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

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 (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/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.

Figure 1. Binding of the locostatin precursor (S)-4-benzyl-2-oxazolidinone (Sigma #294640) to RKIP characterized by NMR chemical shift perturbation.

(A) Chemical structure of locostatin and its precursor (S)-4-benzyl-2-oxazolidinone. (B) Comparison of ¹H,¹⁵N-HSQC spectra of ¹⁵N-enriched RKIP in the absence-black and the presence-red of the compound (1 mM). Each cross peak (dot) represents the correlation between the directly bonded amide ¹H and ¹⁵N atoms of amino acid residue. (C) The locations of amino acid residues whose HSQC peak is significantly affected by compound binding mapped on the RKIP structure. Red, yellow and gray spheres represent residues that have the weighted shift of >0.3 ppm, 0.2> ppm and >0.1 ppm, respectively.

Figure 2. DHPE but not locostatin decreases RKIP interactions with Raf-1 and PKC.

(A) RKIP association with Raf-1 in the presence or increasing concentrations of locostatin or DHPE. Upper panels: Representative western blots. Lower panels: Bar graphs representative of two independent experiments±range. (B) PKC phosphorylation of RKIP in the presence of increasing concentrations of locostatin or DHPE using RKIP or MBP as substrates. Upper panels: Representative autoradiogram is shown. Lower panels: Bar graphs representative of two independent experiments±range.

Figure 3. Locostatin has no effect on EGF-induced MAPK activity.

(A, B) HeLa cells expressing control or depleted RKIP or (C, D) MEFs expressing wild-type RKIP (RKIP^+/+) or (E, F) MEFs not expressing RKIP (RKIP^−/−) were serum starved overnight and then pre-treated for 30 min with DMSO or locostatin (concentrations as indicated). EGF (10 ng/ml) was added in the final 5 minutes of incubation. Whole cell lysates (30 µg) were separated on SDS-PAGE gels (12.5%), transferred to nitrocellulose and analysed by immunoblotting with anti-phospho ERK and anti-total ERK antibodies (A, C and E). ERK phosphorylation was assessed by normalizing phospho-ERK levels to total ERK levels as depicted in bar graphs (B, D and F). The results shown are representative of three independent experiments.

Figure 4. Locostatin impairs wound healing in wild-type and depleted RKIP MEFs.

MEFs expressing wild-type (A, B) or depleted (C, D) RKIP were seeded at 0.5×10⁶/ml and grown for 24 h before treatment with either DMSO (control) or locostatin at 20 (A) and (C) or 50 µM (B) and (D). (E) A standardized scratch (wound) was applied to monolayers and digital images were taken at time points indicated. Cell migration was determined as described in materials and methods. Data represents the mean±SEM of eight wounds from two independent experiments. ^*p<0.05, ^**p<0.01 indicates the significance of the change relative to the corresponding sample in the absence of locostatin.

Figure 5. Locostatin impairs wound healing independent of RKIP.

MEFs expressing wild-type (A, B) or deficient (C, D) RKIP were seeded at 0.5×10⁶/ml and grown for 24 h before treatment with either DMSO (control) or 20 (A) and (C) or 50 µM (B) and (D) locostatin. (E) A standardized scratch (wound) was applied to monolayers and analyzed as in materials and methods. Data represents the mean±SEM of three independent experiments (n = 4 wounds/sample). ^*p<0.05, ^**p<0.01 indicates the significance of the change relative to the corresponding sample in the absence of locostatin.

Figure 6. Locostatin impairs cytoskeletal organization independent of RKIP expression.

MEFs expressing wild-type, depleted or no RKIP were plated at 2×10⁴ cells/coverslip and grown for 48 h before treatment with either DMSO (Control) or locostatin (50 µM) for either 2 or 6 h. Cells were fixed and permeabilized and stained with phalloidin for actin-red. DNA/chromatin was stained with Höechst 33342-blue. Digital photomicrographs were captured using Openlab Darkroom (Improvision) and a Retiga 1300 color digital camera (Q Imaging). Images from MEFs expressing wild-type RKIP were similar to the RKIP^+/+ MEFs which express lentiviral vector control. Intensity of images were adjusted to display qualitative rather than quantitative comparisons so that the intensity of staining in each panel cannot be directly compared.

Figure 7. Locostatin disrupts the mitotic spindle and chromosome organization.

MEFs expressing (A) wild-type (RKIP^+/+) or lacking RKIP were plated at 2×10⁴ cells/coverslip and grown for 48 h before treatment with either DMSO (Control) or locostatin (20 µM) for either 1 or 2 h. (B) Wild-type (RKIP^+/+) or deficient (RKIP^−/−) RKIP were plated as described in (A) and treated with either DMSO (Control), locostatin (20 µM) or paclitaxel (10 µM) for 6 h. Cells were fixed and permeabilized and stained with anti-α-tubulin antibody-green. DNA/chromatin was stained with Höechst 33342-blue. Digital photomicrographs were captured using Openlab Darkroom (Improvision) and a Retiga 1300 color digital camera (Q Imaging). Intensity of images were adjusted to display qualitative rather than quantitative comparisons so that the intensity of staining in each panel cannot be directly compared.