Alignments of the predicted amino acid sequences of human Smurf1 and Smurf2. Identical amino acids are highlighted in black, the critical cysteine at position 716 is marked with an asterisk, and gaps are introduced to optimize the alignment. Conserved domains are underlined; C2 domain, dotted line; WW domains, broken line; Hect domain, solid line.

Physical interactions of Smurf2 with Smads. (A) Full-length Smurf2 interacts with Smad1, Smad2, and Smad3 in mammalian cells. COS-1 cells were transfected with expression plasmids for FLAG (F)-tagged full-size Smad1, Smad2, Smad3, or Smad4 or Myc (M)-tagged Smurf2, as marked. Cell lysates were subjected to anti-FLAG immunoprecipitation followed by anti-Myc immunoblotting. (Top) Coprecipitation of M-Smurf2 with F-Smad1, Smad2, or Smad3 is shown. The levels of F-Smads in the immunoprecipitates (Middle) and M-Smurf2 in total cell lysates (Bottom) are shown as indicated. (B) WW domains of Smurf2 (amino acids 248–369) interact with Smad1, Smad2, or Smad3. Lysates of COS-1 cells, transfected with F-Smad1, Smad2, Smad3, or Smad4 and/or HA-tagged WW domains of Smurf2 expression plasmids, were subjected to anti-FLAG immunoprecipitation followed by anti-HA immunoblotting to detect association of HA-Smurf2 (WW) with F-Smad1, Smad2, or Smad3 (Top). The levels of F-Smads in the immunoprecipitates (Middle) and H-Smurf2 (WW) in the total cell lysates (Bottom) are shown as indicated. (C) WW domains (amino acids 248–369) but not C2 or Hect domains of Smurf2 interact with Smad. COS-1 cells were transfected with HA-tagged Smurf2 (FL), HA-Smurf2 fragments (C2, WW, or Hect) and/or F-Smad2, as indicated. Immunoprecipitation with anti-HA antibodies was followed by anti-FLAG immunostaining to detect Smurf2-associated Smad2 (top). (Middle) The anti-HA immunoprecipitated Smurf2 or its fragments (indicated by arrowheads) is shown. (Bottom) The expression level of Smad2 is shown. (D) Yeast two-hybrid assays demonstrate the interaction of Smad1, Smad2, and Smad3, but not Smad4, with the WW domain segment, and the requirement of the linker (L) region and the PPXY sequence of Smads. Interactions were scored by measuring the β-galactosidase activity from − (negative) to +++ (strongly positive).

Effect of Smurf2 on Smad1 and Smad2 levels. Smurf2 expression results in a dramatic decrease of Smad1 protein level and a slight decrease of Smad2 protein level. COS-1 (A, D) or 293 (B, C) cells were transfected with the indicated mammalian expression plasmids. (A) Smurf2, but not the catalytic inactive mutant of Smurf2 (CG), decreases the Smad1 steady-state levels dramatically and the Smad2 steady-state levels slightly. Smurf2 does not decrease the Smad3 levels. Steady-state protein levels were determined by immunoblotting aliquots of the total cell lysates. (B) The decrease of Smad1 and Smad2 protein levels depends on the expression levels of Smurf2 and can be inhibited by lactacystin. Cells transfected with F-Smad1, HA-Smad2, and increasing amounts of Myc-Smurf2 expression plasmid were treated overnight without (Upper) or with (Lower) lactacystin before lysis of cells and immunostaining for steady-state protein levels. The amounts of Myc-tagged Smurf2 plasmid DNA used in transfections are shown in micrograms. (C) Smurf2 increases Smad1 turnover rate; 293 cells transfected with Smad1 and Smurf2 were pulse-labeled with [³⁵S]methionine and then chased for the indicated times. ³⁵S-labeled Smad1 in anti-Smad1 immunoprecipitates was detected by autoradiography of the gel and quantified by phosphorimaging and plotted relative to the amount present at time 0. (D) Ubiquitination of Smad1 and Smad2 in COS-1 cells in the presence of lactacystin. Cell lysates were subjected to anti-HA immunoprecipitation followed by immunoblotting to detect HA-ubiquitin-conjugated Smads. Multi-ubiquitinated species of Smads are indicated (F-Smad-(HA-Ub)n), whereas the lower band may represent an IgG band. Ubiquitination of Smad1 and Smad2 requires the activity of the Smurf2 Hect domain, as the Smurf2 C716G mutant (CG) does not induce ubiquitination of either Smad.

Smurf2 inhibits the biological functions of Smad1 in Xenopus embryo assays. (A) Alignments of the N-terminal amino acid sequences of Xenopus Smurf1 with Xenopus and human Smurf2. Identical amino acids are highlighted in black, and gaps are introduced to optimize the alignment. (B) Whole-mount in situ hybridization of xSmurf2 in Xenopus embryo. (Left) Gastrula stage; (Right) tailbud stage. (C) Effect of Smurf2 on marker gene expression. Animal poles of two-cell stage embryos were injected with 2 ng of mRNA of Smurf2 or Smurf2C716G (CG), 1 ng of Smad1 mRNA, 0.5 ng of Smad2 mRNA, or combinations of these as indicated. Animal caps were dissected at stage 9 and assayed at tadpole stage 32 by RT-PCR for the expression of the marker genes shown. (D) Comparison of the activities of Smurf2 and Smurf1. Two-cell stage embryos were injected with 2 ng of Smurf1 or Myc-Smurf2 mRNA, 1 ng of Smad1 mRNA, 0.5 ng of Smad2 mRNA plus 0.5 ng, 1 ng, or 2 ng of Smurf1 or Myc-Smurf2 mRNA. The two lanes on the far right in all panels are control reactions of total embryonic RNA, with (+) or without (−) reverse transcription.