Raf Kinase Inhibitory Protein Protects Cells against Locostatin-Mediated Inhibition of Migration Anne N. Shemon 1 , Eva M. Eves1, Matthew C. Clark1,2, Gary Heil3, Alexey Granovsky1, Lingchun Zeng 1 , Akira Imamoto1, Shohei Koide3.*, Marsha Rich Rosner1,2.* 1 Ben May Department for Cancer Research, University of Chicago, Chicago, Illinois, United States of America, 2 Department of Neurobiology, Pharmacology and Physiology, University of Chicago, Chicago, Illinois, United States of America, 3 Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois, United States of America Abstract Background: Raf Kinase Inhibitory Protein (RKIP, also PEBP1), a member of the Phosphatidylethanolamine Binding Protein family, negatively regulates growth factor signaling by the Raf/MAP kinase pathway. Since an organic compound, locostatin, was reported to bind RKIP and inhibit cell migration by a Raf-dependent mechanism, we addressed the role of RKIP in locostatin function. Methods/Findings: We analyzed locostatin interaction with RKIP and examined the biological consequences of locostatin binding on RKIP function. NMR studies show that a locostatin precursor binds to the conserved phosphatidylethanolamine binding pocket of RKIP. However, drug binding to the pocket does not prevent RKIP association with its inhibitory target, Raf-1, nor affect RKIP phosphorylation by Protein Kinase C at a regulatory site. Similarly, exposure of wild type, RKIP- depleted HeLa cells or RKIP-deficient (RKIP2/2) mouse embryonic fibroblasts (MEFs) to locostatin has no effect on MAP kinase activation. Locostatin treatment of wild type MEFs causes inhibition of cell migration following wounding. RKIP deficiency impairs migration further, indicating that RKIP protects cells against locostatin-mediated inhibition of migration. Locostatin treatment of depleted or RKIP2/2 MEFs reveals cytoskeletal disruption and microtubule abnormalities in the spindle. Conclusions/Significance: These results suggest that locostatin’s effects on cytoskeletal structure and migration are caused through mechanisms independent of its binding to RKIP and Raf/MAP kinase signaling. The protective effect of RKIP against drug inhibition of migration suggests a new role for RKIP in potentially sequestering toxic compounds that may have deleterious effects on cells. Citation: Shemon AN, Eves EM, Clark MC, Heil G, Granovsky A, et al. (2009) Raf Kinase Inhibitory Protein Protects Cells against Locostatin-Mediated Inhibition of Migration. PLoS ONE 4(6): e6028. doi:10.1371/journal.pone.0006028 Editor: Mikhail V. Blagosklonny, Roswell Park Cancer Institute, United States of America Received March 17, 2009; Accepted May 20, 2009; Published June 24, 2009 Copyright: 2009 Shemon et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by NIH grants NS033858 and CA112310 to MRR, a gift from the Cornelius Crane Trust for Eczema Research to MRR, and a CJ Martin postdoctoral fellowship from the Australian National Health and Medical Research Council to ANS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: skoide@uchicago.edu (SK); m-rosner@uchicago.edu (MRR) . These authors contributed equally to this work. Introduction Raf Kinase Inhibitory Protein (RKIP) is an inhibitor of key signal transduction cascades in mammalian cells that regulate growth and differentiation (reviewed in [1,2]). RKIP binding to Raf-1 prevents MAP kinase signaling in response to growth factors [3]. Following stimulation of growth factor receptors, RKIP is phosphorylated at Ser-153 by protein kinase C (PKC), enabling RKIP dissociation and subsequent phosphorylation of Raf-1 at activating sites [4,5]. RKIP binding to G-protein coupled receptor kinase 2, a kinase that phosphorylates and downregulates G- protein-coupled receptors, causes up-regulation of G protein- coupled receptors such as the b-adrenergic receptor [6]. RKIP has also been shown to bind NF-kB-inducing enzyme, NIK and inhibit signaling mediated by NF-kB [7]. Finally, RKIP regulates the spindle checkpoint through inhibition of the MAP kinase cascade [8]. RKIP is also missing or depleted in a number of metastatic tumors (reviewed in [1]), and has been implicated as a metastasis suppressor through regulation of one or more of these signaling cascades [9,10]. RKIP, also termed phosphatidylethanolamine binding protein- 1 (PEBP-1), is a 20–25 kDa globular protein that belongs to the PEBP family comprised of over 400 members. The crystal structures of PEPBs from bacteria to humans have revealed a remarkably conserved ligand-binding pocket [1]. In these crystal structures, phosphorylethanolamine as well as other ions are found in the ligand-binding pocket [11], and the pocket has been shown to bind hydrophobic ligands such as phospholipids [12]. While other ligands have also been reported to bind RKIP, the specific ligands that bind RKIP at physiological pH are yet to be defined. Since RKIP regulates key physiological processes, it is of interest for therapeutic purposes to identify agents that either prevent or potentiate its ability to inhibit target proteins such as Raf-1. Recently, locostatin ((S)-(+)-4-benzyl-3-crotonyl-2-oxazolidinone) PLoS ONE | www.plosone.org 1 June 2009 | Volume 4 | Issue 6 | e6028