A binding motif for Siah ligase

Colin M. House*, Ian J. Frew*, Huei-Luen Huang†, Gerhard Wiche†, Nadia Traficante*, Edouard Nice‡, Bruno Catimel‡, and David D. L. Bowtell*§

*Trescowthick Research Laboratories, Peter MacCallum Cancer Institute, Melbourne 8006, Victoria, Australia; †Institute of Biochemistry and Molecular Biology, Vienna Biocenter, A-1030 Vienna, Austria; and ‡Ludwig Institute for Cancer Research, Parkville 3152, Victoria, Australia

Edited by Alexander Varshavsky, California Institute of Technology, Pasadena, CA, and approved January 6, 2003 (received for review August 8, 2002) The Drosophila SINA (seven in absentia) and its mammalian (19, 20). Both SIP and pAPC interact with the C terminus of orthologs (Siah, seven in absentia homolog) are RING domain Siah, although no direct interaction with the substrate, ␤-cate- that function in E3 complexes and facili- nin, was reported. Siah’s ability to act as single E3 ligase and also tate ubiquitination and degradation of a wide range of cellular to participate in a variant SCF complex is very unusual (reviewed proteins, including ␤-. Despite these diverse targets, the in ref. 21) and highlights the importance of understanding how means by which SINA͞Siah recognize substrates or binding pro- Siah SBD interacts with its partners. teins has remained unknown. Here we identify a peptide motif We have previously focused on the Siah SBD and showed that (RPVAxVxPxxR) that mediates the interaction of Siah protein with the crystal structure of that domain displays a fold similar to the a range of protein partners. Sequence alignment and mutagenesis C-terminal domain of tumor necrosis factor receptor associated scanning revealed residues that are important to this interaction. factor proteins (22). Given the diverse interactions of the Siah This consensus sequence correctly predicted a high-affinity inter- SBD with a range of cellular proteins, we have sought to define action with a peptide from the cytoskeletal protein plectin-1 the molecular basis of these interactions. Here we describe a (residues 95–117). The unusually high-affinity binding obtained high-affinity binding peptide, present in the Drosophila protein ؍ with a 23-residue peptide (KDapp 29 nM with SINA) suggests that PHYL, which binds with high affinity to the SINA and Siah it may serve as a useful dominant negative reagent for SINA͞Siah SBDs. Mutagenesis of this peptide has revealed a binding motif proteins. that is conserved and functional in diverse Siah-interacting proteins. roteasomal degradation of proteins requires recognition of a Ppolyubiquitin signal on the targeted protein. Ubiquitination Materials and Methods is a multistep process that involves at least three classes of Plasmid Construction. Mouse Siah1a, Siah2, and Drosophila SINA transfer proteins, E1 (ubiquitin activating proteins), E2 (ubiq- (full length and the SBDs, lacking the N termini and RING uitin conjugating proteins), and E3 (ubiquitin ligases) (1). A domains) were cloned into the bacterial expression vector subset of E3s transfer ubiquitin from the E2 directly to the pMalC2 (New England Biolabs) at the BamHI and HindIII sites, substrate. These comprise both single subunit and multiprotein utilizing sequence specific oligonucleotide primers and PCR complexes, which are characterized by the presence of a RING amplification. For Siah1a, the SBD consisted of residues 80–282, (really interesting new ) domain. Recent structures of a for Siah2, residues 116–325 and for SINA, residues 108–314. cCbl-UbcH7 complex (2) and an SCF complex (3) suggest RING Fragments of PHYL, DCC, and SIP (defined in the text) were domain proteins function as part of the scaffold to optimally cloned and expressed by using pGEX2T (Amersham Pharma- position substrate and E2 for transfer of ubiquitin. cia). The Kid H16 construct, in pGEX-4T2, was a gift from Members of the highly conserved SINA (seven in absentia)/ A. Germani and F. Calvo (Saint-Louis Hospital, Paris). GST- Siah (seven in absentia homologue) family of proteins contain a TIEG-1 expression vector was a gift from Steven Johnsen and RING domain and function as E3 ligases (4). This protein family Thomas Spelsberg (Mayo Clinic and Foundation, Rochester, was first defined in Drosophila, where SINA is required for R7 NY). Mutagenesis of constructs was performed by using the cell determination in the developing eye, downstream of the QuikChange site-directed mutagenesis kit (Stratagene). Sevenless͞Ras pathway (5). Genetic and biochemical evidence support a model where an E3 comprising SINA͞PHYL͞EBI Protein Expression. Proteins were expressed as GST or maltose- interacts with the transcriptional repressor TTK88 and the binding protein (MBP) fusions in Escherichia coli BL21(DE3) ubiquitin-conjugating enzyme UBCD1, leading to the ubiquiti- cells at 22°C for 5 h. Cells were lysed and sonicated (three times nation and proteasomal degradation of TTK88 (6–9). The for 30 sec on ice) in 50 mM Tris, pH 8.0͞200 mM NaCl͞15 Drosophila SINA E3 complex is the best described, both genet- mM 2-mercaptoethanol (␤-ME)͞0.2 mg/ml lysozyme͞0.5% ically and biochemically, suggesting that it can provide clues to Triton X-100͞10 ␮g/ml leupeptin͞10 ␮g/ml aprotinin͞1 ␮g/ml the function of mammalian Siah proteins. pepstatin͞0.5 mM PMSF before purification with either amylose SINA͞Siah sequences are highly conserved from plants to (for MBP proteins) or glutathione (for GST proteins) on Sepha- . Whereas the N terminus and RING domain of Siah rose-4B solid supports. MBP-fusion proteins were eluted with 10 bind E2 proteins (10) (11), the C terminus can be considered as mM maltose in 50 mM Tris, pH 8.0͞200 mM NaCl͞15 mM a substrate- and cofactor-interaction domain (substrate-binding ␤-ME. For Biacore analysis, MBP-Siah-SBD, MBP-Sina-SBD domain, SBD) that interacts with a number of proteins, some of and Siah-SBD were further purified before kinetic studies by which are degraded. Degraded proteins include netrin-1 recep- using size exclusion chromatography (Superose 12 HR 3.2͞30, ͞ tor deleted in colorectal cancer, DCC (10); the nuclear receptor Amersham Pharmacia) equilibrated in 10 mM Hepes, pH 7.4, BIOCHEMISTRY corepressor, N-CoR (12); the motor protein, Kid (13); the containing 3.4 mM EDTA, 0.15 mM NaCl, and 0.005% (vol͞vol) transcriptional activator, OBF-1 (14, 15); the developmental Tween 20 (HBS). The protein concentration was determined by regulator, NUMB (16); the neural transmitter protein, synapto- absorbance at 280 nm using an extinction coefficient calculated physin (17); and the transcriptional repressor, TIEG-1 (18). In these cases, Siah may function alone as a targeting, single subunit E3 ligase, but Siah has also been shown to interact in an This paper was submitted directly (Track II) to the PNAS office. SCF-type complex including Skp1, Ebi, Siah interacting protein Abbreviations: SBD, substrate-binding domain; MBP, maltose-binding protein; pAPC, ad- (SIP), and adenomatous polyposis coli protein (pAPC) to facil- enomatous polyposis coli protein; SIP, Siah interacting protein. itate the degradation of ␤-catenin in a p53-dependent manner §To whom correspondence should be addressed. E-mail: [email protected].

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0534783100 PNAS ͉ March 18, 2003 ͉ vol. 100 ͉ no. 6 ͉ 3101–3106 Downloaded by guest on September 23, 2021 from the amino acid composition. GFP fusions of plectin exons concentrations of proteins (25–1,000 nM) over the sensor sur- 1 and 1c were stably expressed in Chinese hamster ovary cells. face at a flow rate of 10 ␮l͞min. After completion of the injection Total cell lysates, in a buffer of 50 mM Tris, pH 7.5͞0.1M phase, dissociation was monitored in HBS buffer for 300 s at the NaCl͞5% glycerol͞2.5 mM sodium orthovanadate͞2.5 mM so- same flow rate. Bound proteins were eluted, and the surface was dium metavanadate͞0.1% Triton X-100͞0.1% Nonidet P-40͞ regenerated between injections with 10 mM HCl. Regeneration 0.1% sodium deoxycholate͞1 mM PMSF were used for interac- conditions did not denature the immobilized antigen as shown by tion studies with GST-Siah proteins. equivalent signals on reinjection of a ligand-containing sample. The apparent association (ka) and dissociation (kd) rate con- Protein–Protein Interaction Studies (Coprecipitation). GST fusions stants were calculated as described previously (26) (27) by of Siah-binding proteins were expressed as described above and nonlinear least squares regression by using BIAEVALUATION used in binding assays with MBP-Siah-SBD. A total of 0.2–2 ␮g version 3.0 software into which the appropriate iterative curve- of each protein, bound to GSH beads, was incubated with 3 fitting equations have been installed. When applicable, the ␮ ͞ g/ml MBP-Siah-SBD in 50 mM Tris-HCl, pH 8.0 200 mM affinity constant KD was also determined by equilibrium binding ͞ ͞ NaCl 15 mM 2-mercaptoethanol 0.1% Nonidet P-40 (TNBN) analysis where KD is obtained from the reciprocal of the slope of at 4°C for 1 h. In cases where binding was competed with free the graph (KA) obtained by plotting the biosensor data in PHYL peptide, 20 ␮M peptide was added to the MBP-Siah-SBD Scatchard format [(Req͞nC) versus Req, where Req is the for 15 min before the 1 h incubation. After washing four times biosensor response at equilibrium, n the valency and C the in 1 ml of TNBN, the beads and bound proteins were recovered concentration] (26, 28). Calculations for the goodness of fit by centrifugation and boiled in 100 ␮l of Laemmli sample buffer are described in Supporting Materials and Methods, which is before separation by SDS͞PAGE. For Siah interactions with published as supporting information on the PNAS web site, plectin exons 1 and 1c (as GFP fusions), the cell lysis buffer (see www.pnas.org. Protein Expression) was used and interaction was overnight at 4°C. Detection of protein–protein interaction was achieved Results either by Coomassie staining of gels or Western blotting (using A 22-aa Peptide Is Sufficient to Mediate PHYL Binding to Siah and a rabbit anti-MBP polyclonal antibody raised in our laboratory SINA. Although a number of binding partners have been defined or a monoclonal anti-GFP antibody from Roche Diagnostics). for SINA or Siah proteins, we found that fragments of PHYL Westerns were developed by using horseradish peroxidase- bound avidly to both SINA and Siah in coprecipitation experi- conjugated secondary antibody and enhanced chemilumines- ments (data not shown). Additionally, a previous report mapped cence (Amersham Pharmacia). a SINA-binding domain to the vicinity of residues 108–130 (29). The PHYL protein therefore served as a useful starting point for Peptide Synthesis. Peptides were synthesized by using Fmoc analysis of Siah and SINA binding partners. We tested the chemistry on a 96-well format (Mimotopes, Clayton, Victoria, binding of a protein deletion series of PHYL, expressed as Australia) and cleaved from the solid phase support. Peptides GST-fusions, to the Siah1a SBD by using an in vitro pull-down ͞ were dissolved in DMSO before dilution in H2OorPBS 0.1% assay. A fragment corresponding to amino acids 108–130 of Tween 20. Purity and quantitation of peptides was assessed by PHYL was sufficient for the Siah binding under these conditions mass spectrometry and reversed-phase HPLC. Purity of peptides (Fig. 1A). The MBP-Siah-SBD fused protein bound equally well was above 70% in all cases and consistent across the mutagenesis to full-length PHYL (1–400) and fragments corresponding to set. For Biacore analysis, peptides were purified immediately residues 1–130, 1–198 or 108–130 (Fig. 1A, lanes 9–12). MBP before immobilization by using a C18 micropreparative reverse- alone did not bind (Fig. 1A, lanes 2–6). These results suggested phase high-pressure liquid chromatography column and ana- that the peptide 108–130 is sufficient for maximal binding of lyzed by matrix-assisted laser desorption͞ionization time-of- PHYL to the Siah͞SINA family of proteins. flight-mass spectrometry (Kratos IV). Mapping the Siah Interaction Site in Kid. A C-terminal deletion Peptide Binding Assay. Synthetic peptides were synthesized with an analysis of the Kid protein was also performed, because it was N-terminal biotin residue and captured (100 ng͞well) on ELISA previously reported that Siah-binding resided in the C-terminal plates coated with Neutravidin (Pierce, 0.5 ␮g͞well). MBP-Siah- residues 404–665 (13). Siah binding was observed for the SBD (1 ␮g͞ml) was bound to the peptides for 20 min at room constructs 1–665, 1–560, and 1–540, but was substantially re- temperature and quantitated by using anti-MBP antibodies, duced in the 1–530 construct and shorter fragments (Fig. 1B). horseradish peroxidase-goat-anti-rabbit secondary antibody This result suggests that at least part of the Siah binding is (Sigma) and 2,2Ј-azino-bis(3-ethyl)benzthiazoline-6-sulfonic present in the Kid residues 531–540. acid as substrate, monitored at 405 nm after 10–15 min. MBP- Siah-SBD protein bound to mutant peptides was compared with Identification of a Functionally Conserved Binding Motif in Siah-SBD the protein bound to the PHYL108–130 parent peptide. Binding Proteins. On the basis of the avid binding between Siah SBD and the PHYL108–130 peptide, and the mapping of the Protein Alignment. Siah-binding fragments of various proteins, Siah-binding region in Kid, we sought to ascertain whether corresponding to the published minimal interacting fragments, elements of these peptides were present in other Siah SBD- were aligned by using the CLUSTALW program (23). The align- binding proteins. Beginning with PHYL peptide and the region ment was initiated by using short PHYL and OBF-1 sequences. around Val-51 in OBF-1, alignment with fragments of reported Siah SBD-binding partners demonstrated the presence of a Biacore Analysis. Surface plasmon resonance analyses were per- possible binding motif present in many of these proteins (Fig. 2). formed by using a BIAcore 2000 Biosensor (Uppsala). Purified, The core sequence PxAxVxP was found in the Siah interacting N-terminally biotinylated PHYL108–130 and plectin-195–117 proteins SIP, OBF-1, DCC, and TIEG1, with more degenerate peptides were immobilized onto a Neutravidin sensor surface consensus sequences found in NUMB, EF1-␦, Vav, Kid, N-CoR, (N-hydroxy-succinimide-activated CM5 carboxymethylated dex- and FIR. The remaining Siah-SBD-interacting proteins, includ- tran sensor chip) according to described protocols (24, 25). After ing pAPC, synaptophysin, mGlutR1, and ␣-, did not align peptide immobilization, the surface was washed with 10 mM HCl when this method was used. until a stable level of immobilized peptide was achieved. To investigate whether the putative binding motif was func- Binding data were generated by injecting 30 ␮l of varying tional in these proteins, we expressed protein fragments of DCC

3102 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0534783100 House et al. Downloaded by guest on September 23, 2021 Fig. 1. (A) Binding of PHYL to Siah maps to amino acids 108–130 of PHYL. Interaction between GST-PHYL fragments and a soluble fusion protein MBP-Siah-SBD was investigated in an in vitro GST pull-down. PHYL constructs were mixed with MBP alone (lanes 2–6) and MBP-Siah-SBD (lanes 8–12). Lane 1 shows the MBP alone input, and lane 7 shows the MBP-Siah-SBD input. The gels were stained with Coomassie blue. The MBP-Siah-SBD bound to the PHYL fragments is highlighted with an asterisk. (B) Binding of GST-Kid C-terminal deletion mutants to MBP-Siah SBD. Experiment was as in A, although bound proteins were detected by Western blotting using anti-MBP antibody. MBP alone did not bind Kid fragments (data not shown). The full-length GST-Kid is highlighted with an asterisk. The binding͞protein ratio was determined by using densitometry of the bands, excluding the nonspecific bands highlighted by dashes.

(1203–1364), Kid (404–665), and SIP (1–77) reported to contain of biotinylated peptides was synthesized in which alanine was necessary sequences for Siah SBD interaction (10, 13, 19) and substituted at each position across the PHYL108–130 peptide. tested their binding to Siah. Interaction of each fragment with Where alanine occurred in the native sequence, glycine was Siah SBD was observed (Fig. 3A) and was competed with free substituted. The peptide set was captured on a Neutravidin- PHYL peptide, suggesting that these bound the same site on Siah coated ELISA plate, MBP-Siah-SBD was bound directly and SBD. Demonstration that this motif was sufficient for binding measured by using an anti-MBP antibody. Solution-phase bind- was obtained by showing that a 24 residue peptide of DCC ing of Siah-SBD was almost eliminated by substitution of either (1324–1347), which encompassed the motif present in DCC, the Val-120 and Pro-122 residues (Fig. 4A). Whereas mutagen- efficiently bound Siah-SBD. This interaction was also abrogated esis of the conserved Pro-116 and Ala-118 residues partially by free competing PHYL peptide (Fig. 3A). reduced binding, the effects were not as strong as those seen for mutagenesis of Arg-115, Val-117, and Arg-125, which reduced To further investigate the role of the motif in these proteins, Ϸ mutagenesis of the VxP triplet to NxN was performed. The binding by 50% (Fig. 4A). In addition to the alanine mutagenesis, residues in the central results obtained were protein-dependent. Mutation of the Val portion of the peptide were altered to residues with opposite and Pro residues abrogated binding of the full-length proteins characteristics but similar bulk, that is, charge reversal, or PHYL and TIEG-1, and the short DCC peptide (1324–1347) hydrophobic to hydrophilic etc. The results reinforced those (Fig. 3B), and binding was reduced for DCC (1203–1364) and obtained with alanine mutants, although in general the inhibi- SIP (1–77) fragments. Although interaction of Kid with Siah tion of binding was even more pronounced. For example, mapped to the consensus motif (Figs. 1B and 2), mutation of the Met-119Gln, in which a large polar residue (Gln) was less well VxP motif did not affect Siah-binding (Fig. 3B). These findings tolerated than Ala, and Arg-121Glu, where the negative Glu was demonstrate the importance of the consensus binding motif in more inhibitory than Ala substitution (Fig. 4B). As before, mediating interaction of a diverse range of proteins with Siah, mutation of Val-120, Pro-122, Arg-115, and Val-117 were most but indicate that the contribution of specific residues to binding important for strong binding. varies. Database Searches with the Siah Binding Motif Correctly Predict Scanning Mutagenesis of PHYL108–130 Peptide Defines Residues Interaction with a Plectin-1 Peptide. PHYL protein has no mam- Required for Interaction with Siah. Because PHYL108–130 pep- malian ortholog, raising the possibility that another protein may tide bound more strongly to Siah SBD than did any of the form a high-affinity interaction with Siah in mammalian cells. other Siah-interacting proteins, we sought to define further Combining the information from the alignment of Siah-binding binding determinants within the PHYL peptide. A full alanine proteins (P116xAxVxP122) and the mutagenesis scan of the PHYL mutagenesis scan was performed to determine key residues in peptide (R115xVxxVxPxxR125), to generate a consensus motif, we PHYL108–130 required for the interaction with Siah SBD. A set searched protein databases to identify other potential Siah- interacting proteins. A large number of proteins were identified that matched the consensus to varying degrees, including the cytoskeletal linker protein, plectin-1 (31), which was the only protein with a perfect match (A95SLQRVRRPVAMVMPAR- RTPHVQ117) to the proposed motif. We synthesized the plec- tin-1 peptide (residues 95–117) and found that it bound Siah SBD efficiently. Moreover, binding was competed by free PHYL peptide, indicating that the plectin-195–117 peptide also recog- nized the same site on the Siah SBD as did PHYL (Fig. 6, which

is published as supporting information on the PNAS web site). BIOCHEMISTRY GST-Siah SBD efficiently bound a fused protein of mouse plectin-1 (32) residues 1-180(exon 1)-GFP expressed in Chinese hamster ovary cells but not plectin 1c residues 1-66(exon1c)- GFP, a splice variant that lacks the consensus binding motif Fig. 2. Presence of a common motif in many Siah-SBD binding proteins. (Fig. 5K). Alignment of PHYL108–130 with fragments of proteins previously reported to 108–130 95–117 interact with Siah-SBD. Totally conserved residues are highlighted in black and The PHYL and Plectin-1 Peptides Bind SINA-SBD and Siah- residues conserved in Ͼ60% of sequences are in gray. ¶, Y. Hu and D.D.L.B., SBD with Nanomolar Affinity. Biosensor analysis was used to unpublished data. measure the affinity of interactions between peptides and Siah͞

House et al. PNAS ͉ March 18, 2003 ͉ vol. 100 ͉ no. 6 ͉ 3103 Downloaded by guest on September 23, 2021 Fig. 3. Consensus motif confers binding of protein partners to Siah SBD. (A) Relative binding of Siah SBD to fragments of DCC (1203–1364), Kid (404–665), SIP (1–77), PHYL (108–130), and DCC (1324–1347) was assessed in an in vitro GST pull-down assay, in the absence and presence of 20 ␮M free PHYL108–130 peptide. Note that the loading on to the gel for the Western detection of the PHYL interaction was 10% of that for the other proteins, as this interaction was avid and gave an extremely strong signal. For this reason the GST-PHYL108–130 protein was undetectable when probed with anti-GST to show relative loading of solid-phase proteins. The amount of GST-PHYL108–130 fusion protein used in the experiment was approximately equal to that of the GST-Kid (404–665). (B) Effect of mutation of the VxP motif to NxN in a number of GST-fused interacting proteins, assessed by the binding of these proteins to MBP-Siah-SBD. Loading of the PHYL interaction products is 10% of that loaded for the other interactions. The relative loading was assessed by anti-GST immunoblot or Coomassie staining (TIEG-1FL and KidFL). In both panels, the migration of the solid-phase binding proteins are highlighted by an asterisk in the loading control Westerns.

SINA proteins. The results show that both of the peptides, Discussion PHYL108–130 and plectin-195–117, interacted with MBP-SINA- The Siah͞SINA family of proteins has been reported to interact, SBD with higher affinity than MBP-Siah-SBD (Fig. 5 and Table through their SBDs, with various proteins, some of which are 1). The apparent KD values were as low as 29 nM for MBP- degraded in Siah overexpression experiments. Although it is SINA-SBD interacting with the plectin-195–117 peptide and 92– important to know how the Siah SBD recognizes target proteins, 123 nM for the MBP-Siah-SBD interaction with the same the mapping of interacting domains in these proteins has not peptide. The peptide interactions with the isolated Siah-SBD elucidated any common domain or binding mechanism. It is not were of a similar affinity to those seen for the fusion protein known whether substrates and other partners interact at similar MBP-Siah-SBD, showing the validity of working with fusion sites on Siah. Only Siah SBD interactions with PHYL (29), proteins in these experiments and the mutagenesis study. Fur- mGlutR1 (31), and synaptophysin (17) have mapped the inter- ther information concerning the biosensor data (association and action to peptide fragments of the interacting proteins. We have dissociation constants) can be found in Supporting Materials and not investigated binding to synaptophysin, but we could not Methods. detect Siah SBD binding to mGlutR1 peptide under ELISA or Biacore conditions (data not shown). In contrast, we have found that the PHYL108–130 peptide interacts with SINA and Siah with a low nanomolar affinity. Alignment studies and a mutagenesis study of the PHYL peptide have revealed a potential Siah-binding motif, R115PVAxVxPxxR125. The most conserved residues in this motif appear to be Val-120 and Pro-122, and indeed mutagenesis of both of these residues abrogates Siah binding. Identification of the PHYL peptide sequence recognized by SINA͞Siah provided an essential guide to identify potential interacting domains in other known Siah-binding proteins. We investigated some of these interactions and found that the observed binding could be competed by free PHYL peptide and that mutagenesis of the VxP motif in the Siah-binding fragments reduced binding, although not in all proteins. These results suggest that the proteins, whether substrates or interacting partners, are binding at or near the PHYL binding site. It was previously reported that Siah SBD interacted with residues 1–101 of the transcriptional activator OBF-1 (14, 15). Moreover, in a random mutagenesis screen designed to investigate OBF-1 and Oct-1 binding, a Val-51 to Glu substitution abrogated binding to Siah SBD (14, 15). Val-51 lies within the sequence P47TAVV51LP53 (Fig. 2), Fig. 4. Scanning mutagenesis of PHYL peptide identifies core residues suggesting that OBF-1 also binds Siah through a VxP motif. Also required for Siah-SBD binding. Immobilized PHYL mutant peptides were tested for their ability to bind MBP-Siah-SBD in an ELISA-based assay. Binding of interest is that whereas pAPC did not align with the PHYL is compared with the PHYL108–130 parent peptide. The results are averages of motif when the CLUSTALW program was used, visual inspection 2777 2786 five separate experiments, showing SEM. (A) Alanine mutagenesis of individ- has found a V AARVTPFNY sequence, within the Siah ual residues in the PHYL108–130 peptide. (B) Altering the charge and hydro- SBD interacting region (20), that includes part of the PxAxVxP phobicity characteristics of individual residues in the core of the PHYL peptide. motif and is thus a potential binding site for Siah binding. Siah

3104 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0534783100 House et al. Downloaded by guest on September 23, 2021 Fig. 5. Biosensor analysis of the interactions of MBP-SINA-SBD, MBP-Siah-SBD, and Siah-SBD with immobilized PHYL108–130 and plectin-195–117 peptides. Biotinylated peptides were immobilized onto a neutravidin sensor surface as described in Materials and Methods. Varying concentrations of MBP-SINA-SBD (25–800 nM) were injected over immobilized PHYL108–130 (A) and immobilized plectin-195–117 (B). MBP-Siah-SBD (50–1000 nM) was injected over immobilized PHYL108–130 (C) and immobilized plectin-195–117 (D). Siah-SBD (300–1,000 nM) was injected over immobilized PHYL108–130 (E) and immobilized plectin-195–117 (F). The sensorgrams shown have controls subtracted, in which the sample was passed over a control neutravidin surface. Not all traces are shown, to aid clarity. An equilibrium binding analysis of the interaction between MBP-Siah-SBD and Siah-SBD and immobilized peptides PHYL108–130 and plectin-195–117 was performed for the following interactions: MBP-Siah-SBD and PHYL108–130 (G), MBP-Siah-SBD and plectin-195–117 (H), Siah-SBD and PHYL108–130 (I), Siah-SBD and plectin-195–117 (J). KD was obtained from the reciprocal of the slope of the graph (KA) obtained by plotting the biosensor data in Scatchard format [(Req͞nC) versus Req, where Req is the biosensor response at equilibrium, n is the valency, and C is the concentration). The KD values are tabulated in Table 1. (K) Plectin exon1 binds Siah in vitro. GST fusions of Siah1 and Siah2 were used to pull-down recombinant plectin exon 1 or exon 1c-GFP. Bound protein was detected by anti-GFP Western blotting. Lane 1, plectin exon1-GFP; lane 2, plectin exon1c-GFP; lane 3, GST plus exon1-GFP; lane 4, GST-Siah1a plus plectin exon1-GFP; lane 5, GST-Siah2 plus plectin exon1-GFP; lane 6, GST-Siah1a plus plectin exon1c-GFP; lane 7, GST-Siah plectin plus plectin exon1c-GFP. Loading controls are shown in Lower.

is a dimeric protein (22), and as such may be able to accommo- this did not increase binding in the ELISA-based binding assay date both SIP and pAPC binding to equivalent sites on each (data not shown). monomer. Clearly there are residues within PHYL108–130, other than the The Siah͞PHYL interaction that we observe is far stronger core PxAxVxP, that confer high-affinity binding. Although the than the interaction between Siah and the other reported alanine mutagenesis scan did not identify individual residues interacting proteins that we have tested, including DCC, Kid, outside the core PxAxVxP that make contributions greater than OBF-1, SIP, mGlutR1 and pAPC. DCC, OBF-1 and SIP each the VxP residues, mutation of several flanking residues, includ- have a perfect match to the alignment consensus PxAxVxP and ing Arg-115, Val-117, and Arg-125, did reduce binding substan- this region has been shown to be important for interaction with tially. The notion that the combined interaction of residues Siah (this paper and refs. 14 and 15). It is possible that the flanking the core consensus may contribute to a high-affinity binding of Siah to proteins such as SIP or OBF-1 may be interaction with Siah-SBD was supported by the very strong stabilized by factors or modifications not found in the bacterial interaction we identified with the plectin-195–117 peptide (78–123 expression system used here. One possible modification of the nM), which contains a perfect match with the RPVAxVxPxxR PHYL peptide is proline hydroxylation, recently shown to be consensus. required in hypoxia inducible factor-1␣ for recognition by the E3 SINA is part of a complex with EBI and PHYL targeting component, von Hippel-Lindau protein (32–34). We tested the TTK88, but not PHYL, for degradation. Similarly, Siah1 is part effect of hydroxylation of each proline residue in the SIP peptide of an E2͞E3 complex involving SIP, pAPC and an F-Box protein, (P60 and P66 in AELLDNEKP60AAVVAP66ITTGYTVKI), but Ebi, to target the degradation of ␤-catenin (19). Although PHYL

Table 1. Biosensor kinetic analysis of the interaction among MBP-SINA-SBD, MBP-Siah-SBD, and Siah-SBD with immobilized PHYL108–130 and plectin-195–117

Ϫ4 Ϫ1 Ϫ1 3 Ϫ1 Ϫ9 Soluble analyte Immobilized ligand Analysis ka, ϫ10 M ⅐s kd, ϫ10 s KD, ϫ10 M

MBP-SINA-SBD PHYL108–130 NLLS 4.43 2.16 48.8 plectin-195–117 NLLS 4.59 1.32 28.7 MBP-Siah-SBD PHYL108–130 NLLS 2.82 5.65 203 BIOCHEMISTRY Equilibrium 226 plectin-195–117 NLLS 4.43 4.09 92.3 Equilibrium 123 Siah-SBD PHYL108–130 NLLS 6.30 11.1 176 Equilibrium 183 plectin-195–117 NLLS 7.40 6.64 89.7 Equilibrium 78.2

NNLS, nonlinear least squares regression.

House et al. PNAS ͉ March 18, 2003 ͉ vol. 100 ͉ no. 6 ͉ 3105 Downloaded by guest on September 23, 2021 and SIP appear to bind to the same site on Siah, the affinities are Note Added in Proof. During the preparation of this manuscript, Li et al. very different. It may be that a strong interaction of Siah with its (38) reported a strong interaction between SINA and PHYL109–127, with substrate is only achieved in the mammalian setting through the mutagenesis results consistent with those reported in this manuscript. regulated formation of a multiprotein complex. The plectin-195–117 and PHYL108–130 peptides bind to Siah- SBD with apparent affinity constants in the low nanomolar We thank Ken Mitchelhill for HPLC and mass spectrometry, Julie Roth- range, several orders of magnitude lower than those observed acker (Ludwig Institute, Melbourne) for mass spectrometry and Biacore between SH3 domains and polyproline sequences (35). Given analysis, Drs. A. Germani and F. Calvo (Saint-Louis Hospital) for the gift that this interaction maps to a relatively short peptide and of the Kid expression construct, Drs. Steven Johnsen and Thomas Spelsberg involves a small number of key residues it should be possible to (Mayo Clinic and Foundation) for the TIEG-1 expression vector, and Ross generate small molecule inhibitors that block interaction with Dickins, Peter Janes, and Richard Pearson for critical comments on the Siah and a range of protein partners. Such inhibitors would be manuscript. D.D.L.B. and C.M.H. were supported by National Health and of considerable value for investigating the biology of this novel Medical Research Council (Australia). G.W. is supported by Austrian protein family and may have therapeutic uses. Science Research Fund Grant P14520.

1. Ciechanover, A. (1998) EMBO J. 17, 7151–7160. 21. Conaway, R. C., Brower, C. S. & Conaway, J. W. (2002) Science 296, 1254–1258. 2. Zheng, N., Wang, P., Jeffrey, P. D. & Pavletich, N. P. (2000) Cell 102, 533–539. 22. Polekhina, G., House, C. M., Traficante, N., Mackay, J. P., Relaix, F., Sassoon, 3. Zheng, N., Schulman, B. A., Song, L., Miller, J. J., Jeffrey, P. D., Wang, P., Chu, D. A., Parker, M. W. & Bowtell, D. D. (2002) Nat. Struct. Biol. 9, 68–75. C., Koepp, D. M., Elledge, S. J., Pagano, M., et al. (2002) Nature 416, 703–709. 23. Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994) Nucleic Acids Res. 22, 4. Lorick, K. L., Jensen, J. P., Fang, S., Ong, A. M., Hatakeyama, S. & Weissman, 4673–4680. A. M. (1999) Proc. Natl. Acad. Sci. USA 96, 11364–11369. 24. Catimel, B., Teh, T., Fontes, M. R., Jennings, I. G., Jans, D. A., Howlett, G. J., 5. Carthew, R. W. & Rubin, G. M. (1990) Cell 63, 561–577. Nice, E. C. & Kobe, B. (2001) J. Biol. Chem. 276, 34189–34198. 6. Tang, A. H., Neufeld, T. P., Kwan, E. & Rubin, G. M. (1997) Cell 90, 459–467. 25. Mathieu, M. N., Wade, J. D., Catimel, B., Bond, C. P., Nice, E. C., Summers, 7. Li, S., Li, Y., Carthew, R. W. & Lai, Z. C. (1997) Cell 90, 469–478. R. J., Otvos, L., Jr., & Tregear, G. W. (2001) J. Pept. Res. 57, 374–382. 8. Dong, X., Tsuda, L., Zavitz, K. H., Lin, M., Li, S., Carthew, R. W. & Zipursky, 26. Nice, E. C. & Catimel, B. (1999) BioEssays 21, 339–352. S. L. (1999) Dev. 13, 954–965. 27. Catimel, B., Nerrie, M., Lee, F. T., Scott, A. M., Ritter, G., Welt, S., Old, L. J., 9. Boulton, S. J., Brook, A., Staehling-Hampton, K., Heitzler, P. & Dyson, N. Burgess, A. W. & Nice, E. C. (1997) J. Chromatogr. A 776, 15–30. (2000) EMBO J. 19, 5376–5386. 28. Catimel, B., Scott, A. M., Lee, F. T., Hanai, N., Ritter, G., Welt, S., Old, L. J., 10. Hu, G., Zhang, S., Vidal, M., Baer, J. L., Xu, T. & Fearon, E. R. (1997) Genes Burgess, A. W. & Nice, E. C. (1998) Glycobiology 8, 927–938. Dev. 11, 2701–2714. 29. Kauffmann, R. C., Li, S., Gallagher, P. A., Zhang, J. & Carthew, R. W. (1996) 11. Hu, G. & Fearon, E. R. (1999) Mol. Cell. Biol. 19, 724–732. Genes Dev. 10, 2167–2178. 12. Zhang, J., Guenther, M. G., Carthew, R. W. & Lazar, M. A. (1998) Genes Dev. 30. Germani, A., Romero, F., Houlard, M., Camonis, J., Gisselbrecht, S., Fischer, 12, 1775–1780. S. & Varin-Blank, N. (1999) Mol. Cell. Biol. 19, 3798–3807. 13. Germani, A., Bruzzoni-Giovanelli, H., Fellous, A., Gisselbrecht, S., Varin- 31. Liu, C.-G., Maercker, C., Castanon, M. J., Hauptmann, R. & Wiche, G. (1996) Blank, N. & Calvo, F. (2000) Oncogene 19, 5997–6006. Proc. Natl. Acad. Sci. USA 93, 4278–4283. 14. Tiedt, R., Bartholdy, B. A., Matthias, G., Newell, J. W. & Matthias, P. (2001) EMBO J. 20, 4143–4152. 32. Fuchs, P., Zorer, M., Rezniczek, G. A., Spazierer, D., Oehler, S., Castanon, M. 15. Boehm, J., He, Y., Greiner, A., Staudt, L. & Wirth, T. (2001) EMBO J. 20, J., Hauptmann, R. & Wiche, G. (1999) Hum. Mol. Genet. 8, 2461–2472. 4153–4162. 33. Ishikawa, K., Nash, S. R., Nishimune, A., Neki, A., Kaneko, S. & Nakanishi, 16. Susini, L., Passer, B. J., Amzallag-Elbaz, N., Juven-Gershon, T., Prieur, S., S. (1999) Genes Cells 4, 381–390. Privat, N., Tuynder, M., Gendron, M.-C., Israel, A., Amson, R., et al. (2001) 34. Ivan, M., Kondo, K., Yang, H., Kim, W., Valiando, J., Ohh, M., Salic, A., Asara, Proc. Natl. Acad. Sci. USA 98, 15067–15072. J. M., Lane, W. S. & Kaelin, W. G., Jr. (2001) Science 292, 464–468. 17. Wheeler, T. C., Chin, L. S., Li, Y., Roudabush, F. L. & Li, L. (2002) J. Biol. 35. Jaakkola, P., Mole, D. R., Tian, Y. M., Wilson, M. I., Gielbert, J., Gaskell, S. J., Chem. 277, 10273–10282. Kriegsheim, A., Hebestreit, H. F., Mukherji, M., Schofield, C. J., et al. (2001) 18. Johnsen, J. A., Subramaniam, M., Monroe, D. G., Janknecht, R. & Spelsberg, Science 292, 468–472. T. C. (2002) J. Biol. Chem., 277, 30754–30759. 36. Yu, F., White, S. B., Zhao, Q. & Lee, F. S. (2001) Proc. Natl. Acad. Sci. USA 19. Matsuzawa, S. & Reed, J. C. (2001) Mol. Cell 7, 915–926. 98, 9630–9635. 20. Liu, J., Stevens, J., Rote, C. A., Yost, H. J., Hu, Y., Neufeld, K. L., White, R. L. 37. Lim, W. A. (1996) Structure (London) 4, 657–659. & Matsunami, N. (2001) Mol. Cell 7, 927–936. 38. Li, S., Xu, C. & Carthew, R. W. (2002) Mol. Cell. Biol. 22, 6854–6865.

3106 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0534783100 House et al. Downloaded by guest on September 23, 2021