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


RESULTS
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.


CONCLUSIONS
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.
Shemon
Anne N
AN
Ben May Department for Cancer Research, University of Chicago, Chicago, IL, USA.
Eves
Eva M
EM
Clark
Matthew C
MC
Heil
Gary
G
Granovsky
Alexey
A
Zeng
Lingchun
L
Imamoto
Akira
A
Koide
Shohei
S
Rosner
Marsha Rich
MR
D000818:Animals; D045744:Cell Line, Tumor; D002465:Cell Movement; D003599:Cytoskeleton / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism*; D051379:Mice; D008954:Models, Biological; D023303:Oxazolidinones / Q000494:pharmacology*; D049488:Phosphatidylethanolamine Binding Protein / Q000378:metabolism*; D011485:Protein Binding; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction
D036341:Intercellular Signaling Peptides and Proteins; D023303:Oxazolidinones; D049488:Phosphatidylethanolamine Binding Protein; C529490:locostatin; D019908:Proto-Oncogene Proteins c-raf