Extracellular signal-regulated kinase 1/2 (ERK1/2) is activated by various extracellular stimuli including growth factors and cytokines and plays a pivotal role in regulating cell proliferation and differentiation by phosphorylating nuclear transcription factors. Recently, it was reported that activated ERK1/2 also concentrates at adhesion sites and regulates cell spreading and migration. Vinexin is a focal adhesion protein regulating both cell spreading and growth factor signaling. We show here that vinexin was directly phosphorylated by ERK1/2 upon stimulation with growth factors. ERK1/2 phosphorylated the linker region of vinexin between the second and third SH3 domains. Site-directed mutagenesis revealed that ERK2 mainly phosphorylated the serine 189 residue of vinexin . Furthermore, vinexin  interacted with ERK1/2 both in vitro and in vivo. Vinexin interacted with the active but not inactive form of ERK1/2. A putative DEF (docking for ERK FXFP) domain located in the linker region of vinexin was required for the interaction with ERK1/2 and efficient phosphorylation of vinexin  by ERK2. Finally, we showed that cell adhesion to fibronectin also induced the association of vinexin  with ERK2 and the phosphorylation of vinexin . Furthermore, vinexin and ERK were co-localized to the periphery of cells during cell spreading on fibronectin. Together, these results suggest that vinexin is a novel substrate of ERK2 and may play roles in ERK-dependent cell regulation during cell spreading as well as in growth factor-induced responses.Mitogen-activated protein kinase (MAPK) 1 consists of four subfamilies, extracellular signal-regulated kinase 1/2 (ERK1/ 2), c-Jun N-terminal kinase/stress-activated kinase, p38MAPK, and ERK5. ERK1/2 is mainly activated by various growth factors and regulates a diverse array of cellular events including cell proliferation, survival, and differentiation (1, 2). Many extracellular signals activate membrane receptors, including receptor tyrosine kinases, G protein-coupled receptors, or integrins, leading to the activation of Raf. Activated Raf, in turn, activates MEK (MAPK/ERK kinase) by the direct phosphorylation of dual serine residues. Activated MEK also directly phosphorylates and activates ERK1/2. Once activated, ERK1/2 enters the nucleus and phosphorylates nuclear transcription factors, including Elk-1, Sap1, c-Myc, and c-Fos (3).Recently, ERK has been shown to play crucial roles in regulating cell adhesion and cell migration independent of its nuclear functions (4 -7). ERK is involved in the migration of breast cancer cells stimulated by urokinase-type tissue plasminogen activator. Neither de novo gene transcription nor protein synthesis is required for this process (5). Activation of ERK is also suggested to be involved in the chemotaxis or the extension of pseudopodia, probably through the phosphorylation of cytoplasmic proteins (8, 9). Furthermore, ERK is well known to be activated by integrin engagement and to be localized to adhesion sites (7, 10 -14). Although some cytoplasm...