From the
Nencki Institute of Experimental Biology,
3 Pasteur Street, 02-093 Warsaw, Poland, the
¶Department of Molecular Pharmacology, St. Jude
Children's Research Hospital, Memphis, Tennessee 38105-2794, and the
||International Institute of Molecular and Cell
Biology, 4 Trojdena Street, 02-109 Warsaw, Poland
Received for publication, November 12, 2002 , and in revised form, May 12, 2003.
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ABSTRACT |
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INTRODUCTION |
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Attempts to elucidate the function of S100A6 have involved the search for
its calcium-regulated target proteins. Several such proteins have been
identified: glyceraldehyde-3-phosphate dehydrogenase
(7), annexin II
(8), annexin VI
(9), and annexin XI
(10). In 1996, a novel mouse
protein was identified on the basis of its ability to interact with S100A6 in
a calcium-dependent manner
(11). It was named
Calcyclin-Binding Protein
(CacyBP)1
(12). In vitro
characterization of the interaction between S100A6 and CacyBP showed that the
S100A6-binding domain in CacyBP is comprised of 52 C-terminal residues and
that the dissociation constant for the S100A6-CacyBP complex is 1
µM (13). CacyBP
is expressed in many tissues in mice and rats, but its highest expression
level is in the brain (14). In
cortical neurons, upon cell stimulation and elevation of intracellular calcium
concentration, CacyBP undergoes a translocation from the cytoplasm to the
perinuclear space (15).
Recently, a human ortholog of CacyBP, called SIP (Siah-1-Interacting
Protein) was shown to be a component of a novel ubiquitin ligase complex
(16) (hereafter we refer to
both proteins as CacyBP/SIP). In the ubiquitin ligase complex, the N-terminal
region of CacyBP/SIP interacts with Siah-1, and the C-terminal region
interacts with Skp1. Skp1 binds to Ebi, a protein that recognizes
-catenin, which is the substrate that is ubiquitinated and later
degraded.
-catenin is an important oncogene involved in the development
of colon cancer (17,
18). It regulates the activity
of Tcf/Lef transcription factors, which promote cell proliferation. A separate
pool of
-catenin participates in adherens junctions
(19).
At the time of the discovery of CacyBP there were no homologs of this
protein deposited in the sequence data bases. In 1999 a novel yeast protein
called Sgt1 was discovered
(20). Its human homolog is
20% identical to mouse CacyBP. In yeast Sgt1 interacts with Skp1, a
protein component of the SCF ubiquitin ligase and of kinetochore complexes.
Some yeast Sgt1 mutants exhibit defects in chromosome segregation
that result in arrest of the cell cycle at G2 phase. In other
Sgt1 mutants, ubiquitination of Sic1 and Cln1 (two cell cycle
regulators) is impaired, and this impairment results in cell cycle arrest at
G1. These results suggested that Sgt1 is required for normal Skp1
function in kinetochore and SCF. Recently, several studies have shown
involvement of Sgt1 in R gene-mediated plant defense against
pathogens (review Ref. 21).
However, the link between the involvement of Sgt1 in ubiquitination and
pathogen response remains unclear.
The most conserved regions of CacyBP/SIP and Sgt1 are located near their C termini. The S100A6-binding domain is located in this region of CacyBP/SIP (13). Therefore, we determined whether human Sgt1 interacts with S100A6 and other S100 proteins and characterized this interaction. These results, together with our previous data on CacyBP/SIP (22), suggest that S100 proteins can regulate two different ubiquitination pathways in a calcium-dependent manner.
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EXPERIMENTAL PROCEDURES |
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Expression and Purification of Sgt1, Sgt1-(263333), and
Skp1 Expression plasmids were introduced into the E. coli
BL21(DE3) strain (Novagen). The bacteria were cultured in Luria Bertani medium
with kanamycin, and expression was induced by the addition of
isopropyl-1-thio--D-galactopyranoside. The bacteria were
further cultured for
3 h. The expressed His-tagged proteins were purified
on nickel-nitrilotriacetic acid-agarose resin (Qiagen) as previously described
(22). The His tag was removed
by biotinylated thrombin (Novagen) cleavage according to the manufacturer's
instructions. The proteins were dialyzed against a desired buffered
solution.
Affinity ChromatographyThe coupling of Sgt1 and CacyBP/SIP to CNBr-activated-Sepharose was carried out according to the manufacturer's instructions (Amersham Biosciences). CNBr-activated-Sepharose was equilibrated in borate buffer (50 mM boric acid, pH 8.2, 0.4 M NaCl), and Sgt1 or CacyBP/SIP, both without a His tag, was added. After a 2-h incubation at room temperature, the resin was washed, and the sites for nonspecific binding were blocked by incubation in 0.1 M Tris-HCl (pH 8.0) for 2 h. Next, the resin was washed several times with 0.1 M acetate buffer (pH 4.0) containing 0.5 M NaCl and 0.1 M Tris-HCl (pH 8.0) containing 0.5 M NaCl.
Rabbit S100A6 was purified as previously described (8). 7 µg of S100A6 was applied to Sgt1-Sepharose, CacyBP/SIP-Sepharose, and empty resin in buffer A (20 mM Tris-HCl, pH 7.5) containing 1 mM CaCl2. The resin was washed with buffer A containing CaCl2 and 0.5 M NaCl, and bound protein was eluted with buffer A containing 2 mM EGTA. Collected fractions were precipitated with acetone and analyzed by SDS-Tricine-PAGE.
Chemical Cross-linkingHis-tagged Sgt1 (15 µg) or His-tagged Sgt1-(263333) (5 µg) were mixed with 7 µg of S100A6 in the presence of 1 mM CaCl2 or 2 mM EGTA. EDC and NHS cross-linking reagents (Sigma) were added from fresh stocks to 4 and 10 mM final concentration, respectively. In control reactions, Sgt1 and S100A6 were cross-linked alone or both proteins were incubated without cross-linking reagents. After the reaction was carried out for 1 h at room temperature, the proteins were precipitated with acetone and analyzed on SDS-glycine-PAGE or SDS-Tricine-PAGE. Fifteen percent of each reaction mixture was analyzed using Western blot with anti-S100A6 polyclonal antibody, anti-Sgt1 polyclonal antibody, or anti-His tag monoclonal antibody (HIS-1; Sigma). The blots were incubated with a chemiluminescent substrate (Super Signal; Pierce) and then exposed to photographic film (Amersham Biosciences).
Competition AssaySix micrograms of His-tagged human Skp1 in buffer A (20 mM Tris-HCl, pH 7.5) with 1 mM CaCl2 or 2 mM EGTA was applied to Sepharose alone or Sgt1-conjugated-Sepharose. Parallel reactions with Sgt1-conjugated-Sepharose were performed in the presence of 70 µg of S100A6 (6-fold molar excess over Skp1). The unbound fraction was collected, the resin washed, and the bound protein eluted with buffer A containing 2 mM EGTA and 0.5 M NaCl. The proteins were precipitated with trichloroacetic acid and analyzed by SDS-glycine-PAGE.
Interaction of Calcium-binding Proteins with Sgt1-conjugated-SepharoseThe following calcium-binding proteins were used in the assay: parvalbumin, which was a gift from Dr. C. W. Heizmann, Zurich University, Switzerland; calbindin D9k, a gift from Dr. S. Linse, Lund University, Sweden; S100A8, a gift from Dr. C. Kerkhoff, University of Muenster, Germany; and S100A4, a gift from Dr. R. Barraclough, University of Liverpool, Great Britain. The remaining calcium-binding proteins were S100A1, S100B, and calmodulin (each purchased from Sigma). The binding assay was performed as described under "Affinity Chromatography" with 5 µg of each calcium-binding protein in buffer A (20 mM Tris-HCl, pH 7.5) and 1 mM CaCl2 applied to Sgt1-conjugated-Sepharose.
Co-immunoprecipitationHuman epidermal cells (HEp-2 cell
line) were cultured in Dulbecco's modified Eagle's medium supplemented with
10% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100
µg/ml). For co-immunoprecipitation, cells were lysed in buffer I (50
mM Tris-HCl, pH 7.5, 1% Triton X-100, 1 mM EDTA, 1
mM dithiothreitol) with protease inhibitors (CompleteTM
EDTA-free; Roche Applied Science) and next centrifuged at 10,000 x
g for 10 min. Supernatant was then incubated with CaCl2 (2
mM final) for 15 min at room temperature and then monoclonal
antibody against S100A6 (CACY-100 clone; Sigma) was added for 1 h at room
temperature. The reactions were next incubated with protein G-agarose (Sigma)
for 1 h at room temperature. After incubation the beads were washed five times
in buffer I containing 2 mM CaCl2. The proteins bound to
G-agarose were eluted with a buffer containing 100 mM glycine-HCl,
pH 2.7, and precipitated with cold acetone. The pellets were then solubilized
in sample buffer and applied on the SDS-glycine-PAGE. After electrophoresis,
the proteins were transferred to nitrocellulose sheets and analyzed with
antibodies against Sgt1. The blots were developed with -chloronaphtol
and H2O2.
Protein PhosphorylationPhosphorylation of
Sgt1-(263333) (no His tag) with human recombinant casein kinase II
(Calbiochem) was carried out in reactions with 400 ng of the kinase in buffer
containing 20 mM HEPES, pH 7.5, 10 mM MgCl2,
50mM NaCl, 1 mM dithiothreitol, 0.2 mM ATP,
and 1 mM CaCl2 or 2 mM EGTA. The final volume
of the reactions was 50 µl. The reactions were preincubated for 20 min at
30 °C, and 2 µg of Sgt1-(263333), 2 µg of S100A6 or both
proteins were added with [-32P]ATP in final concentration of
0.2 mM. The reaction was carried out for 30 min at 30 °C and
stopped by the addition of PAGE sample buffer and boiling. The proteins were
analyzed in SDS-glycine-PAGE, and the gels were exposed to photographic
film.
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RESULTS |
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To confirm the interaction between human Sgt1 and S100A6, we performed chemical cross-linking experiments using EDC and NHS reagents (Fig. 3A). As control reactions each individual protein was cross-linked alone and both proteins were incubated without EDC and NHS. The products of the reactions were analyzed by PAGE and Western blotting. Coomassie Blue staining of the SDS-glycine-PAGE (Fig. 3A, left panel) revealed a band (designated S) that corresponded to Sgt1 and additional bands (designated SC) not present in control reactions and larger than Sgt1 by 10 kDa, i.e. the mass of S100A6 monomer. Western blot analysis showed that proteins in the SC bands were recognized by antibody to Sgt1 (center panel) and to S100A6 (right panel). This result proved that the SC bands consisted of cross-linked complexes of Sgt1 and S100A6. These complexes were not present in the reaction carried out in the presence of EGTA; therefore, we confirmed that the interaction is calcium-dependent.
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A similar chemical cross-linking experiment was carried out to identify the
S100A6-binding domain in Sgt1 (Fig.
3B). First, we produced a recombinant His-tagged Sgt1
fragment that consisted of residues 263 through 333 (71 residues from C
terminus) and corresponded to the previously identified S100A6-binding domain
in CacyBP/SIP. This tagged fragment has a calculated molecular mass of 10.5
kDa but migrates in SDS-Tricine-PAGE as a band of 13 kDa. We next
subjected the protein to chemical cross-linking and analysis by PAGE.
Coomassie Blue staining revealed a band of 10 kDa corresponding to S100A6, a
band of 13 kDa corresponding to His-tagged Sgt1-(263333), and a band of
20 kDa that corresponded to a cross-linked S100A6 dimer. An additional band
was seen only in the cross-linking reaction containing all components and
CaCl2. Its apparent mass was
10 kDa larger than that of Sgt1
fragment. The additional fragment was recognized by anti-His-tag antibody and
anti-S100A6 antibody, which indicated that it corresponded to a cross-linked
complex of Sgt1-(263333) and S100A6. Therefore, the
Sgt1-(263333) fragment contains the S100A6-binding domain. Little of
the cross-linked complex was detected when the reaction was carried out in the
absence of CaCl2, a result confirming the calcium dependence of the
binding.
The Lack of Influence of S100A6 on Sgt1-Skp1 BindingA prominent biochemical feature of Sgt1 is its ability to interact with Skp1 and regulate Skp1-containing SCF and kinetochore complexes (20). Because Sgt1 also binds to S100A6, we hypothesized that S100A6 influences the Sgt1-Skp1 interaction. The S100A6-binding domain in Sgt1 is located in the C-terminal region, and the Skp1-binding domain in CacyBP/SIP is also located in its C-terminal part, after residue 74 (16). These facts suggested that the Skp1- and S100A6-binding domains in human Sgt1 might overlap. To determine whether mammalian S100A6 and Skp1 can compete in binding to Sgt1, we performed affinity chromatography. His-tagged human Skp1 was applied to a column containing either Sepharose alone or Sgt1-conjugated-Sepharose. The unbound fraction and the fraction eluted with 0.5 M NaCl were collected and analyzed by electrophoresis through SDS-glycine polyacrylamide gels. Skp1 bound to Sgt1-conjugated-Sepharose but not to Sepharose alone (Fig. 4). When Skp1 and a 6-fold molar excess of S100A6 were applied in the presence of CaCl2 to the column containing Sgt1-conjugated-Sepharose, the bound fraction contained both Skp1 and S100A6. Moreover, the amount of His-tagged Skp1 that bound to Sgt1 in the presence of excess S100A6 and CaCl2 did not differ from that bound in the absence of S100A6. This result shows that Skp1 and S100A6 do not compete in binding to Sgt1 and that the Skp1-binding domain and the S100A6-binding domain are probably separate in Sgt1.
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Interaction between Sgt1 and Other Members of the S100 FamilyCacyBP/SIP interacts not only with S100A6 but also with other members of the family of highly homologous S100 proteins (22). To study the possible interaction between Sgt1 and S100 proteins other than S100A6, we again performed affinity chromatography using Sgt1-conjugated-Sepharose (Fig. 5). Different S100 proteins (S100A1, S100A4, S100A6, S100A8, S100B, S100P, and calbindin D9k) and other calcium-binding proteins (parvalbumin and calmodulin) were applied individually to Sgt1-conjugated-Sepharose in the presence of 1 mM CaCl2. Analysis of the unbound fraction and the fraction eluted by EGTA showed that Sgt1 interacts strongly with S100A6 and S100P proteins and more weakly with S100B. S100A1 and S100A4 were also present in the bound fraction, but the interaction was very weak and it might be nonspecific. S100A8, calbindin D9k, calmodulin and parvalbumin did not bind to Sgt1, a result showing that Sgt1 interacts with particular S100 proteins.
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Co-immunoprecipitation of S100A6 and Sgt1To establish whether interaction between S100A6 and Sgt1 can occur in vivo we performed co-immunoprecipitation experiments form cell extracts using monoclonal anti-S100A6 antibody. The human epidermal HEp-2 cell line was selected for these experiments because it expresses both S100A6 and Sgt1. Fig. 6 shows that Sgt1 was co-immunoprecipitated with S100A6 in the presence of 2 mM CaCl2. Sgt1 was not co-immunoprecipitated by a non-relevant monoclonal antibody, and addition of excess recombinant CacyBP/SIP abolished the interaction, which confirms that the observed effect was specific.
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S100A6 Inhibits the Phosphorylation of Sgt1-(263333)It has been previously shown that S100 proteins are able to regulate phosphorylation of their target proteins, most often by binding and blocking the phosphorylation site. Therefore, we hypothesized that the function of Sgt1-S100A6 interaction might be to regulate the phosphorylation of the S100A6-binding domain of Sgt1. A search of PhosphoBase (25) revealed that in Sgt1-(263333) serine 299 can be phosphorylated by casein kinase II. To check whether this phosphorylation can be regulated by S100A6 we performed in vitro phosphorylation assays (Fig. 7). In these assays Sgt1-(263333), but not S100A6, was efficiently phosphorylated by casein kinase II in the presence of 1 mM CaCl2. Addition of S100A6 inhibited phosphorylation of Sgt1-(263333). In the presence of 2 mM EGTA, inhibition of Sgt1 fragment phosphorylation was much weaker. The changes in Sgt1 phosphorylation were not because of the changes in the activity of the kinase, because its autophosphorylation was equal in all the reactions. These results show that S100A6 can regulate the phosphorylation of the Sgt1 region containing the S100A6-binding domain.
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We also performed a phosphorylation assay with Sgt1-(263333) fragment carrying a previously described E303K mutation (see "Discussion"). In this mutant the consensus pattern for casein kinase II site is no longer present. However E303K mutant was efficiently phosphorylated (not shown), suggesting that the casein kinase II phosphorylation site is different from the one identified by computer prediction.
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DISCUSSION |
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Interestingly, a functionally deficient yeast mutant,
Sgt15, has a mutation (E364K) in the S100A6-binding domain
identified in the present study. This mutation together with another point
mutation outside the S100A6-binding domain causes defects in the
ubiquitination of cell cycle regulators; however, the mutant protein retains
the ability to interact with Skp1
(20). A human CacyBP/SIP
mutant modeled after Sgt15 that carries only a single mutation
(E216K) in its S100A6-binding region acts in a dominant-negative fashion and
interferes with -catenin ubiquitination and degradation in vivo
(16). To check whether the
negative effect of these point mutations stems from the fact that they
abrogate the interaction between Sgt1 and S100A6, we produced recombinant
Sgt1-(263333) fragment that carried a mutation corresponding to the
mutation described above (E303K for human Sgt1). This fragment was efficiently
cross-linked with S100A6, which shows that its ability to bind S100A6 was not
affected. However, the properties of mutants indicate that the S100A6-binding
region is essential for the function of CacyBP/SIP and Sgt1 in the
ubiquitination process. When S100A6 interacts in a calcium-dependent manner
with the C-terminal region, the interaction is likely to regulate the activity
of CacyBP/SIP and Sgt1.
To search for possible mechanisms of such regulation we studied the
influence of S100A6 on Sgt1 phosphorylation. Several members of the S100
protein family have been shown to regulate the phosphorylation of their target
proteins (review Refs. 1 and
26)). For example, S100A4 was
shown to inhibit the phosphorylation of its target protein liprin 1 by
protein kinase C and casein kinase II
(27). Our results demonstrated
that the S100A6-binding domain of Sgt1 can be phosphorylated by casein kinase
II and that this phosphorylation is inhibited by S100A6 binding. These
findings suggest that the regulation of phosphorylation might be the
physiological role of Sgt1-S100A6 interaction.
In this work we demonstrated that Sgt1 is capable of interacting with several members of the S100 protein family. S100 proteins are expressed in a strictly tissue- and cell-specific fashion (28). Therefore, the interaction between Sgt1 and S100 proteins will be regulated by the availability of S100 proteins in a given tissue and cell type.
CacyBP/SIP is a component of a novel ubiquitin ligase complex responsible
for ubiquitination and degradation of -catenin
(16). Sgt1 is associated with
a different ubiquitin ligase, SCF
(20). The SCF complex
containing
-TrCP protein is responsible for the ubiquitination of
-catenin. However, SCF- and CacyBP/SIP-dependent pathways are regulated
in a different fashion. Ubiquitination by SCF requires that
-catenin is
phosphorylated on serine residues
(29,
30). In the
CacyBP/SIP-mediated pathway,
-catenin ubiquitination is
phosphorylation-independent and is triggered by the expression of Siah-1, a
limiting component of the ligase complex
(16). Therefore, the two
pathways are activated in different conditions and are likely to play distinct
roles. Because S100A6 and some other S100 proteins can interact with
CacyBP/SIP and Sgt1, these S100 proteins could regulate both pathways in a
similar fashion. The defects in ubiquitination and degradation of
-catenin are key events in the development of colon cancer. It has been
shown that S100A6 may be involved in the progression of colon carcinoma
(3133).
For example, Komatsu et al.
(34) showed that expression of
S100A6 is correlated with the progression and invasive process of human
colorectal carcinoma. Therefore, a very important question is whether S100
proteins, through their interaction with ubiquitin ligases, can regulate the
ubiquitination of
-catenin in vivo and thus participate in the
process of tumor development and progression. Currently, we are performing
experiments to address this question.
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FOOTNOTES |
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Recipient of a stipend from the Polish Foundation for Science.
** To whom correspondence should be addressed. Tel.: 48-22-668-52-20; Fax: 48-22-822-53-42; E-mail: jacek{at}iimcb.gov.pl.
1 The abbreviations used are: CacyBP/SIP, calcyclin (S100A6) binding
protein/Siah-1-interacting protein; EDC,
1-ethyl-3-[3-dimethyl-aminopropyl]carbodiimide hydrochloride; NHS,
N-hydroxysuccinimide.
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ACKNOWLEDGMENTS |
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REFERENCES |
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