Members of the transforming growth factor-beta (TGF-beta) family of peptide growth factors, which include TGF-beta, bone morphogenetic proteins (BMPs) and activins, regulate a broad range of cellular processes from cell growth and differentiation to apoptosis. The signaling responses to TGF-beta and other family members are mediated by a heteromeric complex of two types of transmembrane serine/threonine kinase receptors at the cell surface, and their intracellular substrates, the Smad proteins. Following ligand binding, the type II receptor kinases phosphorylate and thereby activate the type I receptor cytoplasmic domains. The Smads then act as type I receptor-activated signaling effectors, which, following receptor-induced phosphorylation, move into the nucleus to activate transcription of a select set of target genes. The activity of Smad proteins must be tightly regulated to exert the biological effects of different ligands in a timely manner. Any deregulation of these control mechanisms may therefore alter the cellular responses to the ligands and may result in malignant disease as well as abnormal development. Using a combination of yeast two-hybrid interaction assays and EST (expressed sequence tags) database searches, we have identified Smurf2(Smad ubiquitination regulatory factor2), a new member of the Hect family of E3 ubiquitin ligases, which interacts with receptor-activated Smads, and target them for ubiquitination and degradation. The interaction between Smurf and Smad are mediated by the WW domains of Smurf2 and a PPXY motif in the linker regions of the Smads. In order to gain insight of the physiological role of ubiquitin-proteasome-mediated degradation of Smad, we have constructed knockout targeting constructs that disrupt WW domains and Hect domains of both Smurf1 and Smurf2 and have transfected them into ES cells. We are currently in the processing of generating both Smurf1 and Smurf2 knockout mice. Smads are the only known TGF-beta receptor substrates and signal transducers so far, and they are central to most TGF-beta actions. However, numerous reports have indicated that TGF-beta may also elicit some of its effect through a Smad-independent pathway. To investigate the potential Smad-independent TGF-beta signaling pathway, we have constructed mutated TGF-beta type I receptors, which no longer bind Smads nor transmit Smad-dependent signals. We are currently generating stable cells by expressing the mutant receptors in a TGF-beta type I receptor deficient cell line. These cells will enable us to examine the function of the mutant receptors in controlling different aspects of TGF-beta-regulated responses.
