The type I TGF-β receptor TβRI is sumoylated. (a) TβRI, but not TβRII, is sumoylated. Lysates of COS cells, expressing Flag-tagged TβRI or TβRII and myc-tagged SUMO-1, were subjected to immunoprecipitations using anti-Flag, followed by western blotting with anti-myc to detect sumoylated TGF-β receptors. (b) Increasing expression of Ubc9 enhances TβRI sumoylation. COS cells, expressing Flag-tagged TβRI, myc-tagged SUMO-1 and increasing levels of Ubc9, were lysed and subjected to immunoprecipitation, followed by western blotting with the indicated antibodies. (c) In vitro sumoylation of TβRI. Immunopurified Flag-tagged TβRI was incubated with or without recombinant SUMO-1, the E1 enzyme Aos1/Uba2, and the E2 conjugating enzyme Ubc9. The reaction mixture was analyzed by western blotting with anti-Flag. (d) TGF-β induces sumoylation of endogenous TβRI. Lysates of Mv1Lu or MDA-231 cells, treated with or without TGF-β, were immunoprecipitated with anti-TβRI, and immunoblotted with antibody against SUMO-1. (e) TβRI, but not other type I receptors, is sumoylated. 293T cells ectopically expressing the indicated type I receptor, myc-tagged SUMO-1 and Ubc9, were lysed, and sumoylation was analyzed by western blotting.

The kinase activities of TβRI and TβRII are required for TβRI sumoylation. (a) Activated TβRI is more sumoylated than wild-type TβRI. In vitro sumoylation of immunopurified Flag-tagged wild-type and activated (ca) TβRI in the presence or absence of recombinant SUMO-1, Aos1/Uba2 (E1), and Ubc9. The reaction mixture was analyzed by western blotting for TβRI. (b) Effects of the TβRI kinase inhibitor and TβRI dephosphorylation on TβRI sumoylation. In vitro sumoylation was performed as in (a) with wild-type or activated (ca) TβRI, as indicated, in the presence or absence of the TβRI kinase inhibitor SB431542. The phosphates were removed from TβRI using lambda phosphatase, prior to in vitro sumoylation. (c) The kinase activities of TβRII and TβRI are required for efficient TβRI sumoylation. 293T cells co-expressed a cytoplasmic receptor chimera TβRII-RI, in which the TβRI cytoplasmic domain follows the TβRII cytoplasmic domain, or chimeras in which the TβRII and/or TβRI kinase activities were inactivated by point mutation (KR), with myc-tagged SUMO-1 and Ubc9. The chimera sumoylation was analyzed by western blotting. (d) In vitro sumoylation of immunopurified cytoplasmic receptor chimeras or each of the kinase-defective receptor chimeras, used in panel (c). (e) Diagram showing TGF-β-induced sumoylation of TβRI in the receptor complex.

The TβRI receptor is sumoylated on lysine 389. (a) Mutation of Lys³⁸⁹ abolishes TβRI sumoylation. 293T cells expressed Flag-tagged wild-type or mutant TβRI with the indicated lysine-to-arginine mutation, with myc-tagged SUMO-1 and Ubc9. Cell lysates were subjected to immunoprecipitations using anti-Flag, followed by western blotting for SUMO-1. (b) In vitro sumoylation of wild-type TβRI or K389R TβRI. Immunopurified TβRI was subjected to in vitro sumoylation followed by immunoblotting to detect sumoylation. (c) Proposed structure of the TβRI cytoplasmic domain. The Lys³⁸⁹, L45 loop, GS region and ATP binding site are indicated. N and C represent the N- and C-termini. (d) Sequence alignment of the TβRI sequence containing Lys³⁸⁹ and corresponding regions of other type I receptors.

TβRI sumoylation regulates Smad activation and TGF-β responses. (a) Interaction of Smad3 with TβRI. 293T cells were transfected to co-express activated (ca) TβRI or its K389R mutant, with myc-SUMO-1 and Ubc9, and/or Smad3D407E. The lyates were subjected to immunoprecipitation, and analyzed by western blotting. (b) In vitro interaction of Smad3 with TβRI. Immobilized GST or GST-Smad3(D407E) were incubated with in vitro sumoylated and non-sumoylated Flag-tagged TβRI. Adsorbed proteins were subjected to western blotting for TβRI. The lower panel shows Coomassie blue staining of GST and GST-Smad3(D407E) used for the adsorption. (c) Tgfbr1^−/− MEFs stably expressing wild-type or K389R TβRI, or transfected with an empty vector, were subjected to western blotting to assess the expression of TβRI. (d) Biotin-labeled cell surface proteins from the indicated MEFs were subjected to avidin precipitation. Precipitates were analyzed by western blotting to assess the cell surface expression levels of TβRI. (e) Wild-type and K389R TβRI have similar kinase activities. Wild-type or K389R TβRI were expressed in 293T cells, immunopurified and subjected to kinase reactions in the presence or absence of TβRI kinase inhibitor. (f, g) Lack of TβRI sumoylation confers a lower level of Smad3 (f) or Smad2 (g) activation. Tgfbr1^−/− MEFs stably expressing wild-type or K389R TβRI were treated without or with TGF-β for the indicated time. The cell lysates were analyzed by western blotting. (h) Lack of TβRI sumoylation confers a lower level of Smad3-mediated transcription. Tgfbr1^−/− fibroblasts stably expressing wild-type TβRI or K389R mutant TβRI were transfected with the Smad3-responsive (CAGA)₁₂-luciferase reporter. Luciferase activities without or in response to added TGF-β were measured. The error bars represent mean ± s.d. (n = 2) (i) Lack of TβRI sumoylation decreases TGF-β-induced endogenous gene expression. Tgfbr1^−/− fibroblasts stably expressing wild-type TβRI or K389R mutant TβRI were treated with or without added TGF-β. Smad7 mRNA was quantified using real-time PCR and normalized to RPL19 mRNA expression, which is not affected by TGF-β. The error bars represent mean ± s.d. (n = 3) (j) Lack of TβRI sumoylation confers a lower level of TGF-β-induced growth inhibition. Tgfbr1^−/− fibroblasts stably expressing wild-type or K389R TβRI, or with an empty vector, were cultured without or with the indicated dose of added TGF-β for 3 days. The cell numbers were then counted. The error bars represent mean ± s.d. (n = 3). Full scans of a and f are shown in Supplementary Information, Fig. S4.

Lack of TβRI sumoylation decreases TGF-β-regulated invasion and metastasis. (a) Ras-transformed Tgfbr1^−/− fibroblasts stably expressing wild-type or K389R TβRI, or with an empty vector, were subjected to western blotting for TβRI, Ras or phospho-ERK1/2 as marker of Ras activation. (b, c) TβRI-mediated TGF-β responsiveness of Ras-transformed cells promotes invasion, which is decreased by lack of TβRI sumoylation. Cells were seeded onto a Matrigel-coated Transwell filter and incubated for 24 h to allow invasion toward 10% serum. Cells that migrated through the filter were stained with crystal violet. The white stipples represent the pores in the filter. A representative picture and quantification of invaded cells are shown in panels b and c, respectively. Error bars represent mean ± s.d. (n = 4) (d, e) Ras-transformed Tgfbr1^−/− fibroblasts expressing wild-type or K389R TβRI, or with an empty vector, were injected into the tail vein of nude mice. The lung tumor nodules were counted after three weeks. (d) Representative pictures of lungs from mice with Ras-transformed MEFs are shown in upper panels, and corresponding H&E-stained sections of tumor nodules at the same magnification (×10 objective) are shown in the lower panels. (e) Quantification of tumor nodules in the lungs. The error bars represent mean ± s.e.m. (n = 6). The single and double asterisks indicate P < 0.05 and P < 0.01, respectively, compared to wild-type TβRI.

The Ser385Tyr mutation impairs TβRI sumoylation and function. (a, b) Ser385Tyr TβRI is not sumoylated. Panel (a) shows the rat TβRI sequence with Lys³⁸⁹ as sumoylation site four amino acids away from Ser³⁸⁵, which is equivalent to Ser³⁸⁷ in human TβRI. (b) 293T cells co-expressing wild-type, K389R or S385Y TβRI, with Myc-tagged SUMO-1 and Ubc9, were lysed and analyzed by western blotting to detect sumoylation. (c, d) In vitro sumoylation of S385Y mutant TβRI (c) or S385A TβRI (d) in comparison with wild-type TβRI. Immunopurified TβRI was subjected to in vitro sumoylation followed by immunoblotting to detect sumoylation. (e) Tgfbr1^−/− fibroblasts stably expressing wild-type, K389R or S385Y TβRI were transfected with the Smad3-responsive (CAGA)₁₂-luciferase reporter. Luciferase activities without or in response to added TGF-β were measured. Error bars represent mean ± s.d. (n = 3). (f) Representative pictures of lungs from mice with Ras-transformed MEFs are shown in upper panels, and corresponding H&E-stained sections of tumor nodules at the same magnification (×10 objective) are shown in lower panels. (g) Quantification of tumor nodules in the lungs. Error bars represent mean ± s.e.m. (n = 6). Double asterisk indicates P < 0.01 compared to wild-type TβRI.