Controlled proteolysis mediated by Smad ubiquitination regulatory factors (Smurfs) plays a crucial role in modulating cellular responses to signaling of the transforming growth factor-␤ (TGF-␤) superfamily. However, it is not clear what influences the selectivity of Smurfs in the individual signaling pathway, nor is it clear the biological function of Smurfs in vivo. Using a mouse C2C12 myoblast cell differentiation system, which is subject to control by both TGF-␤ and bone morphogenetic protein (BMP), here we examine the role of Smurf1 in myogenic differentiation. We show that increased expression of Smurf1 promotes myogenic differentiation of C2C12 cells and blocks the BMP-induced osteogenic conversion but has no effect on the TGF-␤-induced differentiation arrest. Consistent with an inhibitory role in the BMP signaling pathway, the elevated Smurf1 markedly reduces the level of endogenous Smad5, whereas it leaves unaltered that of Smad2, Smad3, and Smad7, which are components of the TGF-␤ pathway. Adding back Smad5 from a different source to the Smurf1-overexpressing cells restores the BMP-mediated osteoblast conversion. Finally, by depletion of the endogenous Smurf1 through small interfering RNA-mediated RNA interference, we demonstrate that Smurf1 is required for the myogenic differentiation of C2C12 cells and plays an important regulatory role in the BMP-2-mediated osteoblast conversion.Smad proteins are principal intracellular signaling mediators of the TGF-␤ 1 superfamily of peptide growth factors that regulate a wide range of biological processes (1-3). As such, controlling Smad activity and protein levels are crucial for proper signaling by TGF-␤ and its related factors. Recent discoveries of Smad ubiquitination regulatory factors (Smurfs), which are members of the HECT domain family of ubiquitin ligases, have implicated a role of the ubiquitination-mediated protein degradation in the signaling pathways of TGF-␤ superfamily (4 -7). Ubiquitination is a covalent attachment of a chain of the highly conserved, 76-amino acid ubiquitin to selected targets, which are subsequently degraded in the 26 S proteasomes (8, 9). This process is catalyzed by sequential actions of three enzymes, the E1 ubiquitin activating enzyme, the E2 ubiquitin-conjugating enzyme, and the E3 ubiquitin ligase. Ubiquitin ligases physically interact with protein substrates, therefore playing a central role in determining substrate specificity. Despite several reports of biochemical evidence for the Smurf-mediated degradation of Smads, important questions remain with regard to the biological significance of this regulation and the specificity of Smurf action.Signaling by the TGF-␤ superfamily of peptide growth factors is mediated by a complex of two types of transmembrane serine/threonine kinase receptors and the intracellular Smad proteins (for reviews, see Refs. 1-3). Three classes of Smads have been defined based on their differences in sequence and function: the receptor-regulated Smads (R-Smads), the common Smad (Smad4), and the inhib...