The ubiquitin ligase Smurf2 suppresses TGFβ-induced epithelial–mesenchymal transition in a sumoylation-regulated manner

Abstract

Epithelial-mesenchymal transition (EMT) is a fundamental cellular process in epithelial tissue development, and can be reactivated in cancer contributing to tumor invasiveness and metastasis. The cytokine transforming growth factor-β (TGFβ) is a key inducer of EMT, but the mechanisms that regulate TGFβ-induced EMT remain incompletely understood. Here, we report that knockdown of the ubiquitin ligase Smurf2 promotes the ability of TGFβ to induce EMT in a three-dimensional cell culture model of NMuMG mammary epithelial cells. In other studies, we identify Smurf2 as a target of the small ubiquitin like modifier (SUMO) pathway. We find that the SUMO-E2 conjugating enzyme Ubc9 and the SUMO E3 ligase PIAS3 associate with Smurf2 and promote its sumoylation at the distinct sites of Lysines 26 and 369. The sumoylation of Smurf2 enhances its ability to induce the degradation of the TGFβ receptor and thereby suppresses EMT in NMuMG cells. Collectively, our data reveal that Smurf2 acts in a sumoylationregulated manner to suppress TGFβ-induced EMT. These findings have significant implications for our understanding of epithelial tissue development and cancer. Cell Death and Differentiation (2016) 23, 876-888; doi:10.1038/cdd.2015; published online 18 December 2015Epithelial-mesenchymal transition (EMT) is an essential process in epithelial tissue morphogenesis in the developing organism and contributes to postnatal events including mammary postnatal gland development as well as wound healing.1,2 Importantly, EMT can be reactivated during cancer and may contribute to tumor invasiveness.3 Epithelial cells undergoing EMT change phenotypically from cuboidal to fibroblastic morphology, lose epithelial markers including E-cadherin, gain mesenchymal markers, and display increased cell motility and invasiveness. 4,5 The secreted factor transforming growth factor-β (TGFβ) has emerged as a potent inducer of EMT with key roles in development and cancer.6 Thus, there has been interest in the mechanisms that mediate TGFβ-induced EMT.Smurf2 (Smad (Sma and mad) ubiquitination regulatory factor-2) is a HECT (for homology to E6 carboxy terminus domain)-containing E3 ubiquitin ligase that specifies substrates for ubiquitination and degradation by the proteasome.7,8 Smurf2 regulates key biological processes during development and homeostasis including cell polarity, cell cycle, and senescence in an E3 ligase-dependent or -independent manner.9-15 The biological functions of Smurf2 occur via regulation of signaling pathways including the TGFβ-Smad signaling pathway. [16][17][18][19][20][21][22][23] However, the role of Smurf2 in TGFβ-induced EMT has remained to be determined.Sumoylation refers to the covalent attachment of the small ubiquitin-like modifier (SUMO), to protein substrates by the SUMO pathway.24 SUMO is linked to its substrates by an iso-peptide bond between C-terminal carboxyl group of SUMO and ε-amino group of a lysine residue in the substrate. The SUMO E2 conjugating enzyme Ubc9, the second enzyme of a three-step catalytic cascade...

Akt Protein Kinase, miR-200/miR-182 Expression and Epithelial-Mesenchymal Transition Proteins in Hibernating Ground Squirrels

“…MiR-200 family and miR-182 cluster members, therefore, could have a strong influence on the EMT/MET process through controlling SUMOylation levels in TLGS brain during the hibernation cycle. SUMOylation of transcription factors or EMT-related proteins generally leads to EMT inhibition (Wang et al, 2010 ; Ren et al, 2014 ; Chandhoke et al, 2016 ; Zhang et al, 2016 ), which fits with our proposal that the progression of EMT is reversed (or inhibited) at the end of hibernation torpor where SUMOylation returns to basal levels. Unfortunately, we could not yet pinpoint any transcription factors whose SUMOylation levels increased during hibernation torpor.…”

Mechanisms of TGFβ-Induced Epithelial–Mesenchymal Transition

“…Smurf2 is therefore a negative regulator of TGFβ-induced EMT. The enzymatic activity of Smurf2 is induced by sumoylation [ 97 ]. TGFβ signaling downregulates the sumo ligase protein inhibitor of activated STAT (PIAS) 1, which is compatible with a mechanism whereby TGFβ attempts to silence its negative regulators; in addition to Smurf2, PIAS1 sumoylates the transcriptional repressor SnoN, which inactivates nuclear Smad complexes [ 98 ].…”

“…TGFβ signaling downregulates the sumo ligase protein inhibitor of activated STAT (PIAS) 1, which is compatible with a mechanism whereby TGFβ attempts to silence its negative regulators; in addition to Smurf2, PIAS1 sumoylates the transcriptional repressor SnoN, which inactivates nuclear Smad complexes [ 98 ]. Thus, reduction in PIAS1-dependent sumoylation permits the ubiquitylation and degradation of SnoN and inactivates Smurf2; in this manner, Smad complexes are freer to act and regulate genes critical for the EMT response [ 97 , 98 ]. A second ubiquitin ligase, tripartite motif-containing 33 (TRIM33), is activated by as yet unknown mechanisms and then targets Smad4 to negatively regulate the nuclear Smad complexes, thus antagonizing TGFβ-induced EMT [ 99 ].…”

Regulating the human HECT E3 ligases

“…Post-translational modifications (PTMs) are also prevalent in the alteration of HECT E3 ligase functionality such as phosphorylation and the attachment of ubiquitin-like modifiers (UBLs) [13–15]. Various mechanisms other than PTMs and interactions with adaptor proteins also regulate HECT E3 ligases such as intramolecular interactions, oligomerization, and recruitment of the E2 (Fig.…”