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

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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.