TIF1γ requires sumoylation to exert its repressive activity on TGFβ signaling

Abstract

SummaryTIF1c, a new regulator of TGFb signaling, inhibits the Smad4-mediated TGFb response by interaction with Smad2/3 or ubiquitylation of Smad4. We have shown that TIF1c participates in TGFb signaling as a negative regulator of Smad4 during the TGFb-induced epithelial-to-mesenchymal transition (EMT) in mammary epithelial cells, and during terminal differentiation of mammary alveolar epithelial cells and lactation. We demonstrate here that TIF1c is sumoylated and interacts with Ubc9, the only known SUMOconjugating enzyme. Four functional sumoylation sites lie within the middle domain of TIF1c, the Smad interaction domain. We show that a sumoylation-defective TIF1c mutant significantly reduces TIF1c inhibition of Smad complexes and that of the Smad-mediated TGFb transcriptional response. Moreover, chromatin immunoprecipitation experiments indicate that TIF1c sumoylation is required to limit Smad4 binding on the PAI-1 TGFb target gene promoter. Ectopic expression of TIF1c in mammary epithelial cells inhibits TGFbinduced EMT, an effect relieved by expression of non-sumoylated TIF1c. Taken together, our results identify a new TGFb regulatory layer, whereby sumoylation strengthens the TIF1c repressive action on canonical TGFb signaling.

The adenovirus E4-ORF3 protein functions as a SUMO E3 ligase for TIF-1γ sumoylation and poly-SUMO chain elongation

“…To test this, we purified a C-terminal truncated TIF-1γ protein (TIF-1γΔC). TIF-1γΔC contains all four known sumoylation sites (K776, 793, 796, and 839) (28) and the RBCC domain, but not the PHD domain and bromodomain Recombinant empty Ad-CMV (lanes 1 and 2, 900 P/cell) or Ad-CMV-HA-E4-ORF3 expression vector (lanes 3-5, 100, 300, and 900 P/cell) were used to infect His 6 -SUMO3-HeLa cells. At 10 hpi, SUMO conjugates were analyzed as in A.…”

TIF1γ Suppresses Tumor Progression by Regulating Mitotic Checkpoints and Chromosomal Stability

“…The TIF1 proteins are also defined by the presence of a RING domain with E3-ubiquitin ligase activity and belong to the TRIM protein superfamily (tripartite motif). TIF1g, whose activity and stability can be regulated by ubiquitination (3) and sumoylation (4), has been shown to be involved in various biologic functions, such as embryonic development (5-7), hematopoietic differentiation (8,9), DNA repair (10), cell cycle regulation (11), and immune response (12). At the molecular level, TIF1g is involved in chromatin remodeling and subsequent transcription modulation (3,7,10,13,14).…”

Valproic acid (VPA) inhibits the epithelial–mesenchymal transition in prostate carcinoma via the dual suppression of SMAD4

“…This process transforms SMAD4 protein from a deubiquitinated form to a mono-ubiquitinated protein (Xie et al 2014). The E3 ubiquitin ligase Ecto/Tif1-γ and the deubiquitylase FAM/Usp9x induced the mono-ubiquitinated and deubiquitinated forms of SMAD4 protein, respectively, which in turn mediate the ability of SMAD4 to bind to R-SMADs (Fattet et al 2013;Dupont et al 2009). qRT-PCR and Western blotting analysis revealed that VPA down-regulates SMAD4 mRNA (Fig.…”