IIBIO   27936
INSTITUTO DE INVESTIGACIONES BIOTECNOLOGICAS
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Interactions between protein tyrosine phosphatase 1B (PTP1B) and EGFR at ER-PM junctions
Autor/es:
ARREGUI CO; MAR√ćA EUGENIA PEREZ COLLADO
Reunión:
Congreso; LVI SAIB-XV SAMIGE joint meeting online; 2020
Resumen:
Protein-tyrosine phosphatases (PTPs) are relevant negative regulators of receptor tyrosine kinases (RTKs) - mediated signaling. PTP1B, a phosphatase targeted to the cytosolic face of the endoplasmic reticulum (ER) membranes, dephosphorylates several RTKs including the epidermal growth factor receptor (EGFR). Whether this occurs at ER-plasma membrane junctions, or on ER-endosome contact sites is not an established fact yet. To address this issue we used a combination of the bimolecular fluorescence complementation technique (BiFC), confocal fluorescence sectioning, and reflection microscopy. BiFC 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 and EGFR. After stimulation with EGF, BiFC was observed in puncta with increasing size and density in internal locations, coincident with endosome compartments. However, in absence of EGF stimulation, BiFC occurred at the membrane/substrate interface. We have confirmed these results by colocalization analysis of PTP1B and EGFR fused to complete fluorescent proteins and by co-immunoprecipitation. The relative area of the PTP1B/EGFR complexes was significantly reduced in the presence of pervanadate, an inhibitor of PTPs. We observed the same pattern in different epithelial cell lines (CHOK1, MDA-MB231 and HeLa). This pattern does not colocalize with adhesion complexes, other PTP1B interactors (eg. Mena and p130Cas) or actin cytoskeleton, but co-aligns with these elements. We found that PTP1B/EGFR complexes tightly co-localize with STIM1 (stromal-interacting molecule 1), an ER trans-membrane calcium sensor. STIM1 along with the pore-forming channel protein Orai1, residing at the plasma membrane, are essential structural components of the store-operated calcium entry (SOCE), a mechanism controlling calcium signaling and homeostasis that is activated by depletion of calcium stores from the ER. This event produces a fast redistribution of STIM1, from a uniform pattern at the ER to a clustered pattern in ER-plasma membrane (PM) junctions and can be induced by the sarcoendoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin (TG). Remarkably, the magnitude of PTP1B/EGFR complexes containing STIM1 at ER-PM junctions are not further enhanced by addition of TG. Furthermore, we have seen that PTP1B co-localizes with STIM1 at these sites, without the need of EGFR expression. Our results suggest that PTP1B/EGFR complexes and STIM1 are constituents of the same spatially-restricted, temporal transient compartment and we are currently analyzing the role of PTP1B in the regulation of SOCE. Supported by CONICET and ANPCyT.