In the present study, we have characterized an F-actin cross-linking protein, EPLIN, as a novel ERK MAPK substrate. First, ERK phosphorylates EPLIN on Ser360, Ser602, and Ser692 in vitro and in living cells. Second, ERK phosphorylation of EPLIN decreases the affinity of its C-terminal region for actin filaments. Third, EPLIN localizes to actin stress fibers in quiescent cells, and stimulation with PDGF induces relocalization of EPLIN to lamellipodia/membrane ruffles. Fourth, phosphorylated EPLIN localizes to membrane ruffles both upon PDGF stimulation and during wound healing. Fifth, a non-ERK-phosphorylatable mutant of EPLIN inhibits PDGF-dependent actin stress fiber disassembly, membrane ruffling, and cell migration, while RNAi-mediated silencing of EPLIN enhances cell motility. ERK thus controls actin organization and cell motility by phosphorylating EPLIN.
ERK phosphorylation sites within EPLIN were identified by site-directed mutagenesis. We determined that the Ser360, Ser602, and Ser692 residues of EPLIN are the major phosphorylation sites for ERK. These phosphorylation sites were confirmed by LC-MS/MS analysis of in vitro-phosphorylated EPLIN (see Fig. S2A, C, and D in the supplemental material). Immunoblot analysis using phospho-specific antibodies revealed that these three sites are indeed phosphorylated by ERK in intact cells (Fig. and ). Although recent phosphoproteomic studies detected intracellular phosphorylation of mouse EPLIN at Ser360 (43
) and of human EPLIN at Ser604 and Ser698 (corresponding to Ser602 and Ser692 of mouse EPLIN) (27
), spatiotemporal changes had not been reported. Interestingly, PDGF-induced phosphorylation of Ser360 occurred rather slowly compared to the rapid phosphorylation of Ser602, Ser692, and ERK (Fig. ). This raises the possibility that cellular phosphatase activity toward Ser360 is high in the early phase or that ERK indirectly phosphorylates Ser360 through a downstream kinase.
EPLIN contains two actin-binding sites, in the N- and C-terminal halves, and a LIM domain between these sites may allow EPLIN to homodimerize. EPLIN therefore cross-links and bundles actin filaments, but the two actin-binding domains may have different functions in the cell (23
). In cosedimentation assays with F-actin, we found that the C-terminal half of EPLIN, but neither full-length EPLIN nor the N-terminal half of EPLIN, reduces its association with F-actin upon ERK-mediated phosphorylation. This observation was confirmed by an in vivo experiment showing that the amount of actin coimmunoprecipitated with the C-terminal half but neither full-length EPLIN nor the N-terminal half of EPLIN was reduced by activation of ERK. Since EPLIN is supposed to bind to the side of an actin filament through two actin-binding domains (23
), it may be reasonable that a reduction in the actin-binding activity of the C-terminal region does not necessarily lead to a significant decrease in that of full-length EPLIN (Fig. ).
The phosphorylation-dependent reduction of the affinity of the C-terminal region for F-actin may affect the actin-bundling activity of EPLIN to facilitate dynamic remodeling of actin filament networks. Thus, we investigated the effects of EPLIN phosphorylation on its localization, actin dynamics, and cell motility. It has been reported that endogenous EPLIN is distributed predominantly along actin stress fibers in U2OS cells (35
). Consistent with this finding, immunostaining showed that EPLIN colocalized with stress fibers in quiescent NIH 3T3 cells (Fig. ) and primary osteoblasts (Fig. ). Stimulation with PDGF induced stress fiber disassembly and relocalization of EPLIN to membrane ruffles within 15 to 30 min. When cells were treated with PDGF in the presence of U0126, stress fiber disassembly was partly inhibited by blocking the ERK pathway (29
), and a fraction of EPLIN remained localized on the resultant stress fibers.
We further demonstrated by indirect immunofluorescence microscopy that both Ser360 and Ser602 are phosphorylated in specific subcellular areas by PDGF stimulation or during cell migration, suggesting the physiological significance of these phosphorylation sites in cellular processes. Both staining patterns were not detectable when the cells were pretreated with U0126 (Fig. and Fig. ) or transfected with siRNA for ERK2 or ERK1 plus ERK2 (Fig. ), indicating ERK-dependent phosphorylation. PDGF treatment induced the phosphorylation of EPLIN at peripheral and dorsal ruffles. In migrating NIH 3T3 fibroblasts, phosphorylated EPLIN preferentially localized to the leading edge, which is consistent with previous observations that activated ERK is also localized at the leading edge during migration of rat embryo fibroblasts and 3Y1 cells (21
). These findings support the possible involvement of EPLIN phosphorylation by ERK in actin reorganization and cell migration (see below).
To clarify the effects of EPLIN phosphorylation on actin organization and cell motility, we used wild-type EPLIN and a non-ERK-phosphorylatable mutant EPLINα fused to EGFP. The nonphosphorylatable mutant inhibited both cellular processes. The precise molecular mechanism by which ERK promotes ruffle formation and cell migration via phosphorylating EPLIN remains unclear. Since the mutant contains substitutions in both the N- and C-terminal regions, there remained the possibility that the reduction of the affinity of the C-terminal region for F-actin may not participate in this mechanism. However, in a modified Boyden chamber assay, two substitutions in the C-terminal region [EPLINα(S602/692A)-EGFP migration index, 3.53 ± 0.46] showed a similar inhibitory effect to that by three substitutions [EPLINα(S360/602/692A)-EGFP migration index, 3.03 ± 0.35] compared with the wild type (EPLINα-EGFP migration index, 5.17 ± 0.47) or a protein with one substitution in the N-terminal region [EPLINα(S360A)-EGFP migration index, 4.67 ± 0.52], indicating the importance of a phosphorylation-dependent reduction in the C-terminal binding activity. Phosphorylation of Ser360 in the N-terminal region causes an electrophoretic mobility shift (see Fig. S1 in the supplemental material), suggesting conformational and functional changes that should be addressed in future studies. Since nonphosphorylated EPLIN dimers can form thick actin bundles through the N- and C-terminal actin-binding sites, EPLIN in quiescent cells may stabilize stress fibers and inhibit cell migration (Fig. , upper panel). Phosphorylated EPLIN dimers can cross-link actin filaments through only the N-terminal actin-binding sites, and thereby EPLIN in migrating cells may form a dynamic actin meshwork in membrane ruffles (Fig. , lower panel). Taken together, the data show that PDGF stimulation activates ERK, which phosphorylates EPLIN to reduce the affinity of its C-terminal region for actin filaments, and then phosphorylated EPLIN causes destabilization of stress fibers and reorganization of the actin cytoskeleton to form membrane ruffles and to enhance cell migration.
ERK is known to regulate actin organization and cell motility by phosphorylating a number of proteins, including MLCK, FAK, paxillin, actopaxin, and vinexin. We demonstrate in this study that EPLIN is also a mediator of ERK-regulated cytoskeletal dynamics. Because the expression of phosphomimetic mutants of EPLIN had weak effects on these processes (data not shown), many actin-binding proteins phosphorylated by ERK are likely to act in concert to regulate actin dynamics. Furthermore, various extracellular stimuli induce actin reorganization and cell migration through other ERK-independent pathways. For example, it was recently reported that Akt regulates these processes via phosphorylation of girdin, an F-actin cross-linking protein (7
). Other actin cross-linking proteins, such as fascin (42
) and L-plastin (15
), were also shown to be regulated by phosphorylation to control actin cytoskeletal assembly and cell motility.
It has been reported that EPLIN is down-regulated or lost in a number of oral, prostate, and breast cancer cell lines (3
). Since siRNA-mediated depletion of EPLIN enhanced cell motility during wound healing and in PDGF-induced cell migration, the down-regulation of EPLIN expression might be relevant to migration and invasion of these cancer cells. Previously, it was reported that ectopic expression of EPLIN can suppress anchorage-independent growth of NIH 3T3 cells transformed by Cdc42V12 or EWS/Fli-1 but not by RasV12 (35
). This can now be explained by actin reorganization and enhanced cell motility through the Ras-Raf-MEK-ERK-EPLIN pathway. Ras-mediated phosphorylation of EPLIN may be involved in the invasion of tumor cells with Ras mutations. EPLIN is highly conserved from zebra fish to humans and contains multiple Ser/Thr-Pro motifs that can potentially be phosphorylated by ERK. The ERK-EPLIN pathway may play important roles in diverse physiological processes in vertebrates.