TRANSFORMING GROWTH FACTOR BETA SIGNALING PATHWAYS

描述：转化生长因子β(TGFb)是一种强有力的生长 多种细胞的抑制物，包括上皮系的细胞。 事实是，这些正常的大多数转化的对应物 细胞失去了对TGFb的生长抑制反应导致了 假设这种对TGFb的敏感性的丧失可能是 致癌过程。申请人最近的工作产生了一个 TGFb调控的可被诱导增殖或死亡的细胞系 在适当的媒体中以依赖于TGFb的方式。在此应用程序中 提出了产生隐性稳定突变体的建议 对TGFb的响应，随后可用于功能 受影响基因的互补克隆。除了隔离 能够补充TGFb突变体的cDNA，即编码负值的基因 TGFb信号的调节者或抑制者将被隔离并 特色化的。编码补充TGFb突变体的蛋白质的cDNA 或抑制的TGFb信号将被分离、测序和表征 对于保守的功能结构域(即，激酶、磷酸酶、转录 因子、SH2、SH3域等)寻找一些关于它们功能的线索。这些 会被缺失和定点突变所操纵 被引入突变细胞进行结构功能分析。 将产生抗体以进行翻译后修饰的检测(S) 响应生长因子和细胞因子并测定蛋白质-蛋白质 共沉淀分析的相互作用。

DESCRIPTION: Transforming growth factor beta (TGFb) is a potent growth inhibitor for a wide variety of cells including those of epithelial lineage. The fact that the majority of the transformed counterparts of these normal cells have lost their growth inhibitory response to TGFb has led to the hypothesis that this loss of sensitivity to TGFb may be an initial step in the carcinogenic process. Recent work by the applicant has generated a TGFb-regulated cell line which can either be induced to proliferate or die in a TGFb-dependent fashion in the appropriate media. In this application it is proposed to generate recessive stable mutants defective in their response to TGFb which can subsequently be employed for functional complementation cloning of the affected genes. In addition to isolating cDNAs capable of complementing TGFb mutants, genes which encode negative regulators or suppressors of TGFb signaling will be isolated and characterized. cDNAs encoding proteins that either complement TGFb mutants or suppress TGFb signaling will be isolated, sequenced, and characterized for conserved functional domains (i.e., kinases, phosphatases, transcription factors, SH2, SH3 domains, etc.) for some clues to their function. These will then be manipulated by deletional and site directed mutagenesis and introduced into the mutant cells for structure-function analyses. Antibodies will be generated to assay for post-translational modification(s) in response to growth factors and cytokines and to determine protein-protein interactions by co-precipitation analyses.