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. 2005 Apr;25(8):3338–3347. doi: 10.1128/MCB.25.8.3338-3347.2005

FIG. 2.

FIG. 2.

Smad3 regulates Cdc25A degradation in a ubiquitination-dependent manner. (A) TGF-β1 treatment enhances physical interaction of Cdc25A and Smad3. U2OS cells without transfection were treated for 15 h with either 1 μM MG132 or 10 ng of TGF-β1/ml for 24 h plus 1 μM MG132 for the last 15 h (left panels). U2OS cells were also cotransfected with Cdc25A and Smad3 and then treated with MG132 between 9 and 24 h posttransfection (right panels). Cell lysates were subjected to immunoprecipitation by Smad3 antibody or normal control immunoglobulin G (NC), followed by immunoblotting with Cdc25A antibody. Asterisk, immunoglobulin heavy chain. (B) Smad3 can trigger proteasomal degradation of Cdc25A in a dose-dependent manner. U2OS cells were transfected with a constant amount of a Cdc25A plasmid with the CMV promoter and increasing amounts of a Smad3 plasmid with the CMV promoter. The amounts of each plasmid are shown as micrograms. Cells were treated with or without 1 μMMG132 between 9 and 24 h posttransfection, and cell lysates were prepared and immunoblotted for Cdc25A. (C) Smad3 overexpression does not affect Cdc25A mRNA levels. Cells at 24 h posttransfection were analyzed by RT-PCR. cDNA samples from cells with Cdc25A transfection (lanes 4 and 5) were diluted 1:10 for PCR amplification, to ensure semiquantitative analyses. RPS14, rRNA for internal control. (D) Overexpression of Smad3 increases ubiquitination of Cdc25A. U2OS cells were transfected as indicated and were treated with 1 μM MG132 between 9 and 24 h posttransfection. Cdc25A was immunoprecipitated and then immunoblotted with anti-His or anti-Ub antibody to detect ubiquitinated forms [Cdc25A-(Ub)n]. The expression level of transfected Cdc25A was examined by immunoblotting (bottom panel). (E) The MH2 domain of Smad3 is sufficient to trigger Cdc25A degradation. U2OS cells were either transfected with Cdc25A alone or cotransfected with either wt (wild type), SA (SSVS->AAVA mutant), or MH1- or MH2-domain-only Smad3 mutants. Levels of transfected proteins were determined by immunoblotting.