NEDD4-2 (neural precursor cell expressed, developmentally down-regulated 4-2) negatively regulates TGF-β (transforming growth factor-β) signalling by inducing ubiquitin-mediated degradation of Smad2 and TGF-β type I receptor

Abstract

Inhibitory Smad, Smad7, is a potent inhibitor of TGF-β (transforming growth factor-β) superfamily signalling. By binding to activated type I receptors, it prevents the activation of R-Smads (receptor-regulated Smads). To identify new components of the Smad pathway, we performed yeast two-hybrid screening using Smad7 as bait, and identified NEDD4-2 (neural precursor cell expressed, developmentally down-regulated 4-2) as a direct binding partner of Smad7. NEDD4-2 is structurally similar to Smurfs (Smad ubiquitin regulatory factors) 1 and 2, which were identified previously as E3 ubiquitin ligases for R-Smads and TGF-β superfamily receptors. NEDD4-2 functions like Smurfs 1 and 2 in that it associates with TGF-β type I receptor via Smad7, and induces its ubiquitin-dependent degradation. Moreover, NEDD4-2 bound to TGF-β-specific R-Smads, Smads 2 and 3, in a ligand-dependent manner, and induced degradation of Smad2, but not Smad3. However, in contrast with Smurf2, NEDD4-2 failed to induce ubiquitination of SnoN (Ski-related novel protein N), although NEDD4-2 bound to SnoN via Smad2 more strongly than Smurf2. We showed further that overexpressed NEDD4-2 prevents transcriptional activity induced by TGF-β and BMP, whereas silencing of the NEDD4-2 gene by siRNA (small interfering RNA) resulted in enhancement of the responsiveness to TGF-β superfamily cytokines. These data suggest that NEDD4-2 is a member of the Smurf-like C2-WW-HECT (WW is Trp-Trp and HECT is homologous to the E6-accessory protein) type E3 ubiquitin ligases, which negatively regulate TGF-β superfamily signalling through similar, but not identical, mechanisms to those used by Smurfs.

Regulating the stability of TGFβ receptors and Smads

“…This effect is due to the ability of Hsp90 to bind and chaperone the receptors. Earlier work has also established that the [32,33]. Both proteins are structurally similar to the Smurfs and also work similarly to promote receptor downregulation, which results in decreased phosphorylation of R-Smads and negative regulation of TGFb-mediated transcription.…”

Degradation of the Tumor Suppressor Smad4 by WW and HECT Domain Ubiquitin Ligases

“…It has been recently demonstrated that Smad2 and I-Smads can interact not only with WW-HECT domain ligases of the Smurf family, but also with novel related proteins, named WWP1/Tiul1 and NEDD4 -2 (11,12,37). These E3 ligases are larger in size compared with Smurfs and contain more WW domains than Smurfs (Fig.…”

“…A second WW-HECT domain E3 ligase, Tiul1/WWP1, forms complexes with Smad2 after TGF-␤ stimulation via the Smad2-interacting transcriptional co-repressor TGF-␤-induced factor, and leads to Smad2 polyubiquitination and down-regulation of TGF-␤ signaling (11). Similar to WWP1, another E3 ligase that contains WW and HECT domains, NEDD4 -2, interacts with Smad2 and induces its poly-ubiquitination and degradation (12). In contrast, Smad3 is down-regulated by the Roc1⅐SCF ubiquitin ligase complex (13).…”

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

“…Whilst studies concur that WWP1/Tiul1 alone does not induce the degradation of R-Smads, but rather TβRI (see section 7), to negatively regulate TGF-β-signaling [52,53], Seo et al reveal that WWP1/Tiul1 can interact with the TGF-β/Smad transcriptional repressor TGIF to induce ubiquitindependent degradation of Smad2 [53]. Intriguingly NEDD4-2, like Smurf2, was found to bind to Smad2/3 in a ligand dependent manner, but only to induce degradation of Smad2 [54]. Finally, of particular interest, Itch provides a rarer example of how a R-Smad E3 ligase can positively regulate TGF-β signaling, as it appears to enhance Smad2 SXS phosphorylation [55].…”

“…Thus, this is an interesting situation in which Smad7, and presumably Smad6, undergoes post-translational modification itself, whilst working as an adaptor to enable Smurfs to initiate the ubiquitination of TGF-β superfamily receptors and ultimately the termination of signaling upstream of R-Smads. Like Smad2, TβRI can also be degraded by the Smurf-like proteins NEDD4-2 [54] and WWP1/ Tiul1 [52,53]. Similar to Smurfs, WWP1/Tiul1 associates with Smad7 to induce its nuclear export and enhance Smad7-TβRI association, and thus ubiquitination and degradation of the receptor [52,53].…”