Structural Analysis of Siah1 and Its Interactions with Siah-interacting Protein (SIP)

Seven inabsentia homologue (Siah) family proteins bind ubiquitin-conjugating enzymes and target proteins for proteasome-mediated degradation. Recently we identified a novel Siah-interacting protein (SIP) that is a Sgt1-related molecule that provides a physical link between Siah family proteins and the Skp1-Cullin-F-box ubiquitin ligase component Skp1. In the present study, a structure-based approach was used to identify interacting residues in Siah that are required for association with SIP. In Siah1 a large concave surface is formed across the dimer interface. Analysis of the electrostatic surface potential of the Siah1 dimer reveals that the β-sheet concavity is predominately electronegative, suggesting that the protein-protein interactions between Siah1 and SIP are mediated by ionic contacts. The structural prediction was confirmed by site-directed mutagenesis of these electronegative residues, resulting in loss of binding of Siah1 to SIPin vitro and in cells. The results also provide a structural basis for understanding the mechanism by which Siah family proteins interact with partner proteins such as SIP. Seven inabsentia homologue (Siah) family proteins bind ubiquitin-conjugating enzymes and target proteins for proteasome-mediated degradation. Recently we identified a novel Siah-interacting protein (SIP) that is a Sgt1-related molecule that provides a physical link between Siah family proteins and the Skp1-Cullin-F-box ubiquitin ligase component Skp1. In the present study, a structure-based approach was used to identify interacting residues in Siah that are required for association with SIP. In Siah1 a large concave surface is formed across the dimer interface. Analysis of the electrostatic surface potential of the Siah1 dimer reveals that the β-sheet concavity is predominately electronegative, suggesting that the protein-protein interactions between Siah1 and SIP are mediated by ionic contacts. The structural prediction was confirmed by site-directed mutagenesis of these electronegative residues, resulting in loss of binding of Siah1 to SIPin vitro and in cells. The results also provide a structural basis for understanding the mechanism by which Siah family proteins interact with partner proteins such as SIP. seven in absentiahomologue 1 Siah-interacting protein substrate-binding domain human embryonic kidney tumor necrosis factor receptor-associated factor Skp1-Cullin-F-box Siah1/Sina family proteins represent mammalian homologs of the Drosophila Sina (seven inabsentia) protein. Sina is required for R7 photoreceptor cell differentiation within the sevenless pathway (1Carthew R.W. Rubin G.M. Cell. 1990; 63: 561-577Abstract Full Text PDF PubMed Scopus (277) Google Scholar). The members of the family are E3 ubiquitin-protein isopeptide ligases that regulate ubiquitination and protein degradation. For example, Sina binds a ubiquitin-conjugating enzyme (E2). Heterocomplexes of Sina and another protein called Phyllopod form an E3 complex that interacts with a transcriptional repressor called Tramtrack, targeting it for polyubiquitination and proteasome-mediated degradation (2Tang A.H. Neufeld T.P. Kwan E. Rubin G.M. Cell. 1997; 90: 459-467Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 3Li S., Li, Y. Carthew R.W. Lai Z.-C. Cell. 1997; 90: 469-478Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). The destruction of Tramtrack is necessary for differentiation of R7 cells (2Tang A.H. Neufeld T.P. Kwan E. Rubin G.M. Cell. 1997; 90: 459-467Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 3Li S., Li, Y. Carthew R.W. Lai Z.-C. Cell. 1997; 90: 469-478Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). In humans two genes exist that encode Sina-like proteins,SIAH1 and SIAH2 (4Hu G. Chung Y.L. Glover T. Valentine V. Look A.T. Fearon E.R. Genomics. 1997; 46: 103-111Crossref PubMed Scopus (127) Google Scholar). Like theirDrosophila counterpart, the Siah1 and Siah2 proteins contain a N-terminal RING domain that binds E2s followed by a cysteine-rich domain and then a novel domain implicated in binding various substrate proteins and targeting them for degradation. The reported targets of Siah-mediated degradation include DCC (5Hu G. Zhang S. Vidal M. Baer J.L. Fearon E.R. Genes Dev. 1997; 11: 2701-2714Crossref PubMed Scopus (178) Google Scholar), Nco-R (6Zhang J. Guenther M.G. Carthew R.W. Lazar M.A. Genes Dev. 1998; 12: 1775-1780Crossref PubMed Scopus (190) Google Scholar), c-Myb (7Tanikawa J. Ichikawa-Iwata E. Kanei-Ishii C. Nakai A. Matsuzawa S.I. Reed J.C. Ishii S. J. Biol. Chem. 2000; 275: 15578-15585Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), BOB1/OBF1 (8Boehm J., He, Y. Greiner A. Staudt L. Wirth T. EMBO J. 2001; 20: 4153-4162Crossref PubMed Scopus (79) Google Scholar, 9Tiedt R. Bartholdy B.A. Matthias G. Newell J.W. Matthias P. EMBO J. 2001; 20: 4143-4152Crossref PubMed Scopus (71) Google Scholar), Peg3/Pw1 (10Relaix F. Wei X.-J., Li, W. Pan J. Lin Y. Bowtell D.D. Sassoon D.A. Wu X. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2105-2110Crossref PubMed Scopus (144) Google Scholar), APC (11Liu J. Stevens J., Hu, Y. Neufeld K.L. White R. Matsunami N. Mol. Cell. 2001; 7: 927-936Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar), Kid (12Germani A. Bruzzoni-Giovanelli H. Fellous A. Gisselbrecht S. Varin-Blank N. Calvo F. Oncogene. 2000; 19: 5997-6006Crossref PubMed Scopus (78) Google Scholar), Numb (13Susini L. Passer B.J. Amzallag-Elbaz N. Juven-Gershon T. Prieur S. Privat N. Tuynder M. Gendron M.-C. Israel A. Amson R. Oren M. Telerman A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 15067-15072Crossref PubMed Scopus (85) Google Scholar), synaptophysin (14Wheeler T.C. Chin L.S., Li, Y. Roudabush F.L. Li L. J. Biol. Chem. 2002; 277: 10273-10282Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar), and group 1 metabotropic glutamate receptor (15Ishikawa K. Nash S. Nishimune A. Niki A. Kaneko S. Nakanishi S. Genes Cells. 1999; 4: 381-390Crossref PubMed Scopus (72) Google Scholar). In addition, Siah reportedly interacts with Vav, a GDP-exchange factor for Rac/Rho (16Germani A. Romero F. Houlard M. Camonis J. Gisselbrecht S. Fischer S. Varin-Blank N. Mol. Cell. Biol. 1999; 19: 3798-3807Crossref PubMed Scopus (40) Google Scholar), and BAG1, a Hsp70/Hsc70-binding protein that modulates cellular pathways involved in control of cell proliferation, cell death, and cell migration (17Matsuzawa S. Takayama S. Froesch B.A. Zapata J.M. Reed J.C. EMBO J. 1998; 17: 2736-2747Crossref PubMed Scopus (185) Google Scholar). Interestingly, however, Siah does not appear to target Vav or BAG1 for polyubiquitination and degradation despite its ability to bind these proteins. Thus, not all Siah-binding proteins are targets of Siah-mediated degradation. The physiological basis for the broad range of protein-protein interactions with Siah/Sina family members is as yet not clear. Recently, we identified a novel Siah-interacting protein (SIP) by using yeast two-hybrid interaction cloning methods (18Matsuzawa S. Reed J.C. Mol. Cell. 2001; 7: 915-926Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar). SIP is a Sgt1-related protein that provides a physical link between Sina/Siah family proteins and the SCF complex component Skp1. Similar to Siah/Sina family proteins, SCF complexes play a critical role in the ubiquitination and degradation of a variety of target proteins including cyclins, p27 kip1 , p21 waf-1 , E2F, IκB, and β-catenin (19Koepp D.M. Harper J.W. Elledge S.J. Cell. 1999; 97: 431-434Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar, 20Patton E.E. Willems A.R. Tyers M. Trends Genet. 1998; 14: 236-243Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar, 21Winston J.T. Koepp D.M. Zhu C. Elledge S.J. Harper J.W. Curr. Biol. 1999; 9: 1180-1182Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar). We elucidated a network of protein interactions involving Siah and SIP that regulate levels of β-catenin and thus the activity of β-catenin-dependent Tcf/LEF transcription factors. In this network an evolutionarily conserved pattern exists where Siah binds to SIP, which interacts with Skp1, which in turn binds to the F-box protein Ebi (18Matsuzawa S. Reed J.C. Mol. Cell. 2001; 7: 915-926Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar). Here, to begin to dissect this network we have used site-directed mutagenesis to identify the contact surfaces on Siah for SIP. The crystal structure of a fragment encompassing the Cys-rich domain and the substrate-binding domain (SBD) of murine Siah1a has been determined (22Polekhina G. House C.M. Traficante N. Mackay J.P. Relaix F. Sassoon D.A. Parker M.W. Bowtell D.D. Nat. Struct. Biol. 2002; 9: 68-75Crossref PubMed Scopus (118) Google Scholar). The structure reveals that the SBD of Siah1a bears striking structural similarity to the tumor necrosis factor receptor-associated factor (TRAF) domains of TRAFs, a family of adapter proteins that bridges the cytosolic domains of multiple tumor necrosis factor family receptors to intracellular protein kinases (23Arch R.H. Gedrich R.W. Thompson C.B. Genes Dev. 1998; 12: 2821-2830Crossref PubMed Scopus (525) Google Scholar). However, the oligomeric state of Siah1a SBD differs from TRAFs. Siah1a exists as a dimer, whereas TRAFs associate as trimers. In Siah1a a large concave surface is formed across the dimer interface. This region as well as two other clefts have been suggested as sites for protein-protein interaction (22Polekhina G. House C.M. Traficante N. Mackay J.P. Relaix F. Sassoon D.A. Parker M.W. Bowtell D.D. Nat. Struct. Biol. 2002; 9: 68-75Crossref PubMed Scopus (118) Google Scholar, 24Reed J.C. Ely K. Nat. Struct. Biol. 2002; 9: 8-10Crossref PubMed Scopus (39) Google Scholar). The results of the present study identify SIP contact residues in the large concave surface of the Siah1 dimer, which represent critical sites for the first docking event in the Siah/SIP/Skp1/Ebi network. Mutations in Siah1 were generated by two-step PCR-based mutagenesis using a full-length human Siah1 cDNA (17Matsuzawa S. Takayama S. Froesch B.A. Zapata J.M. Reed J.C. EMBO J. 1998; 17: 2736-2747Crossref PubMed Scopus (185) Google Scholar) as a template. Products were purified by the QiaQuick gel extraction kit (Qiagen), digested with EcoRI and XhoI, and then directly subcloned into the EcoRI and XhoI sites of pcDNA3 plasmid (Invitrogen) with a N-terminal Myc epitope-tag (MEQKLISEEDL), thus creating pcDNA3-myc. Alternatively, the cDNAs were subcloned into yeast two-hybrid plasmids pGilda and pJG4–5, which produce fusion proteins with a LexA DNA-binding domain or a B42 transcriptional activation domain, respectively, at the N terminus under the control of a GAL1 promoter. HEK293T cells were maintained in high glucose Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 1 mm l-glutamine, and antibiotics. For transient transfections, cells (∼5 × 105) in 6-well plates were transfected with plasmid DNAs using LipofectAMINE Plus (Invitrogen). HEK293T cells (2 × 106) in 100-mm plates were used directly or transiently transfected with 6 μg (total) of plasmid DNA. After 24 h, cells were treated with 10 μm MG132 for 8 h and lysed in 1 ml of HKMEN solution containing 10 mm HEPES (pH 7.2), 142 mm KCl, 5 mm MgCl2, 2 mm EGTA, 0.2% Nonidet P-40, 0.1 μmphenylmethylsulfonyl fluoride, 5 μg/ml leupeptin, 1 μg/ml aprotinin, 1 μg/ml pepstatin and 20 μm MG132. Immunoprecipitations were performed using agarose-conjugated anti-Myc antibody (9E10, Santa Cruz) at 4 °C for 4 h. After washing in HKMEN solution, immune complexes were analyzed by SDS-PAGE/immunoblotting using anti-hemagglutinin antibodies (3F10, Invitrogen) followed by horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit immunoglobulin (Amersham Biosciences) and detection using enhanced chemiluminescence (Amersham Biosciences). For yeast two-hybrid assays (17Matsuzawa S. Takayama S. Froesch B.A. Zapata J.M. Reed J.C. EMBO J. 1998; 17: 2736-2747Crossref PubMed Scopus (185) Google Scholar), the yeast EGY191 strain was cotransformed with pGilda plasmids encoding wild-type or mutant Siah1/LexA DNA-binding domain fusion proteins, pJG4–5 plasmids encoding SIP/B42 transactivation domain fusion protein, and β-galactosidase reporter plasmids (pRB1840). To detect exogenous cytosolic β-catenin, cells were disrupted in ice-cold hypotonic buffer containing 10 mm Tris-HCl, pH 7.5, 10 mm KCl, 0.1 mm EDTA, and protease inhibitor mixture (Roche Molecular Biochemicals) by passage 15 times through a 26-gauge needle. Cell extracts were clarified by centrifugation at 16,000 × g for 30 min. Resulting supernatants (20 μg of total protein) were separated by SDS-PAGE (10% gels) and transferred to nitrocellulose membranes. Proteins were detected with anti-Myc monoclonal antibody (9E10, Santa Cruz Biotechnology). Tcf/LEF transcriptional activity was measured by transient transfection reporter gene assays using reporter plasmids pFOP-FLASH or pTOP-FLASH (containing wild-type or mutant Tcf/LEF binding sites cloned upstream of a thymidine kinase minimal promoter and luciferase gene, respectively) as described (25Korinek V. Barker N. Morin P.J. van Wichen D. de Weger R. Kinzler K.W. Vogelstein B. Clevers H. Science. 1997; 275: 1784-1787Crossref PubMed Scopus (2974) Google Scholar). The atomic model of Siah1a (22Polekhina G. House C.M. Traficante N. Mackay J.P. Relaix F. Sassoon D.A. Parker M.W. Bowtell D.D. Nat. Struct. Biol. 2002; 9: 68-75Crossref PubMed Scopus (118) Google Scholar) reveals three “clefts” or “grooves” located on the surface of the Siah dimer that have potential as sites for protein-protein interactions (24Reed J.C. Ely K. Nat. Struct. Biol. 2002; 9: 8-10Crossref PubMed Scopus (39) Google Scholar). The most striking is a large concave surface formed at the dimer interface by an antiparallel arrangement of β-sheets from each monomer (Fig. 1). The other two are symmetrically equivalent clefts in each monomer that are located between the Cys-rich domain that contains two zinc fingers and SBD. The electrostatic surface potential of these regions is different (Fig. 1); that is the central large β-sheet concavity is predominately electronegative, whereas the two smaller clefts at the ends of the oligomer are electropositive in nature. Thus, the potential exists for binding at distant sites that differ significantly in overall charge. Because SIP is a basic protein, we tested the role of the negatively charged residues in the concave surface of the Siah1 dimer for binding of SIP, representing a cluster of glutamic acids and aspartic acids in the concave β-sheet structure. In contrast to wild-type Siah1 protein, mutant Siah1 molecules (mutant A) with alanine substituted for Glu-161, Asp-162, Glu-226, and Glu-237 in the concavity failed to bind SIP in co-immunoprecipitation experiments (Fig. 2 A). Because Siah1 exists as a dimer, the large electronegative surface concavity is formed by an antiparallel orientation of identical β-sheets in each monomer. As shown in Fig. 1, this arrangement produces a symmetrical relationship between corresponding structural features of the monomeric subunits. Consequently, eight acidic residues reside in the large concavity; that is four from each monomer. Thus, substitution of alanine for four acidic residues resulted in a loss of eight carboxyl groups at the surface and a dramatic change in the electrostatic character of the concavity. Failure of the mutant with these substitutions to bind SIP identifies this concave region as the SIP-binding interface. Substitution of alanine for Asp-253 and Glu-265 on the surface of the opposite face of the dimer or Asp-142 and Gln-151 did not affect SIP binding. Two other regions were proposed as sites for protein-protein interactions in Siah1a (22Polekhina G. House C.M. Traficante N. Mackay J.P. Relaix F. Sassoon D.A. Parker M.W. Bowtell D.D. Nat. Struct. Biol. 2002; 9: 68-75Crossref PubMed Scopus (118) Google Scholar). These are electropositive clefts between the SBD and two zinc fingers on each monomer (see Fig. 1). These distinct sites are located at each end of the elongated dimer, separated by 46 Å. The mutation of Arg-214, Arg-215, Arg-231, Arg-124, and Arg-232 in these clefts to alanine did not block SIP binding (Fig. 2 A). Similar results with each of those mutant proteins were obtained using yeast two-hybrid assays (Fig. 2 B), confirming that the SIP binding surface is located on one face of the Siah1 dimer in a concave region (420 Å2) that contains a cluster of eight acidic residues. In this predominately negatively charged area, two basic residues (Arg-224 and Arg-233) are also found. To test the role of these positively charged residues on SIP recognition, these two arginines were mutated to alanine. The resulting mutant Siah1 molecule retained the capability to bind SIP, suggesting that the protein-protein interactions between Siah1 and SIP are mediated by ionic contacts involving the negatively charged cluster. Siah participates in a pathway to β-catenin degradation involving Siah and an F-box protein that binds β-catenin of the sites by (18Matsuzawa S. Reed J.C. Mol. Cell. 2001; 7: 915-926Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar). We identified a of protein interactions that link Siah to Ebi by association with SIP and Skp1, a central component of of Siah is by suggesting a mechanism that to destruction of In this we that activity of β-catenin binding Tcf/LEF transcription to cell (18Matsuzawa S. Reed J.C. Mol. Cell. 2001; 7: 915-926Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar). To the present mutagenesis into a cellular we of that of Siah1 degradation of β-catenin (18Matsuzawa S. Reed J.C. Mol. Cell. 2001; 7: 915-926Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar). We performed transient transfection assays in HEK293T cells (Fig. β-catenin protein levels by As shown in Fig. of wild-type Siah1 levels of β-catenin protein. In the Siah1 mutant with alanine substitutions for the cluster (mutant A) did not degradation of it enhanced levels of with the binding the other mutant molecules retained the ability to levels of Because β-catenin is required as a for activation of the transcription factor Tcf/LEF M. P. Science. 2000; PubMed Scopus Google Scholar), we the of wild-type and mutant Siah on Tcf/LEF activity using transient transfection reporter gene assays (25Korinek V. Barker N. Morin P.J. van Wichen D. de Weger R. Kinzler K.W. Vogelstein B. Clevers H. Science. 1997; 275: 1784-1787Crossref PubMed Scopus (2974) Google Scholar, P.J. V. Barker N. Clevers H. Vogelstein B. Kinzler K.W. Science. 1997; 275: PubMed Scopus Google Scholar). of β-catenin a in Tcf/LEF transcriptional activity in HEK293T (Fig. an equivalent of plasmid encoding Siah1 was Tcf/LEF activity was by (Fig. In contrast to wild-type cells with plasmids encoding mutant Siah1 failed to activation of Tcf/LEF and transactivation of the reporter gene thus confirming the of the The other mutant Siah molecules as in this Thus, we that the contact residues required for interaction of Siah to SIP are critical for the of this protein in cells. Siah1 also binds to the BAG1, which has been reported to by Siah1 (17Matsuzawa S. Takayama S. Froesch B.A. Zapata J.M. Reed J.C. EMBO J. 1998; 17: 2736-2747Crossref PubMed Scopus (185) Google Scholar). we tested the of in the acidic on Siah1 interaction with In contrast to SIP, BAG1 with all of the mutant Siah1 proteins in the tested (Fig. suggesting that SIP and BAG1 bind to different surfaces of the Siah The domain of BAG1 is an with an overall electronegative K. Takayama S. L. D.A. J. S. E. J. M. Reed J.C. Ely Nat. Struct. Biol. 2001; PubMed Scopus Google Scholar). Interestingly, basic residues at the two positively charged on Siah1 (Fig. were mutated to binding to BAG1 was the BAG1 binding that is distinct from the SIP interaction region has yet to Recently, it was reported that the Drosophila protein Phyllopod interacts with the SBD region of a homologue of Siah1 S., C. Carthew R.W. Mol. Cell. Biol. 2002; PubMed Scopus Google Scholar). of residues from the terminus of the SBD region of Phyllopod suggesting that Phyllopod interact with the concave in the dimer these residues are located in this large concavity (22Polekhina G. House C.M. Traficante N. Mackay J.P. Relaix F. Sassoon D.A. Parker M.W. Bowtell D.D. Nat. Struct. Biol. 2002; 9: 68-75Crossref PubMed Scopus (118) Google Scholar). the by that a in Phyllopod is necessary for binding to S., C. Carthew R.W. Mol. Cell. Biol. 2002; PubMed Scopus Google Scholar). this in Phyllopod it in a concave region in the Siah1 dimer that we have for SIP binding. Interestingly, is similarity between SIP and the domain of The basis for of SIP and Phyllopod by the structural of The human SIP protein at the protein with a protein, identified as a protein A. J. 1998; PubMed Scopus Google Scholar). Interestingly, the binds to family proteins through its region in a A. B. M. K. M. L. E. J. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). by protein kinase in vitro A. B. M. J. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). is or of its interactions with Skp1, or other proteins. the physiological of family proteins on degradation of β-catenin Because the large SIP-binding identified is formed by a symmetrical arrangement of identical residues from the two Siah1 a as to protein-protein interactions with SIP involved the concave surface or contacts with one that a We a antiparallel arrangement of β-sheets in the repressor R. R. J. Ely Full Text Full Text PDF PubMed Scopus Google Scholar, K. L. K. T. 1990; PubMed Scopus Google Scholar). In the residues that are required for binding the are located on by residues in three from each monomer F. PubMed Scopus Google Scholar). As in a large concave is formed across the dimer interface. In the of the repressor binds one binds across the face of the dimer contacts with residues from K. L. PubMed Scopus Google Scholar). For the Siah1 dimer it is that the contact surface a SIP or those two SIP molecules are to each of the experiments are to the binding of We M. and M. for and R. for