MOLECULAR MECHANISMS OF TGF-BETA SIGNALING PATHWAY

肽生长因子的转化生长因子-β（TGF-β）家族的成员，包括TGF-β、骨形态发生蛋白（BMP）和激活素，调节从细胞生长、分化到凋亡的广泛的细胞过程。 对TGF-β和其他家族成员的信号传导应答由细胞表面的两种类型的跨膜丝氨酸/苏氨酸激酶受体及其细胞内底物Smad蛋白的异聚复合物介导。 配体结合后，II型受体激酶磷酸化，从而激活I型受体胞质结构域。 然后，Smads作为I型受体激活的信号效应物，在受体诱导的磷酸化之后，其移动到细胞核中以激活一组选定的靶基因的转录。 Smad蛋白的活性必须受到严格调控，以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失调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。 最近的一些报道表明，Smads进行泛素-蛋白酶体介导的降解。 使用酵母双杂交相互作用试验与Smad 3作为诱饵和EST（表达序列标签）数据库搜索的组合，我们已经确定了Smurf 2（Smad泛素化调节因子2），Smurf 1相关成员的Hect结构域E3连接酶。 我们证明，Smurf 2与受体调节的Smads，即Smad 1，Smad 2和Smad 3相互作用，但不与共同的调解人Smad 4。 Smurf 2优先靶向Smad 1进行泛素化和降解，对Smad 2蛋白水平的影响要弱得多，但根本不影响Smad 3水平。 非洲爪蟾胚胎中Smurf 2水平的增加通过降低Smad 1信号传导深刻影响腹侧中胚层的形成。 这些发现表明，Smurf 2调节细胞和/或发育生物体对TGF-β超家族信号做出反应的能力。 为了深入了解泛素-蛋白酶体介导的Smad降解的生理作用，我们目前正在与邓初夏（NIDDK）合作制备Smurf 1和Smurf 2基因敲除小鼠。Smad信号通路的不同步骤也可能需要细胞核和细胞质中不同的蛋白质与Smads相互作用，从而在其信号通路中发挥重要作用。 使用组合的分子和生物化学方法，我们已经确定了几个候选蛋白，与不同的Smads。 我们目前正在分析这些候选蛋白在TGF-β/Smad信号通路中的功能。

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, 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. Several recent reports revealed that Smads undergo ubiquitin-proteasome-mediated degradation. Using a combination of yeast two-hybrid interaction assays with Smad3 as bait and EST (expressed sequence tags) database searches, we have identified Smurf2 (Smad ubiquitination regulatory factor 2), a Smurf1-related member of Hect domain E3 ligases. We demonstrate that Smurf2 interacts with receptor-regulated Smads, i.e. Smad1, Smad2 and Smad3, but not with the common mediator Smad, Smad4. Smurf2 preferentially targets Smad1 for ubiquitination and degradation, has a much weaker effect on Smad2 protein levels, but does not affect Smad3 levels at all. Increased Smurf2 levels in Xenopus embryo profoundly affects ventral mesoderm formation by decreasing Smad1 signaling. These findings suggest that Smurf2 regulates the competence of a cell and/or developing organism to respond to TGF-beta superfamily signals. In order to gain insight of the physiological role of ubiquitin-proteasome-mediated degradation of Smad, we are currently in the processing of generating both Smurf1 and Smurf2 knockout mice collaborating with Chuxia Deng (NIDDK).The different steps in Smad signaling pathway are also likely to require different proteins in the nucleus and cytoplasm that interact with Smads and in this way play important roles in their signaling pathway. Using combinational molecular and biochemical approaches, we have identified several candidate proteins that associate with different Smads. We are currently analyzing functions of these candidate proteins in the TGF-beta/Smad signal pathway.