FAM/USP9x, a deubiquitinating enzyme essential for TGFbeta signaling, controls Smad4 monoubiquitination.
The assembly of the Smad complex is critical for TGFbeta signaling, yet the mechanisms that inactivate or empower nuclear Smad complexes are less understood. By means of siRNA screen we identified FAM (USP9x), a deubiquitinase acting as essential and evolutionarily conserved component in TGFbeta and bone morphogenetic protein signaling. Smad4 is monoubiquitinated in lysine 519 in vivo, a modification that inhibits Smad4 by impeding association with phospho-Smad2. FAM reverts this negative modification, re-empowering Smad4 function. FAM opposes the activity of Ectodermin/Tif1gamma (Ecto), a nuclear factor for which we now clarify a prominent role as Smad4 monoubiquitin ligase. Our study points to Smad4 monoubiquitination and deubiquitination as a way for cells to set their TGFbeta responsiveness: loss of FAM disables Smad4-dependent responses in several model systems, with Ecto being epistatic to FAM. This defines a regulative ubiquitination step controlling Smads that is parallel to those impinging on R-Smad phosphorylation.
PubMed ID: 19135894
Article link: Cell.
Grant support: CA095875 NCI NIH HHS , CA095875 NCI NIH HHS , CA095875 NCI NIH HHS , CA095875 NCI NIH HHS , CA095875 NCI NIH HHS , CA095875 NCI NIH HHS
Genes referenced: otx2 smad2 smad4.1 smad4.2 szl t tgfb1 trim33 usp9x
Morpholinos referenced: smad4.1 MO1 smad4.2 MO1 smad4.2 MO2 usp9x MO1
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|Figure 5. FAM and Ecto Operate in the Same Pathway Regulating Smad4(A–L) Panels show in situ hybridizations on Xenopus embryos for the pan-mesodermal marker Xbra (at gastrula stage), for the ventral marker Sizzled and for the dorsoanterior marker Otx2 (at neurula stages). Embryos were microinjected either with morpholino antisense oligonucleotides (MOs), dominant-negative Smad4 mRNA (DN-Smad4), or Ecto mRNA.(M–R) Close-up views of Drosophila wings showing anterior (acv) and posterior (pcv) cross-veins.(N) Mutants for gbb, a BMP ligand, display missing cross-veins.(O and P) Ectopic expression of Ecto (O), but not of the Ecto RING mutant (Ecto-CAmut [P]), in the wing primordium causes loss of the cross-veins.(Q) Expression of Drosophila Fat facets (Faf) induces ectopic wing veins (red box).(R) Expression of Fat facets antagonizes Ecto, rescuing the formation of the cross-veins.(S and T) Ecto is epistatic to FAM. Panels in (S) show immunoblots of HaCaT cells transfected with the indicated combinations of siRNA. Ecto biological function is required downstream of FAM, as also revealed by TGFβ induced transwell migration assays in MDA-MB231 cells (T). Data are represented as mean and SD.|