EAGER: Spatiotemporal Regulation of Receptor-Initiated Intracellular Complexes of Signaling Proteins

1450751 Lazzara，Matthew J.Receptors resident at the cell surface，interact with cognate proteins outside the cell，and transfer information to the cell by activating cellular biochemical pathways that regulate cellular processes including proliferation，differentiation，and migration. 这种“由外向内”的细胞信号传导依赖于激活的受体使细胞内蛋白质的功能性多元复合物成核的能力。 PI实验室的最新数据表明，这些蛋白质复合物的受体介导的组装和维持可以以复杂的、以前没有记录的方式运作，这些方式可以影响细胞信号强度和持续时间，从而对细胞行为产生深远的控制。 PI与表皮生长因子受体（EGFR）系统合作，收集的数据表明EGFR可以通过中间蛋白发挥作用，以调节与受体有一定距离的关键蛋白复合物的功能活性和持久性，该复合物仅限于细胞表面和内化囊泡，可能位于细胞内的任何点。 这一假设与EGFR的经典观点形成鲜明对比。的能力来调节这个复杂的。 为了验证我们的假设，并为其在其他受体系统中的更广泛探索奠定基础，PI将开发新的分子工具，以直接在活细胞中观察EGFR激活后的蛋白质复合物组装过程。 此外，将开发这一过程的数学模型，以解释成像数据，并确定细胞内复杂持久性的关键决定因素。 这一假说的成功验证对于我们理解受体介导的细胞信号传导以及合理调节EGFR和其他信号传导途径的能力具有重要意义。受体酪氨酸激酶（RTK）下游信号传导途径的激活需要磷酸酪氨酸-SH 2结合的信号蛋白的多元复合物的成核（Src同源2）结构域和其他相互作用。 虽然这些复合物通常表示为简单的静态组装，但可逆的结合相互作用、磷酸酶活性和其他系统拓扑可能性允许更复杂的时空蛋白质复合物调控模式，这些模式可以以深刻的方式影响细胞信号传导。 发展这些更复杂的信号调节模式的定量理解是实现我们对各种信号通路组分的分子理解的全部承诺所必需的。 使用表皮生长因子受体（EGFR）作为示例RTK系统，PI已经发现了一种先前未记录的信号传导蛋白复合物调节模式，其中EGFR激活中间胞质Src家族激酶，其放大EGFR活性以抵消GAB 1（Grb 2相关结合物1）去磷酸化并维持SHP 2的结合（含Src同源2结构域的磷酸酶2）与磷酸化GAB 1，其促进EGFR远端胞质区室中的SHP 2活性。 重要的是，激活的SHP 2参与了功能细胞中的一系列关键信号调节过程。 这种信号传导调节模式可以使RTK如EGFR能够调节细胞内位置处的信号传导事件，即使受体由于扩增步骤而通过溶酶体分选过程降解，受体也从细胞内位置被排除。 对于EGFR-GAB 1-SHP 2系统和其他RTK系统，RTK介导的信号复合物调节的这种观点与受体如何使信号蛋白复合物成核的经典理解形成鲜明对比。 拟议的工作的总体目标是直接可视化在活细胞中的空间和时间的持久性GAB 1-SHP 2复合物的EGFR激活相对于受体？的位置，并确定GAB 1-SHP 2复合物持久性的时间和长度尺度的决定因素。 为此，提出了两个具体目标：1。开发荧光融合报告构建体，以使用荧光共振能量转移（FRET）显微镜对GAB 1-SHP 2复合物响应于活细胞中EGFR活化的空间和动态持久性进行成像;以及2.开发并验证响应EGFR激活的GAB 1-SHP 2复合物组装的时空调控的计算模型。 通过开发新的实验和计算工具来探索新发现的信号调节模式，这项工作将奠定一个重要的基础，最终提高工程细胞信号传导的能力，从而提高细胞命运，在预测的方式。这个渴望奖共同-由CBET部门的生物技术和生物化学工程计划以及该部门的合成和系统生物学计划资助分子细胞生物学

1450751Lazzara, Matthew J.Receptors reside at the cell surface, interact with cognate proteins outside the cell, and transmit information to the cell by activating intracellular biochemical pathways that regulate cellular processes including proliferation, differentiation, and migration. This "outside-in" cell signaling depends upon the ability of activated receptors to nucleate functional multi-membered complexes of proteins inside cells. Recent data from the PI's lab suggests that the receptor-mediated assembly and maintenance of these protein complexes can operate in complex, previously undocumented ways that can impact cell signaling strength and duration, and thereby exert profound control over cellular behaviors. Working with the epidermal growth factor receptor (EGFR) system, the PI has gathered data suggesting that EGFR can operate through intermediary proteins to regulate the functional activity and persistence of a key protein complex at a distance from the receptor, which is restricted to the cell surface and internalized vesicles, potentially at any point within the cell. This hypothesis stands in stark contrast to the classical view of EGFR?s ability to regulate this complex. To validate our hypothesis and lay the groundwork for its broader exploration in other receptor systems, the PI will develop new molecular tools to visualize directly in live cells the protein complex assembly process in response to EGFR activation. In addition, a mathematical model of this process will be developed to interpret the imaging data and identify the key determinants of complex persistence within cells. The successful validation of this hypothesis has important implications for our fundamental understanding of receptor-mediated cell signaling and our ability to rationally tune signaling through EGFR and other pathways for a variety of applications.The activation of signaling pathways downstream of receptor tyrosine kinases (RTKs) requires the nucleation of multi-membered complexes of signaling proteins held together by phosphotyrosine-SH2 (Src homology 2) domain and other interactions. While these complexes are typically represented as simple static assemblies, reversible binding interactions, phosphatase activity, and other system topological possibilities allow for more sophisticated modes of spatiotemporal protein complex regulation that can impact cell signaling in profound ways. Developing quantitative understanding of these more complex modes of signaling regulation is required to realize the full promise of our molecular understanding of the components of various signaling pathways. Using the epidermal growth factor receptor (EGFR) as an example RTK system, PI has discovered a previously undocumented mode of signaling protein complex regulation wherein EGFR activates intermediary cytosolic Src family kinases which amplify EGFR activity to counteract GAB1 (Grb2-associated binder 1) dephosphorylation and maintain the association of SHP2 (Src homology 2 domain-containing phosphatase 2) with phosphorylated GAB1, which promotes SHP2 activity, in the cytosolic compartment distal from EGFR. Importantly, activated SHP2 participates in a host of critical signaling regulatory processes in the functioning cell. This mode of signaling regulation could enable RTKs such as EGFR to regulate signaling events at intracellular locations from which the receptor is excluded, even as the receptor degrades through lysosomal sorting processes due to the amplification step. This view of RTK-mediated signaling complex regulation, for the EGFR-GAB1-SHP2 system and other RTK systems in general, stands in stark contrast to the classical understanding of how receptors nucleate complexes of signaling proteins. The overarching goal of the proposed work is to directly visualize in live cells the spatial and temporal persistence of GAB1-SHP2 complexes in response to EGFR activation relative to the receptor?s position and to identify the determinants of the time and length scales of GAB1-SHP2 complex persistence. To do this, two specific aims are proposed: 1. Develop fluorescent fusion reporter constructs to image the spatial and dynamic persistence of GAB1-SHP2 complexes in response to EGFR activation in live cells using fluorescence resonance energy transfer (FRET) microscopy; and 2. Develop and validate a computational model of the spatiotemporal regulation of GAB1-SHP2 complex assembly in response to EGFR activation. By developing new experimental and computational tools to explore a newly discovered mode of signaling regulation, this work will lay an important foundation that will ultimately lead to an improved ability to engineer cell signaling, and therefore cell fates, in a predictive manner.This EAGER award to co-funded by the Biotechnology and Biochemical Engineering Program of the CBET Division and by the Synthetic and Systems Biology Program of the Division of Molecular Cell Biology.