ID OF EFFECTORS FOR RAS FAMILY GTPASES & ELUCIDATION OF MECHANISM OF ACTION

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 Ras信号通路在调节细胞生长和肿瘤发生中起着至关重要的作用。 Ras是相关GTP酶亚家族的成员，其通过调节效应子的多种下游靶标发挥功能。 基于Ras相关结构域的序列同源性，我们已经鉴定了几种新的Ras家族GTP酶效应物，并证实这些新蛋白质在其活性、GTP结合状态下可以与Ras相互作用。 很少，如果有的话，是知道这些新的Ras效应器。 作为深入了解它们的功能和作用机制的一种方式，我们正试图识别它们在细胞内相互作用的蛋白质。 我们已经使用串联亲和纯化（TAP）方法从结肠癌细胞中表达和纯化这些新Ras效应物的TAP标记版本。 通过凝胶电泳和考马斯蓝染色分离蛋白质复合物后，我们可以看到几个共纯化的蛋白质条带，我们现在想通过质谱法鉴定。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The Ras signaling pathway plays a crucial role in regulating cell growth and tumorigenesis. Ras is a member of a sub-family of related GTPases that functions by regulating a variety of downstream targets of effectors. Based on sequence homologies in the Ras Association domain, we have identified serveral new Ras family GTPase effectors and confirmed that these new proteins can interact with Ras when in its active, GTP-bound state. Very little, if anything, is known about these new Ras effectors. As a way of getting some insight into their function and mechanism of action, we are trying to identify the proteins they interact with within the cell. We have used the Tandem Affinity Purification (TAP) methodology to express and purify from colon cancer cells TAP-tagged versions of these new Ras effectors. After separation of the protein complexes by gel electrophoresis and coomassie blue staining, we can visualize several co-purifying protein bands that we would now like to identify by mass spectrometry.