From the Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie an der Universität Hamburg, Martinistrasse 52, D-20251 Hamburg, Germany
Received for publication, January 9, 2001, and in revised form, March 20, 2001
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ABSTRACT |
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The U-box domain has been suggested to be a
modified RING finger motif where the metal-coordinating cysteines and
histidines have been replaced with other amino acids. Known
U-box-containing proteins have been implicated in the
ubiquitin/proteasome system. In a search for proteins interacting with
the ubiquitin-conjugating enzyme UbcM4/UbcH7, we have identified a
novel U-box containing protein, termed UIP5, that is exclusively found
in the nucleus as part of a nuclear dot-like structure. Interaction
between UbcM4 and UIP5 was observed in vivo and in
vitro with bacterially expressed proteins. In addition to UbcM4,
several other ubiquitin-conjugating enzymes (E2s) that share the
same sequence within the L1 loop bind to UIP5. Mutational analysis
showed that the U-box, like the RING finger in other proteins, forms
the physical basis for the interaction with E2 enzymes. Further support
for the structural similarity between U-box and RING finger comes from
the observation that, in both cases, the same regions within the UbcM4
molecule are required for interaction. Our results establish at the
molecular level a link between the U-box and the ubiquitin conjugating
system and strongly suggest that proteins containing U-box domains are functionally closely related to RING finger proteins.
The RING finger motif (for real
interesting new gene) is a
cysteine-rich structure that has been found in many functionally distinct proteins. Because of their widespread occurrence and their
link to human diseases, RING finger proteins have attracted considerable interest (for reviews, see Refs. 1 and 2). Until recently,
very little was known about the molecular function of the RING domain,
except that it was involved in mediating protein-protein interactions.
However, recent results from several laboratories strongly suggest that
RING finger proteins play critical roles in mediating the transfer of
ubiquitin to target proteins (3-5). The ubiquitin pathway generally
involves three types of enzymes (for reviews, see Refs. 6-8). In an
initial step, the ubiquitin-activating enzyme (E1 or Uba) forms a
thioester bond with the C terminus of ubiquitin, which is then
transferred to a specific cysteine residue of a ubiquitin-conjugating
enzyme (E2 or Ubc).1 In the
final step the E2 enzyme donates ubiquitin to a lysine residue of the
target protein, either directly or with the assistance of ubiquitin
protein ligase (E3 or Ubr). Following formation of the polyubiquitin
chain, the protein moiety is in most cases degraded by the 26 S
proteasome complex, and free ubiquitin is released.
The E3 ubiquitin protein ligases play a key role in recognition and
selection of proteins targeted for ubiquitination and subsequent
degradation. Over the past year many RING finger proteins have been
shown to act as E3s, either by themselves (3-5) or as part of a
multisubunit E3 protein complex (9). The RING proteins are thought to
act as scaffolds that bring together the E2 enzyme and the target
protein. In most cases the RING motif itself is needed for the E3
activity and interacts specifically with E2 enzymes. The biological
significance of this interaction is underlined by the fact that
mutations of the RING motif that prevent interaction with E2s can
contribute to oncogenic transformation (10), familial Parkinson disease
(11) and abnormal development (11).
We have recently described a family of proteins, termed UIPs
(UbcM4-interacting proteins), that interact with two closely related E2
enzymes, UbcM4 (identical to UbcH7) and UbcH8 (12). In all cases
interaction occurs through the RING domain of the UIPs, the only
exception being UIP5, where UbcM4 binds to a sequence that has several
amino acids in common with the RING motif; however, the
metal-coordinating cysteine and histidine residues are replaced with
other amino acids. As this modified RING finger motif was first found
in the yeast UFD2 gene, it has been designated U-box (13, 14).
Recently, a data base search initiated with the U-box of UDF2 revealed
the presence of the U-box motif in several other proteins from
eukaryotic organisms (15). In the present report we have characterized
the UIP5 protein and show that the U-box domain can directly interact
with several E2 enzymes and, therefore, is likely to function similar
to the RING finger in the ubiquitination pathway.
Binding Assay Using the Yeast Two-hybrid System--
Yeast
expression plasmids containing the UbcH1/HHR6B (16), UbcM2 (17), UbcM3
(17), UbcM4 (18), UbcH5a (19), UbcH8 (20), or UbcM9 (21) cDNAs
fused in frame to the LexA binding domain were obtained by PCR
amplification of the complete coding regions of the respective
cDNAs followed by ligation into vector pFBL23 (22) or vector
pBTML116. The latter vector was derived from pBTM116 (23) by insertion
of a linker region, consisting of two repeats of four glycine and one
serine residue, adjacent to the LexA protein, thus giving the bait
protein greater flexibility. The plasmid expressing a fusion protein
between the VP16 activation domain and a mouse UIP5 fragment extending
from amino acid 241 to 335 and containing the U-box has been described
earlier (12). A construct containing the complete translated region of
hUIP5 fused to the LexA binding domain was obtained by PCR
amplification of pKIAA0860 (a gift from Dr. T. Nagase, Kazusa DNA
Research Institute, Kisarazu, Japan) followed by cloning of the
amplified fragment into the vector pBTML116. The fusion protein will be
referred to as LexA/hUIP5. Mutations of UbcM4 and hUIP5 were carried
out using the Stratagene QuickChange kit (Stratagene, Amsterdam,
Holland). All constructs were sequenced to confirm their structure.
Sequences of oligonucleotides used for amplification of cDNAs and
for site-directed mutagenesis are available upon request. Expression of
VP16 or LexA fusion proteins in yeast was analyzed by SDS-PAGE of total protein extracts and Western blotting. Fusion proteins were detected by
using anti-VP16 or anti-LexA antibodies (Santa Cruz Biotechnology, Heidelberg, Germany), respectively.
Plasmids containing the proteins of interest fused to the LexA DNA
binding or the VP16 activation domain were co-transformed into the
yeast YRN 974 strain. In this strain, a reporter gene encoding the
green fluorescent protein (GFP), is chromosomally integrated downstream
of a LexA binding site (24). Interaction of two proteins, one with a
LexA binding domain, the other with a transcriptional activation
domain, results in activation of GFP, which was quantified by flow
cytometric analyses. Approximately 20,000 cells of at least three
independent transformants were analyzed for fluorescence intensity
using a Becton Dickinson FACScan flow cytometer.
In Vivo Binding Assay with Tagged Proteins in 293 Cells--
Construction of (His6)-tagged UbcM4 has been
described previously (12). The plasmid phUIP5-HA, containing the
hemagglutinin (HA) tag at the C terminus of the full-length protein,
was obtained by standard PCR techniques using as template the plasmid
pKIAA0860. The amplified fragment was cloned in the EcoRI
site of vector pHCMV-G, from which the insert was removed (25).
Transfection of human 293 kidney cells, purification of
(His6)-tagged proteins by metal ion chromatography,
SDS-PAGE, and Western blotting were performed exactly as described
previously (12).
In Vitro Binding Assay with Bacterially Expressed
Proteins--
UbcM4 with a C-terminal (His6)-tag was
amplified by PCR and cloned in frame with glutathione
S-transferase (GST) of the bacterial expression vector
pGEX-5X-1 (Amersham Pharmacia Biotech, Freiburg, Germany). To obtain
GST-hUIP5-(241-335), a fragment encompassing amino acids
241-335 of hUIP5 was amplified by PCR and cloned in the vector
pGEX-5X-1 in frame with GST. GST fusion proteins were expressed in
Escherichia coli DH5
To obtain purified UbcM4(His6), the
GST-UbcM4-(His6) fusion protein was bound to
glutathione-Sepharose. After several washes, with 50 mM
Tris-HCl, pH 8.5, 150 mM NaCl and then with 1× PBS, UbcM4(His6) was released from the GST tag by incubating the
resin with 50 units of factor Xa (Amersham Pharmacia Biotech) in PBS followed by several washes with lysis buffer. The eluted
UbcM4(His6) was further purified by absorption to TALON
resin (CLONTECH, Heidelberg, Germany) and elution
with imidazole. Binding assays were performed by incubating
GST-hUIP5-(241-335) bound to glutathione-Sepharose with
UbcM4(His6) in 300 µl of PBS. The bound proteins were
then eluted with a buffer containing 50 mM Tris-HCl, pH8.0
and 10 mM reduced glutathione and analyzed by SDS-PAGE and
Western blotting. The GST-tagged hUIP5 protein was detected using a
goat polyclonal anti GST antibody (Amersham Pharmacia Biotech,
Freiburg, Germany). For detection of the (His6)-tagged
UbcM4, BMG-His-1 (Roche Molecular Biochemicals, Mannheim, Germany) was
used as the primary antibody. Primary antibodies were visualized by
peroxidase-conjugated donkey antibodies directed against goat or rabbit
immunoglobulins (Dianova, Hamburg, Germany) and chemiluminescence
detection (ECL kit, Amersham Pharmacia Biotech).
RNA Preparation and Analysis--
Total cellular RNA was
isolated by using the RNeasy total RNA isolation system according to
the manufacturer's protocol (Qiagen GmbH, Hilden, Germany),
fractionated (10 µg per lane) by electrophoresis in 1% agarose gels
containing formaldehyde and transferred by capillary blotting onto
Nylon Plus membranes (Qiagen GmbH, Hilden, Germany). Preparation of
32P-labeled radioactive probe, hybridization, and washing
of filters have been described previously (26). The cDNA insert
from the UIP5 plasmid initially isolated from the mouse embryo cDNA
(12) library was used for preparing the radioactive probe.
Transfection of HeLa Cells and Immunohistochemical
Staining--
HeLa cells were maintained in Dulbecco's modified
Eagle's medium supplemented with 10% fetal calf serum. The plasmids
phUIP5-HA and pSG5-LINK-Sp100, encoding Sp100 (27), were introduced
into HeLa cells by using Effectene Transfection Reagent (Qiagen,
Hilden, Germany) according to the manufacturer's protocol. For
indirect immunofluorescence staining, cells were grown on coverslips
and fixed for 5 min in methanol and for 30 s in acetone. The
HA-tagged hUIP5 was detected using a 1:200 dilution of the rat
monoclonal anti-HA high affinity antibody 3F10 (Roche Molecular
Biochemicals, Mannheim, Germany), and Sp100 was detected using a 1:200
dilution of a rabbit anti-Sp100 antibody (27). Antibodies were
incubated with substrate for 1 h at room temperature and unbound
antibodies removed by three successive washes with PBS. To visualize
the primary antibodies, rhodamine RedX-conjugated goat anti-rat IgG antibodies or dichlorotriazinylfluorescein-conjugated goat anti-rabbit IgG antibodies (Dianova, Hamburg, Germany) were diluted 1:200 in PBS.
DAPI (4',6'-diamidino-2-phenylindole) (Roche Molecular Biochemicals,
Mannheim, Germany) was used for staining of nuclear DNA. Cell imaging
was performed with a Leica DMRA Microscope (Leica, Bensheim, Germany)
and the Spot Fluorescence Imaging Program (Intas, Heidelberg, Germany).
Structure, Expression, and Cellular Location of hUIP5--
UIP5
was originally isolated as a partial cDNA clone from a mouse embryo
yeast two-hybrid library using UbcM4 as a bait (12). The amino acid
sequence predicted from its open reading frame is 84% identical with
the protein encoded by the human gene KIAA0860 (GenBankTM accession number AB020667), for which the
complete amino acid sequence is known. This protein, which will be
referred to in this paper as human UIP5 (hUIP5), was used in all
subsequent experiments where the complete UIP5 protein was analyzed. As
shown schematically in Fig. 1, hUIP5
contains both a RING-HC domain and an U-box domain. Based on sequence
profile analysis, the U-box of hUIP5 (KIAA0860) has been suggested to
be a modified RING finger domain that lacks the hallmark
metal-chelating residues of the latter but is likely to adopt a RING
finger-like conformation (15). The amino acids replacing the
metal-chelating cysteines and histidines of the RING finger are
indicated by asterisks in the U-box sequence of Fig. 1.
The function of hUIP5 or its mouse homolog, mUIP5, is presently
unknown. As a first step to characterize the gene, we analyzed its
transcription pattern in mouse and the cellular location of its
protein. At least two transcripts, about 4.5 and 4.2 kilobases in length, can be detected by Northern blot analysis. They occur predominantly in embryonic stem cells, testis, and embryos and placentas at day 14.5 postcoitus (Fig.
2). However, mUIP5-specific RNA was also
found in other tissues, in particular in brain, when the more sensitive
reverse transcriptase-PCR method was used (data not shown). The
presence of a potential nuclear localization signal (360PSQKRKI), which is related to that of simian virus 40 large T antigen (28), suggested that hUIP5 might be a nuclear protein. To investigate this, a plasmid encoding HA-tagged hUIP5 was transiently transfected into HeLa cells and the protein stained with anti-HA antibody. As shown in Fig. 3, hUIP5
(red fluorescence) was localized exclusively in the nucleus
where it is associated with nuclear body-like structures. As the
staining pattern was very similar to that of the previously described
promyelocytic leukemia protein PML-Sp100 nuclear bodies
(reviewed in Ref. 29), we determined the localization of hUIP5 with
respect to Sp100. For this purpose HeLa cells co-transfected with
HA-tagged hUIP5, and Sp100 were double-immunostained with anti-HA
(red fluorescence) and anti-Sp100 (green
fluorescence) antibodies. An overlay of both pictures showed only
very limited co-localization of the two dot-like structures, indicating
that the nuclear bodies containing hUIP5 are different from those
containing Sp100 (Fig. 3).
Interaction of UIP5 with E2 Enzymes--
A mouse UIP5 fragment
containing little more than the U-box motif was previously shown to
bind to UbcM4 (12). Here we show that several other E2 enzymes can also
interact with the U-box domain of this protein when analyzed by the
yeast two-hybrid system using GFP as a reporter (Fig.
4). Among the E2s tested, only UbcH1 and
UbcM9 did not bind, the latter being a conjugating enzyme for the
ubiquitin-related SUMO-1 (30, 31).
To determine whether hUIP5 interacts with UbcM4 in mammalian cells,
(His6)-tagged UbcM4 and (HA)-tagged hUIP5 were transiently co-expressed in human 293 kidney cells. After lysis of cells, (His6)-tagged UbcM4 was purified by metal affinity
chromatography and the presence of hUIP5 associated with UbcM4 analyzed
by SDS-PAGE, Western blotting, and immunostaining with HA antibodies.
The results in Fig. 5 show that hUIP5
co-elutes with the metal resin-bound UbcM4 (lane 5). In the
absence of (His6)-tagged UbcM4, hUIP5 is not bound to the
resin (lane 4). These results clearly indicate that hUIP5
can associate with UbcM4 in mammalian cells.
U-box of hUIP5 Is Required for Interaction with E2 Enzyme--
As
described above, results obtained with partial cDNA clones
indirectly suggested that the U-box of UIP5 and not the RING-HC domain
was involved in the interaction with UbcM4. To directly show that the
U-box provides the physical basis for binding to UbcM4, some of the
amino acid residues in positions occupied by cysteines and histidine in
classical RING fingers (marked by asterisks in Fig. 1) were
mutated by site-directed mutagenesis. Two mutant hUIP5 proteins were
generated, one with both, Asp-265 and Thr-268, mutated to Gly
(hUIP5[D265G,T268G]) and the other with Ser-280 converted to Leu and
Lys-282 to Gly (hUIP5[S280L,K282G]). Both mutant proteins did not
interact with UbcM4 when analyzed in the yeast two-hybrid system using
GFP as a reporter (Fig. 6). Western blot
analysis showed that the mutant UIP5 proteins are synthesized at levels
comparable with that of the wild-type protein. Therefore, lack
of interaction is not due to the absence or to reduced amounts of the
mutant protein. Mutation of the U-box also prevented binding to other
E2 enzymes (data not shown). These results clearly show that the U-box
is required for hUIP5/E2 interaction.
Interaction of hUIP5 with UbcM4 in Vitro--
To test if there is
a direct interaction between UIP5 and UbcM4, hUIP5 was expressed as a
GST fusion protein in E. coli. As the complete protein was
not soluble under nondenaturating conditions, a 95-amino acid-long
fragment containing the U-box was synthesized as a GST fusion protein
(GST-hUIP5-(241-335)) and bound to glutathione-Sepharose beads.
Binding to UbcM4 was analyzed by adding (His6)-tagged UbcM4 also purified from bacterial extracts. As shown in Fig.
7, lane 3, UbcM4 bound to
hUIP5 under these in vitro conditions.
Mapping of Regions of UbcM4 That Are Required for Interaction with
the U-box of UIP5--
To determine which amino acids of UbcM4 are
necessary for binding to the U-box, mutations were introduced into the
UbcM4 molecule and their effect on interaction analyzed with the help
of the yeast two-hybrid system. The amino acid sequence of UbcM4 and an
alignment of secondary structure features is shown in Fig. 8A. UbcM4, which consists of
little more than the conserved UBC domain, has an
In a further set of experiments, the loop regions were mutated. The
sequence of loop L1 is highly conserved, and Phe-63 at the tip of L1
has previously been shown to be essential for interaction with Hect
domain proteins (35) and with RING finger
proteins.2 The results in
Fig. 8C show that mutation of Phe-63 to Asn (UbcM4[F63N]) abolished interaction with UIP5. To analyze the effect of loop L2
mutations on UbcM4/U-box interaction, several amino acid changes were
introduced into this region. Whereas mutation of Asn-94 to Asp
(UbcM4[N94D]) had no effect, mutation of the adjacent Trp-95, which
is present in all E2 enzymes, to Gly (UbcM4[W95G]) and mutation of
Pro-97 to Gly (UbcM4[P97G]) abolished binding to hUIP5, clearly indicating that these residues are required for interaction with the
U-box. Mutant proteins that do not interact with UIP5 were shown to be
present in yeast protein extracts at levels comparable with
wild-type UbcM4 (Fig. 8D). Binding was not affected
when Lys-96, Thr-99, and Lys-100 were changed to Ser, Leu, and Thr, respectively (UbcM4[K96S,T99L,K100T]), so that loop L2 of UbcM4 looked like that of UbcH5. In contrast to the effects of mutations in
loops L1 and L2, no interference with binding to UIP5 was observed when
either the catalytically active cysteine residue (Cys-86) was mutated
to alanine (UbcM4[C86A]) or Leu-33 was changed to Phe
(UbcM4[L33F]). In summary, amino acids in loops L1 and L2 as well as
an intact N-terminal The most significant result of the present report is the
observation that the U-box domain can directly interact with E2
enzymes. No other proteins seem to be necessary for this interaction,
as bacterially expressed E2 and UIP5 proteins can associate with each
other in vitro. Therefore, for the first time a molecular link has been established between the U-box motif and the
ubiquitin-conjugating system. Our results strongly suggest that
proteins containing U-box domains are functionally closely related to
RING finger proteins, whose importance for the ubiquitin-conjugating
system has been firmly established (4, 5, 9). This functional similarity is in agreement with the structural similarity, which was
suggested by sequence alignment of U-box motifs with a selection of
RING finger motifs (12, 15). Except for the metal-coordinating cysteine
and histidine residues, which are the hallmark of the RING finger, the
U-box retains a similar pattern of amino acid conservation. Therefore,
it has been postulated that the U-box and RING finger domains have
closely related three-dimensional structures consisting of a
three-stranded Further support for the structural similarity between U-box and RING
finger comes from the observation that both interact with the same
regions of UbcM4. We show here that the Evidence from several laboratories suggests that U-box proteins are
involved in the ubiquitin/proteasome pathway. The most prominent
example is UFD2 of Saccharomyces cerevisiae, which was originally identified as a component of the ubiquitin fusion
degradation pathway (13) and which was recently shown to act as a
so-called E4 enzyme, which is required for multiubiquitination of
ubiquitin fusion proteins, a process necessary for efficient
degradation of such proteins by the proteasome (14). In yeast, UFD2 is
needed for cell survival under stress conditions, suggesting that it mediates degradation of stress-induced aberrant proteins. The UFD2
homolog in Dictyostelium discoideum, NOSA, is required for normal differentiation as disruption of the nosA gene causes
developmental arrest at the aggregate stage (37). Another example for
an U-box protein is CHIP, which negatively regulates chaperone activity (38). Our observation that U-box domains can bind to E2 enzymes supports the suggestion that CHIP mediates interactions between the
chaperone and the ubiquitin/proteasome system.
The function of UIP5 is presently unknown. Most likely it is not a
substrate for ubiquitination as it is not ubiquitinated in
vitro (data not shown). However, the fact that it can associate with E2 enzymes strongly suggests that it is involved in the
ubiquitination of as yet unidentified target proteins. Of particular
interest is the observation that hUIP5 is found exclusively in the
nucleus, where it is part of a nuclear dot-like structure. As it does
not co-localize with Sp100, it is most likely not a component of the promyelocytic leukemia protein PML bodies. Therefore, it might be associated with other nuclear dot proteins like PLZF (39). Very
little is known about the physiological role of nuclear bodies. Some
indirect evidence suggests that they might be involved in various
aspects of transcriptional regulation and are targets of viral
infection (reviewed in Ref. 29). In view of the recently described
observation that ubiquitin-mediated proteolysis can be restricted to a
certain cellular compartment (36), one could speculate that UIP5
is needed specifically for the ubiquitination and degradation of
regulatory proteins in the nucleus. Experiments are under way to test
this hypothesis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
induced with 0.1 mM
isopropyl-
-D-thiogalactopyranoside. Cell extracts
were prepared by resuspending the bacterial pellet in lysis buffer (1%
Nonidet P-40, 50 mM sodium phosphate, pH 7.0, 150 mM NaCl, protease inhibitor mixture (Complete, EDTA-free, Roche Molecular Biochemicals)), and sonication. Cell debris was removed
by centrifugation and the cleared lysate stored at
70 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Schematic representation of the structural
arrangement of hUIP5. Positions of the U-box motif, RING-HC finger
motif, and potential nuclear location signal (NLS) are
indicated. The amino acid sequence of the U-box is shown underneath.
Amino acids of the U-box replacing metal-coordinating cysteines and
histidines of the RING finger motif as suggested by a PSI-BLAST search
(15) are marked by asterisks. Amino acids are given in the
single letter code. The numbers at the left and
right indicate amino acid positions at the termini of the
U-box. These numbers are based on the sequence deposited in the
GenBankTM data base under the accession number AB020667.
Amino acids mutated in experiments shown in Fig. 6 are indicated by
arrows.
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Fig. 2.
Expression of the UIP5
gene. Northern blot containing total RNA from the CCE
embryonal stem cell line, from mouse embryos and placentas from day
14.5 of gestation, and from the indicated tissues of adult animals were
hybridized with a radioactive probe derived from a partial mouse UIP5
cDNA. The positions of the 18 and 28 S rRNA are depicted on the
right. The amount of RNA was controlled by hybridization
with the glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) probe.
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Fig. 3.
Cellular location of hUIP5. HeLa
cells, transiently co-transfected with plasmids expressing HA-tagged
hUIP5 and Sp100, were stained with anti-HA antibody (A,
red fluorescence) or anti-Sp100 antibody (B,
green fluorescence). C, DAPI staining. D, for
co-localization the red fluorescence of hUIP5, the green fluorescence
of Sp100, and the DAPI staining were merged.
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Fig. 4.
Interaction between the U-box domain and
different E2 enzymes. Binding of a mouse UIP5 fragment containing
the U-box motif to indicated E2s was analyzed by the yeast two-hybrid
system using GFP as a marker. Fluorescence of GFP was measured by flow
cytometry. Fluorescence intensities are expressed relative to that
obtained with UbcM4, which was arbitrarily set at 100. Data are the
means ± S.D. of three independent clones.
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Fig. 5.
Association of hUIP5 with UbcM4 in mammalian
cells. Cell extracts were prepared by standard Nonidet P-40 lysis
from 293 cells transfected with HA-tagged hUIP5 alone (lanes
1 and 4), together with (His6)-tagged UbcM4
(lanes 2 and 5), or with
(His6)-tagged UbcM4 alone (lanes 3 and
6). Proteins were analyzed directly (lanes 1-3)
or after metal affinity chromatography (lanes4-6) by
SDS-PAGE and Western blotting using a monoclonal antibody directed
against the HA peptide. 3% of total input lysate was loaded on to
lanes 1-3. Lower panel, the blot shown in the upper
panel was stripped and rehybridized with a monoclonal antibody
directed against the His6 peptide.
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Fig. 6.
Mutation of U-box of hUIP5 prevents
interaction with UbcM4. Interaction of UbcM4 fused in frame to the
VP16 activation domain with wild-type or mutant hUIP5s fused to the
LexA binding domain was analyzed in the yeast two-hybrid system using
GFP as a reporter and the fluorescence quantified by flow cytometry.
A, yeast cells expressing either VP16/UbcM4 (continuos
line) or LexA/hUIP5 (shaded peak). B, yeast
cells co-expressing VP16/UbcM4 + LexA/hUIP5 (shaded peak) or
VP16/UbcM4 + LexA/hUIP5[D265G,T268G] (continuos line) or
VP16/UbcM4 + LexA/hUIP5[S280L,K282G] (dotted line).
C, synthesis of LexA fusion proteins in yeast cells
transformed with plasmids encoding LexA/hUIP5 (lane 1),
LexA/hUIP5[D265G,T268G] (lane 2), or
LexA/hUIP5[S280L,K282G] (lane 3). As a control,
untransformed cells were analyzed (lane 4). Fusion proteins
were detected on Western blots by using anti-LexA antibodies.
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Fig. 7.
Interaction of hUIP5 with UbcM4 in
vitro. GST-hUIP5[241-335] bound to glutathione-Sepharose was
incubated alone (lane 2) or with UbcM4(His6)
(lane 3). As a control glutathione-Sepharose was incubated
with UbcM4(His6) (lane 1). After incubation
proteins were eluted with reduced glutathione and analyzed by SDS-PAGE
and Western blotting. Lower panel, the blot was hybridized
with an antibody directed against the (His6)-tag of UbcM4.
Upper panel, the blot shown in the lower panel
was rehybridized with an antibody directed against GST to detect UIP5.
No binding was observed when GST bound to glutathione-Sepharose was
incubated with UbcM4(His6) (data not shown).
/
structure
similar to the structures of other E2s (32). The loop region connecting
the third and fourth strand of the
-sheet and the loop located
between the fourth
-strand and the second
-helix will be referred
to as loop L1 and loop L2, respectively, in accordance with the
nomenclature used in a previous publication (33). Based on the crystal
structure analysis of complexes between UbcH7 and the Hect domain
protein E6-AP or the RING finger protein c-Cbl, the N-terminal
-helix H1 and loops L1 and L2 of UbcH7 are involved in interaction
with these proteins (33, 34). Therefore, in our initial experiments, amino acids in these regions were mutated or deleted and the effect on
interaction with the U-box of UIP5 tested. The results are summarized
in Fig. 8C. When the N-terminal 12 amino acids comprising the H1
-helix of UbcM4 were deleted (UbcM4-(13-154)), interaction with UIP5 was abolished. However, as shown by the hybrid protein UbcH5-(1-14)/UbcM4-(16-154) interaction could be restored by adding the corresponding region of UbcH5. At the C terminus, deletion of 35 residues (UbcM4-(4-119)) did not affect binding; however, larger
deletions like in UbcM4-(2-110) abolished interactions. As shown in
Fig. 8C, the truncated UbcM4 proteins that do not interact
with UIP5 are synthesized at levels comparable with wild-type UbcM4, ruling out the possibility that lack of interaction is due to
the absence of these proteins.
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Fig. 8.
Interaction of wild-type and mutant UbcM4
proteins with U-box of UIP5. A, amino acid sequence and
secondary structural elements of UbcM4. Dark boxes indicate
-helical regions (H1-H4) and arrows indicate
-sheets. Amino acids of loop L1 and loop L2 are
underlined. The catalytically active cysteine residue is
marked by an asterisk. B, comparison of amino
acid sequences of loops L1 and L2 of the indicated E2 enzymes. Amino
acid positions for UbcH5 and UbcH1 are based on published sequences
(16, 19). C, effect of UbcM4 mutations on interaction with
U-box. UbcM4 and mutant UbcM4 proteins were fused to the LexA DNA
binding domain and the U-box of mUIP5 to the VP16 activation domain.
Interaction was analyzed by the yeast two-hybrid system using GFP as a
marker. Fluorescence intensities are expressed relative to that
obtained with UbcM4, which was arbitrarily set at 100. Data are the
means ± S.D. of three independent clones. Mutated amino acids are
indicated in brackets by their position number and their
single letter code. In the case of truncated or chimeric proteins the
amino acids present in the respective E2 enzymes are indicated in
brackets by their position number. The amino acid numbering
system is based on the UbcM4 sequence shown in A or on the
published UbcH5 sequence (19). D, synthesis of LexA fusion
proteins in yeast cells transformed with plasmids encoding LexA fused
to UbcM4 (lane 1), UbcM4[F63N] (lane 2),
UbcM4[W95G] (lane 3), UbcM4[P97G] (lane 4),
UbcM4[2-110] (lane 5), or UbcM4[13-154] (lane
6). Fusion proteins were detected on Western blots by using
anti-LexA antibody.
-helix are required for interaction of UbcM4
with the U-box of UIP5.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sheet, an
-helix, and two large loops (15).
Whereas the structure of the RING domain is stabilized by two
tetrahedrally coordinated Zn2+ ions, salt bridges and
hydrogen bonds are most likely responsible for maintaining the
structural arrangement of the U-box domain. Both loops of the U-box
seem to be required for interaction as mutation of the amino acids
replacing the cysteine or histidine residues in classical RING fingers
in one loop, either the 1st or the 2nd, are
sufficient for preventing interaction with UbcM4.
-helix H1 and amino acid
residues within loops L1 and L2 of UbcM4 are required for interaction
with UIP5. These same regions were shown by mutational
analysis2 and by crystal structure analysis to be important
also for complex formation of UbcM4/UbcH7 with the RING domain (34).
Although closely related in structure, the U-box of UIP5 and the RING
finger of several other UIPs differ in respect to their specificity of interaction with E2 enzymes. Whereas the RING motif of the UIPs binds
only to UbcM4 and the closely related UbcH8 (12), the U-box of UIP5 can
interact with UbcM2, UbcM3, UbcM4, UbcH5, and UbcH8, all of which are
members of a subfamily of E2s that is characterized by the presence of
a phenylalanine at the tip of loop L1. We show here that mutation of
this phenylalanine residue to asparagine abolishes interaction.
Furthermore, E2 enzymes, like UbcH1, which do not contain a
phenylalanine in loop L1, are not capable of binding to the U-box.
Results from other U-box proteins are needed to confirm that the
specificity described here is a general property of U-box/E2 interactions.
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ACKNOWLEDGEMENTS |
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We thank Drs. H. Will and T. Sternsdorf for providing the Sp100 cDNA and the rabbit anti-Sp100 antibody and Dr. J. Camonis for the plasmid pFBL23.
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FOOTNOTES |
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* The Heinrich-Pette-Institut is supported by Freie und Hansestadt Hamburg and the Bundesministerium für Gesundheit.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.
This article is part of a doctoral study by Ekaterini Pringa and
Gustavo Martinez-Noel in the Faculty of Biology, University of Hamburg.
§ To whom correspondence should be addressed. Tel.: 49-40-48051287; Fax: 49-40-48051188; E-mail: harbers@hpi.uni-hamburg.de.
Published, JBC Papers in Press, March 23, 2001, DOI 10.1074/jbc.M100192200
2 G. Martinez-Noel, U. Müller, and K. Harbers, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: E2/Ubc, ubiquitin-conjugating enzyme; E3/Ubr, ubiquitin-protein ligase; E1/Uba, ubiquitin-activating enzyme; GFP, green fluorescent protein; GST, glutathione S-transferase; HA, hemagglutinin; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; UIP, UbcM4 interacting protein; DAPI, 4',6'-diamidino-2-phenylindole.
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