title: Spatially defined EGF receptor activation reveals an F-actin-dependent phospho-Erk signaling complex.
abstract: We investigated the association of signaling proteins with epidermal growth factor (EGF) receptors (EGFR) using biotinylated EGF bound to streptavidin that is covalently coupled in an ordered array of micron-sized features on silicon surfaces. Using NIH-3T3 cells stably expressing EGFR, we observe concentration of fluorescently labeled receptors and stimulated tyrosine phosphorylation that are spatially confined to the regions of immobilized EGF and quantified by cross-correlation analysis. We observe recruitment of phosphorylated paxillin to activated EGFR at these patterned features, as well as β1-containing integrins that preferentially localize to more peripheral EGF features, as quantified by radial fluorescence analysis. In addition, we detect recruitment of EGFP-Ras, MEK, and phosphorylated Erk to patterned EGF in a process that depends on F-actin and phosphoinositides. These studies reveal and quantify the coformation of multiprotein EGFR signaling complexes at the plasma membrane in response to micropatterned growth factors.
authors.affiliation: Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York.
authors.forename: Amit
authors.initials: A
authors.lastname: Singhai
authors.affiliation: Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York.
authors.forename: Devin L
authors.initials: DL
authors.lastname: Wakefield
authors.affiliation: Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York.
authors.forename: Kirsten L
authors.initials: KL
authors.lastname: Bryant
authors.affiliation: University of Rochester Medical Center, Rochester, New York.
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Hammes
authors.affiliation: Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York.
authors.forename: David
authors.initials: D
authors.lastname: Holowka
authors.affiliation: Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York. Electronic address: bab13@cornell.edu.
authors.forename: Barbara
authors.initials: B
authors.lastname: Baird
mesh_terms: D008841:Actin Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D000199:Actins / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D034285:Dynamin II / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism*; D019012:Integrin beta1 / Q000378:metabolism; D008024:Ligands; D020935:MAP Kinase Signaling System / Q000187:drug effects; D051379:Mice; D041681:NIH 3T3 Cells; D051419:Paxillin / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000378:metabolism; D058105:Polymerization / Q000187:drug effects; D020123:Sirolimus / Q000494:pharmacology
chemical_list: D000199:Actins; D019012:Integrin beta1; D008024:Ligands; D051419:Paxillin; D019269:Phosphatidylinositol 4,5-Diphosphate; C030473:biotinyl epidermal growth factor; D019000:Phosphotyrosine; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D034285:Dynamin II; D020123:Sirolimus