Itch E3 Ligase-Mediated Regulation of TGF-β Signaling by Modulating Smad2 Phosphorylation

Abstract

Protein ubiquitination has been implicated in the intracellular biochemical events transduced by TGF-beta receptor via different mechanisms including the degradation of Smads or their binding proteins. Here we show that loss of Itch E3 ligase in mouse embryonic fibroblasts (MEFs) results in reduced susceptibility of TGF-beta-induced cell growth arrest and decreased phosphorylation of Smad2, without apparent alteration in protein levels for Smad2, Smad4, and Smad7 in Itch-/- MEFs. Itch promotes ubiquitination of Smad2 and augments Smad2 phosphorylation that requires an intact ligase activity of Itch. Moreover, Itch facilitates complex formation between TGF-beta receptor and Smad2 and enhances TGF-beta-induced transcription. This study reveals a previously unrecognized positive TGF-beta signaling pathway via proteolysis-independent ubiquitination.

Ubiquitin E3 Ligase Itch Negatively Regulates Osteoclast Formation by Promoting Deubiquitination of Tumor Necrosis Factor (TNF) Receptor-associated Factor 6

“…Ubiquitinated proteins were blotted with anti-TRAF6 antibody. phogenetic protein (40) and TGF␤ signaling proteins (41). Consequently, increased osteoblastic bone formation compensates for increased osteoclastic bone resorption in Itch Ϫ/Ϫ mice.…”

Regulating the stability of TGFβ receptors and Smads

“…All inhibitory arrows represent poly-ubiquitination followed by proteasomal degradation. this pathway due to its ability to promote TbRI-Smad2 association, ubiquitination of Smad2 and receptormediated phosphorylation of Smad2 [51]. On the basis of this mechanism, AIP4/Itch ubiquitinates Smad2 in a manner that promotes its C-terminal phosphorylation by the type I receptor.…”

Regulation of Smad activities

“…In embryonic fibroblasts isolated from mice with null deletion of Itch, a gene encoding an E3 ligase, the sensitivity to TGF-β stimulation was much diminished, as evidenced by decrease in R-Smad phosphorylation and nuclear translocation [94]. The most imminent issue is whether the pro-TGF-β function of Itch is direct, perhaps through ubiquitination of Smads or TGF-β receptor kinases.…”

“…The most imminent issue is whether the pro-TGF-β function of Itch is direct, perhaps through ubiquitination of Smads or TGF-β receptor kinases. Although in vitro Itch can ubiquitinate Smad2 directly, whether this is the case in vivo and how such ubiquitination benefits Smad2 phosphorylation by TGF-β still needs to be verified [94].…”