Function and Mechanism of the Hippo-PI5P4K Axis for Growth Control

项目概要 我们的长期目标是破译 PI5P4K 驱动生长的分子机制并阐明 它们的失调如何导致乳腺肿瘤的发生。在本提案中，我们将着手提供 全面了解新的信号网络，这对于整合代谢信息和 确定相关蛋白质和途径如何相互作用以维持细胞稳态。 磷脂酰肌醇 5 磷酸 4 激酶 (PI5P4K) 是一个非经典磷酸肌醇激酶家族 我们已经证明这对于保持养分可用性和燃料生长至关重要，但它们是如何监管的 他们自己在很大程度上是未知的。通过全面的体外激酶筛选，我们发现了核心 Hippo 通路激酶、MST1 和 MST2 作为 PI5P4K 以及脂质的直接负调节因子 PI5P4Ks、PI-5-P 的底物与 Hippo 接头蛋白 MOB1 相互作用，进而影响活性 Hippo 下游效应子 YAP。众所周知，Hippo 途径对于发育、生长非常重要 和器官发生，该途径的失调会导致肿瘤进展。 虽然 PI5P4K 与肿瘤生长有关，但人们对 PI5P4K 的确切调控方式知之甚少。 此外，PI5P4K 在生理条件和 乳腺癌等病理背景是不完整的。为了回答这些重要问题，我们将执行 以下目标： 在目标 1 中，我们将使用非常可行的生物物理和结构生物学来确定原子分辨率细节 PI-5-P 和 MOB1 之间的界面，并定量 PI-5-P 如何调节 MOB1 与其他 Hippo 通路的结合 体外成分。 在目标 2 中，我们将研究 PI5P4K 的活性如何控制 MOB-LATS 复合物的激活 MST1/2 下游，探索 PI5P4K 致瘤特性的一种潜在机制。 在目标 3 中，我们将使用一组数据来确定 PI5P4K-Hippo 轴在乳腺癌中的生物学意义 TNBC 细胞系、新型小鼠乳腺肿瘤模型和患者乳腺肿瘤样本。 该项目旨在将定量结构生物学与分子细胞遗传学相结合，以破译 PI-5- P 调节乳腺癌细胞生长的 Hippo 通路。鉴于 PI5P4K 是令人兴奋的“可成药”靶标 对于乳腺癌，表征 PI5P4K 如何连接到 Hippo 通路揭示了一种可能的策略 未来的抗癌疗法。

PROJECT SUMMARY Our long-term goal is to decipher the molecular mechanisms underlying PI5P4K-driven growth and to elucidate how their dysregulation contributes to breast tumorigenesis. In this proposal, we will set out to provide a comprehensive understanding of a new signaling network that is critical to integrate metabolic information and determine how the related proteins and pathways interact to maintain cellular homeostasis. Phosphatidylinositol-5-phosphate 4-kinases (PI5P4Ks) are a family of non-canonical phosphoinositide kinases which we have shown to be critical for preserving nutrient availability and fuel growth yet how they are regulated themselves is largely unknown. Using a comprehensive in vitro kinase screen, we have discovered the core kinases of the Hippo pathway, MST1 and MST2 as direct negative regulators of the PI5P4Ks as well as the lipid substrate of the PI5P4Ks, PI-5-P to interact with the Hippo adaptor protein MOB1 which in turn effects the activity of the Hippo downstream effector YAP. The Hippo pathway is known to be important for development, growth and organogenesis, and dysregulation of this pathway leads to tumor progression. While PI5P4Ks have been linked to tumor growth, exactly how the PI5P4Ks are regulated is poorly understood. Furthermore, how the PI5P4Ks actually command cell growth under both physiological conditions and pathological contexts such as breast cancer is incomplete. To answer these important questions, we will perform the following Aims: In Aim 1 we will use very feasible biophysical and structural biology to determine atomic-resolution details of the interface between PI-5-P and MOB1 and quantitate how PI-5-P regulates MOB1 binding to other Hippo pathway components in vitro. In Aim 2 we will investigate how the activity of the PI5P4Ks control the activation of the MOB-LATS complex downstream of MST1/2, exploring one potential mechanism for PI5P4K tumorigenic properties. In Aim 3 we will establish the biological significance of the PI5P4K-Hippo axis in breast cancer using a panel of TNBC cell lines, novel mouse breast tumor models, and patient breast tumor samples. This project seeks to combine quantitative structural biology with molecular cell genetics to decipher how PI-5- P regulates the Hippo pathway for breast cancer cell growth. Given that PI5P4Ks are exciting ‘druggable’ targets for breast cancer, characterizing how PI5P4Ks connect to the Hippo pathway reveals a possible strategy for future anti-cancer therapies.