Core B Proteomics & Biostatistics

描述 核心B的目的是提供创新技术来分析RAS突变特异性效应信号。 RAS效应子信号传导是复杂的，并且涉及RAS与多种（>10）功能多样的 下游效应器。虽然我们目前对RAS效应子利用率的理解是先进的，但它也远没有达到预期的水平。 从完成。到目前为止，四个效应子家族与驱动RAS依赖性癌症的发生有关。 和增长每个效应子网络包括蛋白激酶。还存在在所述信号之间的显著串扰。 效应器网络此外，这些网络是高度动态的，具有复杂的前馈和反馈 机制等经典地，RAS效应子信号传导通过评估两种典型效应子信号传导来分析。 RAF-MEK-ERK丝裂原活化蛋白激酶级联和PI 3 K-AKT-mTOR促生存 信号网络，使用ERK和AKT的磷酸化状态作为读数。然而，现在很明显， 这些分析单独不能提供RAS效应信号传导的充分测定。因为一个主要目标 本计划项目的一个重要部分是确定RAS突变体特异性效应子信号传导， 要实现这一目标，需要进行分析。核心B提供了两个创新的基于蛋白质组学的实验 平台来实现这一点。首先，多重抑制剂珠（MIB）和质谱（MIB/MS） 分析提供了蛋白激酶活性动态变化的全激酶组谱。我们的初步研究 应用MIB/MS来表征KRAS抑制后的这种变化， 以前被称为RAS效应器信号传导的组分，证明了该平台的潜力， 鉴定新RAS效应物信号传导输出。第二，反相蛋白质阵列（RPPA）分析将 癌细胞信号传导中蛋白磷酸化和活化状态的RAS依赖性变化 网络.此外，最近在RPPA中开发的创新性进展使其能够分析 还将应用相互作用蛋白质的激活状态。使用每种方法生成的数据类型 实验平台具有很强的互补性。我们希望他们一起定义新的RAS突变- 特异性效应器信号网络。

DESCRIPTION The aim of Core B is to provide innovative technologies to profile RAS mutation-specific effector signaling. RAS effector signaling is complex and involves RAS interaction with a multitude (>10) of functionally diverse downstream effectors. While our current understanding of RAS effector utilization is advanced, it is also far from complete. To date, four effector families have been implicated in driving RAS-dependent cancer initiation and growth. Each effector network includes protein kinases. There is also significant crosstalk between the effector networks. Furthermore, these networks are highly dynamic, with complex feed-forward and feedback mechanisms. Classically, RAS effector signaling is profiled by evaluation of the two canonical effector pathways, the RAF-MEK-ERK mitogen-activated protein kinase cascade and the PI3K-AKT-mTOR prosurvival signaling network, using the phosphorylated state of ERK and AKT as readouts. However, it is now clear that these analyses alone fail to provide an adequate determination of RAS effector signaling. Since a major goal of this Program Project is the determination of RAS mutant-specific effector signaling, unbiased kinome-wide analyses are needed to accomplish this goal. Core B provides two innovative proteomics-based experimental platforms to accomplish this. First, Multiplexed Inhibitor Beads (MIBs) and Mass Spectroscopy (MIB/MS) analyses provide kinome-wide profiling of dynamic changes in protein kinase activity. Our preliminary studies applying MIB/MS to characterize such changes upon KRAS suppression identified protein kinases not previously known as components of RAS effector signaling, demonstrating the potential for this platform to identify novel RAS effector signaling outputs. Second, Reverse Phase Protein Array (RPPA) analyses will profile RAS-dependent changes in protein phosphorylation and activation states in cancer cell signaling networks. Additionally, a recently developed innovative advance in RPPA enabling the profiling of the activation state of interacting proteins will also be applied. The types of data generated using each experimental platform are highly complementary. We expect that together they will define novel RAS mutation- specific effector signaling networks.