Raf Kinase Inhibitory Protein protects cells against locostatin-mediated inhibition of migration

• Published in: PloS one 2009-06, Vol.4 (6), p.e6028
• Main Authors: Shemon, Anne N, Eves, Eva M, Clark, Matthew C, Heil, Gary, Granovsky, Alexey, Zeng, Lingchun, Imamoto, Akira, Koide, Shohei, Rosner, Marsha Rich
• Format: Article
• Language: English
• Subjects: Abnormalities; Alcohol; Animals; Binding; Biochemistry; Biochemistry/Cell Signaling and Trafficking Structures; Biochemistry/Drug Discovery; Brain research; Cell adhesion & migration; Cell Biology/Cell Signaling; Cell growth; Cell Line, Tumor; Cell migration; Cell Movement; Cytoskeleton; Cytoskeleton - metabolism; Dehydrogenases; Disruption; Embryo fibroblasts; Embryos; Enzymes; Fibroblasts; Fibroblasts - metabolism; HeLa Cells; Humans; Inhibition; Intercellular Signaling Peptides and Proteins - metabolism; Kinases; Leukocyte migration; Ligands; Liver cancer; MAP kinase; Medical research; Mice; Models, Biological; Molecular biology; Neurobiology; Neurosciences; NMR; Nuclear magnetic resonance; Oxazolidinones - pharmacology; Phosphatidylethanolamine; Phosphatidylethanolamine Binding Protein - metabolism; Phosphorylation; Physiology; Protein Binding; Protein kinase C; Protein kinases; Proteins; Proto-Oncogene Proteins c-raf - metabolism; Raf protein; Rats; Sequestering; Signal Transduction; Signaling; Stem cells; Wounding
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0006028

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. 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 (RKIP(-/-)) 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 RKIP(-/-) MEFs reveals cytoskeletal disruption and microtubule abnormalities in the spindle. 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.