Molecular mechanisms of TGF-beta signaling pathway

转化生长因子- β (tgf - β)肽生长因子家族的成员，包括tgf - β、骨形态发生蛋白（BMPs）和激活素，调节从细胞生长、分化到细胞凋亡的广泛细胞过程。tgf - β和其他家族成员的信号反应是由细胞表面的两种跨膜丝氨酸/苏氨酸激酶受体及其细胞内底物Smad蛋白的异质复合物介导的。配体结合后，II型受体激酶磷酸化，从而激活I型受体细胞质结构域。然后，smad作为I型受体激活的信号效应器，在受体诱导的磷酸化之后，进入细胞核激活一组选定的靶基因的转录。必须严格调控Smad蛋白的活性，才能及时发挥不同配体的生物学效应。因此，这些控制机制的任何解除都可能改变细胞对配体的反应，并可能导致恶性疾病以及异常发育。利用酵母双杂交相互作用试验和EST（表达序列标签）数据库搜索，我们确定了Smurf2(Smad泛素化调节因子2)，这是E3泛素连接酶Hect家族的一个新成员，它与受体激活的Smad相互作用，并靶向它们的泛素化和降解。Smurf和Smad之间的相互作用是由Smurf2的WW结构域和Smad连接区域的PPXY基序介导的。为了深入了解泛素蛋白酶体介导的Smad降解的生理作用，我们构建了敲除靶向构建物，破坏Smurf1和Smurf2的WW结构域和Hect结构域，并将其转染到ES细胞中。我们目前正在培育Smurf1和Smurf2敲除小鼠。

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.