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

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Methods to Study Mrp4-containing Macromolecular Complexes in the Regulation of Fibroblast Migration
10:43

Methods to Study Mrp4-containing Macromolecular Complexes in the Regulation of Fibroblast Migration

Published on: May 19, 2016

Anne N Shemon1, Eva M Eves, Matthew C Clark

  • 1Ben May Department for Cancer Research, University of Chicago, Chicago, IL, USA.

Plos One
|June 25, 2009
PubMed
Abstract

Insights

Locostatin does not inhibit cell migration by binding Raf Kinase Inhibitory Protein (RKIP). RKIP actually protects cells from locostatin

Area of Science:

  • Cell biology
  • Molecular signaling
  • Drug discovery

Background:

  • Raf Kinase Inhibitory Protein (RKIP) regulates growth factor signaling via the Raf/MAP kinase pathway.
  • Locostatin, an organic compound, was previously thought to inhibit cell migration by binding RKIP.
  • The precise mechanism of locostatin's action and RKIP's role remained unclear.

Purpose of the Study:

  • To investigate the interaction between locostatin and RKIP.
  • To determine the biological consequences of locostatin binding on RKIP function.
  • To elucidate the role of RKIP in locostatin-mediated inhibition of cell migration.

Main Methods:

  • Nuclear Magnetic Resonance (NMR) studies to analyze locostatin-RKIP binding.
  • Experiments using RKIP-depleted and RKIP-deficient cells (HeLa and mouse embryonic fibroblasts).
  • Assessment of MAP kinase activation, cell migration, and cytoskeletal structure.

Main Results:

  • Locostatin precursor binds to RKIP's phosphatidylethanolamine binding pocket.
  • Locostatin binding does not inhibit RKIP's interaction with Raf-1 or its phosphorylation.
  • Locostatin treatment did not affect MAP kinase activation but inhibited cell migration; RKIP deficiency exacerbated this inhibition.
  • Locostatin induced cytoskeletal and microtubule abnormalities in RKIP-deficient cells.

Conclusions:

  • Locostatin's effects on cell migration and cytoskeleton are independent of RKIP and Raf/MAP kinase signaling.
  • RKIP plays a protective role against locostatin-induced migration inhibition.
  • RKIP may function in sequestering potentially toxic compounds, suggesting a novel cellular protective mechanism.

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