Extracellular Signal-Regulated Kinase/Mitogen-Activated Protein Kinase Regulates Actin Organization and Cell Motility by Phosphorylating the Actin Cross-Linking Protein EPLIN

Abstract

Extracellular signal-regulated kinase (ERK) is important for various cellular processes, including cell migration. However, the detailed molecular mechanism by which ERK promotes cell motility remains elusive. Here we characterize epithelial protein lost in neoplasm (EPLIN), an F-actin cross-linking protein, as a novel substrate for ERK. ERK phosphorylates Ser360, Ser602, and Ser692 on EPLIN in vitro and in intact cells. Phosphorylation of the C-terminal region of EPLIN reduces its affinity for actin filaments. EPLIN colocalizes with actin stress fibers in quiescent cells, and stimulation with platelet-derived growth factor (PDGF) induces stress fiber disassembly and relocalization of EPLIN to peripheral and dorsal ruffles, wherein phosphorylation of Ser360 and Ser602 is observed. Phosphorylation of these two residues is also evident during wound healing at the leading edge of migrating cells. Moreover, expression of a non-ERK-phosphorylatable mutant, but not wild-type EPLIN, prevents PDGF-induced stress fiber disassembly and membrane ruffling and also inhibits wound healing and PDGF-induced cell migration. We propose that ERK-mediated phosphorylation of EPLIN contributes to actin filament reorganization and enhanced cell motility.

SUPPLEMENTAL MATERIAL

We thank Michimoto Kobayashi for his help during the initial stages of this work, Yutaka Harita for help with rabbit immunization, Makoto Watanabe for help with isolating primary calvarial osteoblasts, Hiroyuki Fukuda for LC-MS/MS analysis, Miho Ohsugi and Noriko Tokai-Nishizumi for help with time-lapse video microscopy, Masanori Mishima and Max Douglas for critical readings of the manuscript, and Shiro Suetsugu and Eisuke Nishida for helpful discussions and advice. We also thank Martin McMahon for kindly providing ΔB-Raf:ER cells.

This work was supported in part by grants-in-aid for scientific research from the Japan Society for the Promotion of Science (to H.K.) and the Encouraging Development Strategic Research Centers Program, Special Coordination Funds for Promoting Science and Technology, the Ministry of Education, Culture, Sports, Science, and Technology (to S.H.). This work was also supported by grants from the Nakajima Foundation (to H.K.) and the Novartis Foundation (Japan) for the Promotion of Science (to S.H.). This work was developed and coordinated under the framework of the program for the International Research and Educational Institute for Integrated Medical Sciences (IREIIMS).