Volume 19, Issue 3, Pages 297-308 (August 2005) Regulation of Smurf2 Ubiquitin Ligase Activity by Anchoring the E2 to the HECT Domain  Abiodun A. Ogunjimi, Douglas J. Briant, Nadia Pece-Barbara, Christine Le Roy, Gianni M. Di Guglielmo, Peter Kavsak, Richele K. Rasmussen, Bruce T. Seet, Frank Sicheri, Jeffrey L. Wrana  Molecular Cell  Volume 19, Issue 3, Pages 297-308 (August 2005) DOI: 10.1016/j.molcel.2005.06.028 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 Smad7 Stimulates Smurf2 Ubiquitin Ligase Activity Both In Vivo and In Vitro (A) Smad7 expression decreases Smurf2 steady-state levels. HEK293T cells were transfected with either a constant amount of Smad7-HA and increasing amounts of Flag-Smurf2 (top panel), or a constant amount of Flag-Smurf2 and increasing amounts of Smad7-HA (bottom panel; plasmid DNA amount in μg). Aliquots of total lysates were immunoblotted to detect expression of Flag-Smurf2 and Smad7 (indicated). (B) HEK293T cells expressing Flag-Smurf2 WT either with or without Smad7 were labeled with 100 μCi [35S] methionine (pulse) and chased in regular media for the indicated times, at which point cell lysate was subjected to an anti-Flag immunoprecipitation followed by SDS-PAGE. The amount of Flag-Smurf2 was quantified with a Phosphorimager and is plotted as the percentage of Flag-Smurf2 relative to time 0. (C) Smad7 increases Smurf2 autoubiquitination in vivo. HEK293T cells were transfected with the indicated expression vectors and cell lysates subjected to immunoprecipitation with anti-Flag antibody, boiled in SDS, and then reprecipitated with anti-Flag antibody prior to immunoblotting with the indicated antibodies. (D) Smurf2 autoubiquitination is potentiated by Smad7 in vitro. In vitro ubiquitination reactions using bacterially produced WT Smurf2 together with the indicated combinations of E1, UbcH7 (E2), and Smad7 were analyzed by immunoblotting with anti-ubiquitin, anti-Smurf2, and anti-Smad7(N19) antibodies (“In vitro Reaction” panels). Equal protein amounts in the reactions were confirmed by immunoblotting an aliquot of the starting reaction with anti-Smurf2 or anti-Smad7 (“Totals” panels). Molecular Cell 2005 19, 297-308DOI: (10.1016/j.molcel.2005.06.028) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 The NTD of Smad7 Interacts with the Smurf2-HECT Domain and Stimulates Its Autocatalytic Activity and Degradation (A) Schematic representation of full-length Smad7 (WT) and the mutants used in this study. (B) The N-terminal domain of Smad7 is required for Smad7-dependent decreases in Smurf2 steady-state levels. Lysates from HEK293T cells expressing increasing amounts of Smad7-HA or Smad7 PY-MH2-HA in either the absence or presence of a constant amount of Flag-Smurf2, as indicated, were analyzed by immunoblotting to detect expression of Smurf2 and Smad7 using anti-Flag and anti-HA antibodies, respectively. (C) Smad7 enhances Smurf2 turnover through its NTD. HEK293T cells expressing either WT or CA (Cys716Ala) Flag-Smurf2, with either Smad7 WT or Smad7-PY-MH2, as indicated, were labeled with 100 μCi [35S] methionine (pulse) and chased in regular media for the indicated times. Labeled Flag-Smurf2 in an anti-Flag immunoprecipitation was quantified from SDS-PAGE gels (middle panels) with a Phosphorimager and is plotted as the percentage of Flag-Smurf2 relative to time 0 (top panel). A representative experiment from three experiments is shown. Expression of the overexpressed proteins was confirmed by immunoblotting with anti-HA and anti-Flag antibodies (lower panel). (D) Smurf2 autoubiquitination is enhanced by the Smad7 N-terminal domain. In vitro ubiquitination reactions of bacterially produced Smurf2 in the presence of the indicated combinations of WT and various mutants of Smad7 (NTD, NTD-PY, and PY-MH2) were analyzed by immunoblotting with anti-ubiquitin antibody (top panel). Protein levels were confirmed by immunoblotting an aliquot of the starting reaction with anti-Smurf2 antibody (middle panel) or anti-Smad7 antibody (N19) and anti-HA antibody (lower panel). Molecular Cell 2005 19, 297-308DOI: (10.1016/j.molcel.2005.06.028) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Smad7 Binds and Recruits UbcH7 to Smurf2 via Its NTD (A) The Smad7 NTD interacts with the HECT domain of Smurf2. Equivalent levels of indicated GST fusion proteins were incubated with WT, PY-MH2 and NTD Smad7 expressed in cells, affinity purified, and bound Smad7 detected by immunoblotting. Equivalent protein levels were assessed by immunoblotting aliquots of total cell lysates with the appropriate antibody. (B) Smad7 NTD binding to Smurf2 is dependent on the HECT domain. Full-length or the NTD of Smad7 were incubated with GST, GST-Smurf2 or GST-Smurf2(ΔHECT) and bound Smad7 detected by immunoblotting with an anti-Smad7 antibody (α-N19). Total protein levels were confirmed by immunoblotting binding reactions. (C) UbcH7 interaction with Smurf2 is enhanced by Smad7. Bacterially expressed proteins were incubated at 4°C with increasing concentrations of UbcH7 in the absence (-) or presence of WT Smad7 and either the PY-MH2 (left panel) or NTD-PY (right panel), as indicated. Smurf2 was immunoprecipitated (α-Smurf2 IP) and then immunoblotted to detect anti-UbcH7 (top panel). The levels of proteins in the starting material were confirmed by immunoblotting with respective antisera (“Totals” panels). (D) Interaction of UbcH7 with Smad7 requires the NTD. Bacterially expressed GST-UbcH7, wild-type (WT) and the indicated mutants of Smad7 were incubated at 4°C, purified and then immunoblotted with Smad7 antisera (N19) or anti-HA antibodies to detect bound Smad7. The levels of proteins in the starting material were confirmed by immunoblot (“Totals” panels). Molecular Cell 2005 19, 297-308DOI: (10.1016/j.molcel.2005.06.028) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 UbcH7 Interaction with a Leucine-Rich Repeat in the NTD of Smad7 (A) Identification of the LRM. A filter array of 12 amino acid peptides that walk along the Smad7 NTD-PY (amino acids 1–220) at 2 amino acid intervals was probed with UbcH7. Spot B22 (arrow; LRM) reproducibly bound UbcH7. (B) Alanine scanning mutagenesis of the LRM. LRMs with alanine substitutions (bold, italic) were synthesized and probed with UbcH7. Residues essential for UbcH7 binding are indicated (bottom arrows), as is the mutation corresponding to L108A (left arrow). (C and D) Smad7(L108A) interferes with binding to UbcH7 but not to the Smurf2 HECT. The indicated GST fusions of Smad7 were incubated with UbcH7 (C) and UbcH7 bound to Smad7 was assessed by affinity purification and immunoblotting (top panels). In (D), WT Smad7 or Smad7(L108A) bound to the GST-Smurf2 HECT domain was visualized by immunoblotting with antibody to Smad7. Equal amounts of the corresponding protein were confirmed in (C) and (D) by blotting as indicated (Totals). (E) Smad7(L108A) does not stimulate Smurf2 activity. In vitro autoubiquitination reactions in the presence of Smad7 or Smad7(L108A), as indicated, were analyzed for autoubiquitinated Smurf2 (α-ubiquitin). Equivalent levels of Smurf2 and Smad7 were confirmed by immunoblotting aliquots of the reaction (totals). (F) The L108A mutation interferes with Smad7-dependent inhibition of TGFβ signaling. The TGFβ reporter, ARE-Luc, was cotransfected with FoxH1, β-galactosidase control and increasing amounts of either WT Smad7 or Smad7(L108A) (α-Smad7 immunoblot, lower panel) and were left untreated (white bar) or treated (black bar) with TGFβ for 16 hr. Luciferase activity, normalized to β-galactosidase, is plotted as the mean of triplicates ± standard deviation from a representative experiment. Molecular Cell 2005 19, 297-308DOI: (10.1016/j.molcel.2005.06.028) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 Crystal Structure of Smurf2 HECT Domain (A) Overall structure of Smurf2 HECT domain with modeled UbcH7 (yellow). (Ai) The E2, UbcH7 was docked on the Smurf2 HECT domain based on E6AP-UbcH7 complex structure. Smurf2 HECT domain revealed an open L-shaped conformation for the two lobes, N- and C-terminal. The N-terminal lobe (residues 369–624) consists of two subdomains: the large subdomain in red (residues 369–514 and 597–624) and the small subdomain that contains the E2 binding groove in pink. The two subdomains of the N-terminal lobe are connected by short linker sequences between residues 515–517 and 593–596. The C-terminal lobe (residues 628–741, colored blue) contains the catalytic Cys-716. The structure of E6AP-UbcH7 complex with the C lobe (blue) and the N lobe (red and pink) is shown in panel (Aii). In panel (Aiii), the structure of WWP1 with modeled UbcH7 revealed a T-shaped conformation of WWP1 with the N lobe colored red and pink and the C-lobe in blue. (B) Superimposed ribbon diagrams of Smurf2 HECT (red-pink-blue) showing the motion of the C lobe (blue) and the E2 binding lobe (pink) about the hinge region when compared to (Aii) E6AP (cyan) and (Aiii) WWP1 (cyan). Schematics of the superimposed structures are represented to the right of each ribbon diagram. Note the rotation of the E2 binding lobe by 24.1° and the translation of the C lobe by 13 Å when compared to E6AP (i). Molecular Cell 2005 19, 297-308DOI: (10.1016/j.molcel.2005.06.028) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 6 Stereoview of E2 Binding Groove of Smurf2 HECT Ubiquitin Protein Ligase (A) Close-up view of the suboptimal interface between the UbcH7 L1 and L2 loops with the Smurf2 HECT domain E2 binding groove. The interacting secondary structural elements of the groove are colored pink and the modeled UbcH7 loops are colored yellow. The structural features, as well as the amino acids that are making contact in the groove are indicated in the corresponding color. In green are the side-chains of the residues that contribute to the interaction between the E2 and the E2 binding pocket of the HECT domain. Note that in Smurf2, His547, and Tyr581 (which, in E6AP, are Iso and Phe, respectively), interfere with the hydrophobic character of the pocket that contacts Phe63 in UbcH7. (B) Alignment of UbcH7 interacting loops L1 and L2 sequences with the corresponding regions of UbcH5c and UbcH8, the two other E2s that are known to function with the HECT domain E3 ligases. Residues on the loops that make contact with E2 binding hydrophobic groove on the HECT domain are shown in the blue boxes and are conserved. Molecular Cell 2005 19, 297-308DOI: (10.1016/j.molcel.2005.06.028) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 7 Functional Analysis of Smurf2 E2 Binding Pocket (A) In vitro ubiquitination assay with E2 binding mutants. Autoubiquitination assays using bacterially produced wild-type (WT) Smurf2 or the E2 binding mutants, Trp535Ala (WA), Trp535Asp (WD), Tyr581Ala (YA), Tyr581Phe (YF), His547Ala (HA), or His547Ile (HI) were analyzed by immunoblotting with anti-ubiquitin antibody and with anti-Smurf2 antibody (“In vitro Reaction” panels). Equivalent levels of Smurf2 were confirmed by immunoblotting the starting material (“Totals”). (B) In vitro activation of Smurf2 mutants by Smad7. Bacterially produced WT Smurf2 and E2 binding mutants (WA, WD, YF, YA, HA, and HI) were incubated with or without Smad7 in ubiquitination buffer and analyzed as in 7A above. (C) In vivo activity of Smurf2 E2 binding pocket mutants on TGFβ-dependent activation of transcription. Luciferase activity (normalized to an internal β-galactosidase control) was measured in untreated (white) or TGFβ-treated (black) HepG2 cells transfected with the 3TP-Luc reporter and 12.5 ng or 25 ng, of either wild-type Smurf2, or the indicated mutants and is plotted as the mean ± SD of triplicates from a representative experiment. (D) HEK293T cells were transfected with TβRI and TβRII alone or in the presence of Smad7 and the indicated Smurf2 mutants. Cells were then affinity labeled with 250 pM [125I] TGFβ at 4°C, incubated at 37°C for the indicated times, and receptor complexes were visualized by autoradiography (top panels). Three experiments were quantitated and are plotted in the graph as the mean ± SD TβRI receptor level normalized to time 0. Protein expression was confirmed by immunoblotting total cell lysates (bottom panels). Molecular Cell 2005 19, 297-308DOI: (10.1016/j.molcel.2005.06.028) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 8 Model of Smurf2 HECT Domain Activation by Smad7 TGFβ receptor complexes (shown in blue) bind the MH2 domain of Smad7 (purple), recruits Smurf2 (green), and through its NTD can recruit E2s (red) to the HECT via the LRM. Molecular Cell 2005 19, 297-308DOI: (10.1016/j.molcel.2005.06.028) Copyright © 2005 Elsevier Inc. Terms and Conditions