Overexpression Of Smurf2 Research Articles

SMAD specific E3 ubiquitin protein ligase1 promotes ovarian cancer cell migration and invasion via the activation of the RhoA/ROCK signaling pathway.

SMAD specific E3 ubiquitin protein ligase1 (SMURF1) serves a pivotal role in a variety of pathological processes and in tumor cell migration and invasion; however, its functional mechanism in ovarian cancer (OC) remains unknown. Previously, we observed overexpression of SMURF1 in OC tissues. In the present study, the role of SMURF1 in OC metastasis was investigated. The results revealed that SMURF1 was upregulated in OC cell lines of greater aggression than less aggressive cells. Downregulation of SMURF1 significantly inhibited OC cell invasion and migration, whereas upregulation of SMURF1 promoted OC cell invasion and migration. Investigation of the mechanism underlying the effects of SMURF1 in OC revealed that SMURF1 induced OC cell migration and invasion via activation of the Ras homolog family member A/Rho‑associated protein kinase signaling pathway. Further analysis demonstrated that higher levels of SMURF1 expression were associated with shorter overall survival in patients with OC. The findings of the present study indicated that overexpression of SMURF1 may contribute to the malignancy and metastasis of OC. The inhibition of SMURF1 expression may be a promising strategy for the treatment of patients with OC.

TGF-β Initiates β-Catenin-Mediated CTGF Secretory Pathway in Old Bovine Nucleus Pulposus Cells: A Potential Mechanism for Intervertebral Disc Degeneration.

ABSTRACTWe have recently demonstrated that overexpression of Smurf2 under the control of type II collagen alpha 1 (Col2a1) promoter induces an intervertebral disc degeneration phenotype in Col2a1‐Smurf2 transgenic mice. The chondrocyte‐like cells that express type II collagen and Smurf2 in the transgenic mouse discs are prone to degenerate. However, how the chondrocyte‐like cells contribute to disc degeneration is not known. Here, we utilized primary old bovine nucleus pulposus (NP) cells as substitutes for the chondrocyte‐like cells in Col2a1‐Smurf2 transgenic mouse discs to identify mechanism. We found that 35% of the cells were senescent; TGF‐β treatment of the cells induced a rapid moderate accumulation of β‐catenin, which interacted with connective tissue growth factor (CTGF/CCN2) in the cytoplasm and recruited it to the membrane for secretion. The TGF‐β‐initiated β‐catenin‐mediated CTGF secretory cascade did not occur in primary young bovine NP cells; however, when Smurf2 was overexpressed in young bovine NP cells, the cells became senescent and allowed this cascade to occur. These results suggest that Smurf2‐induced disc degeneration in Col2a1‐Smurf2 transgenic mice occurs through activation of CTGF secretory pathway in senescent disc cells. © 2018 The Authors JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Intracellular attenuation of BMP signaling via CKIP-1/Smurf1 is essential during neural crest induction.

The neural crest is induced at the neural plate border during gastrulation by combined bone morphogenetic protein (BMP), fibroblast growth factor (FGF), and Wnt signaling. While intermediate BMP levels are critical for this induction, secreted BMP inhibitors are largely absent from the neural plate border. Here, we propose a morphogen model in which intracellular attenuation of BMP signaling sets the required intermediate levels to maintain neural crest induction. We show that the scaffold protein casein kinase interacting protein 1 (CKIP-1) and ubiquitin ligase Smad ubiquitin regulatory factor 1 (Smurf1) are coexpressed with BMP4 at the chick neural plate border. Knockdown of CKIP-1 during a critical period between gastrulation and neurulation causes neural crest loss. Consistent with specific BMP modulation, CKIP-1 loss suppresses phospho-Smads 1/5/8 (pSmad1/5/8) and BMP reporter output but has no effect on Wnt signaling; Smurf1 overexpression (OE) acts similarly. Epistasis experiments further show that CKIP-1 rescues Smurf1-mediated neural crest loss. The results support a model in which CKIP-1 suppresses Smurf1-mediated degradation of Smads, uncovering an intracellular mechanism for attenuation of BMP signaling to the intermediate levels required for maintenance of neural crest induction.

Regulation of CNKSR2 protein stability by the HECT E3 ubiquitin ligase Smurf2, and its role in breast cancer progression

BackgroundSmurf2 E3 ubiquitin ligase physically associates with and regulate the stability of distinct cellular protein substrates. The multi-functional scaffold protein Connector enhancer of kinase suppressor of ras 2 (CNKSR2) plays a key role in regulating cell proliferation, and differentiation through multiple receptor tyrosine kinase pathways. The aim of this study was to investigate whether the interaction between Smurf2 and CNKSR2 has any significant role in the post transcriptional regulation of CNKSR2 expression in breast cancer. Methods: Here we demonstrate a novel interaction of CNKSR2 with Smurf2 by co-immunoprecipitation, indirect immunofluorescence studies, and surface plasmon resonance (SPR) analysis, which can ubiquitinate, but stabilize CNKSR2 by protecting it from proteasome mediated degradation.ResultsCNKSR2 protein levels were significantly increased upon forced overexpression of Smurf2, indicating the role of Smurf2 in regulating the stability of CNKSR2. Conversely, Smurf2 knockdown resulted in a marked decrease in the protein level expression of CNKSR2 by facilitating enhanced polyubiquitination and proteasomal degradation and reduced the proliferation and clonogenic survival of MDA-MB-231 breast cancer cell lines. Tissue microarray data from 84 patients with various stages of mammary carcinoma, including (in order of increasing malignant potential) normal, usual hyperplasia, fibrocystic changes, fibroadenoma, carcinoma-in-situ, and invasive ductal carcinoma showed a statistically significant association between Smurf2 and CNKSR2 expression, which is also well correlated with the ER, PR, and HER2 status of the tissue samples. A comparatively high expression of Smurf2 and CNKSR2 was observed when the expression of ER and PR was low, and HER2 was high. Consistently, both Smurf2 and CNKSR2 showed an integrated expression in MCF10 breast progression model cell lines.ConclusionsAltogether, our findings reveal that Smurf2 is a novel positive regulator of CNKSR2 and suggest that Smurf2-CNKSR2 interaction may serve as a common strategy to control proliferation of human breast cancer cells by modulating CNKSR2 protein stability.

Smurf1 restricts the antiviral function mediated by USP25 through promoting its ubiquitination and degradation

Protein ubiquitination and deubiquitination enzymes are widely involved in innate immune responses. The ubiquitin specific protease 25 (USP25), a deubiquitinating enzyme, has been demonstrated to play an important role in virus infection and immunity. However, how USP25 is degraded and regulated by E3 ubiquitin ligases remains poorly understood. Here, we identified Smad ubiquitin regulatory factor 1(Smurf1) as a first novel E3 ubiquitin ligase of USP25. Smurf1 overexpression decreases USP25 protein turnover, and the E3 ligase enzymatic activity of Smurf1 is required for USP25 degradation. Additionally, Smurf1-mediated degradation of USP25 is via promoting the K48-linkage polyubiquitination of USP25 in an ubiquitin proteasome dependent pathway. Importantly, USP25 overexpression restricts vesicular stomatitis virus (VSV) replication and the restriction of VSV replication by USP25 is enhanced in Smurf1 stable knock down cells. Therefore, our study firstly identified that Smurf1 negatively regulated the antiviral function mediated by USP25. Our findings revealed a previously unrecognized role of Smurf1 acting on USP25 and also their roles in the regulation of VSV replications.

Smurf2 regulates stability and the autophagic-lysosomal turnover of lamin A and its disease-associated form progerin.

SummaryA‐lamins, encoded by the LMNA gene, are major structural components of the nuclear lamina coordinating essential cellular processes. Mutations in the LMNA gene and/or alterations in its expression levels have been linked to a distinct subset of human disorders, collectively known as laminopathies, and to cancer. Mechanisms regulating A‐lamins are mostly obscure. Here, we identified E3 ubiquitin ligase Smurf2 as a physiological regulator of lamin A and its disease‐associated mutant form progerin (LAΔ50), whose expression underlies the development of Hutchinson‐Gilford progeria syndrome (HGPS), a devastating premature aging syndrome. We show that Smurf2 directly binds, ubiquitinates, and negatively regulates the expression of lamin A and progerin in Smurf2 dose‐ and E3 ligase‐dependent manners. Overexpression of catalytically active Smurf2 promotes the autophagic–lysosomal breakdown of lamin A and progerin, whereas Smurf2 depletion increases lamin A levels. Remarkably, acute overexpression of Smurf2 in progeria fibroblasts was able to significantly reduce the nuclear deformability. Furthermore, we demonstrate that the reciprocal relationship between Smurf2 and A‐lamins is preserved in different types of mouse and human normal and cancer tissues. These findings establish Smurf2 as an essential regulator of lamin A and progerin and lay a foundation for evaluating the efficiency of progerin clearance by Smurf2 in HGPS, and targeting of the Smurf2–lamin A axis in age‐related diseases such as cancer.

CKIP-1 affects the polyubiquitination of Nrf2 and Keap1 via mediating Smurf1 to resist HG-induced renal fibrosis in GMCs and diabetic mice kidneys.

Our previous study indicated that Casein kinase 2 interacting protein-1 (CKIP-1) could promote the activation of the nuclear factor E2-related factor 2 (Nrf2)/ antioxidant response element (ARE) pathway, playing a significant role in inhibiting the fibrosis of diabetic nephropathy (DN). However, the underlying mechanism is still unknown. Here, we investigated whether CKIP-1 affects the polyubiquitination of Nrf2 and its cytosolic inhibitor kelch like ECH-associated protein 1 (Keap1) via mediating Smad ubiquitylation regulatory factor-1 (Smurf1) to promote the activation of the Nrf2/ARE signaling and resist high glucose (HG)-induced renal fibrosis in glomerular mesangial cells (GMCs) and diabetic mice kidneys. Results showed that the expression of Smurf1 increased in HG-induced GMCs, with a paramount upregulation at 1h. Overexpression of wild-type Smurf1 plasmid further promoted the HG-induced the over-production of fibronectin (FN) and intercellular adhesionmolecule-1 (ICAM-1), and depletion of Smurf1 dramatically reduced the expression of FN and ICAM-1. Overexpression of CKIP-1 decreased the K48-linked polyubiquitination and increased the K63-linked polyubiquitination of Nrf2 as well as enhanced the K48-linked polyubiquitination and reduced K63-linked polyubiquitination of Keap1, promoting the activation of the Nrf2/ARE pathway. Overexpression of Smurf1 increased the K48-linked polyubiquitination and decreased the K63-linked polyubiquitination of Nrf2, and down-regulated the K48-linked polyubiquitination and up-regulated the K63-linked polyubiquitination of Keap1, inhibiting the activation of the Nrf2/ARE pathway. CKIP-1 promoted the degradation of Smurf1 by increasing the ubiquitination of Smurf1. Treatment of CKIP-1 adenovirus infection reduced the Smurf1 levels, promoted the activation of the Nrf2/ARE pathway as well as suppressed the production of reactive oxygen species (ROS), and then improved the failure of renal function of diabetic mice. Experiments above suggested that CKIP-1 affects the polyubiquitination of Nrf2 and Keap1 and promotes the Nrf2-ARE pathway through down-regulating Smurf1 to resist HG-induced up-regulation of FN and ICAM-1 in GMCs and diabetic mice kidneys.

Role of tumor necrosis factor-α in epithelial-to-mesenchymal transition in transplanted kidney cells in recipients with chronic allograft dysfunction

BackgroundChronic allograft dysfunction (CAD) is characterized by allograft kidney interstitial fibrosis, the underlying mechanism of which is unclear. Our aim was to elucidate the role and mechanism of TNF-α-induced epithelial-to-mesenchymal transition (EMT) in transplant kidney tubular interstitial fibrosis. MethodsHuman kidney tissues from normal volunteers and CAD patients were assessed using periodic acid-Schiff, Masson trichrome and immunohistochemical staining. mRNA and protein expression of E-cadherin, α-smooth muscle actin (SMA) and fibronectin(FN) in renal proximal tubule epithelial (HK-2) cells after treatment with TNF-α under different conditions were assessed using western blot and qRT-PCR analysis. Cell motility and migration were assessed using wound healing and transwell assays. Expression of Smurf2 and TNF-α-signaling pathway-related proteins in HK-2 cells treated with TNF-α was detected by western blotting. E-cadherin and α-SMA expression was also assessed in Smurf2 plasmid-transfected or Smurf2 siRNA-treated HK-2 cells. ResultsThe expression of TNF-α, Smurf2, α-SMA, and fibronectin was significantly upregulated, while the expression of E-cad was downregulated in the CAD group compared with the normal group. The in vitro results showed that TNF-α remarkably upregulated the expression of Smurf2, α-SMA and fibronectin and downregulated the expression of E-cadherin in HK-2 cells and enhanced motility and migration in HK-2 cells. Overexpression of Smurf2 could promote the expression of α-SMA and inhibit the expression of E-cad, whereas knockdown of Smurf2 expression reversed TNF-α-induced upregulation of α-SMA and prohibited the reduction of E-cad expression. Furthermore, TNF-α-induced Smurf2 expression promoted EMT through the Akt signaling pathway. ConclusionsTNF-α induced EMT via the TNF-α/Akt/Smurf2 signaling pathways, and it may play a role in aggravating allograft kidney interstitial fibrosis in CAD patients.

Transcription factor Six2 mediates the protection of GDNF on 6-OHDA lesioned dopaminergic neurons by regulating Smurf1 expression.

Glial cell line-derived neurotrophic factor (GDNF) has strong neuroprotective and neurorestorative effects on dopaminergic (DA) neurons in the substantia nigra (SN); however, the underlying molecular mechanisms remain to be fully elucidated. In this study, we found that the expression level of transcription factor Six2 was increased in damaged DA neurons after GDNF rescue in vivo and in vitro. Knockdown of Six2 resulted in decreased cell viability and increased the apoptosis of damaged DA neurons after GDNF treatment in vitro. In contrast, Six2 overexpression increased cell viability and decreased cell apoptosis. Furthermore, genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) indicated that Six2 directly bound to the promoter CAGCTG sequence of smad ubiquitylation regulatory factor 1 (Smurf1). ChIP-quantitative polymerase chain reaction (qPCR) analysis showed that Smurf1 expression was significantly upregulated after GDNF rescue. Moreover, knockdown of Six2 decreased Smurf1 expression, whereas overexpression of Six2 increased Smurf1 expression in damaged DA neurons after GDNF rescue. Meanwhile, knockdown and overexpression of Smurf1 increased and decreased p53 expression, respectively. Taken together, our results from in vitro and in vivo analysis indicate that Six2 mediates the protective effects of GDNF on damaged DA neurons by regulating Smurf1 expression, which could be useful in identifying potential drug targets for injured DA neurons.

EGF Inhibits Wnt/β-Catenin-Induced Osteoblast Differentiation by Promoting β-Catenin Degradation.

Bone morphogenetic protein (BMP) and canonical Wnts are representative developmental signals that enhance osteoblast differentiation and bone formation. Previously, we demonstrated that epidermal growth factor (EGF) inhibits BMP2-induced osteoblast differentiation by inducing Smurf1 expression. However, the regulatory role of EGF in Wnt/β-catenin-induced osteoblast differentiation has not been elucidated. In this study, we investigated the effect of EGF on Wnt/β-catenin signaling-induced osteoblast differentiation using the C2C12 cell line. EGF significantly suppressed the expression of osteoblast marker genes, which were induced by Wnt3a and a GSK-3β inhibitor. EGF increased the expression levels of Smurf1 mRNA and protein. Smurf1 knockdown rescued Wnt/β-catenin-induced osteogenic marker gene expression in the presence of EGF. EGF treatment or Smurf1 overexpression did not affect β-catenin mRNA expression levels, but reduced β-catenin protein levels and TOP-Flash activity. EGF and Smurf1 promoted β-catenin ubiquitination. Co-immunoprecipitation and GST pull-down assays showed that Smurf1 associates with β-catenin. These results suggest that EGF/Smurf1 inhibits Wnt/β-catenin-induced osteogenic differentiation and that Smurf1 downregulates Wnt/β-catenin signaling by enhancing proteasomal degradation of β-catenin.

MiR-15b promotes epithelial-mesenchymal transition by inhibiting SMURF2 in pancreatic cancer.

The epithelial-mesenchymal transition (EMT) is a key developmental program that is often activated during cancer invasion and metastasis. We report here that silencing SMURF2 (SMAD specific E3 ubiquitin protein ligase 2) promoted EMT in HPDE6c7 normal pancreas cells and overexpression of SMURF2 inhibited TGF-β-mediated EMT in the cells. Subsequent studies showed that SMURF2 was downregulated in pancreatic cancer tissues and it promoted mesenchymal-epithelial transition (MET) in pancreatic cancer cells as well as its expression negatively associated with gemcitabine-resistance, but it did not alter cell viability, cell cycle and cell senescence. In addition, we demonstrated that miR‑15b degraded SMURF2 and its overexpression promoted EMT in pancreatic cancer, and its expression was associated with metastasis in the disease. Elucidating molecular mechanism of EMT in pancreatic cancer not only will help us to further understand the pathogenesis and progression of the disease, but also offers new targets for effective therapies.

Smurf2 negatively modulates RIG-I-dependent antiviral response by targeting VISA/MAVS for ubiquitination and degradation.

VISA (also known as MAVS, Cardif, IPS-1) is the essential adaptor protein for virus-induced activation of IFN regulatory factors 3 and 7 and production of type I IFNs. Understanding the regulatory mechanisms for VISA will provide detailed insights into the positive or negative regulation of innate immune responses. In this study, we identified Smad ubiquitin regulatory factor (Smurf) 2, one of the Smad ubiquitin regulator factor proteins, as an important negative regulator of virus-triggered type I IFN signaling, which targets at the VISA level. Overexpression of Smurf2 inhibits virus-induced IFN-β and IFN-stimulated response element activation. The E3 ligase defective mutant Smurf2/C716A loses the ability to suppress virus-induced type I IFN signaling, suggesting that the negative regulation is dependent on the ubiquitin E3 ligase activity of Smurf2. Further studies demonstrated that Smurf2 interacted with VISA and targeted VISA for K48-linked ubiquitination, which promoted the degradation of VISA. Consistently, knockout or knockdown of Smurf2 expression therefore promoted antiviral signaling, which was correlated with the increase in protein stability of VISA. Our findings suggest that Smurf2 is an important nonredundant negative regulator of virus-triggered type I IFN signaling by targeting VISA for K48-linked ubiquitination and degradation.

Ubiquitin E3 ligase SCFβ-TRCP regulates TRIB2 stability in liver cancer cells

Tribbles homolog 2 (TRIB2) is functionally important for liver cancer cell survival and transformation. Our previous study demonstrates TRIB2 is stable in liver cancer cells due to the impaired ubiquitination by Smurf1. However, overexpression of Smurf1 alone cannot completely abolish TRIB2 protein expression, whether other potential factors involved in the degradation of TRIB2 still remains unclear. In the present study, we reveal that the stability and ubiquitination of TRIB2 can also be controlled by ubiquitin E3 ligase SCFβ-TRCP. Depletion of either Cullin1 or β-TRCP up-regulates TRIB2 protein expression. Moreover, knockdown of β-TRCP extends the half-life, whereas reduces ubiquitylation of TRIB2. Similar to Smurf1, β-TRCP exerts its role through the TRIB2 Degradation Domain (TDD) at the N-terminus of the TRIB2 protein. Hence, we add TRIB2 to the substrate list of SCFβ-TRCP and the finding may be helpful in the treatment of TRIB2 dependent liver cancer.

Smurf2 negatively regulates IFN-β production through mediating IRF3 ubiquitination and degradation

Viral and bacterial infection activate several transcription factors including NF-κB and IRF3, which collaborate to induce type I interferons (IFNs). However, the mechanisms involved in terminating the production of type I IFNs are not well defined. Here we identified Smurf2 as a negative regulator in TLR3/4- and RIG-I-mediated IFN-β signaling. Knockdown of Smurf2 expression by small interfering RNA resulted in augmented activation of IRF3 and enhanced expression of IFN-β, whereas overexpression of Smurf2 had opposite effects. As an HECT domain E3 ligase, Smurf2 interacted with activated IRF3 directly and catalyzed K48-linked polyubiquitination of phosphorylated IRF3. These findings suggest that Smurf2 is a negative regulator for TLR3/4- and RIG-I-mediated IFN-β production by targeting phosphorylated IRF3 for ubiquitination and subsequent proteasome-mediated degradation. Structured summary of protein interactionsIrf3physically interacts with Smurf2 by anti tag coimmunoprecipitation (View interaction)Smurf2physically interacts with Irf3 by anti bait coimmunoprecipitation (View interactions: 1, 2)Smurf2physically interacts with Irf3 by pull down (View interaction)

Regulation of early xenopus embryogenesis by smad ubiquitination regulatory factor 2

Smad ubiquitination regulatory factor (Smurf) 1 and 2 are E3 ubiquitin ligases originally identified as inhibitors of transforming growth factor beta signaling and are shown to modulate multiple cellular activities. The roles of Smurfs in vertebrate embryogenesis, however, are not completely understood. Here we investigate the function of Smurf2 during early Xenopus development. We show that distinctly from Smurf1, overexpression of Smurf2 in presumptive mesoderm interfered with mesoderm induction and caused axial defects, whereas knockdown of Smurf2 with antisense morpholino oligonucleotides resulted in expansion of the mesoderm. These results imply that Smurf2 may modulate nodal-mediated mesodermal induction. Consistently, ventral expression of Smurf2 induced a partial secondary axis with head structures. In the ectoderm, Smurf2 resembled Smurf1 in controlling neural and epidermal marker expression and influencing head formation. Smurf1, but not Smurf2, additionally affected neural tube closure. Interestingly, both Smurfs could enhance as well as repress neural crest markers, implying that they modulate their targets dynamically during neural plate border specification. Our data demonstrate that Smurf1 and Smurf2 have overlapping and distinct functionalities during early frog embryogenesis; collectively, they regulate ectodermal and mesodermal induction and patterning to ensure normal development of Xenopus embryos.

Smurf1 Protein Negatively Regulates Interferon-γ Signaling through Promoting STAT1 Protein Ubiquitination and Degradation

Interferons are important cytokines that mediate antiviral, antiproliferative, antitumor, and immunoregulatory activities. However, uncontrolled IFN signaling may lead to autoimmune diseases. Here we identified Smurf1 as a negative regulator for IFN-γ signaling by targeting STAT1 for ubiquitination and proteasomal degradation. Smurf1 interacted with STAT1 through the WW domains of Smurf1 and the PY motif in STAT1 and catalyzed K48-linked polyubiquitination of STAT1. Interestingly, the Smurf1-mediated ubiquitination and degradation did not require STAT1 tyrosine and serine phosphorylation. Subsequently, overexpression of Smurf1 attenuated IFN-γ-mediated STAT1 activation and antiviral immune responses, whereas knockdown of Smurf1 enhanced IFN-γ-mediated STAT1 activation, expression of STAT1 target genes, and antiviral immune responses. Furthermore, IFN-γ stimulation led to enhanced expression of Smurf1. Therefore, our results demonstrate that Smurf1 is a negative feedback regulator for IFN-γ signaling by targeting STAT1 for ubiquitination and proteasomal degradation.

TGFβ and BMP-2 regulate epicardial cell invasion via TGFβR3 activation of the Par6/Smurf1/RhoA pathway

Coronary vessel development requires transfer of mesothelial cells to the heart surface to form the epicardium where some cells subsequently undergo epithelial–mesenchymal transformation (EMT) and invade the subepicardial matrix. Tgfbr3−/− mice die due to failed coronary vessel formation associated with decreased epicardial cell invasion but the mediators downstream of TGFβR3 are not well described. TGFβR3-dependent endocardial EMT stimulated by either TGFβ2 or BMP-2 requires activation of the Par6/Smurf1/RhoA 1pathway where Activin Receptor Like Kinase (ALK5) signals Par6 to act downstream of TGFβ to recruit Smurf1 to target RhoA for degradation to regulate apical-basal polarity and tight junction dissolution. Here we asked if this pathway was operant in epicardial cells and if TGFβR3 was required to access this pathway. Targeting of ALK5 in Tgfbr3+/+ cells inhibited loss of epithelial character and invasion. Overexpression of wild-type (wt) Par6, but not dominant negative (dn) Par6, induced EMT and invasion while targeting Par6 by siRNA inhibited EMT and invasion. Overexpression of Smurf1 and dnRhoA induced loss of epithelial character and invasion. Targeting of Smurf1 by siRNA or overexpression of constitutively active (ca) RhoA inhibited EMT and invasion. In Tgfbr3−/− epicardial cells which have a decreased ability to invade collagen gels in response to TGFβ2, overexpression of wtPar6, Smurf1, or dnRhoA had a diminished ability to induce invasion. Overexpression of TGFβR3 in Tgfbr3−/− cells, followed by siRNA targeting of Par6 or Smurf1, diminished the ability of TGFβR3 to rescue invasion demonstrating that the Par6/Smurf1/RhoA pathway is activated downstream of TGFβR3 in epicardial cells.

Ubiquitination of heat shock protein 27 is mediated by its interaction with Smad ubiquitination regulatory factor 2 in A549 cells

ABSTRACTSmad ubiquitination regulatory factor 2 (Smurf2) is a crucial part of the ubiquitin-proteasome pathway (UPP) that regulates cellular signal transduction via ubiquitin-dependent degradation of some substrates and receptors. The biological function of Smurf2 in lung diseases, however, is not clear. In this study, the authors found that overexpression of Smurf2 altered the subcellular localization and distribution of heat shock protein 27 (HSP27), and induced a decrease of HSP27 protein levels through HSP27 degradation by the UPP in human lung adenocarcinoma epithelial cell line A549. Colocalized assay using confocal microscopy and coimmunoprecipitated reciprocally by either antibody indicated the interaction between Smurf2 and HSP27, which suggested that Smurf2 mediated ubiquitylation-dependent degradation of HSP27 through their interaction in A549 cells.

SMURF1 Amplification Promotes Invasiveness in Pancreatic Cancer

Pancreatic cancer is a deadly disease, and new therapeutic targets are urgently needed. We previously identified DNA amplification at 7q21-q22 in pancreatic cancer cell lines. Now, by high-resolution genomic profiling of human pancreatic cancer cell lines and human tumors (engrafted in immunodeficient mice to enrich the cancer epithelial fraction), we define a 325 Kb minimal amplicon spanning SMURF1, an E3 ubiquitin ligase and known negative regulator of transforming growth factor β (TGFβ) growth inhibitory signaling. SMURF1 amplification was confirmed in primary human pancreatic cancers by fluorescence in situ hybridization (FISH), where 4 of 95 cases (4.2%) exhibited amplification. By RNA interference (RNAi), knockdown of SMURF1 in a human pancreatic cancer line with focal amplification (AsPC-1) did not alter cell growth, but led to reduced cell invasion and anchorage-independent growth. Interestingly, this effect was not mediated through altered TGFβ signaling, assayed by transcriptional reporter. Finally, overexpression of SMURF1 (but not a catalytic mutant) led to loss of contact inhibition in NIH-3T3 mouse embryo fibroblast cells. Together, these findings identify SMURF1 as an amplified oncogene driving multiple tumorigenic phenotypes in pancreatic cancer, and provide a new druggable target for molecularly directed therapy.

The WW-HECT protein Smurf2 interacts with the Docking Protein NEDD9/HEF1 for Aurora A activation

The multi-functional adaptor protein NEDD9/HEF1/Cas-L regulates cell motility, invasion and cell cycle progression, and plays key roles in cancer progression and metastasis. NEDD9 is localized to the centrosome and is required for activation of Aurora A kinase in mitosis. Here we demonstrate that the HECT-WW protein Smurf2 physically associates with NEDD9 and is required for the stability of NEDD9 protein. Smurf2 depletion results in a marked decrease in NEDD9 protein levels, by facilitating polyubiquitination and proteasomal degradation of NEDD9. Conversely, forced overexpression of Smurf2 results in upregulation of endogenous NEDD9 protein, confirming the role for Smurf2 in NEDD9 stability. Cells with Smurf2 depletion fail to activate Aurora A at the G2/M boundary, leading to a marked delay in mitotic entry. These observations suggest that the stable complex of Smurf2 and NEDD9 is required for timely entry into mitosis via Aurora A activation.