Xenbase Image ID: 147667

Figure 5. Ectodermin is a Ubiquitin Ligase for Smad4 (A) The biological activity of Ectodermin relies on an enzymatically active RING finger. Two-cell-stage embryos were injected in the marginal zone with mRNAs (800 pg) encoding wild-type or RING-finger point mutant (EctoCAmut) Ecto proteins and processed by in situ hybridization for paraxial mesoderm marker MyoD. On the right is Western blotting of embryo lysates showing similar protein levels. (B) Expression of Ectodermin decreases the steady-state level of Smad4 via the proteasome pathway. Xenopus embryos were radially injected with 200 pg mRNAs coding for Flag-tagged Smads together with 1 ng of mRNAs for β-gal (−) or xEcto (+). Steady-state protein levels were determined by Western blotting of total embryo lysates. Lanes 7 and 8: mRNAs were coinjected with the proteasome inhibitor MG132 (10 μM/embryo volume). Note that the inhibition of proteasome reverts the effect of Ectodermin on Smad4. (C) Overexpression of Ectodermin causes an increased rate of Smad4 protein degradation. HEK293T cells were transfected with HA-tagged Smad4 expression plasmid alone (1 μg/10 cm dish) or in combination with human or Xenopus Ectodermin vectors (10 μg). After labeling with 35S-Met/Cys and chasing for the indicated times, cell lysates were immunoprecipitated with anti-HA antibody and labeled proteins were resolved by SDS-PAGE and visualized by autoradiography. (D) Endogenous Smad4 protein is downregulated by raising the level of Ectodermin in human cells. HEK293T cells were transfected with pCS2-hEcto (lanes 2 and 5: 7 μg; lanes 3 and 6: 20 μg) and treated for 6 hr with vehicle (DMSO, lanes 1–3) or 30 μM MG132 (lanes 4–6), and Smad4 steady-state levels were determined by Western blotting. Note that downregulation of Smad4 by hEcto (compare lanes 1–3) is inhibited in lanes 5 and 6. (E) Ectodermin enhances ubiquitination of Smad4. HEK293T cells were transfected with combinations of pCS2-HA-ubiquitin (1 μg), pCDNA-Smad4 (1 μg), and pCS2-hEcto (15 μg). Cells were harvested after overnight treatment with 10 μM MG132, and lysates were boiled in 1% SDS before immunoprecipitation with anti-Smad4 polyclonal antibody in 0.1% SDS. Ubiquitin-conjugated Smad4 [(Ub)n-Smad4] was detected by Western blotting with anti-Smad4 monoclonal antibody. The panel is composed by three parts (>120 KDa, 84–120 KDa, and free Smad4 at the bottom) corresponding to different exposition times. Plasmid doses are for transfection of 10 cm petri dish. (F) Ectodermin ubiquitin ligase activity depends on a catalytically active RING finger. Experimental settings were as described in (E). (G) The endogenous machinery that ubiquitinates Smad4 in Xenopus relies on Ectodermin. Two-cell-stage embryos were injected with the indicated combinations of morpholinos (60 ng) and mRNAs (HA-ubiquitin, 1 ng; Flag-Smad4, 1 ng) and harvested at the gastrula stage for immunoprecipitation with anti-Flag antibody. Ubiquitin-conjugated Smad4 [(Ub)n-Smad4] was detected by Western blotting with anti-HA antibody (upper IP panel). Equal levels of precipitated free Smad4 were checked by anti-Flag immunoblotting (lower IP panel). At the bottom, anti-Ecto and anti-actin Western blots of total lysates used for IPs in lanes 3 and 4. (H) Depletion of Ectodermin increases the steady-state levels of injected XSmad4α in Xenopus embryos. Increasing doses of Myc-tagged XSmad4α mRNA were coinjected with 50 ng of control-MO (−) or Ecto-MO (+). Embryos were harvested at the gastrula stage for Western blotting. Epitope-tagged Smad4 was used because antibodies against Xenopus Smad4 are not available. (I) Ectodermin knockdown in human cells rescues the stability of cancer-derived mutant Smad4(R100T). HEK293T cells were transfected with Flag-tagged Smad4(R100T) expression plasmid (4 μg/10 cm dish) and treated as follows. Lane 1: cotransfection of control-MO; lane 2: cotransfection of hEcto-MO; lanes 3 and 4: treatment with vehicle (DMSO) or MG132. Western blotting of total cell lysates shows that depletion of Ecto, just like inhibition of the proteasome, raises Smad4(R100T) level. Image published in: Dupont S et al. (2005) Copyright © 2005. Image reproduced with permission of the Publisher, Elsevier B. V. Larger Image Printer Friendly View

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