From the Department of Biochemistry, Nmegen Center
for Molecular Life Sciences, University of Nijmegen,
6500 HB Nijmegen, The Netherlands
Received for publication, November 8, 2002, and in revised form, December 3, 2002
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ABSTRACT |
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A missense mutation in The ubiquitin/proteasome pathway plays a prominent role in the
pathogenesis of most of these neurodegenerative disorders (19). Ubiquitin-positive proteins are consistently found in the inclusion bodies that are characteristic of these diseases (20, 21). This
suggests that the proteins responsible for aggregate formation may have
a role in ubiquitin-dependent degradation (21, 22). Rapid
protein degradation via the ubiquitin/proteasome pathway is dependent
on the ubiquitination of the substrate protein by a ubiquitin ligase
(23, 24). There are many different ubiquitin ligases, which can be
classified into two families, those containing either RING finger
domain proteins or HECT domain proteins (25). The ubiquitin-protein
isopeptide ligase SCF
(SKP1/CUL1/F-box) complex,
which also contains the RING finger protein RBX1, captures the
target protein via the F-box protein (26-30). The F-box is a conserved
domain of ~40 residues that is responsible for recruiting bound
target proteins to the SCF complex by interacting with SKP1 (27). Ubiquitin moieties are then transferred through the SCF complex
to the target protein, ultimately resulting in the attachment of a
polyubiquitin chain (31). This tag is readily recognized by the 26 S
proteasome and leads to the degradation of the target protein (32).
F-box proteins capture their targets often in a
phospho-dependent manner (27, 29) and generally have
obvious domains for target protein interactions such as leucine-rich
repeats or WD40 repeats (26, 27, 29).
In this study, we identify the F-box protein FBX4 as an
interactor of Plasmids, Cell Culture, Transfections, and Antibodies--
The
coding regions of wild-type human
HeLa cells were grown at 37 °C in Dulbecco's modified Eagle's
medium (Invitrogen) supplemented with 10% fetal calf serum (PAA Laboratories), 100 units/ml penicillin, and 200 µg/ml streptomycin in
the presence of 5% CO2. Transfections of plasmids into
HeLa cells were performed by lipofection using the FuGENETM
6 system (Roche Molecular Biochemicals) as described by the
manufacturer. Immunoblotting was performed with mouse monoclonal
anti- Two-hybrid Screening--
For the interaction screening, yeast
strain EGY48 (ura3 trp1 his3 3lexA-operator-LEU2;
Clontech), containing bait plasmid pEG- Northern Blot Analysis--
The tissue distribution of FBX4 and
Detergent Solubility--
Cells were harvested by trypsinization
and subsequent centrifugation in a Minifuge RF (Hereaus Instruments,
Inc.) at 1000 rpm for 5 min. Cells were washed once with medium and
twice with phosphate-buffered saline, resuspended in ice-cold lysis
buffer (10 mM Tris (pH 7.5), 100 mM KCl, 1 mM dithiothreitol, 1 mM EDTA, 5 mM
MgCl2, 1 mM phenylmethanesulfonyl fluoride, and
0.5% Nonidet P-40), and centrifuged at 3000 rpm for 15 min at 4 °C.
The supernatant containing the detergent-soluble proteins was
collected, and the pellet containing the detergent-insoluble proteins
was obtained after washing once with lysis buffer.
Electrophoresis and Western Blot Analysis--
SDS-PAGE was
performed according to standard procedures. Proteins were transferred
to nitrocellulose membranes (Schleicher & Schüll) for Western
blot analysis by electroblotting. The membranes were incubated
successively with primary antibodies and horseradish peroxidase-conjugated secondary antibodies (Dako Corp.).
For isoelectric focusing, the detergent-insoluble pellets (see above)
were taken up in 100 µl of 2 M thiourea, 6 M
urea, and 2% CHAPS and solubilized by sonication. Protein
concentrations were determined using the 2-D Quant kit (Amersham
Biosciences), and the samples were further diluted to appropriate
concentrations in 2 M thiourea, 6 M urea, 0.8%
Immobilized pH gradient buffer 3-10 (Amersham Biosciences), 2%
CHAPS, 10 mM dithiothreitol, and bromphenol blue. Four
micrograms of protein was analyzed on pH 3-10 gradient strips (7 cm;
Amersham Biosciences) by rehydration loading. After isoelectric
focusing, the strips were equilibrated two times for 10 min in 50 mM Tris-HCl (pH 8.8), 6 M urea, 30% glycerol,
2% SDS, and bromphenol blue containing 10 mg/ml dithiothreitol and 25 mg/ml iodoacetamide, respectively. The strips were placed on small
conventional 12% SDS-polyacrylamide gels. These gels were subsequently
blotted and stained for ubiquitin conjugates and Yeast Two-hybrid Screening for Mimicking Phosphorylation of Expression of FBX4--
A physiologically meaningful interaction
between FBX4 Cotranslocates with
We wanted next to analyze the effect of Coexpression of FBX4 with
Importantly, the ubiquitination was dependent on the presence of FBX4
because coexpression of HSP70 Is Not Induced upon Overexpression of Analysis of Ubiquitination by Two-dimensional
Electrophoresis--
To determine the number and sizes of proteins
that are ubiquitinated by FBX4 and We have characterized a new interactor of We made the interesting observation that not only the
phosphorylation-mimicking mutations stimulate the interaction of
We found that N-terminally truncated FBX4, FBX4-(179-387), interacted
efficiently not only with The interaction between FBX4 and The accumulation of ubiquitinated products only when FBX4 is present
with The protein(s) ubiquitinated by the FBX4-ubiquitin ligase in
combination with phosphorylated B-Crystallin is a small heat-shock protein in
which three serine residues (positions 19, 45, and 59) can be
phosphorylated under various conditions. We describe here the
interaction of
B-crystallin with FBX4, an F-box-containing protein
that is a component of the ubiquitin-protein isopeptide ligase
SCF (SKP1/CUL1/F-box). The
interaction with FBX4 was enhanced by mimicking phosphorylation of
B-crystallin at both Ser-19 and Ser-45 (S19D/S45D), but
not at other combinations. Ser-19 and Ser-45 are preferentially
phosphorylated during the mitotic phase of the cell cycle. Also
B-crystallin R120G, a mutant found to co-segregate with a
desmin-related myopathy, displayed increased interaction with FBX4.
Both
B-crystallin S19D/S45D and R120G specifically translocated FBX4
to the detergent-insoluble fraction and stimulated the ubiquitination
of one or a few yet unknown proteins. These findings implicate the
involvement of
B-crystallin in the ubiquitin/proteasome pathway in a
phosphorylation- and cell cycle-dependent manner and may
provide new insights into the
B-crystallin-induced aggregation in
desmin-related myopathy.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B-Crystallin is a 20-kDa protein that is highly expressed in
eye lens and muscle tissues and to a lesser extent in many other tissues such as brain, skin, and kidney (1). It is a member of the
family of small heat-shock proteins
(HSPs),1 which are
characterized by the presence of a conserved
-crystallin domain (2).
Ten different small HSPs are expressed in human (3), mostly in muscle
tissues (1). An important property of
B-crystallin is its ability to
bind unfolding proteins (4). This chaperone-like activity might help to
prevent protein aggregation during stress conditions (5) and thus
increase the stress resistance of the cell (6, 7). More specific
functions of
B-crystallin are its apparent inhibition of apoptosis,
possibly by preventing the activation of procaspase-3 (8), and its
association with cytoskeletal components under normal conditions (9),
which is more pronounced during stress (10-12). The interaction with type III intermediate filaments might modulate the assembly of these
proteins in the cell and prevent inappropriate interactions between
bundled intermediate filaments (13).
B-crystallin can be
phosphorylated at three different positions: Ser-19 and Ser-45 are
mainly phosphorylated during mitosis, and Ser-59 under various stress
conditions (14, 15). Although the phosphorylation of
B-crystallin is
thus clearly regulated by the cellular conditions, it is not clear what
role phosphorylation plays in the functioning of
B-crystallin.
B-crystallin (R120G) has been shown to
co-segregate with desmin-related myopathy in a French family (16).
Desmin-related myopathies are usually adult-onset neuromuscular diseases characterized by the accumulation of aggregates of cytoplasmic desmin in conjunction with other proteins. In this French family, these
inclusion bodies were found to contain large amounts of
B-crystallin
R120G. Interestingly,
B-crystallin is also found in cytoplasmic
inclusions in various neurological disorders such as Alzheimer's,
Parkinson's, Huntington's, Alexander's, and diffuse Lewy body
disease (5, 17, 18), but its role in these diseases remains elusive.
B-crystallin. The interaction with FBX4 seems
to depend on the phosphorylation status of
B-crystallin, but is also
enhanced by the mutation R120G. Binding of FBX4 to
B-crystallin
stimulates the ubiquitination of a detergent-insoluble protein, which
probably destines this protein for ubiquitin-dependent degradation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B-crystallin,
B-crystallin
S19A/S45A (kindly provided by Paul Muchowski, University of
Washington, Seattle),
B-crystallin S19D/S45D,
B-crystallin R120G, wild-type human
A-crystallin (kindly provided by Jack Liang,
Harvard Medical School, Boston), and human
A-crystallin R116C
cDNAs were cloned into the polylinker region of the expression vector pIRES (Clontech) and the yeast two-hybrid
vectors pEG202 and pJG4-5. Mutations were introduced by site-directed
mutagenesis (Stratagene). The complete coding region of FBX4 cDNA
was obtained by reverse transcription-PCR from total HeLa RNA and
cloned into the pIRES, pEG202, and pJG4-5 vectors. SKP1 cDNA
was obtained by PCR using the HeLa library as a cDNA source and
cloned into the pEG202 and pJG4-5 vectors. The cDNAs coding for
vimentin and desmin were cloned into the pEG202 and pJG4-5 vectors. DNA
fragments encoding the sequence of HA-ubiquitin were cloned in
the pBSSK
vector (kindly provided by Dirk Bohmann,
European Molecular Biology Laboratory, Heidelberg, Germany).
B-crystallin antibody (Riken Cell Bank), monoclonal anti-HA
antibody (Roche Molecular Biochemicals), monoclonal anti-HSP70 antibody
(Stressgen Biotech Corp.), monoclonal anti-
-actin antibody (Sigma),
and rabbit polyclonal anti-FBX4 serum (obtained from a rabbit immunized with purified recombinant FBX4 protein).
B and
LacZ reporter plasmid pJK103, was transformed with the pJG-HeLa library (kindly provided by Roger Brent, The
Molecular Sciences Institute, Berkeley, CA). Transformants were
selected on plates lacking histidine, uracil, and tryptophan. A total
of ~1 × 106 transformants were obtained. After
induction of prey expression, ~3 × 106
colony-forming units were plated on galactose plates lacking leucine to
select for clones coding for proteins able to interact with the
B-crystallin fusion protein.
B-crystallin mRNAs was analyzed using human multiple-tissue
Northern blots (Clontech). A 710-bp
BglII-NcoI cDNA fragment of FBX4 and a 342-bp
BamHI cDNA fragment of human
B-crystallin were
labeled with [
-32P]dCTP using a random primer
kit (Invitrogen). Hybridization was done at 65 °C in 0.25 M Na2HPO4, 7% SDS, 1% bovine
serum albumin, 1 mM EDTA, and 100 µg or herring sperm
DNA. Following hybridization, filters were washed under high stringency
conditions (0.025 M Na2HPO4, 1%
SDS, and 1 mM EDTA at 65 °C) and visualized by
autoradiography. The blots were first hybridized with the FBX4 probe,
stripped, and subsequently reprobed for
B-crystallin.
B-crystallin.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B-Crystallin-interacting
Proteins--
Yeast two-hybrid screening was performed with an
B-crystallin-LexA fusion protein (bait) to select proteins
(preys) that are able to interact with
B-crystallin. We have shown
before that the
B-crystallin bait is not hampered by its
chaperone-like activity and can interact specifically with well known
interactors such as
A-crystallin and HSP27 (33). The cDNA
library used for screening was derived from HeLa cells. HeLa cells have
a low expression of
B-crystallin and thus may contain
B-crystallin-binding proteins. Most of the selected clones contained
the cDNA coding for the
-type proteasomal subunit
7/C8, which
has previously been shown to be a specific interactor of
B-crystallin (33). One of the selected clones contained a cDNA
coding for the C-terminal part (residues 179-387) of the F-box protein
FBX4. This protein belongs to a large family of F-box-containing
proteins, which are components of the SCF ubiquitin-protein isopeptide
ligases (26, 28) and function in phosphorylation-dependent
ubiquitination of specific substrate proteins. The interaction between
B-crystallin and the truncated FBX4 protein seemed to be specific
and thus not due to improper folding of the prey because
FBX4-(179-387) did not interact with the
B-crystallin homolog
A-crystallin or with desmin (Table I).
However, no interaction with
B-crystallin could be detected when
full-length FBX4 was used (Table I). Full-length FBX4 seemed to be
properly expressed in the two-hybrid system because it was able to bind
SKP1 (Table I), which is the F-box-binding component of the SCF
complex. Thus, the N-terminal region of FBX4 strongly reduces the
affinity of its C-terminal region for
B-crystallin.
Effect of mimicking phosphorylation of B-crystallin on the
interaction efficiency with wild-type or truncated FBX4 in the yeast
two-hybrid system
-galactosidase
expression in arbitrary units:
, 0-20 units; +, 20-100 units; ++,
100-500 units; +++, >500 units.
B-Crystallin at Ser-19 and Ser-45
Stimulates Interaction with FBX4--
F-box proteins bind their
substrate proteins often in a phosphorylation-dependent
manner. Because
B-crystallin can be phosphorylated at Ser-19,
Ser-45, and Ser-59 (14), it is possible that phosphorylation of
B-crystallin is needed to allow interaction with full-length FBX4.
These serines were therefore replaced, in different combinations, with
negatively charged aspartic acid residues, which mimic phosphorylation (34). As controls, non-phosphorylatable mutants were made in which the
serines were replaced with alanines. Interestingly,
B-crystallin
S19D/S45D showed a weak but specific interaction with full-length FBX4
compared with wild-type
B-crystallin and S19A/S45A (Table I).
B-Crystallin mutants in which a single serine or a combination of
two other serine residues was replaced did not show any detectable
interaction. Furthermore, the S19D/S45D/S59D mutant had a very similar
affinity compared with the S19D/S45D mutant. These results indicate
that the interaction with FBX4 might be regulated by phosphorylation of
B-crystallin at Ser-19 and Ser-45. It is of interest that
phosphorylation of
B-crystallin at these two serine residues is
specifically enhanced during the mitotic phase of the cell cycle,
whereas phosphorylation of Ser-59 is reduced (15).
B-Crystallin R120G Stimulates FBX4 Interaction--
Aggregate
formation might well be affected by the ubiquitination process. For
this reason, we were interested whether
B-crystallin R120G, which
causes intracellular aggregates, enhances the interaction with FBX4.
Interestingly,
B-crystallin R120G was able to bind FBX4 with a
similar affinity compared with the S19D/S45D mutant (Table I). A
corresponding missense mutation in
A-crystallin (R116C) that causes
congenital cataracts (35) did not stimulate the interaction of
A-crystallin with FBX4 (Table I). This result suggests that FBX4
might play a role in the aggregate formation caused by the R120G
mutation of
B-crystallin.
B-Crystallin S19D/S45D and R120G Interact with FBX4
in HeLa Cell Extracts--
We next wanted to confirm the association
of full-length FBX4 with
B-crystallin S19D/S45D and R120G in another
protein-protein interaction assay. To this end, co-immunoprecipitation
experiments were performed with lysates of HeLa cells cotransfected
with different eukaryotic expression constructs (see "Experimental
Procedures"). FBX4 was immunoprecipitated from the cell lysates with
anti-FBX4 antibodies coupled to protein A-Sepharose beads, and the
immunoprecipitates were analyzed on Western blots stained with a
monoclonal antibody directed against
B-crystallin (Fig.
1). About 1% of the expressed
B-crystallin S19D/S45D and R120G mutants coprecipitated with FBX4.
The control proteins (wild-type
B-crystallin and S19A/S45A) did not
coprecipitate or hardly coprecipitated with FBX4. These results confirm
that FBX4 is able to interact specifically with both
B-crystallin
S19D/S45D and R120G. However, the recovery of the coprecipitation was
low, indicating that the interaction between the proteins is relatively
weak.
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Fig. 1.
Co-immunoprecipitation of
B-crystallin with FBX4. Extracts of HeLa
cells, non-transfected (
) or cotransfected with pIRES-FBX4 and a
pIRES construct coding for wild-type
B-crystallin (WT),
S19D/S45D (SD), S19A/S45A (SA), or R120G
(RG), were subjected to immunoprecipitation (IP)
with a polyclonal antibody against FBX4. The immunoprecipitates were
analyzed by immunoblotting using a monoclonal antibody against
B-crystallin. In the lower panel (Input), each
lane contained 1% of the HeLa extract used for the
immunoprecipitation.
B-crystallin and FBX4 requires that both proteins are
expressed in similar tissue types. We therefore compared the tissue
distribution of FBX4 and
B-crystallin by Northern blotting (Fig.
2). The transcript of FBX4 was found to
be ~1.8 kb, which is in good agreement with the full-length 1.5-kb
FBX4 cDNA without a poly(A) tail (GenBankTM/EBI
accession number NM_012176), and showed a tissue expression profile
similar to that presented by Cenciarelli et al. (28). Importantly, most tissues expressing
B-crystallin transcripts also
expressed FBX4, although the ratio varied between the different tissues.
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Fig. 2.
Tissue distribution of FBX4 and
B-crystallin mRNAs. Human multiple-tissue
Northern blots were probed with a 32P-labeled 710-bp
BglII-NcoI cDNA fragment of FBX4 (upper
panel) and with a 32P-labeled 342-bp BamHI
cDNA fragment of
B-crystallin (lower panel). Each
lane contains ~2 µg of purified poly(A)+ RNA isolated
from the indicated tissues. The exposure times for FBX4 and
B-crystallin were 11 and 7 days, respectively.
B-Crystallin to the Detergent-insoluble
Fraction--
Under stress conditions,
B-crystallin translocates
from the detergent-soluble to the detergent-insoluble fraction in a
phosphorylation-independent manner (12, 36, 37). However, there are
indications that, under non-stress conditions, phosphorylation of
B-crystallin, at least at Ser-59, might have an effect on detergent
solubility (38). For this reason, we were interested in the detergent
solubility of
B-crystallin S19D/S45D in HeLa cells under regular
culture conditions. We found about twice as much
B-crystallin
S19D/S45D in the detergent-insoluble fraction compared with wild-type
B-crystallin or S19A/S45A as determined by quantification (Fig.
3, lower left panel; and data
not shown). This suggests that, upon phosphorylation at Ser-19 and
Ser-45,
B-crystallin might have an increased affinity for a
detergent-insoluble structure. In good agreement with the fact that the
R120G substitution stimulates the formation of
B-crystallin-containing aggregates, the pathological hallmark of the
disease it causes, we found this
B-crystallin mutant predominantly
in the detergent-insoluble fraction (Fig. 3, lower left
panel).
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Fig. 3.
FBX4 cotranslocates to the
detergent-insoluble fraction together with
B-crystallin. HeLa cells were transfected with
a pIRES construct coding for wild-type
B-crystallin (WT),
S19D/S45D (SD), S19A/S45A (SA), or R120G
(RG) with either an empty pIRES vector (
; left
panels) or pIRES-FBX4 (+; right panels). Cells were
harvested and lysed after 2 days and fractionated into
detergent-insoluble and -soluble fractions (i and s
lanes, respectively). Each fraction was subjected to SDS-PAGE and
immunoblotting with anti-
B-crystallin (lower panels) and
anti-FBX4 (upper panels) antibodies.
B-crystallin on the
solubility of FBX4. For this purpose, we cotransfected FBX4 with wild-type
B-crystallin or its mutants in HeLa cells (Fig. 3, right panels). When FBX4 was transfected alone, it was
completely soluble, and it remained soluble when cotransfected with
wild-type
B-crystallin or S19A/S45A. However, when FBX4 was
cotransfected with either
B-crystallin S19D/S45D or R120G, a
proportional fraction of FBX4 also became detergent-insoluble (Fig. 3,
upper right panel). Thus,
B-crystallin S19D/S45D and
R120G seem to recruit FBX4 to a detergent-insoluble structure.
B-Crystallin S19D/S45D or
R120G Induces Ubiquitination of Detergent-insoluble
Proteins--
FBX4, like other F-box proteins, can function as an
adaptor molecule to induce the ubiquitination of a bound protein (28). It might thus well be that FBX4 induces the ubiquitination of
B-crystallin S19D/S45D and R120G or of proteins bound to these isoforms of
B-crystallin. To test this, HeLa cells were transfected with a combination of three expression constructs coding for one of the
isoforms of
B-crystallin, for FBX4, and for HA-tagged ubiquitin (see
"Experimental Procedures"). The HA tag enabled us to determine the
total amount of ubiquitinated proteins present in the transfected HeLa
cells by Western blotting (39). The distribution of the different
B-crystallin isoforms and FBX4 between detergent-soluble and
-insoluble fractions was similar to that found without coexpression of
HA-tagged ubiquitin (cf. Fig. 3, right panels;
and data not shown). Interestingly, upon coexpression of FBX4 with
B-crystallin S19D/S45D or R120G, a strong signal of ubiquitinated
proteins could be detected in the detergent-insoluble fraction, but not
in the detergent-soluble fraction (Fig.
4, compare lanes 15 and
16 and lanes 19 and 20). These
ubiquitinated proteins were not detected when FBX4 was coexpressed with
wild-type
B-crystallin or S19A/S45A (Fig. 4, lanes 13,
14, 17, and 18). Also other control
proteins such as
B2-crystallin and HSP27 were tested and
found not to stimulate ubiquitination by FBX4 (data not shown),
underlining the specificity of the ubiquitination reaction.
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Fig. 4.
Ubiquitination is stimulated in the presence
of FBX4 and B-crystallin S19D/S45D or
R120G. HeLa cells were cotransfected with
pBSSK
-HA-ubiquitin (HA-Ub) and either a pIRES
construct coding for wild-type
B-crystallin (WT),
S19D/S45D (SD), S19A/S45A (SA), or R120G
(RG) alone (lanes 3-10) or additionally with
pIRES-FBX4 (lanes 13-20). After 2 days, the cells were
lysed, fractionated into detergent-insoluble and -soluble fractions
(i and s lanes, respectively), subjected to
SDS-PAGE and Western blotting, and subsequently stained for the HA
epitope (upper panel) and FBX4 (lower panel). The
ubiquitinated proteins (X-Ub(n)) range in size from
34 kDa to much larger than 98 kDa. The asterisk indicates an
unidentified endogenous HA-immunopositive soluble protein.
B-crystallin S19D/S45D or R120G with
HA-tagged ubiquitin alone gave no or very little ubiquitinated protein
in the detergent-insoluble fraction (Fig. 4, compare lanes 5 and 15 and lanes 9 and 19). No higher
molecular mass species of
B-crystallin or FBX4 could be detected
upon Western blotting with their respective antisera (data not shown),
making it unlikely that one of these proteins itself becomes
ubiquitinated. These results indicate that
B-crystallin S19D/S45D
and R120G are able to ubiquitinate one or more proteins concomitantly
with the recruitment of FBX4 to the detergent-insoluble fraction.
Similar ubiquitinated products were seen in a mouse C2 cell line (data
not shown); thus, the target protein is present in different cell lines.
B-Crystallin and
FBX4--
One could imagine that overexpression of
B-crystallin
S19D/S45D or R120G with FBX4 and HA-ubiquitin somehow induces in itself a stress response and that this stress reaction is responsible for the
observed ubiquitination. To exclude this possibility, we estimated the
levels of the endogenous stress-inducible HSP70 in the transfected
cells by Western blotting (Fig. 5). No
significant increase in HSP70 levels could be detected upon
coexpression with FBX4 and
B-crystallin S19D/S45D or R120G compared
with the different control transfected HeLa cells (Fig. 5A,
middle panel), whereas upon heat stress, HSP70 was induced
(Fig. 5B). A similar negative result was obtained by
cotransfecting a stress-inducible reporter construct (data not shown).
Thus, overexpression of
B-crystallin S19D/S45D or R120G together
with FBX4 induced the ubiquitination of one or more proteins in
non-stressed cells (Fig. 5A, upper panel).
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Fig. 5.
HSP70 is not induced upon cotransfection of
the different isoforms of B-crystallin
together with FBX4 and HA-ubiquitin. A, HeLa cells were
cotransfected with pBSSK
-HA-ubiquitin and either a pIRES
construct coding for wild-type
B-crystallin (WT),
S19D/S45D (SD), S19A/S45A (SA), or R120G
(RG) alone or additionally with pIRES-FBX4. Total cell
extracts were prepared after 2 days and analyzed by SDS-PAGE and
subsequent immunoblotting with anti-
-actin (lower panel),
anti-HSP70 (middle panel), and anti-HA (upper
panel) antibodies. The ubiquitinated proteins are indicated by
X-Ub(n). The sizes of the protein markers are shown
on the left in kilodaltons. The asterisk indicates an
unidentified endogenous HA-immunopositive protein. B, HeLa
cells were subjected to a heat shock at 45 °C for 30 min and
analyzed for HSP70 induction: no heat shock (0), 1-h
recovery (1), 3-h recovery (3), 6-h recovery
(6), and 18-h recovery (18).
-Actin was used
as a loading control.
B-crystallin S19D/S45D or R120G,
the ubiquitinated proteins were analyzed by two-dimensional gel
electrophoresis. Each target protein to which an increasing number of
ubiquitin moieties are bound should give a curved pattern of spots,
starting from the mono-ubiquitinated form toward highly
polyubiquitinated forms (40). The shape of the curve is dependent on
the difference in pI of ubiquitin and the target protein.
Detergent-insoluble fractions, in which the ubiquitinated proteins were
enriched, were isolated from HeLa cells transfected with HA-ubiquitin,
FBX4, and wild-type or mutant
B-crystallin; subjected to
two-dimensional electrophoresis; and analyzed by Western blotting. The
two-dimensional Western blots were first stained for
B-crystallin
and then for the HA epitope (Fig. 6).
B-Crystallin R120G (arrows) gave the strongest signal
(Fig. 6D), as was expected, because this mutant was more
abundantly present in the insoluble fraction than the other isoforms.
In the extracts of HeLa cells cotransfected with wild-type
B-crystallin or S19A/S45A, no ubiquitinated products were detected
(Fig. 6, A and C), in agreement with the findings in Fig. 4. The major ubiquitinated products formed a single curved pattern, indicating that the number of proteins that have been ubiquitinated is very limited, perhaps just one. Importantly, a similar
but less pronounced pattern was obtained with
B-crystallin S19D/S45D
(Fig. 6B), indicating that
B-crystallin S19D/S45D and R120G most probably stimulate the ubiquitination of the same
protein(s). The fastest migrating ubiquitinated product
(arrowheads) has a molecular mass of ~35 kDa.
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Fig. 6.
Ubiquitin-protein conjugates resolved by
two-dimensional electrophoresis. HeLa cells were cotransfected
with pBSSK -HA-ubiquitin, pIRES-FBX4, and a pIRES
construct coding for wild-type
B-crystallin (A),
S19D/S45D (B), S19A/S45A (C), or R120G
(D). After 2 days, the cells were lysed and fractionated
into detergent-soluble and -insoluble fractions. The
detergent-insoluble fraction was dissolved by ultrasonication
in buffer containing 6 M urea, 2 M thiourea,
and 2% CHAPS and separated by isoelectric focusing and in the second
dimension by 12% SDS-PAGE. After blotting onto nitrocellulose
membranes, immunostaining was successively performed for
B-crystallin and the HA epitope. Proteins staining for
B-crystallin are indicated, as well as the region in which ubiquitin
staining of unknown protein(s) X was observed
(X-Ub(n)).
B-Crystallin displays charge
heterogeneity, which may be due to modifications such as
phosphorylation or deamidation (44). The arrowheads indicate
the smallest ubiquitinated protein (~35 kDa).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B-crystallin, the
F-box protein FBX4. The interaction between FBX4 and
B-crystallin is
most likely regulated by phosphorylation at Ser-19 and Ser-45. This
combination is of particular interest because these serines are mainly
phosphorylated during mitosis (15), suggesting that the interaction
with FBX4 might play a role during cell division. In our experiments,
phosphorylation at Ser-19 and Ser-45 was mimicked by replacing these
residues with negatively charged aspartic acids (Fig. 1 and Table I).
It is possible that phosphorylated serines give a stronger interaction
than aspartic acid residues because a phosphate group has two negative
charges. For this reason, it cannot be excluded that phosphorylation of
just one of the two serines is already sufficient to stimulate the
interaction with FBX4. Because Ser-19 and Ser-45 are phosphorylated by
different kinases (15), it would be interesting to determine whether
both phosphoserines are indeed necessary for FBX4 interaction.
B-crystallin with FBX4, but also the mutation R120G, which causes a
desmin-related myopathy. Ser-19 and Ser-45 are localized in the
N-terminal domain, and R120G in the
-crystallin domain. The
interaction of
B-crystallin R120G with FBX4 could be the result of a
conformational change induced by the mutation and resembling the
phosphorylated form. Because the structure of
B-crystallin is not
known, it cannot be excluded that these residues are actually close to
each other in the tertiary structure. In wheat HSP16.9, the crystal
structure of which is known, Arg-108 is located at a position
equivalent to Arg-120 in
B-crystallin and forms a salt bridge with a
negatively charged residue (41). Thus, due to the loss of Arg-120, a
negative charge might become available and, if at the right position,
can mimic phosphorylation.
B-crystallin S19D/S45D and R120G, but also
with wild-type
B-crystallin (Table I). This is remarkable because
full-length FBX4 did not interact with wild-type
B-crystallin, but
only with S19D/S45D and R120G. Also for some other proteins, it has
been shown that the truncated version is a stronger interactor than the
full-length protein (42). It should be emphasized that, in the case of
FBX4-(179-387), this is not due to improper folding because the
interactions are specific (Table I). At the moment, we cannot explain
this result. It could be an artifact of the two-hybrid system, but it
is also possible that it reflects a specific functional aspect. One
could hypothesize that, under certain conditions, FBX4 can change its
conformation in such a manner that it resembles FBX4-(179-387),
allowing interaction not only with phosphorylated but also
non-phosphorylated
B- crystallin.
B-crystallin S19D/S45D or R120G was
relatively weak in both the two-hybrid system and the co-immunoprecipitation assay (Fig. 1 and Table I). However, upon coexpression of FBX4 with these
B-crystallin mutants, a large proportion of the FBX4 protein followed the translocation of the mutants to the detergent-insoluble fraction (Fig. 4). It is possible that the interaction between FBX4 and the
B-crystallin mutants is
stabilized in the detergent-insoluble fraction by a yet unknown protein. It would be interesting to find out if this protein is the
target protein ubiquitinated by the FBX4-ubiquitin ligase complex.
B-crystallin S19D/S45D or R120G (Figs. 4-6) is probably a
result of the ubiquitin ligase activity of FBX4. However, we cannot
exclude the possibility that the ubiquitinated products accumulate due
to indirect effects such as inhibition of protein degradation by
aggregate formation (43) or induction of a stress response. The latter
is unlikely because we were unable to detect a stress response due to
coexpression of FBX4, HA-ubiquitin, and
B-crystallin S19D/S45D or
R120G (Fig. 5). Furthermore, it is not very likely that inhibition of
protein degradation would cause ubiquitination of just one or two proteins.
B-crystallin remain to be
determined. Identification of the ubiquitinated protein(s) might be an
important step in understanding the role of phosphorylation of
B-crystallin during mitosis and may give insight into the
B-crystallin R120G-induced aggregates found in a desmin-related
myopathy. It has been shown that, in many different cell lines,
B-crystallin is specifically phosphorylated during mitosis (15).
Both
B-crystallin and FBX4 are expressed together in a variety of
tissues (Fig. 2). It may thus well be that the ubiquitination induced
by the regulated interaction between FBX4 and
B-crystallin is a
generally occurring process.
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FOOTNOTES |
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* This work was supported by Grant NWO-MW 902-27-227 from the Netherlands Organization for Scientific Research.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.
Present address: Intervet Nederland B. V., 5831 AP
Boxmeer, The Netherlands.
§ To whom correspondence should be addressed: Dept. of Biochemistry 161, NCMLS, University of Nijmegen, P. O. Box 9101, 6500 HB Nijmegen, The Netherlands. Tel.: 31-24-361-6753; Fax: 31-24-354-0525; E-mail: W.Boelens@ncmls.kun.nl.
Published, JBC Papers in Press, December 4, 2002, DOI 10.1074/jbc.M211403200
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ABBREVIATIONS |
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The abbreviations used are: HSPs, heat-shock proteins; HA, hemagglutinin; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
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