Single Cell Analysis of Cross Talk Among Kinase Pathways

描述（由申请人提供）：信号蛋白的相互作用级联，特别是激酶，在癌症的发展和维持中起着至关重要的作用。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，这些信号级联不仅仅是一系列线性的酶促反应，从离散的刺激到确定的细胞反应。相反，它们在复杂的网络中相互关联，因此它们在活细胞内的生化特性不是直观的。传统的生物化学研究依赖于大量细胞群体的裂解物，由于在整个群体中平均异质细胞反应，无法破译细胞信号传导的固有特性。为了理解诸如通路间的串扰、激活阈值和双稳态等生化现象，必须在完整的单细胞中进行激酶测定。目前的工作是针对解码ras相关途径的生化行为-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，在单细胞中对具有组成性活性或非活性信号蛋白的独特系列肿瘤细胞系进行激酶检测。这些细胞系显示出一系列的致瘤性特征，这取决于ras相关信号通路的组成活性。在这些细胞中，我们的数据支持这样一种观点，即这些通路之间的阈值行为和串扰与它们致瘤表型的相对侵袭性密切相关。目前的研究将揭示包括磷酸肌醇-3激酶（PI3K）、Akt、p21活化激酶和Erkl/2激酶在内的信号级联的行为如何在活的单细胞中相互关联。该项目的具体目的是：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)确定这些激酶途径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于Akt， 4)确定MAP激酶级联的串扰激活在PI3K刺激退出后是可逆的还是不可逆的。这些研究将为我们理解激酶信号通路激活对恶性转化、侵袭性肿瘤生长和生存的影响提供突破性的答案。

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.