Single Cell Analysis of Cross Talk Among Kinase Pathways

描述(由申请人提供)：相互作用的信号蛋白级联，特别是激酶，在癌症的发展和维持中发挥关键作用。尽管它们在致癌信号转导途径中很重要，但人们对这些蛋白质在活细胞中的生化行为知之甚少。现在很清楚，这些信号级联不只是一系列线性的酶反应，导致从离散的刺激到明确的细胞反应。相反，它们在复杂的网络中相互关联，以至于它们在活细胞内的生化特性并不直观。传统的生化研究依赖于大量细胞群体的裂解产物，由于群体中异质细胞反应的平均化，无法破译细胞信号的内在属性。为了了解生物化学现象，如通路之间的串扰、激活阈值和双稳态，必须在完整的单细胞中进行激酶分析。目前的工作旨在破译RAS相关信号通路的生化行为--对癌症生物学特别重要的信号通路。这些研究将应用一种强大的新技术，在单细胞中对一系列独特的具有结构性活性或非活性信号蛋白的肿瘤细胞株进行激酶分析。这些细胞系表现出一系列的致癌特征，这取决于RAS相关信号通路中的结构性活性。在这些细胞中，我们的数据支持这样的观点，即阈值行为和这些通路之间的串扰与它们的致瘤表型的相对侵袭性密切相关。目前的研究将揭示在活的单个细胞中，涉及磷酸肌醇-3-激酶(PI3K)、Akt、p21-激活的激酶和Erkl/2的信号级联的行为是如何相互关联的。这个项目的具体目的是：1)确定由PI3K活性产生的串扰的诱导是否需要激活PI3K的阈值水平；2)确定这些激酶通路的交叉激活是协调的还是顺序的；3)确定串扰的诱导是否依赖Akt；以及4)确定在PI3K刺激取消后，MAP激酶级联的串扰激活是可逆的还是不可逆的。这些研究将为一些问题提供开创性的答案，这些问题对于我们理解激活激酶信号通路在恶性转化、侵袭性肿瘤生长和生存方面的后果至关重要。

DESCRIPTION (provided by applicant): Interacting cascades of signaling proteins, particularly kinases, play crucial roles in the development and maintenance of cancer. Despite their importance in oncogenic signal transduction pathways, little is understood about the biochemical behavior of these proteins within the context of the living cell. It is now clear that these signaling cascades are not merely a linear series of enzymatic reactions leading from discrete stimuli to defined cellular responses. Rather they are interrelated in complex networks such that their biochemical properties within the living cell are not intuitive. Traditional biochemical studies that rely on lysates of bulk cell populations can not decipher the inherent properties of cell signaling due to averaging of heterogeneous cellular responses across the population. To understand such biochemical phenomena as cross-talk between pathways, thresholds of activation, and bistable states, kinase assays must be performed in intact, single cells. The current work is directed at decoding the biochemical behaviors of the Ras-related pathways- signaling pathways of particular import to cancer biology. These studies will apply a powerful new technology for kinase assays in single cells to a unique series of tumor cell lines possessing constitutively active or inactive signaling proteins. The cell lines display a range of tumorigenic characteristics dependent on the repertoire of constitutive activity in Ras-related signaling pathways. In these cells our data support the notion that threshold behavior and crosstalk among these pathways closely relate to the relative aggressiveness of their tumorigenic phenotype. The current studies will reveal how the behavior of signaling cascades involving the kinases phosphoinositide-3-kinase (PI3K), Akt, p21-activated kinase, and Erkl/2 interrelate in living, single cells. The specific aims for this project are to: 1) determine whether induction of cross talk, generated by PI3K activity, requires a threshold level of activated PI3K, 2) determine if cross-activation of these kinase pathways is coordinate or sequential, 3) determine whether induction of cross talk is dependent on Akt, and 4) determine if cross-talk activation of MAP kinase cascades is reversible or irreversible after withdrawal of the PI3K stimulus. These studies will provide ground breaking answers to questions critical for our understanding of the consequences of activation of kinase signaling pathways with respect to malignant transformation, aggressive tumor growth and survival.