differences in their effects on cell migration or cytoskeleton, unfortunaletly solubility constraints for the precursor limited our study in an intact cell system. The effect of locostatin is highly concentration-dependent; just a two-fold decrease in dose caused loss of inhibition. Thus, a reduction of the effective concentration of locostatin in cells could significantly diminish its efficacy. RKIP may be present in the cell at sufficient concentrations to promote RKIP-locostatin interaction and sequester the toxic drug. Alternatively, it is also possible that RKIP plays a discrete role in cytoskeletal organization and migration that counteracts the effects of locostatin. Cell migration is a key step underlying cellular development as well as other physiological and disease processes but whether RKIP plays a role in regulating this process is still unresolved. In some studies, RKIP overexpression inhibits migration and invasion [18,19]. In other reports, RKIP promotes cell migration by downregulating E cadherin and upregulating b1 integrin [13,14]. In the present study using MEFs, the presence or absence of RKIP has no effect on the rate of wound healing. The inhibitory action of locostatin on wound healing in the MEFs appears to be associated with changes in the cytoskeleton. We stained cells of varying RKIP genotypes for actin with phalloidin and observed the shortening of stress fibers within the cytoplasm and a concentration of actin around the nucleus. These were exacer- bated in MEFs with depleted or absent RKIP and these cells had an increased amount of nuclear bodies with very little actin association compared to their wild-type counterparts. Similarly, the inhibitory effect of locostatin on migration and cytoskeleton was more pronounced when cells were grown to a lower density as opposed to the confluent monolayers described by [17], and we also observed interbatch variations of locostatin potency. Collec- tively, these discrepancies may reflect differences in cell type and/ or assays and will require further analysis of multiple tissues from RKIP knockout mice. At the doses of locostatin used in this study, we have shown that RKIP may not be the only locostatin target regulating cell migration. Previously, [3H]locostatin was shown in an association assay to bind to four proteins only, one of which was RKIP [13]. The other proteins were omega 1-1, prolyl endopeptidase and aldehyde dehydrogenase (ALDH1A1). However, they reported that ALDH1A1, which belongs to the ALDH family of enzymes and is known to clear the cell of toxic reactive oxygen species byproducts predominantly in the liver [20], bound to locostatin but was not involved in cell migration. Interestingly, ALDH1A1 was recently shown to mediate cell invasion in pancreatic cancer cells [21] suggesting that it does play a role in cytoskeletal rearrangement. Therefore, these results raise the question whether ALDH1A1 might function to bind locostatin as a toxic byproduct for its removal from the cell. If this were true, ALDH1A1 function would complement a role for RKIP in binding to locostatin to mop up the drug and subsequently dampen its anti-migratory effect on the cell. To our knowledge this report provides the first evidence that locostatin can alter both microtubule (tubulin) and actin-based cytoskeletal elements. We observed that locostatin interferes with the cytoskeleton of MEFs since it caused the collapse of the actin cytoskeleton within the cell and localized actin to perinuclear regions. Locostatin also disrupted mitotic spindle formation in MEFs similar to the effects of Taxol. These results suggest that locostatin plays a role in altering the cytoskeleton via a yet to be defined mechanism warranting future investigation. The present study of locostatin binding to RKIP also provides insight into the role of the pocket in Raf-1 interactions. The precursor bound directly to the pocket as shown by the Locostatin Impairs Migration perturbation of key residues in the RKIP ligand binding pocket. Although locostatin’s binding to RKIP could not be examined directly by NMR due to its aggregation at high concentrations, it is likely that locostatin binds to the same RKIP pocket residues. The data show precursor binding to the RKIP pocket is not sufficient to interfere with Raf binding, consistent with the observed MAPK signaling in locostatin-treated cells. This result is in contrast to the lipid DHPE that not only binds the pocket but also inhibits RKIP- Raf association. Comparison of these different outcomes suggests that other residues and/or steric features of RKIP as well as size of the ligand are critical for Raf-1 binding [12]. RKIP is a negative regulator of major signaling cascades including MAPK; therefore, the identification of drugs that bind to RKIP and influence its inhibitory function could represent an important therapeutic strategy. Although locostatin is the first described drug to bind RKIP, it is likely that other chemicals also interact with RKIP via the ligand binding pocket. The studies here suggest that it may be possible to find therapeutic agents that either potentiate or suppress RKIP association with kinases and its inhibitory targets. Further studies to characterize the nature of the interactions will be needed to identify the most effective RKIP- modulating drugs. This work also suggests that RKIP might serve as a ‘‘reservoir’’ for hydrophobic chemicals circulating in the blood. Materials and Methods Reagents and antibodies (S)-(+)-4-benzyl-3-crotonyl-2-oxazolidinone (locostatin) and its precursor, (S)-4-benzyl-2-oxazolidinone (Fig. 1A) used in NMR studies were from Aldrich (St. Louis, MO). Protease inhibitor cocktails and locostatin unless otherwise indicated were from Calbiochem. All cell culture media and supplements were from GIBCO (Invitrogen, Carlsbad, CA). 15N-ammonium chloride was from Cambridge Isotope Laboratories (Andover, MA). Anti- phospho and -total ERK antibodies were from Cell Signaling Technology (Beverly, MA). Anti-rabbit and –mouse IR dye secondary antibodies as well as Odyssey blocking buffer used for Western blotting were purchased from LI-COR Biosciences (Lincoln, NE). Anti-a tubulin (B-7) mouse antibody was purchased from Santa Cruz Technologies (Santa Cruz, CA). Anti-RKIP antibody was developed as described [4]. FITC-conjugated donkey anti-mouse secondary antibody was from Jackson Immunolabs (West Grove, PA). Ho¨ echst 33342 nucleic acid stain and Alexafluor 594-tagged phalloidin were from Molecular Probes (Eugene, OR) and mounting media was Vectashield Hardset from Vector Labs (Burlingame, CA). IgG sepharose beads were from Amersham Biosciences (GE Healthcare, Piscataway, NJ). Biotiny- lated thrombin (50 U) was from Novagen (Madison, WI). Cell lines and cell culture HeLa cells stably expressing short hairpin RNA vectors for depleting human RKIP or rat RKIP control were maintained at 37uC/5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 50 U/ml penicillin and 50 mg/ml streptomycin under puromycin (2 mg/ml) selection as previously described [5]. Mouse embryonic fibroblasts (MEFs) expressing varying genotypes of RKIP (wild-type RKIP+/+ or homozygous RKIP2/2) were isolated from knockout RKIP mice that were generated using embryonic stem cells established by the Sanger Center using a gene targeting approach similar to that previously described [22]. MEFs were isolated from 12.5-day-old embryos that were derived from intercross of RKIP+/2 mice. The generation and characterization of RKIP knockout mice and PLoS ONE | www.plosone.org 11 June 2009 | Volume 4 | Issue 6 | e6028