Signal Transduction Pathways of Transforming Growth Factor-beta

tgf - β家族配体通过一种独特的异聚体受体复合物进行信号传递，其特征是丝氨酸-苏氨酸激酶活性。最近，从这些受体到核靶基因的直接信号转导途径已经被阐明，其中涉及一个新的蛋白质家族，称为Smads。在这一途径中，受体激活的Smads直接被I型受体激酶磷酸化，并与共同的介质Smad4/ DPC4结合，转运到细胞核，参与转录复合物。我们采取了多方面的方法来描绘这一途径的体外生物化学，并了解其在体内的意义。一种方法是确定调节受体或Smad活性的独特因素。我们利用酵母双杂交系统，以Smad1为诱饵，鉴定了两个新的无性系。一种蛋白质SNX6是受体相互作用蛋白分选连接蛋白家族的成员，可能在丝氨酸-苏氨酸激酶受体的运输中发挥作用。另一个是SNIP1，它是一种新的Smad信号的核抑制因子，可能整合不同信号通路之间的交叉对话，这些信号通路聚集在依赖于转录共激活因子p300和CBP的转录复合物上。我们还阐明了先前鉴定的tgf - β受体相互作用蛋白TRAP1的新作用，该蛋白作为义务伴侣Smad4的伴侣，将Smad4呈现给受体激活的Smads。这些研究将进一步扩展，以确定这些分子的作用，并包括额外的酵母双杂交筛选，以确定这些蛋白质是否反过来与信号复合物的其他成分相互作用。我们还使用微阵列分析来鉴定小鼠胚胎成纤维细胞中由Smad3或smad2依赖途径唯一诱导的cdna。这些研究已经确定了这些信号通路的靶点，其特征确定为tgf - β的即时早期和二级基因靶点提供了新的视角。为了补充上述基础科学方法，我们还开发了一个强大的研究计划，该计划基于以下假设：从概念上讲，体内特定信号成分的删除应该比涉及靶向删除或过表达配体或受体的更广泛的方法具有更少的严重和更多的选择性作用。我们很幸运地与NIDDK的邓楚霞合作，对Smad3敲除小鼠进行了表征，这为tgf - β在造血、伤口愈合和纤维化中的作用提供了新的见解。tgf - β依赖于这一特定途径的发病机制的阐明表明，Smad3抑制剂的开发将具有广泛的临床应用。为了研究bmp依赖的信号通路，我们创建了Smad1基因的条件敲除，以克服Smad1敲除小鼠的早期胚胎致死性，这也是在我们的小组中产生的。

The TGF-beta family of ligands signal through a unique heteromeric receptor complex distinguished by its serine-threonine kinase activity. Recently, a direct signal transduction pathway from these receptors to nuclear target genes has been elucidated which involves a novel family of proteins termed Smads. In this pathway, receptor-activated Smads are phosphorylated directly by the type I receptor kinase and, in association with a common mediator Smad4/ DPC4, translocate to the nucleus where they participate in transcriptional complexes. We have taken a multi-faceted approach to delineate the biochemistry of this pathway in vitro and to understand its significance in vivo. One approach has been to identify unique factors which modulate receptor or Smad activity. We have utilized a yeast two-hybrid system using Smad1 as bait to identify two novel clones. One protein, SNX6, a member of the sorting nexin family of receptor interacting proteins, is likely to play a role in trafficking of serine-threonine kinase receptors. The other, SNIP1, is a novel nuclear repressor of Smad signaling, and may integrate cross-talk between different signaling pathways converging on transcriptional complexes dependent on the transcriptional coactivators p300 and CBP. We have also elucidated a novel role for the previously identified TGF-beta-receptor-interacting protein, TRAP1, which acts as a chaperone for the obligate partner, Smad4, presenting Smad4 to receptor-activated Smads. These studies will be expanded to define the roles of these molecules further and to include additional yeast two-hybrid screens to determine whether these proteins, in turn, interact with yet other components of the signaling complex. We are also using microarray analysis to identify cDNAs uniquely induced by Smad3- or Smad2-dependent pathways in mouse embryo fibroblasts. These studies have resulted in identification of targets of these signaling pathways, the characterization of which is certain to provide a new perspective on immediate-early and second-order gene targets of TGF-beta. To complement the above basic science approaches, we have also developed a strong program of research based on the hypothesis that deletion of specific signaling components in vivo should, conceptually, have a less severe and more selective effect than broader-based approaches involving targeted deletion or overexpression of ligand or receptors. We have been fortunate to collaborate with Chuxia Deng, NIDDK, on the characterization of a Smad3 knockout mouse, which is now providing new insights into the roles of TGF-beta in hematopoiesis, in wound healing, and in fibrosis. Elucidation of pathogenetic mechanisms of TGF-beta dependent on this specific pathway now suggest that development of a Smad3 inhibitor will have wide-ranging clinical applications. To investigate BMP-dependent signaling pathways, we have created a conditional knockout of the Smad1 gene to overcome the early embryonic lethality of the Smad1 knockout mouse, also generated in our group.