Eps8, a Tyrosine Kinase Substrate, Is Recruited to the Cell Cortex and Dynamic F-Actin upon Cytoskeleton Remodeling

• Published in: Experimental cell research 1998-07, Vol.242 (1), p.186-200
• Main Authors: Provenzano, Claudia, Gallo, Rita, Carbone, Roberta, Di Fiore, Pier Paolo, Falcone, Germana, Castellani, Loriana, Alemà, Stefano
• Format: Article
• Language: English
• Subjects: 3T3 Cells; actin cytoskeleton; Actins - analysis; Adaptor Proteins, Signal Transducing; Animals; Blood; Catenins; Cell Adhesion Molecules - analysis; Cell Line; Cortactin; Cytoskeletal Proteins; Cytoskeleton - chemistry; Cytoskeleton - metabolism; Detergents; Dogs; Epidermal Growth Factor - pharmacology; Epithelial Cells - cytology; Eps8; ErbB Receptors - analysis; ErbB Receptors - physiology; Fibroblasts - cytology; Mice; Microfilament Proteins - analysis; Muscles - cytology; Octoxynol; Oncogene Protein pp60(v-src) - genetics; Oncogene Protein pp60(v-src) - metabolism; p120 cas; Phosphoproteins - analysis; Proteins - analysis; Quail; Receptor, ErbB-2 - analysis; Receptor, ErbB-2 - physiology; SH3 domain; Tetradecanoylphorbol Acetate - pharmacology; tyrosine kinase
• ISSN: 0014-4827; 1090-2422
• DOI: 10.1006/excr.1998.4095

Eps8 is a recently identified substrate of receptor and nonreceptor tyrosine kinases implicated in the control of cell proliferation. To investigate potential functions of Eps8, its intracellular localization has been examined in several cell types. In cycling fibroblasts immunolabeling with antibodies to Eps8 reveals a punctate pattern within the perinuclear region and staining of motile peripheral cell extensions and cell–cell contact regions. Stimulation of quiescent Swiss 3T3 fibroblasts with serum induces a striking reorganization of the actin cytoskeleton which is accompanied by the enrichment of Eps8 and cortactin in membrane ruffles and lamellipodia. A similar accumulation of Eps8 to membrane ruffles is observed in cells treated with phorbol esters, which also induce marked changes of the F-actin cytoskeleton. The localization of Eps8 at the cell cortex is largely independent from the binding of Eps8 to an EGFR/ErbB-2 chimeric receptor. Moreover, fractionation studies reveal that a portion of the Eps8 molecules present in the cell periphery, unlike cortactin and the receptor, is resistant to mild extraction with detergent. Upon cellular transformation by the tyrosine kinase v-Src, a pool of Eps8 is recruited to newly formed specialized regions of the cytoskeleton, such as actin bodies in terminally differentiated myotubes and podosomes in fibroblasts, where cortactin and a variety of cytoskeletal proteins are also found. Extraction with Triton X-100 preserves the association of Eps8 to podosomes and leaves the majority of the v-Src tyrosine-phosphorylated Eps8 in the detergent-resistant fraction. The observed recruitment of Eps8 to highly dynamic cytoskeletal structures of normal and transformed cells suggests that Eps8 may play a role in the reorganization of the cytoskeleton, perhaps acting as a docking site for other signaling molecules.