Presentation description

The Ras/ERK pathway is canonical within cancer cell biology and is well-understood to promote proliferation, survival, and migration. Unregulated cell migration, due to mutations in the RAS-ERK pathway, drives cancer cell metastasis. ERK signaling regulates migration by driving protrusion of the cell edge, forming lamellipodia, and influencing the assembly and disassembly of adhesions that provide traction with the extracellular matrix. These ERK-regulated mechanisms are necessarily coordinated to produce efficient cell migration; however, it is unknown how ERK may be a coordinating factor. Preliminary to this project, our lab used a variety of fluorescent imaging techniques to quantify ERK signaling that is spatially localized within adhesions in lamellipodia. We found that ERK activity is highest in the membrane-proximal region of newly forming adhesions using TIRF imaging with a targeted FRET biosensor as well as immunofluorescent labeling.
This project investigates whether the adhesion proteins paxillin (PXN) and GIT1 increase ERK activity within adhesions through the scaffolding of essential pathway proteins, especially MEK, the kinase responsible for phosphorylating ERK. Using bimolecular fluorescence complementation (BiFC) reporter and fluorescent imaging, this study assessed interactions between MEK and the scaffold proteins. Image analysis determined that MEK is co-recruited to both PXN and GIT1. We quantified ERK activity in adhesions of CRISPR knockouts of PXN and GIT1 to determine if the absence of these scaffolds altered ERK activity. Our results indicate that paxillin, but not GIT1, is critical to ERK activation within adhesions.