Specificity and autoregulation of Notch binding by tandem WW domains in suppressor of Deltex

WW domains target proline-tyrosine (PY) motifs and frequently function as tandem pairs. When studied in isolation, single WW domains are notably promiscuous and regulatory mechanisms are undoubtedly required to ensure selective interactions. Here, we show that the fourth WW domain (WW4) of Suppressor of Deltex, a modular Nedd4-like protein that down-regulates the Notch receptor, is the primary mediator of a direct interaction with a Notch-PY motif. A natural Trp to Phe substitution in WW4 reduces its affinity for general PY sequences and enhances selective interaction with the Notch-PY motif via compensatory specificity-determining interactions with PY-flanking residues. When WW4 is paired with WW3, domain-domain association, impeding proper folding, competes with Notch-PY binding to WW4. This novel mode of autoinhibition is relieved by binding of another ligand to WW3. Such cooperativity may facilitate the transient regulatory interactions observed in vivo between Su(dx) and Notch in the endocytic pathway. The highly conserved tandem arrangement of WW domains in Nedd4 proteins, and similar arrangements in more diverse proteins, suggests domain-domain communication may be integral to regulation of their associated cellular activities.     

Modeling and analysis of molecularinteraction between Smurf1-WW2 domain and various isoforms of LIM mineralization protein

LIM Mineralization Protein-1 (LMP-1) has been cloned and shown to be osteoinductive. Our efforts to understand the mode of action of LMP-1 led to the determination that LMP-1 interacts with Smad Ubiquitin Regulatory Factor-1 (Smurf1). Smurf1 targets osteogenic Smads, Smad1/5, for ubiquitin-mediated proteasomal degradation. Smurf1 interaction with LMP-1 or Smads is based on the presence of unique WW-domain interacting motif in these target molecules. By performing site-directed mutagenesis and binding studies in vitro on purified recombinant proteins, we identified a specific motif within the osteogenic region of several LMP isoforms that is necessary for Smurf1 interaction. Similarly, we have identified that the WW2 domain of Smurf1 is necessary for target protein interaction. Here, we present a homology-based modeling of the Smurf1 WW2 domain and its interacting motif of LMP-1. We performed computational docking of the interacting domains in Smurf1 and LMPs to identify the key amino acid residues involved in their binding regions. In support of the computational predictions, we also present biochemical evidence supporting the hypothesis that the physical interaction of Smurf1 and osteoinductive forms of LMP may prevent Smurf1 from targeting osteogenic Smads by ubiquitin-mediated proteasomal degradation.     

Identification of the WW domain-interaction sites in the unstructured N-terminal domain of EBV LMP 2A

Epstein-Barr virus latency is maintained by the latent membrane protein (LMP) 2A, which mimics the B-cell receptor (BCR) and perturbs BCR signaling. The cytoplasmic N-terminal domain of LMP2A is composed of 119 amino acids. The N-terminal domain of LMP2A (LMP2A NTD) contains two PY motifs (PPPPY) that interact with the WW domains of Nedd4 family ubiquitin-protein ligases. Based on our analysis of NMR data, we found that the LMP2A NTD adopts an overall random-coil structure in its native state. However, the region between residues 60 and 90 was relatively ordered, and seemed to form the hydrophobic core of the LMP2A NTD. This region resides between two PY motifs and is important for WW domain binding. Mapping of the residues involved in the interaction between the LMP2A NTD and WW domains was achieved by chemical shift perturbation, by the addition of WW2 and WW3 peptides. Interestingly, the binding of the WW domains mainly occurred in the hydrophobic core of the LMP2A NTD. In addition, we detected a difference in the binding modes of the two PY motifs against the two WW peptides. The binding of the WW3 peptide caused the resonances of five residues (Tyr(60), Glu(61), Asp(62), Trp(65), and Gly(66)) just behind the N-terminal PY motif of the LMP2A NTD to disappear. A similar result was obtained with WW2 binding. However, near the C-terminal PY motif, the chemical shift perturbation caused by WW2 binding was different from that due to WW3 binding, indicating that the residues near the PY motifs are involved in selective binding of WW domains. The present work represents the first structural study of the LMP2A NTD and provides fundamental structural information about its interaction with ubiquitin-protein ligase.     

LIM mineralization protein-1 potentiates bone morphogenetic protein responsiveness via a novel interaction with Smurf1 resulting in decreased ubiquitination of Smads

Development and repair of the skeletal system and other organs is highly dependent on precise regulation of bone morphogenetic proteins (BMPs), their receptors, and their intracellular signaling proteins known as Smads. The use of BMPs clinically to induce bone formation has been limited in part by the requirement of much higher doses of recombinant proteins in primates than were needed in cell culture or rodents. Therefore, control of cellular responsiveness to BMPs is now a critical area that is poorly understood. We determined that LMP-1, a LIM domain protein capable of inducing de novo bone formation, interacts with Smurf1 (Smad ubiquitin regulatory factor 1) and prevents ubiquitination of Smads. In the region of LMP responsible for bone formation, there is a motif that directly interacts with the Smurf1 WW2 domain and can effectively compete with Smad1 and Smad5 for binding. We have shown that small peptides containing this motif can mimic the ability to block Smurf1 from binding Smads. This novel interaction of LMP-1 with the WW2 domain of Smurf1 to block Smad binding results in increased cellular responsiveness to exogenous BMP and demonstrates a novel regulatory mechanism for the BMP signaling pathway.     

Smad7 binds to Smurf2 to form an E3 ubiquitin ligase that targets the TGF beta receptor for degradation

Ubiquitin-mediated proteolysis regulates the activity of diverse receptor systems. Here, we identify Smurf2, a C2-WW-HECT domain ubiquitin ligase and show that Smurf2 associates constitutively with Smad7. Smurf2 is nuclear, but binding to Smad7 induces export and recruitment to the activated TGF beta receptor, where it causes degradation of receptors and Smad7 via proteasomal and lysosomal pathways. IFN gamma, which stimulates expression of Smad7, induces Smad7-Smurf2 complex formation and increases TGF beta receptor turnover, which is stabilized by blocking Smad7 or Smurf2 expression. Furthermore, Smad7 mutants that interfere with recruitment of Smurf2 to the receptors are compromised in their inhibitory activity. These studies thus define Smad7 as an adaptor in an E3 ubiquitin-ligase complex that targets the TGF beta receptor for degradation.     

Smurf1-mediated Axin Ubiquitination Requires Smurf1 C2 Domain and Is Cell Cycle-dependent

Previously, Smad ubiquitination regulatory factor 1 (Smurf1)-mediated Lys29 (K29)-linked poly-ubiquitination of Axin has been identified as a novel regulatory process in Wnt/β-catenin signaling. In this work, we discovered that the C2 domain of Smurf1 is critical for targeting Axin for ubiquitination. We found that the C2 domain-mediated plasma membrane localization of Smurf1 is required for Axin ubiquitination, and interfering with that disturbs the co-localization of Smurf1 and Axin around the plasma membrane. Moreover, the C2 domain of Smurf1, rather than its WW domains, is involved in Smurf1''s interaction with Axin; and the putative PPXY motifs (PY motif) of Axin are not essential for such an interaction, indicating that Smurf1 binds to Axin in a non-canonical way independent of WW-PY interaction. Further, we found that Smurf1-Axin interaction and Axin ubiquitination are attenuated in the G2/M phase of cell cycle, contributing to an increased cell response to Wnt stimulation at that stage. Collectively, we uncovered a dual role of Smurf1 C2 domain, recruiting Smurf1 to membrane for accessing Axin and mediating its interaction with Axin, and that Smurf1-mediated Axin ubiquitination is subjected to the regulation of cell cycle.     

Degradation of the tumor suppressor Smad4 by WW and HECT domain ubiquitin ligases

Smad4 mediates signaling by the transforming growth factor-beta (TGF-beta) superfamily of cytokines. Smad signaling is negatively regulated by inhibitory (I) Smads and ubiquitin-mediated processes. Known mechanisms of proteasomal degradation of Smads depend on the direct interaction of specific E3 ligases with Smads. Alternatively, I-Smads elicit degradation of the TGF-beta receptor by recruiting the WW and HECT domain E3 ligases, Smurfs, WWP1, or NEDD4-2. We describe an equivalent mechanism of degradation of Smad4 by the above E3 ligases, via formation of ternary complexes between Smad4 and Smurfs, mediated by R-Smads (Smad2) or I-Smads (Smad6/7), acting as adaptors. Smurfs, which otherwise cannot directly bind to Smad4, mediated poly-ubiquitination of Smad4 in the presence of Smad6 or Smad7. Smad4 co-localized with Smad7 and Smurf1 primarily in the cytoplasm and in peripheral cell protrusions. Smad2 or Smad7 mutants defective in Smad4 interaction failed to induce Smurf1-mediated down-regulation of Smad4. A Smad4 mutant defective in Smad2 or Smad7 interaction could not be effectively down-regulated by Smurf1. We propose that Smad4 is targeted for degradation by multiple ubiquitin ligases that can simultaneously act on R-Smads and signaling receptors. Such mechanisms of down-regulation of TGF-beta signaling may be critical for proper physiological response to this pathway.     

Recognition of p63 by the E3 ligase ITCH: Effect of an ectodermal dysplasia mutant

The E3 ubiquitin ligase Itch mediates the degradation of the p63 protein. Itch contains four WW domains which are pivotal for the substrate recognition process. Indeed, this domain is implicated in several signalling complexes crucially involved in human diseases including Muscular Dystrophy, Alzheimer's Disease, and Huntington Disease. WW domains are highly compact protein-protein binding modules that interact with short proline-rich sequences. The four WW domains present in Itch belong to the Group I type, which binds polypeptides with a PY motif characterized by a PPxY consensus sequence, where x can be any residue. Accordingly, the Itch-p63 interaction results from a direct binding of Itch-WW2 domain with the PY motif of p63. Here, we report a structural analysis of the Itch-p63 interaction by fluorescence, CD and NMR spectroscopy. Indeed, we studied the in vitro interaction between Itch-WW2 domain and p63(534-551), an 18-mer peptide encompassing a fragment of the p63 protein including the PY motif. In addition, we evaluated the conformation and the interaction with Itch-WW2 of a site specific mutant of p63, I549T, that has been reported in both Hay–Wells syndrome and Rapp–Hodgkin syndrome. Based on our results, we propose an extended PPxY motif for the Itch recognition motif (P-P-P-Y-x(4)-[ST]-[ILV]), which includes these C-terminal residues to the PPxY motif.     

Ubiquitin ligase Smurf1 targets TRAF family proteins for ubiquitination and degradation

The HECT-type E3 Smad ubiquitination regulation factor 1 (Smurf1) functions in regulation of cell polarity and bone homeostasis by targeting Smads, Runx2, RhoA and MEKK2 for ubiquitination and degradation. In a yeast two-hybrid screening, we identified TNF receptor-associated factor 4 (TRAF4) as a candidate substrate and was further validated. The PY motifs of TRAF4 mediated the interaction with the second WW domain of Smurf1. Overexpression of Smurf1 reduced the protein levels of TRAF4 dependent of its E3 activity and the proteasome. Further, we showed that all six members of TRAF family could be ubiquitinated by Smurf1. Consequently, Smurf1 interfered with the functions of TRAFs in NF-κB signaling under stimulation or not. These results suggested a new role of Smurf1 in inflammation and immunity through controlling the degradation of TRAFs.     

All three WW domains of murine Nedd4 are involved in the regulation of epithelial sodium channels by intracellular Na+

The amiloride-sensitive epithelial sodium channel (ENaC) plays a critical role in fluid and electrolyte homeostasis and consists of alpha, beta, and gamma subunits. The carboxyl terminus of each ENaC subunit contains a PPxY motif which is necessary for interaction with the WW domains of the ubiquitin-protein ligase, Nedd4. Disruption of this interaction, as in Liddle's syndrome where mutations delete or alter the PY motif of either the beta or gamma subunits, results in increased ENaC activity. We have recently shown using the whole-cell patch clamp technique that Nedd4 mediates the ubiquitin-dependent down-regulation of Na+ channel activity in response to increased intracellular Na+. In this paper, we demonstrate that WW domains 2 and 3 bind alpha-, beta-, and gamma-ENaC with varying degrees of affinity, whereas WW domain 1 does not bind to any of the subunits. We further show using whole-cell patch clamp techniques that Nedd4-mediated down-regulation of ENaC in mouse mandibular duct cells involves binding of the WW domains of Nedd4 to three distinct sites. We propose that Nedd4-mediated down-regulation of Na+ channels involves the binding of WW domains 2 and 3 to the Na+ channel and of WW domain 1 to an unknown associated protein.     

Structural determinants for high-affinity binding in a Nedd4 WW3* domain-Comm PY motif complex

Interactions between the WW domains of Drosophila Nedd4 (dNedd4) and Commissureless (Comm) PY motifs promote axon crossing at the CNS midline and muscle synaptogenesis. Here we report the solution structure of the dNedd4 WW3* domain complexed to the second PY motif (227'TGLPSYDEALH237') of Comm. Unexpectedly, there are interactions between WW3* and ligand residues both N- and C-terminal to the PY motif. Residues Y232'-L236' form a helical turn, following the PPII helical PY motif. Mutagenesis and binding studies confirm the importance of these extensive contacts, not simultaneously observed in other WW domain complexes, and identify a variable loop in WW3* responsible for its high-affinity interaction. These studies expand our general understanding of the molecular determinants involved in WW domain-ligand recognition. In addition, they provide insights into the specific regulation of dNedd4-mediated ubiquitination of Comm and subsequent internalization of Comm or the Comm/Roundabout complex, critical for CNS and muscle development.     

Affinity and specificity of interactions between Nedd4 isoforms and the epithelial Na+ channel

The epithelial Na+ channel (alphabetagammaENaC) regulates salt and fluid homeostasis and blood pressure. Each ENaC subunit contains a PY motif (PPXY) that binds to the WW domains of Nedd4, a Hect family ubiquitin ligase containing 3-4 WW domains and usually a C2 domain. It has been proposed that Nedd4-2, but not Nedd4-1, isoforms can bind to and suppress ENaC activity. Here we challenge this notion and show that, instead, the presence of a unique WW domain (WW3*) in either Nedd4-2 or Nedd4-1 determines high affinity interactions and the ability to suppress ENaC. WW3* from either Nedd4-2 or Nedd4-1 binds ENaC-PY motifs equally well (e.g. Kd approximately 10 microm for alpha- or betaENaC, 3-6-fold higher affinity than WW4), as determined by intrinsic tryptophan fluorescence. Moreover, dNedd4-1, which naturally contains a WW3* instead of WW2, is able to suppress ENaC function equally well as Nedd4-2. Homology models of the WW3*.betaENaC-PY complex revealed that a Pro and Ala conserved in all WW3*, but not other Nedd4-WW domains, help form the binding pocket for PY motif prolines. Extensive contacts are formed between the betaENaC-PY motif and the Pro in WW3*, and the small Ala creates a large pocket to accommodate the peptide. Indeed, mutating the conserved Pro and Ala in WW3* reduces binding affinity 2-3-fold. Additionally, we demonstrate that mutations in PY motif residues that form contacts with the WW domain based on our previously solved structure either abolish or severely reduce binding affinity to the WW domain and that the extent of binding correlates with the level of ENaC suppression. Independently, we show that a peptide encompassing the PY motif of sgk1, previously proposed to bind to Nedd4-2 and alter its ability to regulate ENaC, does not bind (or binds poorly) the WW domains of Nedd4-2. Collectively, these results suggest that high affinity of WW domain-PY-motif interactions rather than affiliation with Nedd4-1/Nedd-2 is critical for ENaC suppression by Nedd4 proteins.     

Characterization of the structure and function of W --> F WW domain variants: identification of a natively unfolded protein that folds upon ligand binding

The WW domain adopts a compact, three-stranded, antiparallel beta-sheet structure that mediates protein-protein interactions by binding to xPPxY-based protein ligands, such as the PY-ligand (EYPPYPPPPYPSG) derived from p53 binding protein-2. The conserved Trp residues, after which this domain was named, were replaced with Phe so their importance in structural integrity and for ligand binding could be evaluated. A biophysical approach was employed to compare the W17F, W39F, and W17F/W39F WW domains to the wild-type protein. The data demonstrate that replacement of Trp39 with Phe (W39F) does not disrupt the structure of the WW domain variant, but does abolish ligand binding. In contrast, the W17F WW domain variant is largely if not completely unfolded; however, this variant undergoes a PY-ligand induced disorder to order (folding) transition. The dissociation constant for the W17F WW domain-PY-ligand interaction is 15.1 +/- 1.2 microM, only slightly higher than that observed for the wild-type WW domain interaction (5.9 +/- 0.33 microM). The W17F WW domain is a natively unfolded protein which adopts a native conformation upon PY-ligand binding.     

Smad7 antagonizes transforming growth factor beta signaling in the nucleus by interfering with functional Smad-DNA complex formation

Smad7 plays an essential role in the negative-feedback regulation of transforming growth factor beta (TGF-beta) signaling by inhibiting TGF-beta signaling at the receptor level. It can interfere with binding to type I receptors and thus activation of receptor-regulated Smads or recruit the E3 ubiquitin ligase Smurf to receptors and thus target them for degradation. Here, we report that Smad7 is predominantly localized in the nucleus of Hep3B cells. The targeted expression of Smad7 in the nucleus conferred superior inhibitory activity on TGF-beta signaling, as determined by reporter assay in mammalian cells and by its effect on zebrafish embryogenesis. Furthermore, Smad7 repressed Smad3/4-, Smad2/4-, and Smad1/4-enhanced reporter gene expression, indicating that Smad7 can function independently of type I receptors. An oligonucleotide precipitation assay revealed that Smad7 can specifically bind to the Smad-responsive element via its MH2 domain, and DNA-binding activity was further confirmed in vivo with the promoter of PAI-1, a TGF-beta target gene, by chromatin immunoprecipitation. Finally, we provide evidence that Smad7 disrupts the formation of the TGF-beta-induced functional Smad-DNA complex. Our findings suggest that Smad7 inhibits TGF-beta signaling in the nucleus by a novel mechanism.     

Smurf1 regulates the inhibitory activity of Smad7 by targeting Smad7 to the plasma membrane

Smad ubiquitin regulatory factor 1 (Smurf1), a HECT-type E3 ubiquitin ligase, interacts with inhibitory Smad7 and induces cytoplasmic localization of Smad7. Smurf1 then associates with transforming growth factor-beta type I receptor (TbetaR-I) and enhances the turnover of this receptor. However, the mechanisms of the nuclear export and plasma membrane localization of the Smurf1.Smad7 complex have not been elucidated. We show here that Smurf1 targets Smad7 to the plasma membrane through its N-terminal conserved 2 (C2) domain. Both wild-type Smurf1 (Smurf1(WT)) and Smurf1 lacking the C2 domain (Smurf1(deltaC2)) bound to Smad7 and translocated nuclear Smad7 to the cytoplasm. However, unlike Smurf1(WT), Smurf1(deltaC2) did not move to the plasma membrane and failed to recruit Smad7 to the cell surface TbetaR-II.TbetaR-I complex. Moreover, although Smurf1(deltaC2) induced ubiquitination of Smad7, it failed to induce the ubiquitination and degradation of TbetaR-I and did not enhance the inhibitory activity of Smad7. Thus, these results suggest that the plasma membrane localization of Smad7 by Smurf1 requires the C2 domain of Smurf1 and is essential for the inhibitory effect of Smad7 in the transforming growth factor-beta signaling pathway.     

Regulation of the cardiac voltage-gated Na+ channel (H1) by the ubiquitin-protein ligase Nedd4

The cardiac voltage-gated Na+ channel H1, involved in the generation of cardiac action potential, contains a C-terminal PY motif (xPPxY). Since PY motifs are known ligands to WW domains, we investigated their role for H1 regulation and the possible involvement of the WW domain containing ubiquitin-protein ligase Nedd4, taking advantage of the Xenopus oocyte system. Mutation of the PY motif leads to higher peak currents when compared to wild-type channel. Moreover, co-expression of Nedd4 reduced the peak currents, whereas an enzymatically inactive Nedd4 mutant increased them, likely by competing with endogenous Nedd4. The effect of Nedd4 was not observed in the PY motif mutated channel or in the skeletal muscle voltage-gated Na+ channel, which lacks a PY motif. We conclude that H1 may be regulated by Nedd4 depending on WW-PY interaction, and on an active ubiquitination site.