Interactions between protein tyrosine phosphatase 1B (PTP1B), EGFR and FAK in intact cells

MARÍA EUGENIA PEREZ COLLADO; ANA E. GONZÁLEZ WUSENER; ANA E. GONZÁLEZ S. WUSENER, MELISA MONTELEONE AND CARLOS O. ARREGUI.

Paraná

Congreso; 54th Annual Meeting Argentine Society for Biochemistry and Molecular Biology; 2018

Sociedad Argentina de Investigación Bioquímica y Biología Molecular

Different cell behaviors, such as contractility, migration and proliferation, depend on specific spatiotemporal regulation of adhesion complexes and the cytoskeleton. Previous work from our laboratory revealed that protein PTP1B has a critical role in this regulation. Several substrates of PTP1B have been identified, such as the Epidermal Growth Factor Receptor (EGFR) and the Focal Adhesion Kinase (FAK), which also are able to interact with each other. However the functional relationships between these interactors and their spatial location remains unknown. We used Bimolecular Fluorescence Complementation (BiFC) for direct visualization and analysis of the interactions in intact CHO or PTPWT cells. This approach is based on complementation and restoration of fluorescence when two non-fluorescent fragments of a fluorescent protein are a few nanometers apart. N- and C-fragments of YFP were fused to PTP1B, FAK and EGFR. Confocal fluorescence sectioning and reflection microscopy revealed positive BiFC signal for PTP1B/FAK, PTP1B/EGFR and FAK/EGFR pairs at different cellular compartments. In absence of EGF stimulation PTP1B/EGFR BiFC occurred at the membrane/substrate interface, although not associated with adhesion complexes. In contrast, after stimulation with EGF, BiFC was in puncta with increasing size and density in internal locations. This pattern was altered depending on substrate, temperature and concentration of EGF. On the other hand, BiFC between FAK/PTP1B, and FAK/EGFR was observed at adhesions in the membrane/substrate interface. Our results indicate that PTP1B recognize components of cell-matrix adhesion complexes and EGFR in different membrane subcompartments. Supported by CONICET and ANPCyT.