Smurf Ubiquitin Ligases in Xenopus Development

转化生长因子β超家族调控细胞的生长和分化。在非洲爪蛙发育过程中，Nodal、Vg1和激活素相关信号诱导内胚层和中胚层，BMP信号形成这些组织并调节外胚层细胞的命运。TGFbeta/激活素和BMP途径分别通过Smads 2和3或Smads1、5和8转导信号。关于TGFbeta信号下调的情况知之甚少。我们已经发现了两个E3泛素连接酶，S-1和S-2，它们分别抑制BMP和TGFβ/激活素通路(朱等人，1999，Kavsak等人，2000)。蓝精灵以Smads和受体为目标，进行泛素化和蛋白酶体降解。虽然S-1使胚胎细胞命运背靠背，但S-2在胚胎中的作用尚不清楚。我们假设蓝精灵通过区域特异性拮抗TGFbeta信号成分来调节胚胎发育中的组织诱导和模式形成。我们将研究SMurf1和SMurf2在胚胎发育过程中的作用，并探讨SMurf调控发育所必需的生化机制。深入了解蓝精灵的功能将产生关于TGFbeta信号和早期发育的重要信息，并将解决出生缺陷、组织退化和癌症等病理问题，这些疾病是由于TGFbeta信号或泛素连接酶功能调节缺陷造成的。目的1.蓝精灵在非洲爪哇胚胎发育中起作用吗？我们将建立胚胎发育过程中蓝精灵蛋白的空间分布，并确定干扰蓝精灵蛋白水平或活性对发育的影响。目的2.蓝精灵和转化生长因子β信号是如何相互调节的？为了了解蓝精灵在胚胎中调节哪些信号通路，我们将使用生物学(表型)和生化(相互作用，泛素化)分析来测试蓝精灵对BMP和激活素信号通路成分的影响。我们将确定蓝精灵在发育过程中是否受到TGFbeta或其他诱导信号的调节。目的3.新的蓝精灵相互作用蛋白的鉴定和功能。为了全面了解蓝精灵底物并寻找潜在的蓝精灵调节因子，我们将筛选和鉴定蓝精灵相互作用蛋白。我们已经分离出89个与S-1相互作用的克隆，包括已知的S-1靶标Smad1。

The TGFbeta superfamily regulates cell growth and differentiation. During Xenopus development, Nodal, Vg1 and activin-related signals induce endoderm and mesoderm, and BMP signals pattern these tissues and regulate ectodermal cell fate. The TGFbeta/activin and BMP pathways respectively transduce signals via Smads 2 and 3, or Smads1, 5 and 8. Little is known about TGFbeta signal downregulation. We have discovered two E3 ubiquitin ligases, Smurf1 and Smurf2, that inhibit BMP and TGFbeta/activin pathways, respectively (Zhu et al., 1999, Kavsak et al., 2000). Smurfs target Smads and receptors for ubiquitination and proteasomal degradation. While Smurf1 dorsalizes embryonic cell fates, the action of Smurf2 in embryos is unknown. We hypothesize that the Smurfs regulate tissue induction and pattern formation in embryogenesis by region-specific antagonism of TGFbeta signaling components. We will examine the role of Smurf1 and Smurf2 during embryogenesis and investigate the biochemical mechanisms necessary for Smurf regulation of development. Insight into Smurf functions will yield vital information about TGFbeta signaling and early development and will address pathologies such as birth defects, tissue degeneration and cancer, that result from defective regulation of TGFbeta signals or ubiquitin ligase functioning. Aim 1. Do the Smurfs function in Xenopus embryonic development? We will establish the spatial distribution of Smurf proteins during embryogenesis, and determine the developmental effects of perturbing Smurf protein levels or activity. Aim 2. How do Smurfs and TGFbeta signals regulate each other? To understand which signaling pathways Smurfs regulate in embryos, we will test the effects of Smurfs on components of BMP and activin signaling pathways, using biological (phenotype) and biochemical (interaction, ubiquitination) assays. We will determine whether Smurfs are regulated by TGFbeta, or other inductive signals, during development. Aim 3. Identification and function of new Smurf interacting proteins. To obtain a comprehensive view of Smurf substrates and to find potential Smurf regulators, we will screen for and characterize Smurf-interacting proteins. We have already isolated 89 Smurf1-interacting clones, including Smad1, a known Smurf1 target.