KSR AND REGULATORS OF RAS SIGNALING IN C ELEGANS

我们的长期目标是了解RAS信号如何控制动物的生长和发育。RAS可以刺激一个复杂的信号通路网络，其中之一是Raf/MEK/ERK激酶级联。我们的具体目标是了解KSR(Kinase Suppressor Of RAS)的功能，我首先确定KSR是线虫中RAS信号的正向调节因子。KSR可以激活Raf或通过Raf/MEK/ERK调节信号的传播，或者KSR可能在单独的信号通路中发挥作用。由于我们的研究结果表明，KSR与MEK结合，与蛋白磷酸酶2A(PP2A)和RAC蛋白一起在共同的遗传途径中发挥作用，我们认为KSR的功能是协调两条不同的RAS刺激途径。为了验证这一假设，我们将测试KSR、PP2A、RAC和已知的Raf/MEK/ERK级联之间的物理和调控关系。针对L，我们将通过镶嵌分析检测KSR在与RAS相同或不同的细胞中的功能(通过镶嵌分析)，确定KSR的正常表达模式(通过免疫定位)，并测试KSR膜定位是否受RAS、RAc或PP2A的调节。在目标2中，我们将测试KSR是否与RAC或PP2A共定位或物理上相互作用，测试KSR是否可以调节RAC或PP2A与MEK之间的相互作用(支持支架模型)，以及测试Raf是否是PP2A活动的可能目标(支持Raf激活模型)。在目标3中，我们将测试KSR-1缺失突变株在MEK定位、蛋白复合体形成、ERK磷酸化或靶基因表达方面是否发生变化。根据过度表达的研究，这些都被归因于KSR的活动。我们还将进行转基因结构/功能研究，以确定对KSR-1调控和功能重要的位置，并测试KSR-1是否为PP2A的可能靶点。在目标4中，我们将使用遗传和反向遗传方法来识别更多调节RAS或RAC信号的基因，我们将鉴定/克隆我们已经识别的三个基因。RAS信号失控会导致许多人类疾病，包括癌症。我们对KSR、PP2A和RAC的研究将为了解不同的RAS信号通路在体内如何正常协调提供重要的见解。

Our long term goal is to understand how Ras signaling controls animal growth and development. Ras can stimulate a complex web of signaling pathways, one of which is the Raf/MEK/ERK kinase cascade. Our specific goal is to understand the function of KSR (Kinase Suppressor of Ras), which I first identified as a positive regulator of Ras signaling in C. elegans. KSR may activate Raf or modulate signal propagation through Raf/MEK/ERK, or KSR may function in a separate signaling pathway. Since our data suggest that KSR binds to MEK yet functions in a common genetic pathway with Protein Phosphatase 2A (PP2A) and a Rac protein, we propose that KSR functions to coordinate two different Ras- stimulated pathways. To test this hypothesis, we will test the physical and regulatory relationship among KSR, PP2A and Rac and the known Raf/MEK/ERK cascade. In Aim l, we will test if KSR functions within the same or different cells as Ras (by mosaic analysis), define KSR's normal expression pattern (by immuno-localization) and test if KSR membrane localization is regulated by Ras, Rac or PP2A. In Aim 2, we will test if KSR colocalizes or physically interacts with Rac or PP2A, test if KSR can mediate interactions between Rac or PP2A and MEK (supporting a scaffold model), and test if Raf is the likely target of PP2A activity (supporting a Raf activation model). In Aim 3, we will test if ksr- 1 null mutant strains have alterations in MEK localization, protein complex formation, ERK phosphorylation or target gene expression. These are all activities that have been attributed to KSR based on overexpression studies. We will also conduct transgenic structure/function studies to identify sites important for KSR- 1 regulation and function, and we will test if KSR- 1 is the likely target of PP2A. In Aim 4, we will use genetic and reverse genetic approaches to identify additional genes that regulate Ras or Rac signaling, and we will characterize/clone three genes that we have already identified. Deregulated Ras signaling causes many human pathologies, including cancer. Our studies of KSR, PP2A and Rac will provide important insights into how different Ras signaling pathways are normally coordinated in vivo.