Smurf1-Mediated Lys29-Linked Nonproteolytic Polyubiquitination of Axin Negatively Regulates Wnt/β-Catenin Signaling

Ubiquitination plays important and diverse roles in modulating protein functions. As a C2-WW-HECT-type ubiquitin ligase, Smad ubiquitination regulatory factor 1 (Smurf1) commonly serves to regulate ubiquitin-dependent protein degradation in a number of signaling pathways. Here, we report a novel function of Smurf1 in regulating Wnt/β-catenin signaling through targeting axin for nonproteolytic ubiquitination. Our data unambiguously demonstrate that Smurf1 ubiquitinates axin through Lys 29 (K29)-linked polyubiquitin chains. Unexpectedly, Smurf1-mediated axin ubiquitination does not lead to its degradation but instead disrupts its interaction with the Wnt coreceptors LRP5/6, which subsequently attenuates Wnt-stimulated LRP6 phosphorylation and represses Wnt/β-catenin signaling. The inhibitory function of Smurf1 on Wnt/β-catenin signaling is further evidenced by analysis with Smurf1 knockout murine embryonic fibroblasts. We next identified K789 and K821 in axin as the ubiquitination sites by Smurf1. Consistently, Smurf1 could neither disrupt the interaction of an axin^K789/821R double mutant with LRP5/6 nor attenuate the phosphorylation of LRP6 in axin^K789/821R-expressing cells. Collectively, our studies uncover Smurf1 as a new regulator for the Wnt/β-catenin signaling pathway via modulating the activity of axin.     

Ubiquitin ligase Smurf1 controls osteoblast activity and bone homeostasis by targeting MEKK2 for degradation

Bone is constantly resorbed and formed throughout life by coordinated actions of osteoclasts and osteoblasts. Here we show that Smurf1, a HECT domain ubiquitin ligase, has a specific physiological role in suppressing the osteogenic activity of osteoblasts. Smurf1-deficient mice are born normal but exhibit an age-dependent increase of bone mass. The cause of this increase can be traced to enhanced activities of osteoblasts, which become sensitized to bone morphogenesis protein (BMP) in the absence of Smurf1. However, loss of Smurf1 does not affect the canonical Smad-mediated intracellular TGFbeta or BMP signaling; instead, it leads to accumulation of phosphorylated MEKK2 and activation of the downstream JNK signaling cascade. We demonstrate that Smurf1 physically interacts with MEKK2 and promotes the ubiquitination and turnover of MEKK2. These results indicate that Smurf1 negatively regulates osteoblast activity and response to BMP through controlling MEKK2 degradation.     

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.     

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.     

Protein Arginine Methyltransferase 1 Methylates Smurf2

Smurf2, a member of the HECT domain E3 ligase family, is well known for its role as a negative regulator of TGF-β signaling by targeting Smads and TGF-β receptor. However, the regulatory mechanism of Smurf2 has not been elucidated. Arginine methylation is a type of post-translational modification that produces monomethylated or dimethylated arginine residues. In this report, we demonstrated methylation of Smurf2 by PRMT1. In vitro methylation assay showed that Smurf2, not Smurf1, was methylated by PRMT1. Among the type I PRMT family, only PRMT1 showed activity for Smurf2. Transiently expressed Smurf2 was methylated by PRMT1, indicating Smurf2 is a novel substrate of PRMT1. Using deletion constructs, methylation sites were shown to be located within amino acid region 224–298 of Smurf2. In vitro methylation assay following point mutation of putative methylation sites confirmed the presence of Arg232, Arg234, Arg237, and Arg239. Knockdown of PRMT1 resulted in increased Smurf2 expression as well as inhibition of TGF-β-mediated reporter activity. Although it is unclear whether or not increased Smurf2 expression can be directly attributed to lack of methylation of arginine residues, our results suggest that methylation by PRMT1 may regulate Smurf2 stability and control TGF-β signaling.     

Tumor Necrosis Factor-Receptor–associated Factor-4 Is a Positive Regulator of Transforming Growth Factor-β Signaling That Affects Neural Crest Formation

The transforming growth factor (TGF)-β superfamily regulates cell proliferation, apoptosis, differentiation, migration, and development. Canonical TGFβ signals are transduced to the nucleus via Smads in both major signaling branches, bone morphogenetic protein (BMP) or Activin/Nodal/TGFβ. Smurf ubiquitin (Ub) ligases attenuate these pathways by targeting Smads and other signaling components for degradation by the 26S proteasome. Here, we identify tumor necrosis factor (TNF)-receptor–associated factor-4 (TRAF4) as a new target of Smurf1, which polyubiquitylates TRAF4 to trigger its proteasomal destruction. Unlike other TRAF family members, which mediate signal transduction by TNF, interleukin, or Toll-like receptors, we find that TRAF4 potentiates BMP and Nodal signaling. In the frog Xenopus laevis, TRAF4 mRNA is stored maternally in the egg animal pole, and in the embryo it is expressed in the gastrula marginal zone, neural plate, and cranial and trunk neural crest. Knockdown of embryonic TRAF4 impairs signaling, neural crest development and neural folding, whereas TRAF4 overexpression boosts signaling and expands the neural crest. In human embryonic kidney 293 cells, small interfering RNA knockdown of Smurf1 elevates TRAF4 levels, indicating endogenous regulation of TRAF4 by Smurf1. Our results uncover new functions for TRAF4 as a Smurf1-regulated mediator of BMP and Nodal signaling that are essential for neural crest development and neural plate morphogenesis.     

Cooperative inhibition of bone morphogenetic protein signaling by Smurf1 and inhibitory Smads

Smad ubiquitin regulatory factor (Smurf) 1 binds to receptor-regulated Smads for bone morphogenetic proteins (BMPs) Smad1/5 and promotes their degradation. In addition, Smurf1 associates with transforming growth factor-beta type I receptor through the inhibitory Smad (I-Smad) Smad7 and induces their degradation. Herein, we examined whether Smurf1 negatively regulates BMP signaling together with the I-Smads Smad6/7. Smurf1 and Smad6 cooperatively induced secondary axes in Xenopus embryos. Using a BMP-responsive promoter-reporter construct in mammalian cells, we found that Smurf1 cooperated with I-Smad in inhibiting BMP signaling and that the inhibitory activity of Smurf1 was not necessarily correlated with its ability to bind to Smad1/5 directly. Smurf1 bound to BMP type I receptors via I-Smads and induced ubiquitination and degradation of these receptors. Moreover, Smurf1 associated with Smad1/5 indirectly through I-Smads and induced their ubiquitination and degradation. Smurf1 thus controls BMP signaling with and without I-Smads through multiple mechanisms.     

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.     

AIMP1/p43 downregulates TGF-beta signaling via stabilization of smurf2

AIMP1 (also known as p43) is a factor associated with a macromolecular aminoacyl-tRNA synthetase (ARS) complex but also plays diverse regulatory roles in various physiological processes. Here, we report that AIMP1 negatively regulates TGF-β signaling via stabilization of Smurf2. TGF-β-dependent phosphorylation and nuclear localization of R-Smads, induction of target genes, and growth arrest were increased in AIMP1-deficient or -suppressed cells. In AIMP1-deficient or suppressed cells, the Smurf2 level was decreased. Various binding assays demonstrated the direction interaction of the C-terminal region of AIMP1 directly with the Smad7-binding region of Smurf2. The association of Smurf2 with Smad7 and its ubiquitination were inhibited by AIMP1, thereby protecting its autocatalytic degradation stimulated by Smad7. Thus, this work suggests the novel activity of AIMP1 as a component of negative feedback loop of TGF-β signaling.     

Smurf2 E3 ubiquitin ligase modulates proliferation and invasiveness of breast cancer cells in a CNKSR2 dependent manner

Background

Smurf2 is a member of the HECT family of E3 ubiquitin ligases that play important roles in determining the competence of cells to respond to TGF- β/BMP signaling pathway. However, besides TGF-β/BMP pathway, Smurf2 regulates a repertoire of other signaling pathways ranging from planar cell polarity during embryonic development to cell proliferation, migration, differentiation and senescence. Expression of Smurf2 is found to be dysregulated in many cancers including breast cancer. The purpose of the present study is to examine the effect of Smurf2 knockdown on the tumorigenic potential of human breast cancer cells emphasizing more on proliferative signaling pathway.

Methods

siRNAs targeting different regions of the Smurf2 mRNA were employed to knockdown the expression of Smurf2. The biological effects of synthetic siRNAs on human breast cancer cells were investigated by examining the cell proliferation, migration, invasion, focus formation, anchorage-independent growth, cell cycle arrest, and cell cycle and cell proliferation related protein expressions upon Smurf2 silencing.

Results

Smurf2 silencing in human breast cancer cells resulted in a decreased focus formation potential and clonogenicity as well as in vitro cell migration/invasion capabilities. Moreover, knockdown of Smurf2 suppressed cell proliferation. Cell cycle analysis showed that the anti-proliferative effect of Smurf2 siRNA was mediated by arresting cells in the G0/G1 phase, which was caused by decreased expression of cyclin D1and cdk4, followed by upregulation p21 and p27. Furthermore, we demonstrated that silencing of Smurf2 downregulated the proliferation of breast cancer cells by modulating the PI3K- PTEN-AKT-FoxO3a pathway via the scaffold protein CNKSR2 which is involved in RAS-dependent signaling pathways. The present study provides the first evidence that silencing Smurf2 using synthetic siRNAs can regulate the tumorigenic properties of human breast cancer cells in a CNKSR2 dependent manner.

Conclusions

Our results therefore suggest a novel relation between Smurf2 and CNKSR2 thereby regulating AKT-dependent cell proliferation and invasion. Owing to the fact that PI3K-AKT signaling is hyperactivated in various human cancers and that Smurf2 also regulates cellular transformation, our results indicate that Smurf2 may serve as a potential molecule for targeted cancer therapy of certain tumour types including breast cancer.

     

CD109-mediated degradation of TGF-β receptors and inhibition of TGF-β responses involve regulation of SMAD7 and Smurf2 localization and function

Transforming growth factor-β (TGF-β) is a multifunctional cytokine that regulates a wide variety of cellular processes including proliferation, differentiation, and extracellular matrix deposition. Dysregulation of TGF-β signaling is associated with several diseases such as cancer and tissue fibrosis. TGF-β signals through two transmembrane proteins known as the type I (TGFBR1) and type II (TGFBR2) receptors. The levels of these receptors at the cell surface are tightly regulated by several mechanisms, including degradation following recruitment of the E3 ubiquitin ligase SMAD ubiquitination regulatory factor (Smurf) 2 by SMAD7. In addition, TGF-β co-receptors can modulate TGF-β signaling receptor activity in a cell-specific manner. We have previously identified a novel TGF-β co-receptor, CD109, a glycosyl phosphatidylinositol (GPI)-anchored protein that negatively regulates TGF-β signaling. Despite CD109's potential relevance as a regulator of TGF-β action in vivo, the mechanisms by which CD109 regulates TGF-β signaling are still incompletely understood. Previously, we have shown that CD109 downregulates TGF-β signaling by promoting TGF-β receptor localization into the lipid raft/caveolae compartment and by enhancing TGF-β receptor degradation. Here, we demonstrate that CD109 enhances SMAD7/Smurf2-mediated degradation of TGFBR1 in a ligand-dependent manner. Moreover, we show that CD109 regulates the localization and the association of SMAD7/Smurf2 with TGFBR1. Finally, we demonstrate that CD109's inhibitory effect on TGF-β signaling and responses require SMAD7 expression and Smurf2 ubiquitin ligase activity. Taken together, these results suggest that CD109 is an important regulator of SMAD7/Smurf2-mediated degradation of TGFBR1.     

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.     

Cdh1 regulates osteoblast function through an APC/C-independent modulation of Smurf1

The APC/Cdh1 E3 ubiquitin ligase plays an essential role in both mitotic exit and G1/S transition by targeting key cell-cycle regulators for destruction. There is mounting evidence indicating that Cdh1 has other functions in addition to cell-cycle regulation. However, it remains unclear whether these additional functions depend on its E3 ligase activity. Here, we report that Cdh1, but not Cdc20, promotes the E3 ligase activity of Smurf1. This is mediated by disruption of an autoinhibitory Smurf1 homodimer and is independent of APC/Cdh1 E3 ligase activity. As a result, depletion of Cdh1 leads to reduced Smurf1 activity and subsequent activation of multiple downstream targets, including the MEKK2 signaling pathway, inducing osteoblast differentiation. Our studies uncover a cell-cycle-independent function of Cdh1, establishing Cdh1 as an upstream component that governs Smurf1 activity. They further suggest that modulation of Cdh1 is a potential therapeutic option for treatment of osteoporosis.     

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.     

Transcriptional induction of Smurf2 ubiquitin ligase by TGF-beta

Smad ubiquitination regulatory factor 2 (Smurf2), a ubiquitin ligase for Smads, plays critical roles in the regulation of transforming growth factor-beta (TGF-beta)-Smad signaling via ubiquitin-dependent degradation of Smad2 and Smad7. We found that TGF-beta stimulates Smurf2 expression. TGF-beta activated the Smurf2 promoter in a TGF-beta responsive cell lines, whereas IL-1alpha, PDGF and epidermal growth factor did not. TGF-beta-mediated Smurf2 promoter activation was inhibited by Smad7 or an activin receptor-like kinase 5 inhibitor but not by dominant negative Smad or disruption of Smad-binding elements in the promoter. Moreover, inhibition of the phosphatidil inositol 3 kinase (PI3K)/Akt pathway suppressed TGF-beta-mediated Smurf2 induction. These results suggest that TGF-beta stimulates Smurf2 expression by Smad-independent pathway such as PI3K/Akt pathway via TGF-beta receptor.