From the 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
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ABSTRACT |
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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.
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-1 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 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
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
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).
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
( STRA13 Is a Hypoxia-inducible Gene--
Due to the fact that pVHL
orchestrates cellular responses to hypoxia through regulation of
HIF-1- 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).
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).
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).
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.
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.
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).
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 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-1 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 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- 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-1 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 MAT 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 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-1 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.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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/HIF-1
heterodimer. Under normoxic conditions, the
subunit interacts with the pVHL-elongin B-elongin C complex and
is rapidly degraded (1). Hypoxia or pVHL deficiency stabilizes
HIF-1
, 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-
(5), and transforming growth factor-
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).
-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
-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).
-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.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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
-galactosidase activity by the filter assay according to the manufacturer.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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
-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
-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 -actin
probe shows even loading.
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.
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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.
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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.
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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.
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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.
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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.
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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.
-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
-helices were dispensable.
(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
-domain of pVHL
(Fig. 1). pVHL plays a critical role in hypoxia-regulated proteolysis
of the HIF-
subunits of the transcriptional heterodimer HIF-1.
HIF-
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.
and importin-
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.
(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.
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.
-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.
, 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-1
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-1
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.
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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.
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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.
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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.
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