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.     

Autoinhibition of the HECT-type ubiquitin ligase Smurf2 through its C2 domain

Ubiquitination of proteins is an abundant modification that controls numerous cellular processes. Many Ubiquitin (Ub) protein ligases (E3s) target both their substrates and themselves for degradation. However, the mechanisms regulating their catalytic activity are largely unknown. The C2-WW-HECT-domain E3 Smurf2 downregulates transforming growth factor-beta (TGF-beta) signaling by targeting itself, the adaptor protein Smad7, and TGF-beta receptor kinases for degradation. Here, we demonstrate that an intramolecular interaction between the C2 and HECT domains inhibits Smurf2 activity, stabilizes Smurf2 levels in cells, and similarly inhibits certain other C2-WW-HECT-domain E3s. Using NMR analysis the C2 domain was shown to bind in the vicinity of the catalytic cysteine, where it interferes with Ub thioester formation. The HECT-binding domain of Smad7, which activates Smurf2, antagonizes this inhibitory interaction. Thus, interactions between C2 and HECT domains autoinhibit a subset of HECT-type E3s to protect them and their substrates from futile degradation in cells.     

Smurf1 directly targets hPEM-2, a GEF for Cdc42, via a novel combination of protein interaction modules in the ubiquitin-proteasome pathway

Smurf1, a member of HECT-type E3 ubiquitin ligases, regulates cell polarity and protrusive activity by inducing ubiquitination and subsequent proteasomal degradation of the small GTPase RhoA. We report here that hPEM-2, a guanine nucleotide exchange factor for the small GTPase Cdc42, is a novel target of Smurf1. Pulse-chase labeling and a ubiquitination experiment using MG132, a proteasomal inhibitor, indicate that Smurf1 induces proteasomal degradation of hPEM-2 in cells. GST pull-down assays with heterologously expressed firefly luciferase-fusion proteins that include partial sequences of hPEM-2 reveal that part of the PH domain (residues 318-343) of hPEM-2 is sufficient for binding to Smurf1. In contrast, the hPEM-2 binding domain in Smurf1 was mapped to the C2 domain. Although it has been reported that the binding activities of some C2 domains to target proteins are regulated by Ca2+, Smurf1 interacts with hPEM-2 in a Ca2+-independent manner. Our discovery that hPEM-2 is, in addition to RhoA, a target protein of Smurf1 suggests that Smurf1 plays a crucial role in the spatiotemporal regulation of Rho GTPase family members.     

Smad Ubiquitylation Regulatory Factor 1/2 (Smurf1/2) Promotes p53 Degradation by Stabilizing the E3 Ligase MDM2

The tumor suppressor p53 protein is tightly regulated by a ubiquitin-proteasomal degradation mechanism. Several E3 ubiquitin ligases, including MDM2 (mouse double minute 2), have been reported to play an essential role in the regulation of p53 stability. However, it remains unclear how the activity of these E3 ligases is regulated. Here, we show that the HECT-type E3 ligase Smurf1/2 (Smad ubiquitylation regulatory factor 1/2) promotes p53 degradation by enhancing the activity of the E3 ligase MDM2. We provide evidence that the role of Smurf1/2 on the p53 stability is not dependent on the E3 activity of Smurf1/2 but rather is dependent on the activity of MDM2. We find that Smurf1/2 stabilizes MDM2 by enhancing the heterodimerization of MDM2 with MDMX, during which Smurf1/2 interacts with MDM2 and MDMX. We finally provide evidence that Smurf1/2 regulates apoptosis through p53. To our knowledge, this is the first report to demonstrate that Smurf1/2 functions as a factor to stabilize MDM2 protein rather than as a direct E3 ligase in regulation of p53 degradation.     

IRAK2 counterbalances oncogenic Smurf1 in colon cancer cells by dictating ER stress

The endoplasmic reticulum (ER) is a cellular organelle with central roles in maintaining proteostasis. The accumulation of misfolded proteins in the ER lumen causes ER stress. Cells evoke an evolutionarily conserved adaptive signaling network “unfolded protein response” to restore ER homeostasis, however, how the signaling network is delicately orchestrated remains largely unrevealed. Meanwhile, the HECT type E3 ligase Smad ubiquitylation regulatory factor 1 (Smurf1) has been reported to play critical roles in several important biological pathways by targeting distinct substrates for ubiquitylation, including WFS1, a critical mediator of ER stress, whereas the regulation of Smurf1 activity and abundance upon ER stress are poorly understood. Here, we identified Interleukin-1 Receptor Associated Kinase 2 (IRAK2) as a novel modulator of Smurf1 in response to ER stress induced cell death. Mechanistically, IRAK2 phosphorylates Smurf1 at threonine residues to promote its self-degradation by ubiquitylation, resulting in altered cascade of ER effectors to induce apoptosis. Reduced IRAK2 expression correlates with increased Smurf1 abundance in human colorectal cancer cells. Taken together, these findings demonstrate a novel mechanism of interplays for the different branches of ER stress signaling network and highlight IRAK2 as a potential tumor suppressor to counterbalance oncogenic Smurf1.     

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.     

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.     

The WW1 Domain Enhances Autoinhibition in Smurf Ubiquitin Ligases

Downregulation of ubiquitin (Ub) ligase activity prevents premature ubiquitination and is critical for cellular homeostasis. Nedd4 Ub ligases share a common domain architecture and yet are regulated in distinct ways through interactions of the catalytic HECT domain with the N-terminal C2 domain or the central WW domain region. Smurf1 and Smurf2 are two highly related Nedd4 ligases with ~70% overall sequence identity. Here, we show that the Smurf1 C2 domain interacts with the HECT domain and inhibits ligase activity in trans. However, in contrast to Smurf2, we find that full-length Smurf1 is a highly active Ub ligase, and we can attribute this striking difference in regulation to the lack of one WW domain (WW1) in Smurf1. Using NMR spectroscopy and biochemical assays, we identified the WW1 region as an additional inhibitory element in Smurf2 that cooperates with the C2 domain to enhance HECT domain binding and Smurf2 inhibition. Our work provides important insights into Smurf regulation and highlights that the activities of highly related proteins can be controlled in distinct ways.     

Smurf2 induces ubiquitin-dependent degradation of Smurf1 to prevent migration of breast cancer cells

Ubiquitin-dependent protein degradation is involved in various biological processes, and accumulating evidence suggests that E3 ubiquitin ligases play important roles in cancer development. Smad ubiquitin regulatory factor 1 (Smurf1) and Smurf2 are E3 ubiquitin ligases, which suppress transforming growth factor-beta (TGF-beta) family signaling through degradation of Smads and receptors for TGF-beta and bone morphogenetic proteins. In addition, Smurf1 has been reported to promote RhoA ubiquitination and degradation and regulate cell motility, suggesting the involvement of Smurf1 in cancer progression. However, the regulation and biological function of Smurf1 and Smurf2 in cancer development remain to be elucidated. In the present study, we show the post-translational regulation of Smurf1 by Smurf2 and the functional differences between Smurf1 and Smurf2 in the progression of breast cancer cells. Smurf2 interacted with Smurf1 and induced its ubiquitination and degradation, whereas Smurf1 failed to induce degradation of Smurf2. Knockdown of Smurf2 in human breast cancer MDA-MB-231 cells resulted in increases in the levels of Smurf1 protein, and enhancement of cell migration in vitro and bone metastasis in vivo. Of note, knockdown of Smurf1, but not of Smurf2, enhanced TGF-beta signaling in MDA-MB-231 cells, suggesting that increased an protein level of Smurf1 offsets the effect of Smurf2 knockdown on TGF-beta signaling. These results indicate that two related E3 ubiquitin ligases, Smurf1 and Smurf2, act in the same direction in TGF-beta family signaling but play opposite roles in cell migration.     

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.     

SCFFBXL¹⁵ regulates BMP signalling by directing the degradation of HECT-type ubiquitin ligase Smurf1

Smad ubiquitination regulatory factor 1 (Smurf1), an homologous to E6AP C-terminus (HECT)-type E3 ubiquitin ligase, performs a crucial role in the regulation of the bone morphogenetic protein (BMP) signalling pathway in both embryonic development and bone remodelling. How the stability and activity of Smurf1 are negatively regulated remains largely unclear. Here, we report that F-box and LRR domain-containing protein 15 (FBXL15), an F-box protein of the FBXL family, forms an Skp1-Cullin1-F-box protein-Roc1 (SCF)(FBXL15) ubiquitin ligase complex and targets Smurf1 for ubiquitination and proteasomal degradation. FBXL15, through its leucine-rich repeat domain, specifically recognizes the large subdomain within the N-lobe of the Smurf1 HECT domain and promotes the ubiquitination of Smurf1 on K355 and K357 within the WW-HECT linker region. In this way, FBXL15 positively regulates BMP signalling in mammalian cells. Knockdown of fbxl15 expression in zebrafish embryos by specific antisense morpholinos causes embryonic dorsalization phenocoping BMP-deficient mutants. Injection of FBXL15 siRNAs into rat bone tissues leads to a significant loss of bone mass and decrease in bone mineral density. Collectively, our results demonstrate that Smurf1 stability is suppressed by SCF(FBXL15)-mediated ubiquitination and that FBXL15 is a key regulator of BMP signalling during embryonic development and adult bone formation.     

Pivotal role of the C2 domain of the Smurf1 ubiquitin ligase in substrate selection

The C2-WW-HECT-type ubiquitin ligases Smurf1 and Smurf2 play a critical role in embryogenesis and adult bone homeostasis via regulation of bone morphogenetic protein, Wnt, and RhoA signaling pathways. The intramolecular interaction between C2 and HECT domains autoinhibits the ligase activity of Smurf2. However, the role of the Smurf1 C2 domain remains elusive. Here, we show that the C2-HECT autoinhibition mechanism is not observed in Smurf1, and instead its C2 domain functions in substrate selection. The Smurf1 C2 domain exerts a key role in localization to the plasma membrane and endows Smurf1 with differential activity toward RhoA versus Smad5 and Runx2. Crystal structure analysis reveals that the Smurf1 C2 domain possesses a typical anti-parallel β-sandwich fold. Examination of the sulfate-binding site analysis reveals two key lysine residues, Lys-28 and Lys-85, within the C2 domain that are important for Smurf1 localization at the plasma membrane, regulation on cell migration, and robust ligase activity toward RhoA, which further supports a Ca(2+)-independent localization mechanism for Smurf1. These findings demonstrate a previously unidentified role of the Smurf1 C2 domain in substrate selection and cellular localization.     

CNF1-induced ubiquitylation and proteasome destruction of activated RhoA is impaired in Smurf1-/- cells

Ubiquitylation of RhoA has emerged as an important aspect of both the virulence of Escherichia coli producing cytotoxic necrotizing factor (CNF) 1 toxin and the establishment of the polarity of eukaryotic cells. Owing to the molecular activity of CNF1, we have investigated the relationship between permanent activation of RhoA catalyzed by CNF1 and subsequent ubiquitylation of RhoA by Smurf1. Using Smurf1-deficient cells and by RNA interference (RNAi)-mediated Smurf1 knockdown, we demonstrate that Smurf1 is a rate-limiting and specific factor of the ubiquitin-mediated proteasomal degradation of activated RhoA. We further show that the cancer cell lines HEp-2, human embryonic kidney 293 and Vero are specifically deficient in ubiquitylation of either activated Rac, Cdc42, or Rho, respectively. In contrast, CNF1 produced the cellular depletion of all three isoforms of Rho proteins in the primary human cell types we have tested. We demonstrate that ectopic expression of Smurf1 in Vero cells, deficient for RhoA ubiquitylation, restores ubiquitylation of the activated forms of RhoA. We conclude here that Smurf1 ubiquitylates activated RhoA and that, in contrast to human primary cell types, some cancer cell lines have a lower ubiquitylation capacity of specific Rho proteins. Thus, both CNF1 and transforming growth factor-beta trigger activated RhoA ubiquitylation through Smurf1 ubiquitin-ligase.     

A synthetic compound that potentiates bone morphogenetic protein-2-induced transdifferentiation of myoblasts into the osteoblastic phenotype

There is an urgent need to develop methods that lower costs of using recombinant human bone morphogenetic proteins (BMPs) to promote bone induction. In this study, we demonstrate the osteogenic effect of a low-molecular weight compound, SVAK-12, that potentiated the effects of BMP-2 in inducing transdifferentiation of C2C12 myoblasts into the osteoblastic phenotype. Here, we report a specific compound, SVAK-12, which was selected based on in silico screenings of small-molecule databases using the homology modeled interaction motif of Smurf1-WW2 domain. The enhancement of BMP-2 activity by SVAK-12 was characterized by evaluating a BMP-specific reporter activity and by monitoring the BMP-2-induced expression of mRNA for osteocalcin and alkaline phosphatase (ALP), which are widely accepted marker genes of osteoblast differentiation. Finally, we confirmed these results by also measuring the enhancement of BMP-2-induced activity of ALP. Smurf1 is an E3 ligase that targets osteogenic Smads for ubiquitin-mediated proteasomal degradation. Smurf1 is an interesting potential target to enhance bone formation based on the positive effects on bone of proteins that block Smurf1-binding to Smad targets or in Smurf1-/- knockout mice. Since Smads bind Smurf1 via its WW2 domain, we performed in silico screening to identify compounds that might interact with the Smurf1-WW2 domain. We recently reported the activity of a compound, SVAK-3. However, SVAK-3, while exhibiting BMP-potentiating activity, was not stable and thus warranted a new search for a more stable and efficacious compound among a selected group of candidates. In addition to being more stable, SVAK-12 exhibited a dose-dependent activity in inducing osteoblastic differentiation of myoblastic C2C12 cells even when multiple markers of the osteoblastic phenotype were parallelly monitored.