'Study of the Grb7/Fliamin-A protein-protein interaction and colocalization in SKBR3 breast cancer cells

Grb7 is an intracellular adaptor protein that mediates receptor tyrosine kinase activated signaling to downstream pathways. Grb7 is overexpressed in a variety of invasive and metastatic cancers where it is considered an indicator of poor prognosis. Different Grb7 domains are known to interact with many signaling molecules including, transmembrane receptors, intracellular kinases and other scaffold proteins. Grb7 has also been established as a component involved in ErbB2 receptor and FAK mediated regulation of cell proliferation and migration. Different Grb7 domains have been shown to mediate protein-protein interactions thus modulating downstream signaling.The C-terminal Grb7-SH2 domain has been studied with respect to its interaction with receptor tyrosine kinases whereas the central GM (Grb and Mig-10) region has increasingly been reported to be involved in cell migration functions. Although a number of upstream Grb7 binding partners have been reported, little is known regarding downstream signaling interactions and mechanisms of Grb7-mediated signal regulation. Here, through yeast two hybrid screening of mouse embryonic stem cell proteins, Filamin-a has been discovered as a novel Grb7 binding protein. Filamin-a is a cytoplasmic actin crosslinking protein that functions as an important intracellular scaffold for coupling a diverse range of signaling molecules. The Grb7 GM region appears to mediate the interaction with immunoglobulin like Filamin-a repeats, 16-19 (lgFln16-19). Mutation of Grb7 phophotyrosine residues-pY188 and pY338 to phenylalanine affects binding with Filamin-a. Grb7 and Filamin-a colocalization observed in SKBR3 cell membrane ruffles upon EGF stimulation suggests an EGFR/ErbB2 mediated response and consequent cytoskeletal reorganization. Grb7shRNA knockdown studies in SKBR3 cells demonstrated decreased cell migration, thus potentially indicating a role for Grb7/Filamin-a colocalization and cell motility responses. Grb7 and Filamin-a colocalization observed in heat shock induced cell stress granules suggests an additional function for Grb7 in stress granule (SG) dynamics and associated cell signaling.