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

1450751 Lazzara，Matthew J.Receptors位于细胞表面，与细胞外的同源蛋白相互作用，通过激活细胞内调节细胞过程(包括增殖、分化和迁移)的生化途径向细胞传递信息。这种“由外而内”的细胞信号传递依赖于激活的受体在细胞内形成功能性多元蛋白质复合体的能力。PI实验室的最新数据表明，受体介导的这些蛋白质复合体的组装和维护可以以复杂的、以前未见文献报道的方式运作，可以影响细胞信号强度和持续时间，从而对细胞行为施加深刻控制。与表皮生长因子受体(EGFR)系统合作，PI收集的数据表明，EGFR可以通过中间蛋白来调节距离受体一段距离的关键蛋白复合体的功能活性和持久性，该复合体限制在细胞表面和内化小泡，可能在细胞内的任何位置。这一假说与EGFR的经典观点形成鲜明对比？S有能力调控这一复合体。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，直接在活细胞中可视化响应EGFR激活的蛋白质复合体组装过程。此外，将建立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从根本上理解受体介导的细胞信号以及通过EGFR和其他途径合理调节信号的能力具有重要意义。受体酪氨酸激酶(RTK)下游信号通路的激活需要通过磷酸化酪氨酸-SH2(Src Homology 2)结构域和其他相互作用来实现信号蛋白的多成员复合体的成核。虽然这些复合体通常被表示为简单的静态组装，但可逆的结合作用、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体调节模式，可以深刻地影响细胞信号转导。发展对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成部分的分子理解的全部承诺所必需的。以表皮生长因子受体(EGFR)为例，PI发现了一种以前未见文献报道的信号蛋白复合体调控模式，其中EGFR激活胞浆中的中间Src家族激酶，从而放大EGFR的活性，以抵消GAB1(Grb2相关结合蛋白1)的去磷酸化，并维持SHP2(Src Homology 2结构域含磷酸酶2)与磷酸化的GAB1的结合，后者促进了EGFR远端的细胞室中SHP2的活性。重要的是，激活的SHP2参与了功能细胞中的一系列关键信号调节过程。这种信号调节模式可以使RTK，如EGFR，在受体被排除在外的细胞内位置调节信号事件，即使受体由于扩增步骤而通过溶酶体分选过程降解。对于EGFR-GAB1-SHP2系统和其他RTK系统来说，RTK介导的信号复合体调控的这种观点与经典的关于受体如何使信号蛋白复合体成核的理解形成鲜明对比。这项工作的首要目标是在活细胞中直观地显示GAB1-SHP2复合体在EGFR激活时相对于受体S位置的时空持续性，并确定GAb1-SHP2复合体持续性的时间和长度尺度的决定因素。为此，提出了两个具体的目标：1.开发荧光融合报告构建物，利用荧光共振能量转移(FRET)显微镜成像GAB1-SHP2复合体在活细胞中响应EGFR激活的空间和动态持久性；2.建立并验证GAB1-SHP2复合体组装响应EGFR激活的时空调节的计算模型。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，这项工作将奠定重要的基础，最终将以预测的方式提高设计细胞信号的能力，从而提高细胞命运。这一热切的奖项由CBET部门的生物技术和生化工程计划以及分子细胞生物学部门的合成和系统生物学计划共同资助。

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.