From the Center for Nutrition and Toxicology,
Department of Bioscience at NOVUM, Karolinska Institutet, SE-141
57 Huddinge, Sweden, and § Laboratory of Molecular Genetics,
National Institute of Chemical Physics and Biophysics and Department of
Gene Technology, Tallinn Technical University, Akadeemia 23, 12618 Tallinn, Estonia
Received for publication, September 16, 2002, and in revised form, November 7, 2002
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ABSTRACT |
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The Hedgehog signaling pathway is involved
in both development and cancer induction in a wide range of organisms.
The end point of the Hedgehog signal-transduction cascade is the
Gli/Ci, zinc-finger transcription factors. Proteins such as Fused,
Suppressor of fused (SUFU), Costal-2, and protein kinase A are
essential for regulation of Gli/Ci processing, activity, and
localization. Coimmunoprecipitation and Far Western assays, coupled
with truncation analysis and mutagenesis have been used to define the
region of interaction between Gli proteins and SUFU. We identify a
novel motif SYGH in Gli/Ci family proteins, which is required for the interaction with SUFU. Mutational studies revealed that
Gly122 and His123 are crucial for
binding to SUFU, suggesting the importance of hydrophobicity for the
correct binding conformation. Functional analysis revealed that the
activity of GLI transcription factors with mutations in this motif is
no longer suppressed by co-expression of SUFU. Moreover, we have found
that a C-terminal 19-amino acid deletion in SUFU ( GLI1 was originally isolated as a highly amplified gene
in a malignant glioma and subsequently implicated in the development of
other tumor types, including liposarcoma, rhabdomyosarcoma, osteosarcoma, and astrocytoma (1-3). Later it was shown that GLI1 encodes a transcription factor that is a downstream
nuclear component of the Sonic hedgehog-patched signaling pathway. This pathway is evolutionary conserved and plays important roles in tissue
patterning during early embryogenesis in vertebrates and invertebrates
(4, 5). In addition to GLI1, two other isoforms have been identified in
vertebrates, Gli2 and Gli3, each encoded by separate genes (6). All Gli
proteins bind to DNA through five Zn-finger domains that recognize the
consensus sequence 5'-TGGGTGGTC-3' (7). The role of each Gli isoform in
mediating the hedgehog signal is not clear yet but recent studies have
shown overlapping roles and indicated some functional redundancy (8,
9). There are indications that Gli1 is mainly a transcriptional
activator, whereas Gli2 and Gli3 can act both as activators and
repressors depending on specific post-translational modifications (10). Gli proteins include both a nuclear export signal and a nuclear localization signal, suggesting that they shuttle between the nucleus
and the cytoplasm.
Gli and Ci (Drosophila homolog of Gli proteins) are known to
interact with a number of proteins such as the Ser/Thr kinase Fused
(11), Suppressor of Fused
(SUFU)1 (12, 13), and a
kinesin-like protein Costal-2 in Drosophila (14). This
complex is assumed to receive and transmit a signal induced by hedgehog
ligands to activate or repress certain genes. Moreover, Gli2 and Gli3
have potential binding sites for interaction with cAMP-response
element-binding protein/p300 (a coactivator-acetyltransferase), whereas Gli1 lacks such a site (15).
SUFU encodes a 484-amino acid cytoplasmic protein. Genetic screens in
Drosophila first identified Su(fu) as a suppressor of mutants in the Fused serine/threonine kinase, a positive regulator of
Hh signaling (12). It has recently been demonstrated that SUFU acts as
a negative regulator of Hh signaling. SUFU might inhibit the Hh
signaling pathway through two distinct mechanisms: sequestration of
Gli/Ci proteins in the cytoplasm (13-17) and direct interaction with
Gli proteins on DNA (13, 14, 18, 19). However, the precise role of SUFU
is still unknown. Interestingly, loss-of-function mutations in SUFU
were recently reported in medulloblastomas showing that SUFU is a human
tumor-suppressor gene most likely because of its normal role as a
negative regulator of hedgehog signaling (20).
Three alternatively spliced transcripts of SUFU with tissue specific
expression have been identified. One SUFU variant has a trinucleotide
insertion resulting in an extra glutamine and a protein of 485 amino
acids. A second variant, SUFU-Lk, lacks exon 10 encoding a protein of
388 amino acids. The third variant, SUFU-Tt, has an extra exon after
exon 8 (exon 8a) resulting in a protein containing 359 amino acids. The
latter two of these variants have lost the ability to interact with
GLI1 (21). In addition, an alternative splice variant of 433 amino acids lacking the C terminus was identified and reported to
retain ability to suppress Gli-mediated activation of transcription
(16). These observations underscore the importance of the C-terminal
domain of SUFU for biological function.
Interaction domains for each individual protein involved in the
intracellular signaling complex have not yet been precisely mapped, and
no known protein-protein interaction motifs have been found by sequence
analysis. Therefore, we decided to pinpoint domains of Gli proteins and
SUFU, which specifically contribute to their interaction. We have used
a combination of immunoprecipitation in vivo and in
vitro and Far Western blot analysis to define the required
fragment and specific amino acids of GLI1 that are essential for
full SUFU-binding activity. We show that this fragment comprises residues 116-125, which are necessary for SUFU association. Designed mutant proteins corresponding to alterations in the SYGH amino acid
motif within the interaction region selectively disrupted this
protein-protein interaction. Functional analysis revealed that the
activity of GLI transcription factors with mutations in this motif
is no longer suppressed by co-expression of SUFU. We have also
demonstrated the requirement of the C-terminal domain (19 amino acids)
of SUFU for the interaction with GLI1. Deletion of these amino
acids led to relocation of SUFU from the cytoplasm to the nucleus. We
assume that this effect can be explained by loss of sequestering of
SUFU in the cytoplasm.
Plasmids--
To express MYC-tagged and hemagglutinin
(HA)-tagged proteins, relevant sequences were amplified by PCR and
subcloned into pCMV and pcDNA3 vectors. Sequences included
full-length human GLI1 and SUFU. The deletion series of GLI1
such as GLI1-(55-407), -(78-407), -(111-407), -(125-407),
-(424-1111) were also cloned in the same vectors. The following
regions GLI1-(55-116), -(55-125), -(111-407), Gli2-(205-300);
GLI3-(250-350) were used to generate thioredoxin fusion proteins. The
fragments were PCR-amplified using specific primers and cloned into the
pBAD/TOPO vector (Invitrogen). All constructs were sequence verified.
Immunoprecipitation Studies and Western Blot Analysis--
Human
293T cells (embryonic kidney epithelial cell line) were maintained in
Dulbecco's modified Eagle's medium containing 10% fetal calf serum.
Cells were transiently transfected with expression constructs encoding
HA-tagged GLI1 or Myc-tagged SUFU using the Superfect transfection
reagent (Qiagen) according to the manufacturer's instructions. Before
harvesting, cells were washed twice with 1× PBS and lysed in 1 ml of
immunoprecipitation buffer (1× PBS, 1% Nonidet P-40, complete
protease inhibitor mixture (Roche)). Cell suspensions were centrifuged
at 14,000 rpm for 20 min to remove nuclei and unbroken cells.
Immunoprecipitations were performed using antibodies to the HA- or
c-Myc epitopes conjugated with agarose. Proteins were separated by
SDS-PAGE, transferred to nitrocellulose, and detected with antibodies
to the HA or Myc epitopes, using the chemiluminescence detection system
(PerkinElmer Life Sciences).
Immunoprecipitation in vitro was performed as follows; GLI1
fragments and SUFU were synthesized by in vitro translation
in the rabbit reticulocyte lysate system (TNT, Promega). The in
vitro translation mixture was diluted with 1× PBS buffer and
immunoprecipitated with anti-Myc antibodies. Pellets were washed four
times with 1× PBS or other washing conditions as indicated,
resuspended in sample buffer, and resolved by SDS-PAGE.
Expression of Thioredoxin Fusion Proteins--
The
Escherichia coli Top10 strain was transformed with different
deletion constructs of GLI1. Fusion proteins were generated with the
N-terminal-attached thioredoxin according to the manufacturer's protocol (pBAD/Topo®ThioFusionTM Expression System, Invitrogen). After induction with different concentrations of arabinose for 4 h, the cells were harvested by centrifugation and resuspended in lysis
buffer (50 mM NaH2PO4, 300 mM NaCl, 1 mM imidazole). The cells were
disrupted by sonication, and the lysate was subjected to centrifugation
for 30 min at 14,000 rpm.
Far Western Blot--
E. coli cells expressing GLI1
fragments were collected by centrifugation and lysed by boiling in SDS
sample buffer. Lysates were resolved by SDS-PAGE and transferred onto
nitrocellulose membranes (Amersham Biosciences). Proteins were
visualized by staining with Ponceau-S (0.5% in 1% acetic acid).
Membranes were blocked in buffer A containing 10% glycerol, 100 mM NaCl, 20 mM Tris-HCl at pH 7.5, 0.5 mM EDTA, 0.1% Tween 20, 5% skim milk powder for 1 h
at room temperature.
SUFU probes were synthesized by coupled in vitro
translation/transcription (Promega TNT with T7 RNA polymerase) in the
presence of [35S]methionine. Reactions were diluted with
buffer and passed over Millipore spin columns to remove unincorporated
methionine. Subsequently, the blocked membranes were incubated for
1 h in the same buffer containing 35S-labeled SUFU.
Filters were washed three times with buffer A, allowed to dry and
exposed to x-ray film (Eastman Kodak).
Site-directed Mutagenesis--
Point mutations were introduced
by site-directed mutagenesis using the QuikChange site-directed
mutagenesis kit (Stratagene) according to the manufacturer's
instruction. A fragment of GLI1 (55-407) in pBAD or full-length GLI1
in pEGFP-C2 (Clontech) were used as the template
for mutagenesis. For all constructs, mutagenesis was confirmed by
sequence analysis.
Luciferase Assay--
The expression vectors for GLI1 and SUFU
used in this study have been described previously (13). All constructs
were verified by DNA sequencing. The NIH-3T3 cell clone Shh-LIGHT
having a stably incorporated GLI-luc reporter (22) was used for the
luciferase reporter assay. One day before transfection the cells were
plated onto 24-well plates so that on the next day the cell density was ~80%. Cells were transfected for 3 h using 0.4 µg of GLI1
wild-type or mutant DNA and 0.2 µg of SUFU DNA or empty vector using
Biofect (InBio) according to the manufacturer's instructions at a
ratio of DNA to Biofect of 1:3 (w/w). After 24 h the medium was
replaced with low serum media and cells were incubated for an
additional 24 h. Subsequently cells were lysed and luciferase
activity was measured with a luciferase kit from Tropix (Bedford)
according to the manufacturer's instructions using an Ascent Fluoroscan combined fluori- and luminometer (Thermo LabSystems). All experiments were repeated three times and normalized for alkaline phosphatase activity.
Cell Fractionation Experiments--
293T cells were transfected
with full-length SUFU or SUFU- Mapping the GLI1 Domain That Is Responsible for SUFU Interaction in
Vivo--
As a first step we determined the affinity of the GLI1-SUFU
interaction by examination of its sensitivity to elevated salt concentration. An N-terminal deletion construct (1-407) of GLI1 and
Myc-tagged SUFU were produced by in vitro translation in the presence of [35S]methionine, and the translation mixture
was subjected to immunoprecipitation using an anti-Myc antibody. GLI1
and SUFU associate with high affinity, as these associations occur in a
600 mM NaCl buffer (Fig.
1A).
It was previously shown that the GLI1 (1-407) amino acid region is
capable of binding SUFU (13). To further analyze this interaction, we
mapped the region of GLI1 required for interaction with SUFU.
Expression constructs encoding different lengths of HA-tagged GLI1 were
cotransfected into 293T cells with Myc-tagged SUFU. Fig. 1 shows a
schematic diagram of the amino acid regions of GLI1 used for the
experiments. GLI1 fragments and SUFU were coimmunoprecipitated from
lysates of transfected cells using monoclonal antibodies specific for
the Myc and HA epitope tags. The presence of the proteins in the
immunoprecipitates was monitored by Western blot analysis (Fig. 1,
A and C). Proteins containing GLI1 fragments 111-407 and 78-407 interacted with SUFU, whereas GLI1 fragments containing amino acids 125-407 or 143-407 failed to maintain binding to SUFU. Thus, our results indicate that the N-terminal region of GLI1
including residues 111-125 defines a SUFU-binding domain.
Far Western Binding Assay with N-terminal Fragments of
GLI1--
To confirm the importance of the 111-125 amino acid domain
of GLI1 for the binding of SUFU, we constructed and expressed in E. coli a series of deletion derivatives of GLI1 fused with
thioredoxin. Cleared bacterial lysates of each fusion protein were
resolved by SDS-PAGE and transferred onto nitrocellulose membranes. The interaction of GLI1 fragments with SUFU was determined by Far-Western blot analysis using in vitro translated
35S-labeled SUFU as a probe (Fig.
2). The GLI1 fragment including amino
acids 55-407 was used as a positive control. Thioredoxin itself was
used as a negative control. The GLI1 fragment including amino acids
55-116 failed to interact with SUFU. Inclusion of the 116-125 amino
acid domain recovered SUFU binding. Thus, the results show that the
minimal required region for GLI1-SUFU binding is amino acid residues
116-125 of GLI1.
Interaction of SUFU with Other Members of the Gli Family--
We
aligned the pinpointed region to reveal the homology with other Gli/Ci
proteins, as shown in Fig. 3A.
In addition, we used the PSI-BLAST program to detect weak but
biologically relevant sequence similarities (23). All identified
proteins belong to the Gli/Ci transcription factor family. Four amino
acids, SYGH, were highly conserved in all analyzed proteins including
Ci. To confirm that similar regions of Gli2/Gli3 would be responsible for SUFU interaction, we designed deletion constructs of Gli2 and GLI3.
The derivatives including amino acids 205-300 of Gli2 and amino acids
250-350 of GLI3, respectively, were positive in the Far Western assay
and bound SUFU with similar efficiency (Fig. 3B). Both
fragments encompass the SYGH motif. Thus, SYGH is implicated as a
required motif in all members of the Gli/Ci family to allow SUFU
binding.
Mutation Analysis of the SYGH Motif in GLI1--
To determine
which amino acids in the SYGH motif of GLI1 are essential for the
interaction with SUFU, we used site-directed mutagenesis to change
conserved amino acids to alanine. Mutants were expressed in E. coli and tested for interaction by the Far Western assay. The
results of the mutagenesis study are summarized in Fig.
4A. Substitution of
Ser120 to alanine resulted in SUFU binding with higher
affinity. Mutation of the tyrosine at position 121 had no effect on the
interaction. Mutations of the glycine residue at position 122 and the
histidine residue at position 123 to alanine almost eliminated
GLI1-SUFU interaction. In addition, we introduced double alanine
substitutions of S120A and Y121A, which also led to reduced binding.
These results indicate that amino acids Gly122 and
His123 are critical for GLI1-SUFU interaction.
The NetPhos 2.0 prediction program showed that the serine at position
120 is a good candidate for phosphorylation by serine kinases (score
0.814). Therefore, we assumed that phosphorylation of GLI1 might be
involved in regulation of its regulation and localization. To test this
hypothesis, we substituted Ser120 to glutamic acid, which
would mimic a phosphorylated state of GLI1. Surprisingly, this
replacement led to an increased affinity in comparison to wild-type, as
did an alanine substitution (Fig. 4B). Therefore, we
conclude that most likely this substitution led to the alteration of
protein folding.
To further strengthen the functional relevance of the GLI1 mutations
crucial for SUFU interaction, we used a reporter gene assay. SUFU,
mutated G122A, and H123A GLI1 full-length proteins were co-expressed in
NIH3T3 cells, and the effect of these mutations on GLI1-activated
transcription was examined. This reporter was activated by wild-type
GLI1, whereas SUFU itself had no effect (Fig. 4C).
Co-transfection of SUFU led to suppression of the reporter gene
activation by GLI1. However, SUFU had no effect on activation of
reporter gene activity by mutated G122A or H123A GLI1. Thus, these
results confirm the importance of these amino acid residues of GLI1 for
SUFU interaction. In addition, we observed that the activity of mutated
forms of GLI1 was higher in comparison with wild-type. We assume that
wild-type GLI1 transfected into NIH3T3 cells can form a complex with
endogenous Sufu and can be sequestered in the cytoplasm, because NIH3T3
cells do express Sufu. Mutated GLI1 does not bind Sufu and may escape
cytoplasmic retention.
Mapping the SUFU Domain That Is Responsible for GLI
Interaction--
To map the region of SUFU required for interaction
with GLI1, SUFU deletion constructs were tested using
immunoprecipitation (Fig. 5, A
and B). Myc-tagged SUFU or its deletion constructs were
cotransfected into 293T cells with HA-tagged GLI1 and
immunoprecipitated with HA or c-Myc antibodies. The immunoprepicitates
were separated by SDS-PAGE, blotted, and detected with c-Myc or HA
antibodies. The expression of all proteins was confirmed by Western
blotting of whole-cell extracts (data not shown). C-terminal deletion
constructs of SUFU (
To confirm the importance of the C-terminal domain of SUFU for the
binding of GLI proteins, we constructed two N-terminal deletion
derivatives of SUFU referred to as 100-484 and 200-484, respectively.
Their interaction with GLI proteins was determined by Far Western blot
analysis using in vitro translation for labeling (Fig.
5B). We tested the following constructs of GLI proteins, GLI1 55-125, GLI2 205-300, and GLI3 250-350, expressed in E. coli. We detected an interaction of both SUFU-deletion derivatives
with all GLI proteins. However, deletion of the SUFU N terminus led to
reduced binding compared with wild-type. A possible explanation for
this result could be that any truncations of SUFU affect the overall
conformation of the protein and that tertiary structure is critical for
interaction with GLI proteins.
Subcellular Distribution of SUFU and LMB Treatment--
SUFU is
assumed to be a cytoplasmic-nuclear shuttling protein, because it is
detected in both cytoplasm and nucleus (13, 16). Its localization might
be an important factor in regulation of the Hh signaling pathway. To
examine subcellular distribution of SUFU, we transfected 293T cells
with either wild-type or C-terminally truncated SUFU
(
The surprising pattern of nuclear localization of the SUFU ( In this study, using deletion analysis coupled with in
vivo coexpression and Far Western assays, we have identified the
required region, the 116-125 amino acid residues, within the GLI1
N-terminal region responsible for interaction with SUFU. Deletion of
this domain resulted in a complete loss of its association with SUFU. Although N-terminal residue conservation in the Gli/Ci protein family
is quite modest, a motif containing the SYGH amino acids is
specifically conserved from fly to man (25; Fig. 3). Analysis of
alanine-substitution mutants of the GLI1 SYGH motif strengthened its
important role for SUFU interaction. We demonstrated that substitutions
of the glycine residue at position 122 and the histidine residue at
position 123 as well as the double mutant of Ser120 and
Tyr121 led to a dramatic reduction in SUFU binding,
indicating that they are essential for the GLI1-SUFU interaction. Thus,
we have identified a novel motif, which is involved in protein-protein interactions within the biologically very important Hh signaling pathway.
Next, our results demonstrated that substitution of Ser120
to alanine enhanced binding of GLI1 to SUFU in comparison with
wild-type GLI1. Because phosphorylation is a common mechanism for
regulation of protein function, we assumed that phosphorylation of
Ser120 would be functionally important for SUFU
interaction. It has been demonstrated in Drosophila that Hh
regulates the phosphorylation of downstream signaling proteins such as
Fused (26), Costal-2 (27), Ci (28, 29), and Smoothened (30). However,
binding to SUFU of GLI1 with a substitution of Ser120 to
glutamic acid, which would mimic a phosphorylated state of GLI1, was
also significantly increased. Therefore, it is more likely that mutated
GLI1 undergoes a significant conformational rearrangement leading to
stabilization of protein-protein binding. However, involvement of
phosphorylation in regulation of GLI-SUFU interaction cannot be
excluded. Thus, the specific roles of individual phosphorylation sites
remain to be elucidated.
Gli2 and GLI3 share many similar characteristics, whereas GLI1 is
different. Whereas Gli2 and GLI3 contain both transcriptional activation and repression domains, GLI1 appears to contain only an
activation domain (31). Truncation of the activation domain in the C
terminus of Gli2 or GLI3 results in a protein with repressor activity,
whereas removal of the repression domain at the N terminus converts
them into strong activators (10). The N-terminal repression domains of
Gli2 and Gli3 do not overlap with their SUFU-binding sites. Therefore,
it is unlikely that SUFU binding significantly contributes to the
repressive activity of Gli2 and Gli3. Rather, the repressive activity
might be associated with another conserved motif present in the
N-terminal portions of Gli2 and Gli3 but not Gli1 (10).
Some reports indicate that both N- and C-terminal domains of GLI1 can
be involved in interaction with SUFU. Reporter gene assays, using
full-length Gli1 or a Gli1 deletion mutant lacking the N terminus,
revealed that transcriptional transactivation was inhibited by SUFU in
both cases (32). Ci, the Drosophila homolog of Gli proteins,
interacts with SUFU within an N-terminal region that encompasses amino
acids 244-346 (254SYGH257) (32, 33). Moreover,
the N-terminal domain of GLI3, referred to as r1 (amino acids 274-353;
333SYGH336), was reported to be sufficient for
SUFU interaction (18). In this study we performed a functional
luciferase reporter gene assay to examine transcriptional repression of
point-mutated forms of GLI1 (G122A or H123A) by SUFU. These mutations
essentially eliminate SUFU-GLI1 biochemical interaction. Our results
revealed that cotransfection of SUFU did not inhibit transactivation by mutated forms of GLI1 in comparison with wild-type GLI1. Thus, substitution of one amino acid crucial for SUFU-GLI1 binding led to
complete abrogation of SUFU suppression. These data suggest that this
domain is the only one, which is functionally important in GLI-SUFU interaction.
We also analyzed amino acid residues of SUFU involved in
Gli binding. Deletion of 19 amino acids at the C terminus caused abrogation of SUFU-GLI1 interaction as shown by immunoprecipitation. Moreover, Far Western assay revealed that the N-terminal deletion derivatives of SUFU containing 100-484 and 200-484 amino acids retained the ability to bind Gli although with lower affinity. Our data
strongly indicate that the C terminus of SUFU is required for
interaction with Gli proteins.
The results reported in the literature from analysis of SUFU domains
involved in the interaction with Gli appear to be variable.
1) Using the yeast two-hybrid system, the central domain of mouse Sufu
between residues 109-325 was shown to be required for the interaction.
The results obtained with glutathione S-transferase pull-down assays showed that the N-terminal domain of Sufu (amino acids
13-173) could interact only with GLI3, whereas the C-terminal region
(amino acids 174-484) could bind GLI1 and GLI3, but not Gli2 (35). It
has been speculated that the interaction domains of the Sufu protein
with three Gli proteins are different.
2) An alternatively spliced isoform of SUFU, lacking 52 amino acids at
the C terminus (referred to as SUFU 433), coimmunoprecipitated with
GLI1 in transiently transfected 293 cells (16). Hence, it was concluded
that the C terminus of SUFU (amino acids 433-484) is not involved in
its interaction with Gli proteins.
3) Deletion of the C-terminal domain (amino acids 386-484) led to
disruption of GLI1-SUFU interaction in the same cell type (13). Similar
results were found for two additional SUFU natural isoforms referred to
as SUFU-Lk (amino acids 1-388) and SUFU-Tt (amino acids 1-359)
lacking 96 and 125 amino acids at the C termini (21). These data
suggest that the C-terminal domain (amino acids 386-484) is required
for SUFU-Gli interaction.
4) SUFU lacking the C-terminal half (designated SUFU- A summary of these results is presented in Fig.
7. Based on available data, it is
difficult to draw any firm conclusion about which domains of SUFU is
crucial for the interaction with Gli proteins. The best way to resolve
the discrepancies would be high resolution crystallographic studies of
SUFU and the SUFU-Gli protein complex.
465) is sufficient
to abrogate interaction with GLI1. Interestingly, this SUFU mutant
localizes in the nucleus, most probably because it is not efficiently
sequestered in the cytoplasm. Taken together, we identified a novel
motif in the Gli/Ci family of proteins that is essential both for
protein-protein interaction with SUFU and for functional repression of
GLI1 by SUFU.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
465 deletion constructs. To isolate
nuclear and cytoplasmic fractions, cells were washed twice with cold
PBS and lysed with hypotonic buffer (HEPES pH 8.0, 10 mM
MgCl2, 1 mM dithiothreitol, 0.1 mM EDTA). After incubation for 10 min at 4 °C, cells were treated with
0.5% Nonidet P-40 and centrifuged at 1,000 rpm for 10 min. After
recovery of supernatants representing cytoplasmic components, the
pellets were washed twice with hypotonic buffer, centrifuged at 1,500 rpm for 5 min, and lysed with 2× Laemmli sample buffer. Samples were
analyzed by Western blot analysis using an anti-SUFU polyclonal
antibody (Santa Cruz).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Mapping of the interaction domain of GLI1
with SUFU. A, interaction of GLI1 55-407 domain and
full-length SUFU under different salt concentrations. SUFU and GLI1
were produced by in vitro translation and incubated with
anti-Myc antibodies. Washing was performed with buffer A with
increasing concentration of NaCl. B, coimmunoprecipitation
of GLI1 and SUFU. 293T cells were transfected with plasmids encoding
wild-type HA-GLI1 or HA-GLI1 deletion mutants in combination with
Myc-SUFU. Extracts were assayed by immunoprecipitation of complexes
with anti-Myc or anti-HA antibodies followed by Western blotting.
C, diagram represents GLI1 fragments used in this study and
summarizes the results of the immunoprecipitation assays.
Numbers indicate the amino acids of each fragment. Tags (HA
or Myc) were located at the N terminus of each fragment. The zinc
fingers of Gli1-3 are indicated as black boxes.
+, positive binding; , no detectable binding.
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Fig. 2.
Analysis of GLI1-SUFU interaction by Far
Western analysis. Lysates were prepared from E. coli
expressed fusion proteins of GLI1 deletion derivatives, fractionated by
SDS-PAGE, transferred to nitrocellulose, and probed with
35S-radiolabeled SUFU. After washing, bound SUFU was
detected by autoradiography (left). Ponceau staining shows
loading of protein extracts and molecular sizes of the expressed
proteins (right).
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Fig. 3.
Amino acid sequence alignment of the SUFU
binding domain of GLI1. A, region of GLI1 interacting
with SUFU is aligned with the corresponding regions of other Gli/Ci
family proteins. A consensus sequence is shown at the bottom
(top). B, Far Western analysis of deletion
derivatives of GLI1, Gli2, and GLI3 (right). Ponceau
staining shows loading of bacterial extract (left).
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Fig. 4.
Point mutation analysis of the SUFU binding
domain of GLI1. A, Far Western analysis of point
mutants in the SUFU-binding domain of GLI1. Site-directed mutagenesis
was used to substitute the conserved amino acids to alanine. GLI1
mutants were tested for SUFU binding using the Far Western assay
(right). Ponceau staining shows loading of bacterial extract
(left). B, Far Western analysis of point mutants
of Ser120 in the SUFU-binding domain of GLI1. Site-directed
mutagenesis was used to substitute the conserved Ser120 by
alanine or glutamic acid. The GLI1 mutants were assayed for SUFU
binding using the Far Western assay (right). Ponceau
staining shows loading of bacterial extract (left).
Wild-type GLI1 was used as a positive control. The band corresponding
to GLI1 is indicated by an arrow. C, effect of
SUFU on transcriptional activity of wild-type and mutated forms of GLI1
in NIH3T3 cells. All experiments were repeated three times and
normalized for alkaline phosphatase activity. Standard deviations are
indicated by error bars.
465) and (
425) failed to interact with GLI1.
These results indicate that the C terminus of SUFU is absolutely
required for the formation of a complex with GLI1.
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Fig. 5.
Mapping of the interaction domain of SUFU
with GLI1. A, 293T cells transfected with combinations
of HA-tagged GLI1 55-407 and Myc-tagged wild-type SUFU, SUFU 1-465,
or SUFU 1-425 were lysed, and Myc or HA antibodies were used for
immunoprecipitation. Western blot analysis was performed with the same
antibodies to detect the corresponding proteins. B, lysates
were prepared from E. coli expressing fusion proteins of
GLI1 deletion derivatives, fractionated by SDS-PAGE, transferred to
nitrocellulose, and probed with 35S-radiolabeled SUFU.
After washing, bound SUFU was detected by autoradiography.
Input, 90% of the translation mixture was used for
incubation.
465), and protein levels in cytosolic and nuclear fractions were
detected by Western blot analysis (Fig.
6A). The purity of the
fractions was determined with lamin B antibodies as a marker for
nuclear fractions (Fig. 6A, lower panel). Whereas wild-type SUFU showed a predominant cytosolic localization, the C-terminally truncated form was mainly observed in the nuclear fraction. We also analyzed NIH3T3 cells, which express endogenous Sufu.
The distribution of endogenous Sufu in NIH3T3 cells was similar to that
observed in 293T cells transfected with wild-type SUFU (Fig.
6B).
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Fig. 6.
Subcellular localization of wild type or
truncated ( 465) SUFU. A,
Western blot analysis of cytoplasmic and nuclear proteins from 293T
cells transfected with wild-type or a C-terminal deletion variant
(
465) of SUFU. Western blot analysis with lamin B antibodies is
shown as a fractionation and loading control (lower panel).
B, Western blot analysis with SUFU antibodies shows
endogenous Sufu protein levels in cytoplasmic and nuclear fractions
prepared from NIH3T3 cells. C, subcellular localization of
endogenous Sufu in NIH3T3 cells treated with LMB (final concentration
20 ng/ml) for 2 or 24 h. N denotes the nuclear and
C denotes the cytoplasmic cellular fraction,
respectively.
465)
mutant can be explained either by disruption of one or more SUFU
interactions with other proteins, which might sequester SUFU in the
cytoplasm, or a loss of ability to be exported from the nucleus. To
determine whether the C-terminal deletion interferes with export of
SUFU from the nucleus, we treated NIH3T3 cells with leptomycin B (LMB),
a fungal metabolite that inhibits nuclear protein export by binding to
the CRM1 receptor for nuclear export signals (24). LMB treatment for
either 2 or 24 h had no effect on the subcellular distribution of
endogenous Sufu. Most of the Sufu protein was present in the cytosolic
fraction (Fig. 6C). These results suggest that Sufu is not
exported from the nucleus through the CRM1/exportin pathway in NIH3T3
cells. Thus, it is likely that a motif within the twenty C-terminal
amino acids has a role in mediating subcellular localization.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
ex8) was not
able to bind Gli proteins. In addition, the SUFU (212-484) mutant also
lost this ability (20).
View larger version (9K):
[in a new window]
Fig. 7.
A summary of the results reported in the
literature from analysis of Sufu domains involved in the interaction
with Gli proteins.
As we show here, deletion of 19 amino acids at the C terminus of SUFU
caused its relocalization from the cytoplasm to the nucleus. Similar
results were found in SW-480 cells, where SUFU mutants with C-terminal
deletions showed significant nuclear accumulation (36). Moreover,
SUFU-ex8 was localized in the nucleus as well (20). The SUFU
C-terminal domain shares no apparent sequence homology to any other
proteins. Altered subcellular localization could be explained either by
disruption of one or several interactions with other proteins, which
sequester SUFU in the cytoplasm, or by a loss of ability to be exported
from the nucleus. Meng et al. (36) have presented data
suggesting that SUFU is actively exported from the nucleus, because it
could be readily detected in the nuclear compartment of LMB-treated
SW-480 cells. In contrast, no substantial nuclear accumulation of
Myc-Sufu could be observed in untreated or LMB-treated wing discs in
the Drosophila (17). Moreover, SUFU inhibited nuclear
accumulation of GLI1 in the presence of LMB (13). Our results have
further demonstrated that LMB treatment of NIH3T3 cells did not alter
subcellular localization of endogenous Sufu. In addition, no concensus
leucine-rich nuclear export signal is present in SUFU. Taken together,
these data are most consistent with a model in which SUFU
465
relocates to the nucleus because it cannot be efficiently sequestered
in the cytoplasm. However, additional work is necessary to identify the
mechanism and protein factors involved in SUFU subcellular distribution.
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ACKNOWLEDGEMENTS |
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We thank Thomas Grimm for the constucts used for immunoprecipitation assays. We also thank Peter Zaphiropoulos and Stephan Teglund for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by grants from the Swedish Cancer Fund and from the National Institutes of Health (PO1 AR47898-02).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.: 46-8-6089152; Fax: 46-8-6081501; E-mail: Rune.Toftgard@cnt.ki.se.
Published, JBC Papers in Press, November 7, 2002, DOI 10.1074/jbc.M209492200
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ABBREVIATIONS |
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The abbreviations used are: SUFU, Suppressor of Fused; Hh, hedgehog; HA, hemagglutinin; PBS, phosphate-buffered saline; LMB, leptomycin B.
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