Calcium-regulated Interaction of Sgt1 with S100A6 (Calcyclin) and Other S100 Proteins*

Marcin Nowotny {ddagger} §, Magdalena Spiechowicz {ddagger}, Beata Jastrzebska {ddagger}, Anna Filipek {ddagger}, Katsumi Kitagawa ¶ and Jacek Kuznicki {ddagger} || **

From the {ddagger}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.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
S100A6 (calcyclin), a small calcium-binding protein from the S100 family, interacts with several target proteins in a calcium-regulated manner. One target is Calcyclin-Binding Protein/Siah-1-Interacting Protein (CacyBP/SIP), a component of a novel pathway of {beta}-catenin ubiquitination. A recently discovered yeast homolog of CacyBP/SIP, Sgt1, associates with Skp1 and regulates its function in the Skp1/Cullin1/F-box complex ubiquitin ligase and in kinetochore complexes. S100A6-binding domain of CacyBP/SIP is in its C-terminal region, where the homology between CacyBP/SIP and Sgt1 is the greatest. Therefore, we hypothesized that Sgt1, through its C-terminal region, interacts with S100A6. We tested this hypothesis by performing affinity chromatography and chemical cross-linking experiments. Our results showed that Sgt1 binds to S100A6 in a calcium-regulated manner and that the S100A6-binding domain in Sgt1 is comprised of 71 C-terminal residues. Moreover, S100A6 does not influence Skp1-Sgt1 binding, a result suggesting that separate Sgt1 domains are responsible for interactions with S100A6 and Skp1. Sgt1 binds not only to S100A6 but also to S100B and S100P, other members of the S100 family. The interaction between S100A6 and Sgt1 is likely to be physiologically relevant because both proteins were co-immunoprecipitated from HEp-2 cell line extract using monoclonal anti-S100A6 antibody. Phosphorylation of the S100A6-binding domain of Sgt1 by casein kinase II was inhibited by S100A6, a result suggesting that the role of S100A6 binding is to regulate the phosphorylation of Sgt1. These findings suggest that protein ubiquitination via Sgt1-dependent pathway can be regulated by S100 proteins.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
S100 proteins are a family of small, highly homologous proteins that contain two calcium-binding EF-hand motifs (reviewed in Ref. 1). Because the binding of calcium ions is the most important biochemical feature of S100 proteins, they are thought to function as sensors of calcium ion concentration in the cell. They appear to have several functions, including regulation of protein phosphorylation, cytoskeleton polymerization, and cell cycle progression. S100A6 (calcyclin) is a typical member of the S100 family. The function of S100A6 remains unclear, but evidence suggests that it is involved in cell cycle regulation (2, 3) and exocytosis (4, 5). S100A6 may also be involved in tumorigenesis; the protein is overexpressed in several tumors, e.g. melanoma (6).

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 {beta}-catenin, which is the substrate that is ubiquitinated and later degraded. {beta}-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 {beta}-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.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids—We prepared the plasmids pET28-Sgt1 and pET28-Sgt1-(263–333) by the following methods. The appropriate fragments of human Sgt1 DNA were amplified from the BKK51 plasmid (20) by PCR with Pfu polymerase: forward primers, 5'-TAAATCATATGGCGGCGGCTGCAGCA-3' for pET28-Sgt1 and 5'-GCCCGCATATGAAAGAAGAAGAAAAGAAT-3' for pET28-Sgt1-(263–333), and the same backward primer for both constructs, 5'-CGGCGGGATCCTTAGTACTTTTTCCATTCCA-3' (restriction endonucleases recognition sites in bold). PCR products and vector (pET28a; Novagen) were digested with restriction endonucleases (NdeI and BamHI, both from Promega). The fragments of appropriate size were purified from the agarose gel using the QIAquick gel extraction kit (Qiagen). The appropriate vector and insert were ligated by T4 DNA ligase (Promega), and the ligation products were used to transform Escherichia coli (TOP10F' strain). Single-colony PCR was used to detect colonies that contained the insert. The plasmids were purified and sequenced. The plasmid pET21d-Skp1-H6 (23) was generously provided by Dr. R. Poon from Hong Kong University of Science and Technology, China.

Expression and Purification of Sgt1, Sgt1-(263–333), 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-{beta}-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 Chromatography—The 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-linking—His-tagged Sgt1 (15 µg) or His-tagged Sgt1-(263–333) (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 Assay—Six 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-Sepharose—The 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-immunoprecipitation—Human 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 {beta}-chloronaphtol and H2O2.

Protein Phosphorylation—Phosphorylation of Sgt1-(263–333) (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-(263–333), 2 µg of S100A6 or both proteins were added with [{gamma}-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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Interaction between S100A6 and Sgt1—The S100A6-binding region of human CacyBP/SIP was compared with a corresponding fragment of human Sgt1 using ClustalW software (24) (Fig. 1). In this region the amino acid identity was 29%. Because of this significant similarity between C-terminal regions of CacyBP/SIP and Sgt1, we hypothesized that Sgt1 interacts with S100A6. To test this hypothesis, recombinant human Sgt1 with N-terminal His tag was expressed in E. coli and purified on nickel-nitrilotriacetic acid-agarose resin. This one-step purification procedure yielded protein that was pure enough for biochemical characterization. The tag was cleaved off, and Sgt1 was coupled to CNBr-activated-Sepharose. Affinity chromatography was used to check whether Sgt1 could interact with S100A6 (Fig. 2). S100A6 was applied to Sgt1-Sepharose, CacyBP/SIP-Sepharose, or Sepharose alone in the presence of 1 mM CaCl2, and bound protein was eluted in the presence of 2 mM EGTA. S100A6 was present in the fractions eluted from CacyBP/SIP-bound and Sgt1-bound resins but not from Sepharose alone. This result shows that S100A6 interacts with Sgt1 in a calcium-dependent manner.



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FIG. 1.
Sequence comparison of the S100A6-binding domain of mouse CacyBP/SIP with the corresponding fragment of human Sgt1. The sequence of whole CacyBP/SIP was compared with whole Sgt1 using ClustalW software. Only the alignment of C-terminal sequences corresponding to S100A6-binding domain is shown. Asterisk, double dots, and single dots indicate identical, closely similar, and similar amino acid residues, respectively. The arrow indicates the site of the E216K mutation (residue 217 in mouse CacyBP/SIP) (see "Discussion").

 


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FIG. 2.
The interaction between Sgt1 and S100A6, affinity chromatography. S100A6 was applied to Sepharose alone, Sgt1-Sepharose, or CacyBP/SIP-Sepharose in a buffer containing 1 mM CaCl2. The unbound fraction (U) and the fraction eluted with a buffer with 2 mM EGTA (E) were collected and analyzed by SDS-Tricine-PAGE stained with Coomassie Brilliant Blue.

 

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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FIG. 3.
The interaction of S100A6 with Sgt1 and with Sgt1-(263–333), chemical cross-linking. Proteins were cross-linked by treatment with EDC and NHS in the presence of 1 mM CaCl2 or 2 mM EGTA. The components of each reaction are indicated on the top of the figure. The products were analyzed by SDS-PAGE and were Coomassie Blue-stained (left panels). Fifteen percent of the reaction mixture was analyzed by Western blotting with the indicated antibodies (center and right panels). A, the cross-linking of S100A6 with His-tagged Sgt1 is shown. In addition to a band corresponding to Sgt1 (S), bands designated SC are visible in the reaction performed in the presence of CaCl2. (Diffuse bands at the bottom of the lanes in panel A (anti-Sgt1 and Coomassie Blue-stained) correspond to the products of proteolytic degradation of Sgt1). B, shows the cross-linking of S100A6 with His-tagged Sgt1-(263–333). In addition to the band corresponding to Sgt1-(263–333) (S) and S100A6 (C), a cross-linked dimer of S100A6 (D) and another band present only in the CaCl2-containing reaction (SC) are seen. S100A6 is not detected by Western blotting, because it requires special conditions of electrotransfer to nitrocellulose.

 

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-(263–333), 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-(263–333) and S100A6. Therefore, the Sgt1-(263–333) 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 Binding—A 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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FIG. 4.
The influence of S100A6 on Sgt1-Skp1 interaction, affinity chromatography. Human Skp1 was applied to Sepharose alone or to human Sgt1-conjugated-Sepharose in the presence of 1 mM CaCl2 or 2 mM EGTA. The symbol + indicates that a 6-fold molar excess of S100A6 over Skp1 was added to the reaction mixture. Unbound (U) fractions and fractions eluted with a buffer containing 0.5 M NaCl (E) were collected and analyzed by SDS-glycine-PAGE. The gel was stained with Coomassie Brilliant Blue.

 

Interaction between Sgt1 and Other Members of the S100 Family—CacyBP/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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FIG. 5.
Interaction between Sgt1 and S100 proteins, affinity chromatography. Proteins (5 µg of each) were applied individually to Sgt1-conjugated-Sepharose in the presence of 1 mM CaCl2. The unbound fractions (U) were collected, the resin washed, and the bound protein eluted with a buffer containing 2 mM EGTA (E). The fractions were analyzed by SDS-Tricine-PAGE. The gel was stained with Coomassie Brilliant Blue. D9k, calbindin D9k; PV, parvalbumin; CaM, calmodulin.

 

Co-immunoprecipitation of S100A6 and Sgt1—To 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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FIG. 6.
Co-immunoprecipitation of S100A6 and Sgt1. HEp-2 cell extracts containing 2 mM CaCl2 were immunoprecipitated with monoclonal anti-S100A6 antibody or non-relevant antibody. Immunoprecipitated fractions were analyzed by immunoblotting with anti-Sgt1 antibody. Lane 1, Sgt1 immunoprecipitated with anti-S100A6 antibody. Lane 2, the reaction carried out in the presence of excess recombinant CacyBP/SIP. Lane 3, immunoprecipitate with non-relevant antibody. Lane 4, recombinant His-tagged Sgt1.

 

S100A6 Inhibits the Phosphorylation of Sgt1-(263–333)—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-(263–333) 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-(263–333), but not S100A6, was efficiently phosphorylated by casein kinase II in the presence of 1 mM CaCl2. Addition of S100A6 inhibited phosphorylation of Sgt1-(263–333). 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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FIG. 7.
Phosphorylation of Sgt1-(263–333). Indicated proteins were phosphorylated in vitro with purified recombinant human casein kinase II, using [{gamma}-32P]ATP. Lanes 1 and 2, phosphorylation of S100A6 and Sgt1-(263–333), respectively, in the presence of 1 mM CaCl2. Lanes 3 and 4, phosphorylation of Sgt1-(263–333) in the presence of S100A6 and 1 mM CaCl2 or 2 mM EGTA, respectively. Upper panel, Coomassie Blue-stained gel. Lower panel, autoradiography. Bands corresponding to autophosphorylated casein kinase II are indicated.

 

We also performed a phosphorylation assay with Sgt1-(263–333) 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.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In this work we showed that human Sgt1, like its homolog CacyBP/SIP, interacts in a calcium-dependent manner with S100A6 and other S100 proteins. Moreover, we determined that S100A6 binds to Sgt1-(263–333), a C-terminal fragment corresponding to the domain in CacyBP/SIP that binds to S100A6 and to other S100 proteins (22). Therefore, we assume that this C-terminal region of Sgt1 also binds S100 proteins other than S100A6. Our results also showed that S100A6 does not regulate the interaction between Sgt1 and Skp1 through simple competition. This finding suggests that separate domains for binding to S100A6 and to Skp1 exist in Sgt1.

Interestingly, a functionally deficient yeast mutant, Sgt1–5, 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 Sgt1–5 that carries only a single mutation (E216K) in its S100A6-binding region acts in a dominant-negative fashion and interferes with {beta}-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-(263–333) 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 {beta}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 {beta}-catenin (16). Sgt1 is associated with a different ubiquitin ligase, SCF (20). The SCF complex containing {beta}-TrCP protein is responsible for the ubiquitination of {beta}-catenin. However, SCF- and CacyBP/SIP-dependent pathways are regulated in a different fashion. Ubiquitination by SCF requires that {beta}-catenin is phosphorylated on serine residues (29, 30). In the CacyBP/SIP-mediated pathway, {beta}-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 {beta}-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 {beta}-catenin in vivo and thus participate in the process of tumor development and progression. Currently, we are performing experiments to address this question.


    FOOTNOTES
 
* This work was supported by State Committee of Scientific Research Grants 3 P04A 043 22 (to A. F.) and 6 P04A 039 20 (to J. K.), statutory funds from the Nencki Institute of Experimental Biology, Cancer Center Support Grant CA21765 from the NCI, National Institutes of Health, and funds from the American Lebanese Syrian Associated Charities (ALSAC). Back

§ Recipient of a stipend from the Polish Foundation for Science. Back

** 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. Back


    ACKNOWLEDGMENTS
 
We thank Dr. Wieslawa Lesniak for critical reading of the manuscript.



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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