NANOSCALE ANALYSIS OF PROTEIN-PROTEIN INTERACTIONS IN CELL-MATRIX ADHESIONS: REGULATORY IMPLICATIONS

ÁNGELA GONZÁLEZ, MAURICIO SULLIGOY AND CARLOS O. ARREGUI

Congreso; Molecular mecanisms in cell signaling and gene expresion; 2013

Our previous work suggested that the ER-bound tyrosine phosphatase PTP1B has the potential to modulate the phosphorylation state of multiple components of cell-matrix adhesions. Here we used Bimolecular Fluorescence Complementation (BiFC) for direct visualization of PTP1Bsubstrate complexes in cells. This approach is based on complementation and restoration of fluorescence when two non fluorescent fragments of the yellow fluorescent protein are a few nanometers apart. These fragments were fused to PTP1B and candidate substrates resident of adhesions. Identification of PTP1B substrates was facilitated by using the substrate trapping mutant PTP1B (D181A). We visualized BiFC between PTP1BD181A and α-actinin, paxillin and FAK at peripheral adhesions. BiFC was undetectable in cells pre-incubated with pervanadate, and in cells expressing wild type PTP1B. PTP1BD181A-paxillin and PTP1BD181A-α-actinin BiFC complexes overlapped with the adhesion marker vinculin, but they segregate spatially to the distal and proximal poles, respectively. Elimination of the SH3-ligand motif on PTP1BD181A did not abolished BiFC. We are currently mapping the residues targeted by PTP1B on the substrates. Our results suggest that PTP1B could regulate adhesion-cytoskeleton coupling through dephosphorylation of α-actinin, and signaling through dephosphorylation of FAK and paxillin.