Premium
Micro‐patterned arrays of epidermal growth factor (EGF) reveal stimulated association of paxillin, ERK, and F‐actin with EGF receptors during cell signaling
Author(s) -
Singhai Amit,
Bryant Kirsten L,
Baird Barbara A,
Hammes Stephen R,
Holowka David A
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.971.5
Subject(s) - paxillin , epidermal growth factor , microbiology and biotechnology , tyrosine phosphorylation , phosphorylation , cortactin , proto oncogene tyrosine protein kinase src , actin , signal transducing adaptor protein , epidermal growth factor receptor , tyrosine kinase , actin cytoskeleton , receptor tyrosine kinase , signal transduction , chemistry , receptor , biology , focal adhesion , cell , cytoskeleton , biochemistry
We are investigating the spatiotemporal association of signaling proteins with EGF receptors (EGFR) using micro‐patterned EGF surfaces. We utilize EGF‐biotin bound to streptavidin that is covalently coupled in ordered micron sized square arrays on silicon surfaces. NIH‐3T3 cells stably over‐expressing wt EGFR are added and adhere to these patterned surfaces. We observe concentration of fluorescently labeled EGFR and stimulated tyrosine phosphorylation that are spatially confined to the patterns of immobilized EGF. We also observe recruitment of paxillin‐GFP to the receptor signaling complexes formed on EGF patterns, and this association occurs in the absence of integrin co‐clustering but depends on EGFR tyrosine kinase activity and F‐actin, which also accumulates in these patterned regions. Inhibition of Src kinase with PP2 inhibits tyrosine phosphorylation of associated paxillin but does not prevent its localization with patterned EGF. We further detect recruitment of phospholipase Cγ‐1‐GFP and phosphorylated Erk to the patterned EGF in a ligand‐dependent manner. Our ongoing studies are investigating the roles of paxillin and F‐actin in recruitment of Erk to the EGF‐mediated signaling complex. These studies highlight the utility of micro‐patterned growth factors to characterize the spatially regulated formation of signaling complexes at the plasma membrane. Supported by NIH Grant R01‐AI018306.