EPS8, A TYROSINE KINASE SUBSTRATE, IS RECRUITED TO THE CELL CORTEX AND DYNAMIC F-ACTIN UPON CYTOSKELETON REMODELING

Eps8 is a recently identified substrate of receptor and nonreceptor ty rosine kinases implicated in the control of cell proliferation, To inv estigate potential functions of Eps8, its intracellular localization h as been examined in several cell types. In cycling fibroblasts immunol abeling with antibodies to Eps8 reveals a punctate pattern within the perinuclear region and staining of motile peripheral cell extensions a nd cell-cell contact regions. Stimulation of quiescent Swiss 3T3 fibro blasts with serum induces a striking reorganization of the actin cytos keleton which is accompanied by the enrichment of Eps8 and cortactin i n membrane ruffles and lamellipodia. A similar accumulation of Eps8 to membrane ruffles is observed in cells treated with phorbol esters, wh ich also induce marked changes of the F-actin cytoskeleton. The locali zation of Eps8 at the cell cortex is largely independent from the bind ing of Eps8 to an EGFR/ErbB-2 chimeric receptor. Moreover, fractionati on studies reveal that a portion of the Eps8 molecules present in the cell periphery, unlike cortactin and the receptor, is resistant to mil d extraction with detergent. Upon cellular transformation by the tyros ine kinase v-Src, a pool of Eps8 is recruited to newly formed speciali zed regions of the cytoskeleton, such as actin bodies in terminally di fferentiated myotubes and podosomes in fibroblasts, where cortactin an d a variety of cytoskeletal proteins are also found, Extraction with T riton X-100 preserves the association of Eps8 to podosomes and leaves the majority of the v-Src tyrosine-phosphorylated Eps8 in the detergen t-resistant fraction. The observed recruitment of Eps8 to highly dynam ic cytoskeletal structures of normal and transformed cells suggests th at Eps8 may play a role in the reorganization of the cytoskeleton, per haps acting as a docking site for other signaling molecules, (C) 1998 Academic Press.