured on coverslips overnight. The cells were washed in PBS, fixed in 3% paraformaldehyde for 30 min, and permeabilized with 0.1% Triton X-100 for 5 min. After preincubation with 1% fetal bovine serum, cells were immunostained with anti-FLAG M2 mouse monoclonal Ab, anti-HA rabbit polyclonal Abs (Santa Cruz Biotechnology), or with both antisera at concentrations of 1 µg/ml. Goat anti-mouse antibodies with AlexaTM 594 (red) (Molecular Probes, Inc., Eugene, OR) were used to visualize the anti-FLAG mouse monoclonal Ab. Goat anti-rabbit antibodies labeled AlexaTM 488 (green) (Molecular Probes) were used to visualize the anti-HA rabbit Abs. Cells were mounted in mounting medium with DAPI (Vector).

    RESULTS
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ABSTRACT
INTRODUCTION
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pVHL Suppresses STRA13 Expression in Renal Clear Cell Carcinoma Cell Lines-- We recently reported STRA13 (DEC1) as a novel pVHL target detected by RNA differential display (7). Here we used VHL-deficient cell lines with introduced VHL transgenes (7) shown in Fig. 1A to further investigate pVHL involvement in the regulation of STRA13 expression. The original 786-0 cell line that is devoid of both pVHL protein-binding domains (beta -domain that binds to HIF (14) and the elongin C binding domain (CBD domain) (15)) (7) demonstrated a very high steady-state level of STRA13 expression (Fig. 1B). Stable transfection of this cell line with wild type VHL caused a ~3.5-fold decrease in STRA13 mRNA amount, whereas mutant 2, which possessed an intact beta -domain and lacked the CBD domain, produced no visible effect. Transfection of the 786-0 cell line with mutant 3 or 4 containing an intact CBD domain and a missense mutation in the beta -domain caused an ~2-fold reduction in the expression. Thus, compared with wild type VHL (lane 2) and mutant 2 (lane 3), suppression of STRA13 with mutant 3 or 4 (lanes 3 and 4) produced an intermediate effect. These data demonstrate that both domains of pVHL are involved in the regulation of STRA13 expression on the level of mRNA. Similar results were obtained for the UM-RC-6 clear cell carcinoma cell line (data not shown).


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Fig. 1.   Regulation of STRA13 gene expression on the mRNA level by pVHL transgenic constructs and hypoxia. A, schematic representation of transgenic (wild type VHL (wtVHL), mut2-4) and endogenous (786-0) VHL protein products. The arrows indicate residues involved in naturally occurring mutations. B, Northern analysis of STRA13 expression in the parental (786-0) and wild type VHL-transfected clear cell renal carcinoma cell lines. Even loading on the lanes was monitored by staining the membrane with methylene blue. C, up-regulation of STRA13 expression on the mRNA level by hypoxia in the U-87 glioblastoma cell line. After 20 h of incubation to a constant flow of 1% O2, 5% CO2, and 94% N2, a ~10-fold increase in the STRA13 mRNA level was observed. CA9 expression served as a positive control. The beta -actin probe shows even loading.

STRA13 Is a Hypoxia-inducible Gene-- Due to the fact that pVHL orchestrates cellular responses to hypoxia through regulation of HIF-1-alpha on the level of protein degradation (1), pVHL and HIF-1 targets substantially overlap (1, 4), and many, if not all, pVHL targets are hypoxia-inducible (1, 3). To investigate STRA13 response to oxygen deprivation, we compared mRNAs isolated from normoxic and hypoxic glioblastoma cells U-87. As a positive control, we used another pVHL target, the CA9 (5) that was recently reported as a highly hypoxia-inducible gene with a broad range of expression in many common malignancies, including brain tumors (16). The basal level of STRA13 expression in the U-87 cell line was significantly higher than that of CA9 (Fig. 1C). Upon 20 h of hypoxic treatment, this expression level was dramatically (~10-fold) elevated. As expected, STRA13 up-regulation was paralleled by a dramatic CA9 induction.

STRA13 Is Expressed in Common Tumors-- Steady-state STRA13 expression was detected in U-87 glioblastoma cell line (Fig. 1C, lane 1). However, this cell line has no mutations in the VHL gene (16). This fact prompted us to compare STRA13 expression in normal tissues and tumor cell lines representing common malignancies. A high level of STRA13 expression was observed in several normal tissues such as muscle, liver, placenta, and heart (Fig. 2A). This expression pattern was in agreement with the data obtained for mouse Stra13 (9) and human DEC1 (8). Noteworthy, some tissues, namely pancreas, kidney, and brain, demonstrated only limited level of STRA13 expression, while every kidney and brain tumor cell line demonstrated high level of STRA13 expression (Fig. 2B). Overall, STRA13 transcripts were observed in 28 out of 36 cancer cell lines derived from leukemia, lung adenocarcinoma, and tumors of colon, kidney, and brain (Fig. 2, B and C), clearly indicating that overexpression of the gene is typical for many common tumors and, therefore, may be critical for tumor progression. Northern analysis on poly(A+) mRNA samples isolated from human brain tumors confirmed that STRA13 expression takes place in tumors in vivo (Fig. 2D).


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Fig. 2.   Northern analysis of STRA13 mRNA expression in normal adult tissues (A) and common tumors (B-D). A and C, premade RNA blots (CLONTECH, catalog nos. 7759-1 and 7757-1, respectively). B, mRNA isolated from cancer cell lines. D, mRNA isolated from brain tumors. mel., melanoma; lung ad., lung adenocarcinoma; col. ad., colorectal adenocarcinoma; Burk.ly, Burkitt's lymphoma; lym.le., lymphoblastic leukemia MOLT-4; ch.my.le., chronic myelogenous leukemia K-562; HeLa, HeLa cell S3; pro.leu., promyelocytic leukemia HL-60; men.*, meningioma; gliobl., glioblastoma; hem.*, hemangioblastoma.

STRA13 Interacts with the Ubiquitin-conjugating Enzyme UBC9 in the Yeast Two-hybrid System and in Mammalian Cells-- To study the mechanisms of STRA13 regulation on protein level we searched for STRA13-binding partners using a yeast two-hybrid system. Out of 186 confirmed positive clones, five represented the entire cDNA (1.1 kilobase pair) of the UBC9 (human ubiquitin-conjugating enzyme) gene. Retransformation assay showed that GAL4-AD-UBC9 fusion protein interacted with pSTRA13 specifically, since no binding of AD-UBC9 was detected with either the pAS2-1 vector alone (BD domain) or with pAS2-1-ARNT1 that produces the BD-ARNT1 fusion protein (data not shown). From these experiments, we conclude that the STRA13/UBC9 interaction is specific in the yeast two-hybrid system.

To confirm the interaction between pSTRA13 and UBC9 we cloned the UBC9 cDNA into mammalian pCMV-HA vector to produce the HA-UBC9 fusion protein. STRA13 cDNA was introduced into pCMV2 vector to produce a FLAG-STRA13 fusion protein (Fig. 3A) HA-UBC9 and FLAG-STRA13 or an empty pCMV2 plasmid were cotransfected into HEK 293 cells. After 48 h of incubation, STRA13 was immunoprecipitated from cell lysates with an anti-FLAG monoclonal antibody. The presence of UBC9 in STRA13 precipitates was detected using polyclonal anti-HA antibodies. The fact that UBC9 was co-precipitated with FLAG-STRA13 shows that pSTRA13 and UBC9 interact in mammalian cells (Fig. 3B, line 2). No precipitation products were observed with the empty pFLAG-CMV2 vector (Fig. 3B, line 1).


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Fig. 3.   pSTRA13 and UBC9 interact in vivo. A, schematic representation of pSTRA13 domain structure and its deletion mutants used in this study. The numbers of amino acid (a.a.) sequence included in the deletion constructs are indicated on the left. All deletion mutants are fused to the FLAG tag on their N termini. B, pSTRA13 and UBC9 interact through the C-terminal region of the pSTRA13. STRA13 from HEK 293 cells transiently transfected with the indicated plasmids was immunoprecipitated with anti-FLAG M2 antibodies. Precipitates (IP) were analyzed by 8-16% SDS-PAGE and Western blotting (W). The presence of UBC9 in STRA13 precipitates was detected with anti-HA polyclonal antibodies. The upper bands represent IgG light chain that cross-reacted with the secondary antibody.

Subcellular Localization of pSTRA13-- To localize the STRA13 protein in the HEK 293 cells transiently expressing FLAG-tagged pSTRA13, we used indirect immunofluorescence. A monoclonal anti-FLAG antibody (M2) and AlexaTM-tagged secondary antibodies were used to study FLAG-pSTRA13 distribution in the transfected cells. The STRA13 protein was localized primarily in the nuclei (Fig. 4A), as confirmed by counterstaining with DAPI. No nuclear staining was visible in vector transfected cells (not shown). These results are in an agreement with the nuclear localization of mouse Stra13 protein (9).


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Fig. 4.   Subcellular localization of pSTRA13. A, the pSTRA13 is localized in the nucleus (A), whereas truncation of 113 C-terminal amino acids of pSTRA13 prevents nuclear translocation of the protein (B). The entire pSTRA13 and its C-terminal deletion construct (residues 1-299) fused to the FLAG tag were transiently transfected into HEK 293 cells. The cells were fixed and stained with anti-FLAG M2 antibodies and AlexaTM-conjugated secondary antibodies (red staining). Blue color shows DAPI-stained nucleus. TRITS or DAPI plus TRITS filters were used. n, nucleus; c, cytoplasm.

We further delineated the putative nuclear localization signal of pSTRA13 using a set of deletion mutants (Fig. 3A). Deletions of most of the pSTRA13 N-terminal region (mutants 69-412, 123-412, 184-412, and 259-412) did not change nuclear localization of the resulting products (not shown). In contrast, deletion of the 113 C-terminal amino acids (1-299 mutant) or further truncation of the protein from N terminus (293-412 mutant) prevented nuclear localization (Fig. 4B), demonstrating only cytoplasmic distribution of the truncated proteins. This result indicates that the STRA13 protein possesses nuclear localization signal(s) in its C-terminal region that is necessary for nuclear translocation.

It had been shown previously that pUBC9 is a nuclear protein (17, 18). We confirmed that HA-UBC9 displays nuclear localization (data not shown) as well as the protein of our interest, STRA13. These data provide additional evidence of the possibility of physical interaction between pSTRA13 and pUBC9 in the cell nucleus.

Inhibition of the 26 S Proteasome Activity Leads to Accumulation of pSTRA13-- Association of pSTRA13 and ubiquitiun-conjugating enzyme raised the possibility that pSTRA13 could be degraded through an UBC9-dependent proteasome degradation pathway. Therefore, we asked whether blocking of the major cellular protein degradation pathway (19), i.e. the pathway mediated by the 26 S proteasome, would lead to accumulation of transiently expressed pSTRA13. HEK 293 cells were transfected with FLAG-STRA13-pCMV2 expression vector and exposed to various concentrations of the proteasome inhibitor ALLnL (20). Treatment of transfected cells with ALLnL resulted in accumulation of STRA13 that was dependent on ALLnL concentration (Fig. 5A). Treatment of the cells with vehicle alone (Me2SO) did not produce any effect (not shown). These results demonstrate that the level of STRA13 in the cells is regulated through the 26 S proteasome pathway.


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Fig. 5.   pSTRA13 is accumulated and ubiquitinated upon proteasome inhibitor treatment. A, the proteasome inhibitor ALLnL blocks degradation of the pSTRA13, resulting in accumulation of pSTRA13 and its high molecular protein complexes in a concentration-dependent manner. The FLAG-tagged pSTRA13 was detected with anti-FLAG M2 antibodies in lysates of HEK 293 cells transiently transfected with the pFLAG-STRA13 plasmid and treated for 18 h with various concentrations of ALLnL. B, the pSTRA13 is ubiquitinated in vivo. FLAG-STRA13 from 293 cells transfected with pCMV2-STRA13 plasmid and treated with ALLnL as described above was immunoprecipitated with the anti-FLAG polyclonal Abs. The immunoprecipitates were analyzed by SDS-PAGE and Western blotting with the monoclonal antibodies to ubiquitin or FLAG. The arrow indicates the position of the nonubiquitinated form of pSTRA13. The bracket shows the position of ubiquitinated pSTRA13. IP, immunoprecipitation; W, Western blot. Molecular weights of FLAG-STRA13 molecular complexes are shown on the right.

STRA13 Is Ubiquitinated in Mammalian Cells-- Transiently expressed FLAG-STRA13 protein always produced high molecular mass bands of ~105, 120, 170, and 200 kDa (Fig. 5A) even under reducing conditions of protein electrophoresis. The presence of these bands suggested the pSTRA13 ability to produce a variety of stable protein complexes including ubiquitin-STRA13 complexes.

Protein degradation through the 26 S proteasome pathway requires tagging of a protein by covalent attachment of multiple ubiquitin molecules (19). We next investigated whether pSTRA13 could be ubiquitinated in vivo. FLAG-STRA13-pCMV2 expression plasmid or an empty vector were transiently expressed in HEK 293 cells. Twenty-four h later, the cells were treated with various concentration of proteasome inhibitor ALLnL for 20 h. Cell lysates were prepared, and pSTRA13 was immunoprecipitated with anti-FLAG antibodies. Precipitated proteins were separated by SDS-PAGE, blotted into ECL nitrocellulose membranes, and probed with monoclonal antibodies to either FLAG or ubiquitin. A faint ladder of bands visible above the 55-kDa marker (apparent molecular mass of pSTRA13) indicated the formation of multiple ubiquitin conjugates (Fig. 5B). The ubiquitination was more pronounced after treatment with 100 mM ALLnL.

These results imply that pSTRA13 is ubiquitinated in mammalian cells and that proteasome inhibitor blocks its degradation. Thus, we confirmed that the ubiquitin-proteasome pathway appears to regulate level of this protein.

N- and C-terminal Parts of pSTRA13 Contain Motifs Responsible for Its Degradation-- We used a series of STRA13 deletions (Fig. 3A) to find protein motifs involved in protein degradation. We transiently expressed STRA13 mutants fused to the FLAG epitope in HEK 293 cells. 24 h later, the cells were treated with 100 mM ALLnL overnight and then lysed and analyzed by 8-16% SDS-polyacrylamide gel. Untreated transfected cells served as a control. Mutants lacking the N or C terminus of pSTRA13 showed a significant increase in protein degradation (bands representing truncated proteins were much weaker than that of the entire STRA13 protein; Fig. 6A) and rapid accumulation of the truncated proteins in response to inhibition of proteolysis. Noticeably, the steady-state level of the 1-299 mutant lacking 113 amino acid residues from its C terminus was barely detectable upon transient expression but significantly increased after exposure to proteasome inhibitor.


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Fig. 6.   Deletion mutants of pStra13 are unstable. A, cell treatment with proteasome inhibitor ALLnL causes accumulation of pSTRA13 and pSTRA13 deletion mutants. The aliquots of extracts from HEK 293 cells transiently transfected with the FLAG-pSTRA13 plasmid or with the series of FLAG-fused STRA13 deletion mutants are loaded on odd-numbered lanes; even-numbered lanes are loaded with the lysates from the same set of transfections treated with 100 mM ALLnL for 18 h. The amount of FLAG-STRA13 proteins was detected by immunoblotting with anti-FLAG M2 antibodies. Molecular weights of FLAG-STRA13 truncated proteins are indicated on the right and on the left. B, truncation of either terminus of the pSTRA13 decreased the half-life of the proteins. Cycloheximide at a final concentration of 50 µg/ml was added to HEK 293 cells transiently transfected with the indicated FLAG constructs. Whole cell lysates were prepared at the indicated time points following the addition of cycloheximide. The amount of STRA13 protein was analyzed with the anti-FLAG M2 antibodies. Even loading on the lanes was monitored by staining of the membranes with Ponceau dye reagent (not shown). The level of STRA13 was calculated by measuring of the relative band intensity with "Image 2" software. The half-life was calculated as the mean of two independent experiments. a.a., amino acids.

We also studied degradation of pSTRA13 and its mutants using the protein synthesis inhibitor cycloheximide to block new protein synthesis. Fifty mg/ml of cycloheximide was added to HEK 293 cells transiently transfected with the deletion constructs, and 24 h later lysates were prepared at the indicated intervals. We defined the FLAG-pSTRA13 half-life as ~30 h. In contrast, the deletion mutants' half-life values were significantly lower (Fig. 6B). These data are in agreement with ALLnL treatment experiments described above.

The results indicate that pSTRA13 harbors protein-stabilizing elements at the N- and C-terminal regions, so that truncation of either leads to a significant increase in degradation rate.

Co-expression of UBC9 and STRA13 Increases pSTRA13 Ubiquitination and Turnover Rate-- To demonstrate the role of UBC9 in pSTRA13 degradation directly, we tested the effect of co-expression of these two proteins in HEK 293 cells. The cells were transiently transfected with the FLAG-STRA13 construct alone or together with the HA-UBC9 expression plasmid. Co-expression of FLAG-STRA13 and HA-ATBF (C-terminal region of ATBF protein fused to HA tag) was used as a control. Forty-eight h after transfection, cells were solubilized and analyzed by SDS-PAGE and Western blotting with anti-FLAG antibody. In the absence of exogenously expressed UBC9 or in combination with HA-ATBF protein, pSTRA13 showed high expression level. In contrast, an ~5-fold reduction in the pSTRA13 level was observed upon its co-expression with UBC9 (Fig. 7A).


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Fig. 7.   Stability of pSTRA13 is dependent on pUBC9 and is not dependent on pVHL and hypoxia. A, pSTRA13/UBC9 co-expression decreases the STRA13 protein level. HEK 293 cells were transiently transfected with FLAG-STRA13 alone or co-transfected with HA-UBC9 or HA-ATBF domain as a control. Equal protein amounts from lysates of transfected HEK 293 cell were loaded on a gel. The level of STRA13 was determined by Western blot with the FLAG M2 antibodies. B, co-expression of pSTRA13 and UBC9 decreases the pSTRA13 half-life. Cell transfection, treatment with cycloheximide, and pSTRA13 half-life calculation were performed as described in the legend to Fig. 6B. C, co-expression of pSTRA13 and UBC9 increases pSTRA13 ubiquitination level. The HEK 293 cells were transiently co-transfected with FLAG-pSTRA13 and HA-UBC9 or HA-ATBF domain as a control and treated or not treated with the proteasome inhibitor ALLnL. FLAG-STRA13 was immunoprecipitated (IP) with the polyclonal FLAG antibodies. Co-precipitated ubiquitin was detected with anti-ubiquitin monoclonal antibodies (W). D, VHL and hypoxia have no impact on pSTRA13 stability. 293T cells were transiently transfected with FLAG epitope-tagged STRA13 alone or in combination with a VHL expressing plasmid. Twenty-four h after transfection, cells were split on two equal plates for 20 h of hypoxia treatment and for normoxic control.

To further confirm the effect of UBC9 on pSTRA13 degradation, we applied cycloheximide to cells co-transfected with STRA13 and UBC9. Consequently, the pSTRA13 half-life decreased from ~30 h in cells expressing the protein alone to ~8 h in cells expressing both STRA13 and UBC9 (Fig. 7B).

We also checked whether UBC9 is directly involved in pSTRA13 ubiquitination. We immunoprecipitated FLAG-pSTRA13 with anti-FLAG polyclonal antibodies from lysates of HEK 293 cells co-transfected with FLAG-STRA13 and HA-UBC9 or HA-ATBF. Proteins were separated by SDS-PAGE, blotted onto ECL nitrocellulose membranes, and probed with a monoclonal antibodies to ubiquitin. This experiment demonstrated that the amount of ubiquitinated pSTRA13 was increased in the presence of UBC9 as compared with the ATBF control (Fig. 7C).

Taken together, these results strongly suggest that pSTRA13 is targeted for proteolysis by the ubiquitin-dependent proteasome pathway through association with UBC9.

Mapping pSTRA13/UBC9-interacting Regions-- To identify pSTRA13 domains that bind UBC9, a series of cDNA deletion mutants fused to the FLAG epitope (Fig. 3A) and HA-UBC9 were cotransfected to HEK 293 cells, and proteins were co-immunoprecipitated as described above. Due to extreme instability of STRA13 truncated proteins, the proteasome inhibitor ALLnL was added to the transfected cells to prevent mutant degradation. The anti-FLAG antibodies bind to all truncated proteins with equal efficiency (Fig. 6). The deletion of the C-terminal part of the STRA13 in the 1-299 mutant abolished the pSTRA13/pUBC9 interaction (Fig. 3B, line 7). Subsequent deletions of the N-terminal bHLH domain or three adjacent alpha -helices (Fig. 3, lines 3-6 and 8) did not lead to binding abrogation. Therefore, the region between amino acid residues 299 and 412 is absolutely required for the pSTRA13/pUBC9 interaction, whereas the bHLH domain and all three alpha -helices were dispensable.

VHL and Hypoxia Have No Impact on the STRA13 Protein Degradation-- pVHL is known to regulate its targets either on the mRNA level (2-7) or in an oxygen-dependent manner on the protein level through the proteasome degradation pathway (HIF-1alpha (1, 52)). In this study, we showed that STRA13 is VHL- and hypoxia-regulated on the mRNA level. We also demonstrated that under normoxic conditions, STRA13 protein is degraded through the ubiquitin-proteasome pathway. Therefore, it was of interest to investigate the potential involvement of hypoxia and VHL in STRA13 protein degradation. We transiently transfected 293T cells with FLAG epitope-tagged STRA13 alone or in combination with a VHL-expressing plasmid. Since the STRA13 construct did not contain native 5'- or 3'-regulatory elements, all changes in the expressed STRA13 protein amount would reflect its regulation by protein degradation. Twenty-four h after transfection, cells were split on two equal plates to compare the effect of hypoxia on STRA13 protein stability. After 20 h of hypoxic treatment, no changes in the STRA13 protein amount were detected as compared with normoxic conditions (Fig. 7D, lanes 1 and 2). Transient co-expression of FLAG/STRA13 and VHL also did not result in STRA13 protein accumulation or degradation during hypoxia (Fig. 7D, lanes 3 and 4). These results indicate that stability of STRA13 protein is not affected by hypoxia or VHL.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Involvement of bHLH-containing proteins, such as c-myc, Max (reviewed in Ref. 21), and HIF-1 (22) in carcinogenesis is well established. In this study, we characterized the bHLH-containing pSTRA13 as a new cancer-associated protein. A bHLH domain of pSTRA13 is highly homologous to bHLH domains of hairy and E(spl) proteins involved in Drosophila embryogenesis (23). It was shown that the Stra13 expression during mouse development is restricted to the neuroectoderm and also to a number of mesodermal and endodermal derivatives (9). Overexpression of mouse Stra13 in P19 cells led to repression of mesodermal and endodermal promoting neuronal differentiation (9). Sun and Taneja (11) showed that pStra13 mediates growth arrest in cell culture. Altogether, these data strongly suggest that the protein may play a critical role during embryogenesis and may be involved, like many other genes engaged in differentiation and cell cycle regulation, in carcinogenic pathways.

In our work, we confirmed that the STRA13 gene is down-regulated by pVHL on the mRNA level. Expression of the STRA13 mRNA is dependent on the VHL status in renal cell carcinoma cell lines with involvement of both the elongin-binding domain and beta -domain of pVHL (Fig. 1). pVHL plays a critical role in hypoxia-regulated proteolysis of the HIF-alpha subunits of the transcriptional heterodimer HIF-1. HIF-alpha proteins are significantly stabilized in response to oxygen deprivation. Because pVHL regulates stability of HIF-1, which, in turn, orchestrates expression of hypoxia-sensitive genes, it may be suggested that most, if not all, pVHL targets are also HIF-1 targets and, thus, are hypoxia-up-regulated. We demonstrated that the STRA13 gene is indeed hypoxia-sensitive (Fig. 1C), like previously studied pVHL targets vascular endothelial growth factor (3), glycolytic enzymes lactate dehydrogenase and glycerophosphate dehydrogenase (4), glucose transporter GLUT1 (3), and carbonic anhydrases CA9 and CA12 (7). In addition, STRA13 possesses another feature typical for a number of pVHL targets; it is up-regulated in a large variety of common tumors (Fig. 2). Taken together, our observations on STRA13 expression analysis in different types of malignant cells suggest that the gene may be 1) up-regulated in pVHL-deficient tumors, 2) induced by hypoxia in pVHL-positive tumors, and 3) up-regulated in pVHL-positive tumors by unknown mechanisms. The morphogenic potential of STRA13 (9) combined with its cancer-associated elevated expression warranted further investigation into its regulation on the protein level.

Ubiquitin-dependent proteolysis is a highly complex, temporally controlled, and tightly regulated process that plays important roles in a broad array of basic cellular functions. The list of cellular proteins targeted by ubiquitin is growing rapidly. These are cell cycle regulators, tumor suppressors and oncogenes, transcriptional activators and their inhibitors, cell surface receptors, and endoplasmic reticulum proteins (19, 24). Protein ubiquitination requires the sequential action of three enzymes, ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin-protein isopeptide ligase (E3). Usually there is a single E1, but there are many species of E2s and E3s. While in Saccharomyces cerevisiae 13 genes encode E2 and E2-like proteins, many more ubiquitin-conjugating enzymes have been described in mammals (25). Some E2s are involved in specific cellular processes, while the role of others is obscure. One S. cerevisiae E2 called UBC9 and its ortholog, hus5 (Schizosaccharomyces pombe), have been shown to be essential for cell viability (26) and for normal mitosis (27). Human UBC9 shares 75 and 82% amino acid similarity with S. cerevisiae and S. pombe proteins, respectively (27, 28), and is 100% identical to the product of the mouse Ubc9 gene (29).

Human UBC9 was identified as an interacting protein in yeast two-hybrid screening with a surprisingly large number of partners (17, 18, 30-38). Two major functions for UBC9 were proposed. According to one set of data obtained on Ran GTPase-activating protein (39-41) and other proteins (42, 43), UBC9 is a key conjugating enzyme for the sentrin-dependent pathway. Modification of Ran GTPase-activating protein by sentrin, a small ubiquitin-like protein, does not lead to proteolysis but, instead, appears to modulate the Ran GTPase-activating protein subcellular localization. Other results obtained on E2A (37), ATF2 (17), and MITF (38) support the idea that UBC9 is involved in the ubiquitin-dependent protein degradation.

With the help of a yeast two-hybrid assay, we found that UBC9 interacts with pSTRA13. The data we present here assure that the meaning of this interaction is in maintaining tight control of pSTRA13 turnover, supporting the idea of direct involvement of UBC9 in the protein degradation pathway.

Interaction of UBC9 and STRA13 was demonstrated in vivo in a yeast two-hybrid assay (data not shown) and was further confirmed in mammalian cells by the co-immunoprecipitation assay (Fig. 3B, lanes 1 and 2).

We found that pSTRA13, like pUBC9 (17, 18, 37), is localized primarily in the nucleus that confirms their physical ability to interact. The deletion of 113 C-terminal amino acids or 292 N-terminal amino acids resulted in an entirely cytoplasmic distribution of the protein (Fig. 4B), while subsequent deletions of the 258 N-terminal amino acids did not alter nuclear localization (Fig. 4A). Nuclear proteins like transcription factors and ribosomal proteins are synthesized in the cytoplasm and have to be transported into the nucleus to fulfill their functions. The transport of proteins through the nuclear pore complex is an active, energy-requiring process. The best characterized protein import pathway is the "classical" nuclear localization signal-dependent pathway (44). Proteins that contain a simple or bipartite basic amino acid sequence called the nuclear localization signal are imported into the nucleus by a heterodimeric receptor that consists of the importin-alpha and importin-beta subunits (44). In the past few years, knowledge about protein trafficking across the nuclear membrane has grown exponentially. It was shown that another mechanism such as substrate phosphorylation (reviewed in Ref. 45) or protein dimerization (46) regulates nuclear transport as well. From our data, we may conclude that localization of STRA13 is determined by a specific C-terminal sequence called the nuclear localization signal. In fact, computer prediction of the basic nuclear localization signal at position 286 (PSORT II server) of STRA13 is in agreement with our experimental data. We may also conclude that STRA13 nuclear localization does not depend on the ability of STRA13 to form dimers like in the case of the mitogen-activated protein kinase extracellular signal-regulated kinase 2 (46). We showed that STRA13 needs the entire protein for homodimerization (data not shown), so most of the deletion mutants lost their ability to homodimerize but did not change their distribution in the nucleus.

Since UBC9 is involved in ubiquitin-dependent proteasome degradation of some proteins (17, 37, 38), we checked whether the STRA13 protein is a target for 26 S degradation machinery. We found that exposure of STRA13-transfected HEK 293 cells to 26 S proteasome inhibitor ALLnL led to pSTRA13 ubiquitination and accumulation in a dose-dependent manner. This is the first demonstration of pSTRA13 degradation through the ubiquitin-proteasome pathway.

We showed that pSTRA13 is relatively stable as compared with strikingly short half-lives of other stress-activated transcription factors such as HIF-1alpha (half-life under normoxia is a few minutes) (47), c-Myc (~20-30 min) (48), c-Jun (~90 min) (49), or p53 (~7-8 h) (see Ref. 50 and references therein). We estimated the half-life of pSTRA13 as ~30 h (Fig. 7B), which correlates with the predicted half-life value of ~30 h in vitro and ~20 h in yeast in vivo.

In our attempt to identify the regions of pSTRA13 that are required for its stabilization, we found that the entire STRA13 protein has a much shorter half-life than its C or N terminus-truncated derivatives (Fig. 6B). For instance, the steady-state level of the least stable mutant (1) lacking 113 C-terminal amino acids was hardly detectable upon transient expression but significantly increased after exposure to ALLnL (Fig. 6B). The half-life of the mutant was less than 0.5 h, which is ~60 times shorter than that of the entire pSTRA13. Given that the whole STRA13 protein is needed for homodimerization (data not shown), it is tempting to speculate that pSTRA13 is stabilized by either homo- or heterodimerization. A similar protein stabilization mechanism was demonstrated for two transcription factors, MATa1 and MATalpha 2. Both were stabilized by heterodimerization that interfered with degradation by masking the ubiquitin recognition signal (51). It was reported recently that pVHL proteins with mutations in the CBD domain disrupting elongin B and C binding were highly unstable and degraded in a proteasome-dependent manner (14). It is possible that a similar proteasome-resistant complex may form between pSTRA13 and its yet unidentified partners and that truncation of either terminus would lead to dissociation of such complexes with immediate ubiquitination and degradation of pSTRA13.

Further evidence of UBC9 direct involvement in ubiquitin-dependent degradation of pSTRA13 was obtained in experiments on these two proteins' co-expression. The level of pSTRA13 was significantly decreased upon UBC9/STRA13 co-expression. STRA13 and ATBF domain co-expression did not change the amount of STRA13 protein (Fig. 7A). The half-life of pSTRA13 in the presence of UBC9 was shortened from ~30 to ~8 h (Fig. 7B). Moreover, the ubiquitination level of pSTRA13 was higher when pSTRA13 was co-expressed with UBC9 than when it was co-expressed with the ATBF domain (Fig. 7C).

We have determined that the interaction between STRA13 and UBC9 required the C-terminal end of STRA13 and was unaffected by deletions of the bHLH domain or three N-proximal alpha -helices (Fig. 3B). It has been shown previously that highly unstable helix-loop-helix E2A protein that binds Ubc9 for degradation purposes also does not need the bHLH domain for the interaction (37). On the other hand, interaction of another helix-loop-helix protein, TEL, with UBC9 through the HLH domain modulates TEL transcriptional activity but does not lead to TEL degradation (18). These data suggest that fulfillment by UBC9 of two different functions (protein degradation and modulation of subcellular localization) occurs through interactions with different proteins' domains.

Collectively, these results strongly imply that pSTRA13 is targeted for proteolysis by the ubiquitin-dependent proteasome pathway through association with UBC9.

To address the possible involvement of VHL in pSTRA13 degradation and to find whether protein stability depends on oxygen level as was shown for HIF-1alpha , we set up a hypoxia assay. We did not find any effect of hypoxia or VHL on the degradation of exogenously expressed FLAG-tagged STRA13 protein (Fig. 7D). In contrast, in a similar experiment transiently expressed HIF-1alpha protein was rapidly degraded under normoxic conditions and stabilized under hypoxic conditions in a VHL-dependent manner (52). Thus, STRA13 regulation by VHL is distinct from that of HIF-1alpha and is mainly maintained on the mRNA level, as was shown for a set of other pVHL targets, such as vascular endothelial growth factor (2, 3), GLUT-1 (3), glycolytic enzymes (4), tyrosine hydroxylase (53), and carbonic anhydrases CAIX and CAXII (7). Some of these targets are regulated by VHL on transcription initiation (4-7) or elongation (53) levels. Regulation of other targets is maintained on the mRNA stabilization level (2, 3). The exact mechanism of STRA13 mRNA regulation remains to be elucidated.

Identification and molecular characterization of a new hypoxia-sensitive, cancer-associated pVHL target, the bHLH STRA13 protein, opens new directions for unraveling pVHL functions in carcinogenesis. It further broadens the class of already pleiotropic complex pVHL/HIF-1 targets, implying the existence of pSTRA13 partners and target genes of that potent transcriptional regulator that may be involved in carcinogenic pathways.

    ACKNOWLEDGEMENTS

We thank Dr. Michael T. Chin for providing pBDGAL4-ARNT plasmid and Dr. Vera Matrosova for helpful advice with immunofluorescent microscopy. We are grateful to Dr. Ed Leonard for helpful comments and review of the manuscript.

    FOOTNOTES

* This work was supported by NCI, National Institutes of Health, Contract NO1-CO-56000.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed. Tel.: 301-846-1288; Fax: 301-846-6145; E-mail: ivanova@mail.ncifcrf.gov.

Published, JBC Papers in Press, February 6, 2001, DOI 10.1074/jbc.M010516200

2 Throughout, STRA13 is used instead of DEC1 (differentially expressed in chondrocytes) to avoid confusion with the unrelated gene DEC1 (deleted in esophagus cancer; GenBankTM accession number NM_017418) and highlight its close similarity to the mouse Stra13 gene.

    ABBREVIATIONS

The abbreviations used are: pVHL, von Hippel-Lindau tumor-suppressor protein; HIF-1, hypoxia-inducible factor 1; HLH, helix-loop-helix; bHLH, basic helix-loop-helix; Ab, antibody; HA, hemagglutinin; DAPI, 4',6-diamidino-2-phenylindole; ALLnL, acetyl-leucinyl-leucinyl-norleucinal; E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin-protein isopeptide ligase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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