Activation of Transforming Growth Factor-β Signaling by SUMO-1 Modification of Tumor Suppressor Smad4/DPC4

Abstract

Smads are important intracellular effectors in signaling pathways of the transforming growth factor-␤ (TGF-␤) superfamily. Upon activation by TGF-␤, receptor-phosphorylated Smads form a complex with tumor suppressor Smad4/DPC4, and the Smad complexes then are imported into the nucleus. Although diverse pathways regulate the activity and expression of receptorphosphorylated and inhibitory Smads, cellular factors modulating the activity of the common Smad4 remain unidentified. Here we describe the involvement of the small ubiquitin-like modifier-1 (SUMO-1) conjugation pathway in regulating the growth inhibitory and transcriptional responses of Smad4. The MH1 domain of Smad4 was shown to associate physically with Ubc9, the ubiquitin carrier protein (E2) conjugating enzyme in sumoylation. In cultured cells, Smad4 is modified by SUMO-1 at the endogenous level. The sumoylation sites were identified as two evolutionarily conserved lysine residues, Lys-113 and Lys-159, in the MH1 domain. We found that the mutations at Lys-113 and Lys-159 did not alter the ability of Smad4 to form a complex with Smad2 and FAST on the Mix.2 promoter. Importantly, SUMO-1 overexpression enhanced TGF-␤-induced transcriptional responses. These findings identify sumoylation as a unique mechanism to modulate Smad4-dependent cellular responses.Transforming growth factor-␤ (TGF-␤) 1 is a secreted multifunctional protein that exhibits a diverse set of cellular responses including cell proliferation and differentiation. TGF-␤ functions as a potent growth inhibitor and induces the expression of a variety of genes during growth and development (1-4).TGF-␤ signals are transduced by transmembrane serine/threonine kinase receptors and intracellular effectors called Smads (2,5,6). Upon TGF-␤ stimulation, Smad2 and/or Smad3 are phosphorylated by the activated type I receptor and then form complexes with Smad4. The heteromeric complexes of R-Smads and Smad4 then are translocated into the nucleus, where they exert ligand-induced changes in the transcription of a variety of genes (2, 6, 7). The heteromeric Smad complex activates transcription through its ability to cooperate functionally with several promoter-specific transcription factors and/or to bind specific DNA sequences (7,8).Proper TGF-␤ signaling requires precise control of Smad functions. Recent studies have shown that R-Smads are modified post-translationally by ubiquitin and can be removed irreversibly by the proteasome-mediated degradation system (9 -13). A number of ubiquitin-related proteins also are present in eukaryotic cells. These proteins, including the small ubiquitinlike modifier-1 (SUMO-1), utilize a conjugation system that is similar to ubiquitination (14 -16). SUMO-1 uses Aos1/Uba2 as E1 activating enzymes (17, 18) and Ubc9 as an E2 conjugating enzyme (19,20). Unlike ubiquitination, SUMO-1 modifications of target proteins do not promote their degradation. In contrast, SUMO-1 modifications of I B preclude its ubiquitination (21). Functional consequences of SUMO-1 modification vary dep...

Medea SUMOylation restricts the signaling range of the Dpp morphogen in the Drosophila embryo

“…We next used the in vitro SUMOylation assay (Desterro et al 1998) to investigate Med as a SUMOylation target. Med is SUMO modified in vitro, yet mutation of the two amino acids-K113, K159 (MedAB)-equivalent to those shown to be necessary for SUMOylation of Smad4 (Lin et al 2003a) does not abolish Med SUMOylation (Fig. 2B,D).…”

“…As Smad4 has been shown to be SUMOylated (Lin et al 2003a), we tested whether Drosophila Med is also a target. First, we used the yeast two-hybrid assay to investigate a possible interaction between Lwr and Med.…”

“…During C. elegans development, SUMOylation of the Polycomb group repressor protein SOP-2 is necessary for it to repress Hox genes (Zhang et al 2004), whereas modification of the Drosophila gypsy insulator may influence the establishment of chromatin domains (Capelson and Corces 2006). Smad4, the vertebrate ortholog of Med (Wisotzkey et al 1998), is SUMO modified in tissue culture cells, resulting in either activation (Lin et al 2003a) or repression (Long and Griffith 2000) of its activity depending on the reporter analyzed. Although SUMOylation activates some transcription factors, it is more commonly associated with transcriptional repression.…”

Regulating the stability of TGFβ receptors and Smads

“…In addition to being poly-ubiquitinated and degraded by proteasomes, Smad4 has also been shown to be mono-ubiquitinated and sumoylated [57,[63][64][65][66]. Monoubiquitination of Smad4 occurs on Lys-507 in the MH2 domain and leads to improved transcriptional TGFb signaling [63], but also promotes export of Smad4 to the cytoplasm [67].…”

To (TGF)β or not to (TGF)β: Fine-tuning of Smad signaling via post-translational modifications

“…Smad4 is modified by SUMO-1 on lysines 159 and 113 in/near the MH1 domain (Figure 2), and this SUMOylation has been shown to require the PIAS (Protein Inhibitors of Activated STAT) family member PIASy [72], PIAS1 [73,74] or PIASxβ [74] as an E3 ligase. Smad4 SUMOylation has largely been shown to increase its stability in the nucleus and/or decrease its nuclear export, ultimately stimulating TGF-β signaling [72,[74][75][76]. However, SUMOylation has also been shown to decrease Smad4s transcriptional activity in some reporter assays [77], and the transcriptional co-repressor Daxx can suppress Smad4-mediated transcriptional activity by directly interacting with SUMOylated-Smad4 in a lys159 (but not lys113) SUMO dependent manner [78].…”