Structural analysis of Siah1-Siah-interacting protein interactions and insights into the assembly of an E3 ligase multiprotein complex

Siah1 is the central component of a multiprotein E3 ubiquitin ligase complex that targets beta-catenin for destruction in response to p53 activation. The E3 complex comprises, in addition to Siah1, Siah-interacting protein (SIP), the adaptor protein Skp1, and the F-box protein Ebi. Here we show that SIP engages Siah1 by means of two elements, both of which are required for mediating beta-catenin destruction in cells. An N-terminal dimerization domain of SIP sits across the saddle-shaped upper surface of Siah1, with two extended legs packing against the sides of Siah1 by means of a consensus PXAXVXP motif that is common to a family of Siah-binding proteins. The C-terminal domain of SIP, which binds to Skp1, protrudes from the lower surface of Siah1, and we propose that this surface provides the scaffold for bringing substrate and the E2 enzyme into apposition in the functional complex.     

Structure of a beta-TrCP1-Skp1-beta-catenin complex: destruction motif binding and lysine specificity of the SCF(beta-TrCP1) ubiquitin ligase

The SCF ubiquitin ligases catalyze protein ubiquitination in diverse cellular processes. SCFs bind substrates through the interchangeable F box protein subunit, with the >70 human F box proteins allowing the recognition of a wide range of substrates. The F box protein beta-TrCP1 recognizes the doubly phosphorylated DpSGphiXpS destruction motif, present in beta-catenin and IkappaB, and directs the SCF(beta-TrCP1) to ubiquitinate these proteins at specific lysines. The 3.0 A structure of a beta-TrCP1-Skp1-beta-catenin complex reveals the basis of substrate recognition by the beta-TrCP1 WD40 domain. The structure, together with the previous SCF(Skp2) structure, leads to the model of SCF catalyzing ubiquitination by increasing the effective concentration of the substrate lysine at the E2 active site. The model's prediction that the lysine-destruction motif spacing is a determinant of ubiquitination efficiency is confirmed by measuring ubiquitination rates of mutant beta-catenin peptides, solidifying the model and also providing a mechanistic basis for lysine selection.     

STD and TRNOESY NMR studies on the conformation of the oncogenic protein beta-catenin containing the phosphorylated motif DpSGXXpS bound to the beta-TrCP protein

beta-TrCP is the F-box protein component of an Skp1/Cul1/F-box (SCF)-type ubiquitin ligase complex. Biochemical studies have suggested that beta-TrCP targets the oncogenic protein beta-catenin for ubiquitination and followed by proteasome degradation. To further elucidate the basis of this interaction, a complex between a 32-residue peptide from beta-catenin containing the phosphorylated motif DpSGXXpS (P-beta-Cat17-48) and beta-TrCP was studied using Saturation Transfer Difference (STD) Nuclear Magnetic Resonance (NMR) experiments. These experiments make it possible to identify the binding epitope of a ligand at atomic resolution. An analysis of STD spectra provided clear evidence that only a few of the 32 residues receive the largest saturation transfer. In particular, the amide protons of the residues in the phosphorylated motif appear to be in close contact to the amino acids of the beta-TrCP binding pocket. The amide and aromatic protons of the His24 and Trp25 residues also receive a significant saturation transfer. These findings are in keeping with a recently published x-ray structure of a shorter beta-catenin fragment with the beta-TrCP1-Skp1 complex and with the earlier findings from mutagenesis and activity assays. To better characterize the ligand-protein interaction, the bound conformation of the phosphorylated beta-catenin peptide was obtained using TRansfer Nuclear Overhauser Effect SpectroscopY (TRNOESY) experiments. Finally, we obtained the bound structure of the phosphorylated peptide showing the protons identified by STD NMR as exposed in close proximity to the molecule surface.     

Siah-1 mediates a novel beta-catenin degradation pathway linking p53 to the adenomatous polyposis coli protein

The adenomatous polyposis coli (APC) tumor-suppressor protein, together with Axin and GSK3beta, forms a Wnt-regulated signaling complex that mediates phosphorylation-dependent degradation of beta-catenin by the proteasome. Siah-1, the human homolog of Drosophila seven in absentia, is a p53-inducible mediator of cell cycle arrest, tumor suppression, and apoptosis. We have now found that Siah-1 interacts with the carboxyl terminus of APC and promotes degradation of beta-catenin in mammalian cells. The ability of Siah-1 to downregulate beta-catenin signaling was also demonstrated by hypodorsalization of Xenopus embryos. Unexpectedly, degradation of beta-catenin by Siah-1 was independent of GSK3beta-mediated phosphorylation and did not require the F box protein beta-TrCP. These results indicate that APC and Siah-1 mediate a novel beta-catenin degradation pathway linking p53 activation to cell cycle control.     

Structural analysis of Siah1 and its interactions with Siah-interacting protein (SIP)

Seven in absentia 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 beta-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 SIP in 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.     

Molecular dynamics simulations elucidate the mode of protein recognition by Skp1 and the F‐box domain in the SCF complex

Polyubiquitination of the target protein by a ubiquitin transferring machinery is key to various cellular processes. E3 ligase Skp1‐Cul1‐F‐box (SCF) is one such complex which plays crucial role in substrate recognition and transfer of the ubiquitin molecule. Previous computational studies have focused on S‐phase kinase‐associated protein 2 (Skp2), cullin, and RING‐finger proteins of this complex, but the roles of the adapter protein Skp1 and F‐box domain of Skp2 have not been determined. Using sub‐microsecond molecular dynamics simulations of full‐length Skp1, unbound Skp2, Skp2‐Cks1 (Cks1: Cyclin‐dependent kinases regulatory subunit 1), Skp1‐Skp2, and Skp1‐Skp2‐Cks1 complexes, we have elucidated the function of Skp1 and the F‐box domain of Skp2. We found that the L₁₆ loop of Skp1_, which was deleted in previous X‐ray crystallography studies, can offer additional stability to the ternary complex via its interactions with the C‐terminal tail of Skp2. Moreover, Skp1 helices H6, H7, and H8 display vivid conformational flexibility when not bound to Skp2, suggesting that these helices can recognize and lock the F‐box proteins. Furthermore, we observed that the F‐box domain could rotate (5°–129°), and that the binding partner determined the degree of conformational flexibility. Finally, Skp1 and Skp2 were found to execute a domain motion in Skp1‐Skp2 and Skp1‐Skp2‐Cks1 complexes that could decrease the distance between ubiquitination site of the substrate and the ubiquitin molecule by 3 nm. Thus, we propose that both the F‐box domain of Skp2 and Skp1‐Skp2 domain motions displaying preferential conformational control can together facilitate polyubiquitination of a wide variety of substrates. Proteins 2016; 84:159–171. © 2015 Wiley Periodicals, Inc.     

Wnt-5a inhibits the canonical Wnt pathway by promoting GSK-3-independent beta-catenin degradation

Wnts are secreted signaling molecules that can transduce their signals through several different pathways. Wnt-5a is considered a noncanonical Wnt as it does not signal by stabilizing beta-catenin in many biological systems. We have uncovered a new noncanonical pathway through which Wnt-5a antagonizes the canonical Wnt pathway by promoting the degradation of beta-catenin. This pathway is Siah2 and APC dependent, but GSK-3 and beta-TrCP independent. Furthermore, we provide evidence that Wnt-5a also acts in vivo to promote beta-catenin degradation in regulating mammalian limb development and possibly in suppressing tumor formation.     

Multiple isoforms of beta-TrCP display differential activities in the regulation of Wnt signaling

The F-box proteins beta-TrCP1 and 2 (beta-transducin repeat containing protein) have 2 and 3 isoforms, respectively, due to alternative splicing of exons encoding the N-terminal region. We identified an extra exon in between the previously known exons 1 and 2 of beta-TrCP1 and beta-TrCP2. Interestingly, sequence analysis suggested that many more isoforms are produced than previously identified, via the alternative splicing of all possible combination of exons II to V of beta-TrCP1 and exons II to IV of beta-TrCP2. Different mouse tissues show specific expression patterns of the isoforms, and the level of expression of the isoform that has been used in most published papers was very low. Yeast two-hybrid assays show that beta-TrCP1 isoforms containing exon III, which are the most highly expressed isoforms in most tissues, do not interact with Skp1. Indirect immunofluorescence analysis of transiently expressed beta-TrCP1 isoforms suggests that the presence of exon III causes beta-TrCP1 to localize in nuclei. Consistent with the above findings, isoforms including exon III showed a reduced ability to block ectopic embryonic axes induced via injection of Wnt8 or beta-catenin in Xenopus embryos. Overall, our data suggest that isoforms of beta-TrCPs generated by alternative splicing may have different biological roles.     

Transfer-NMR and docking studies identify the binding of the peptide derived from activating transcription factor 4 to protein ubiquitin ligase beta-TrCP. Competition STD-NMR with beta-catenin

ATF4 plays a crucial role in the cellular response to stress. The E3 ubiquitin ligase, SCF beta-TrCP protein responsible for ATF4 degradation by the proteasome, binds to ATF4 through a DpSGXXXpS phosphorylation motif, which is similar but not identical to the DpSGXXpS motif found in most other substrates of beta-TrCP. NMR studies were performed on the free and bound forms of a peptide derived from this ATF4 motif that enabled the elucidation of the conformation of the ligand complexed to the beta-TrCP protein and its binding mode. Saturation transfer difference (STD) NMR allowed the study of competition for binding to beta-TrCP, between the phosphorylation motifs of ATF4 and beta-catenin, to characterize the ATF4 binding epitope. Docking protocols were performed using the crystal structure of the beta-catenin-beta-TrCP complex as a template and NMR results of the ATF4-beta-TrCP complex. In agreement with the STD results, in order to bind to beta-TrCP, the ATF4 DpSGIXXpSXE motif required the association of two negatively charged areas, in addition to the hydrophobic interaction in the beta-TrCP central channel. Docking studies showed that the ATF4 DpSGIXXpSXE motif fits the binding pocket of beta-TrCP through an S-turning conformation. The distance between the two phosphate groups is 17.8 A, which matched the corresponding distance 17.1 A for the other extended DpSGXXpS motif in the beta-TrCP receptor model. This study identifies the residues of the beta-TrCP receptor involved in ligand recognition. Using a new concept of STD competition experiment, we show that ATF4 competes and inhibits binding of beta-catenin to beta-TrCP.     

Molecular dissection of the interactions among IkappaBalpha, FWD1, and Skp1 required for ubiquitin-mediated proteolysis of IkappaBalpha.

The SCF complex containing Skp1, Cul1, and the F-box protein FWD1 (the mouse homologue of Drosophila Slimb and Xenopus beta-TrCP) functions as the ubiquitin ligase for IkappaBalpha. FWD1 associates with Skp1 through the F-box domain and also recognizes the conserved DSGXXS motif of IkappaBalpha. The structural requirements for the interactions of FWD1 with IkappaBalpha and with Skp1 have now been investigated further. The D31A mutation (but not the G33A mutation) in the DSGXXS motif of IkappaBalpha abolished the binding of IkappaBalpha to FWD1 and its subsequent ubiquitination without affecting the phosphorylation of IkappaBalpha. The IkappaBalpha mutant D31E still exhibited binding to FWD1 and underwent ubiquitination. These results suggest that, in addition to site-specific phosphorylation at Ser(32) and Ser(36), an acidic amino acid at position 31 is required for FWD1-mediated ubiquitination of IkappaBalpha. Deletion analysis of Skp1 revealed that residues 61-143 of this protein are required for binding to FWD1. On the other hand, the highly conserved residues Pro(149), Ile(160), and Leu(164) in the F-box domain of FWD1 were dispensable for binding to Skp1. Together, these data delineate the structural requirements for the interactions among IkappaBalpha, FWD1, and Skp1 that underlie substrate recognition by the SCF ubiquitin ligase complex.     

TIP120A associates with unneddylated cullin 1 and regulates its neddylation

The cullin-containing E3 ubiquitin ligases play an important role in regulating the abundance of key proteins involved in cellular processes such as cell cycle and cytokine signaling. We recently identified TIP120A as a cullin-interacting protein and found that TIP120A functions as a negative regulator of a ubiquitin ligase by interfering with the binding of Skp1 and an F box protein to CUL1. Here we show that TIP120A binds to the unneddylated CUL1 but not the neddylated one. The association of TIP120A with CUL1 requires both the N-terminal stalk and the C-terminal globular domain of CUL1. TIP120A efficiently inhibits neddylation of CUL1 but does not affect substrate-independent ubiquitination by CUL1/Rbx1, implying that it blocks the access of Nedd8 to the conjugation site but does not interfere with the interaction of the ubiquitin-conjugating enzyme with Rbx1. Our data suggest that the association/dissociation of TIP120A coupled to neddylation/deneddylation of CUL1 may play an important role in assembly and disassembly of Skp1-Cdc53/cullin-F box ubiquitin ligases.     

Calcium-regulated interaction of Sgt1 with S100A6 (calcyclin) and other S100 proteins

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.     

An SCF complex containing Fbxl12 mediates DNA damage-induced Ku80 ubiquitylation

The Ku heterodimer, composed of Ku70 and Ku80, is the initiating factor of the nonhomologous end joining (NHEJ) double-strand break (DSB) repair pathway. Ku is also thought to impede the homologous recombination (HR) repair pathway via inhibition of DNA end resection. Using the cell-free Xenopus laevis egg extract system, we had previously discovered that Ku80 becomes polyubiquitylated upon binding to DSBs, leading to its removal from DNA and subsequent proteasomal degradation. Here we show that the Skp1-Cul1-F box (SCF) E3 ubiquitin ligase complex is required for Ku80 ubiquitylation and removal from DNA. A screen for DSB-binding F box proteins revealed that the F box protein Fbxl12 was recruited to DNA in a DSB- and Ku-sensitive manner. Immunodepletion of Fbxl12 prevented Cul1 and Skp1 binding to DSBs and Ku80 ubiquitylation, indicating that Fbxl12 is the F box protein responsible for Ku80 substrate recognition. Unlike typical F box proteins, the F box of Fbxl12 was essential for binding to both Skp1 and its substrate Ku80. Besides Fbxl12, six other chromatin-binding F box proteins were identified in our screen of a subset of Xenopus F box proteins: β-TrCP, Fbh1, Fbxl19, Fbxo24, Fbxo28 and Kdm2b. Our study unveils a novel function for the SCF ubiquitin ligase in regulating the dynamic interaction between DNA repair machineries and DSBs.     

Suprafacial orientation of the SCFCdc4 dimer accommodates multiple geometries for substrate ubiquitination

SCF ubiquitin ligases recruit substrates for degradation via F box protein adaptor subunits. WD40 repeat F box proteins, such as Cdc4 and beta-TrCP, contain a conserved dimerization motif called the D domain. Here, we report that the D domain protomers of yeast Cdc4 and human beta-TrCP form a superhelical homotypic dimer. Disruption of the D domain compromises the activity of yeast SCF(Cdc4) toward the CDK inhibitor Sic1 and other substrates. SCF(Cdc4) dimerization has little effect on the affinity for Sic1 but markedly stimulates ubiquitin conjugation. A model of the dimeric holo-SCF(Cdc4) complex based on small-angle X-ray scatter measurements reveals a suprafacial configuration, in which substrate-binding sites and E2 catalytic sites lie in the same plane with a separation of 64 A within and 102 A between each SCF monomer. This spatial variability may accommodate diverse acceptor lysine geometries in both substrates and the elongating ubiquitin chain and thereby increase catalytic efficiency.     

Skp1p and the F-box protein Rcy1p form a non-SCF complex involved in recycling of the SNARE Snc1p in yeast

Skp1p-cullin-F-box protein (SCF) complexes are ubiquitin-ligases composed of a core complex including Skp1p, Cdc53p, Hrt1p, the E2 enzyme Cdc34p, and one of multiple F-box proteins which are thought to provide substrate specificity to the complex. Here we show that the F-box protein Rcy1p is required for recycling of the v-SNARE Snc1p in Saccharomyces cerevisiae. Rcy1p localized to areas of polarized growth, and this polarized localization required its CAAX box and an intact actin cytoskeleton. Rcy1p interacted with Skp1p in vivo in an F-box-dependent manner, and both deletion of its F box and loss of Skp1p function impaired recycling. In contrast, cells deficient in Cdc53p, Hrt1p, or Cdc34p did not exhibit recycling defects. Unlike the case for F-box proteins that are known to participate in SCF complexes, degradation of Rcy1p required neither its F box nor functional 26S proteasomes or other SCF core subunits. Importantly, Skp1p was the only major partner that copurified with Rcy1p. Our results thus suggest that a complex composed of Rcy1p and Skp1p but not other SCF components may play a direct role in recycling of internalized proteins.     

Targeted degradation of beta-catenin by chimeric F-box fusion proteins

Adenomatous polyposis coli (APC) tumor suppressor protein, together with Axin and glycogen synthase kinase 3beta (GSK-3beta), forms a Wnt-regulated signaling complex that mediates phosphorylation-dependent degradation of cytoplasmic beta-catenin by ubiquitin-dependent proteolysis. Degradation of phosphorylated beta-catenin is initiated by interaction through the WD40-repeat of a F-box protein beta-TrCP, a component of SCF ubiquitin ligase complex. Mutations in APC, Axin, and beta-catenin that prevent down-regulation of cytoplasmic beta-catenin are found in various types of cancers. In the search for efficient treatment and prevention of malignancies associated with increased levels of cytoplasmic beta-catenin, we created chimeric F-box fusion proteins by replacing the WD40-repeat of beta-TrCP with the beta-catenin-binding domains of Tcf4 and E-cadherin. Expression of chimeric F-box fusion proteins successfully promotes degradation of beta-catenin independently of GSK-3beta-mediated phosphorylation. More importantly, this degradation does not require intact APC protein (pAPC).