was shown to bind to RKIP and inhibit migration of Madin-Darby canine kidney (MDCK) epithelial cells [13,14]. In this cell type, RKIP depletion also inhibited migration. Consistent with a role in Raf signaling, locostatin prevented co-immunoprecipitation of RKIP with Raf-1 in vitro. Overexpression of RKIP in these cells reversed the anti-migratory effect of locostatin and cells adopted a fibroblast-like phenotype [13]. More recently, locostatin was also observed to impair migration in another cell type, human breast adenocarcinoma (MCF7), as well as causing these cells to have poor cell-cell adhesive properties [14]. These results suggest that locostatin regulates cell migration via an RKIP-dependent, Raf/ MAPK-regulated mechanism. To address the role of RKIP in locostatin function, we analyzed locostatin interaction with RKIP and the biological consequences of locostatin treatment in both human and murine cell types. Our results suggest that locostatin interacts with the RKIP ligand- binding pocket but its effects on the cytoskeleton, mitotic spindle, and cell migration are independent of RKIP and the MAP kinase signal transduction cascade. Instead, RKIP appears to rescue cells from inhibition of migration by locostatin, raising the possibility that RKIP may function in part to sequester deleterious drugs. Results Locostatin precursor binds to the ligand binding pocket of RKIP To define the RKIP binding site for locostatin, we analyzed their interaction by NMR. We found that locostatin, a suicide inhibitor that irreversibly attached itself to RKIP, had limited solubility, and addition of locostatin to an NMR sample containing 100 mM RKIP caused RKIP precipitation, rendering NMR characterization of the RKIP-locostatin complex impossible. However, a precursor of locostatin that had the same basic structure but lacked the reactive crotonyl group, (S)-4-benzyl-2- oxazolidinone (Fig. 1A), was compatible with NMR studies. To detect perturbations caused by precursor binding at a single amino acid residue resolution, we obtained 1H,15N single-quantum correlation spectra (HSQC) in which each cross peak representing a directly bonded 1H-15N pair functions as an amide-specific probe in 15N-enriched proteins [15,16]. Since we previously obtained the complete sequence-specific resonance assignments of backbone amides for wild-type RKIP [16], this approach enabled us to relate any perturbed HSQC cross peaks to a specific residue within RKIP. A comparison of HSQC spectra of wild-type RKIP in the presence or absence of the locostatin precursor (Fig. 1B) revealed that a majority of peaks affected by ligand binding were associated with the ligand-binding pocket (Fig. 1C). The entire rim of the pocket as well as the C-terminal alpha helix were perturbed, and the strongest perturbations seem to cluster near the C-terminal alpha helix and the conserved P74 residue on the opposite face. However, there are almost no perturbations in the region of S153, the site that, when phosphorylated, prevents RKIP binding to Raf [4,12]. These results indicate that a locostatin- related compound indeed binds RKIP and occupies the evolutionarily conserved ligand-binding pocket of RKIP. Given the structural similarity, these results strongly suggest that locostatin also binds to the RKIP ligand-binding pocket. Locostatin does not alter RKIP binding to Raf-1 Initially we determined whether locostatin affects cellular RKIP association with Raf-1 either directly or indirectly via regulation of RKIP phosphorylation at S153. To test whether locostatin inhibits RKIP binding to Raf-1, we immunoprecipitated TAP-Raf-1 (Tandom Affinity Purification using Protein A and Flag-tagged Locostatin Impairs Migration Raf-1) that was stably expressed in H19-7 cells. The TAP-Raf-1 bound to beads was incubated with soluble, bacterially expressed RKIP in the presence of increasing concentrations of locostatin. We have used this approach previously (confirmed in Fig. 2A) to show that 2-dihexanoyl-sn-glycero-3-phosphoethanolamine (DHPE), a water-soluble, short-chain analog of phosphatidyleth- anolamine, binds to the RKIP pocket and competes with Raf-1 for association [12]. However, in contrast to DHPE, locostatin up to a concentration of 2.5 mM did not significantly inhibit RKIP binding to Raf-1 (Fig. 2A). It is also possible that locostatin binding to RKIP in cells alters PKC phosphorylation of RKIP, thus indirectly affecting associa- tion of RKIP with Raf-1. To test this possibility, we determined the effect of different locostatin concentrations on RKIP phosphorylation at S153 by PKC using an in vitro kinase assay. Again, in contrast to DHPE [12], locostatin had no effect on RKIP phosphorylation at S153 even at higher drug concentrations (Fig. 2B). Taken together, these results indicate that locostatin binding to RKIP does not significantly alter RKIP binding to its inhibitory target, Raf-1. Locostatin does not inhibit ERK We have previously shown that depletion of RKIP in HeLa cells enhances the amplitude of the ERK signal induced by epidermal growth factor (EGF) stimulation [5]. Since locostatin does not prevent RKIP association with Raf, then locostatin treatment of cells should not effect ERK signaling. Therefore, to investigate the role of locostatin in MAPK signaling, we pretreated control and RKIP-depleted HeLa cells with 20 or 50 mM locostatin for 30 min, a concentration range that was previously shown to inhibit cell migration [17]. Cells were then stimulated with EGF (10 ng/ml) in the final five minutes of incubation. As predicted, we observed a 2 to 3-fold increase in MAPK activity (measured as a ratio of phospho-ERK to total ERK) in depleted RKIP cells compared to control cells (Fig. 3A and B). Locostatin (20 or 50 mM) had no effect on basal levels of MAPK activity in these cells (Fig. 3). Similarly, locostatin failed to impair or enhance the EGF-induced MAPK activity independent of RKIP expression. An inactive analogue of locostatin that cannot bind RKIP, UIC1017 [17] also had no effect on EGF-induced MAPK activity in these cells (data not shown). To further confirm that any locostatin effects on cells are independent of RKIP or MAPK activation status, we also tested the effect of locostatin on mouse embryonic fibroblasts (MEFs) isolated from either wild type (RKIP+/+) or homozygous (RKIP2/ 2) mice. As observed with HeLa cells, neither 20 mM nor 50 mM locostatin had any effect on EGF-induced MAPK activity in MEFs that express or are deficient in RKIP (Fig. 3C–F). These results indicate that locostatin binding to the RKIP ligand pocket does not perturb RKIP/Raf-1 association nor alter MAPK activity. RKIP protects against locostatin inhibition of cellular migration after wound healing in MEFs Previous reports using MDCK and MCF-7 cells have shown that 50 mM locostatin inhibits the rate of cell migration of cultured cells following wounding [14]. To determine whether locostatin inhibits migration in another cell type, we established a scratch wound assay using RKIP+/+ MEFs (Fig. 4). MEFs were treated with DMSO (vehicle control) or two different doses of locostatin and the wounds were visualized using digital images at 0, 4, 8 and 24 h time points. In the presence of vehicle (0.25% DMSO), the cells healed within 24 h (Fig. 4A) with no signs of cell toxicity as determined by trypan blue exclusion (data not shown). Similarly, 20 mM locostatin had no effect on migration (Fig. 4A), as opposed PLoS ONE | www.plosone.org 2 June 2009 | Volume 4 | Issue 6 | e6028