NudC L279P Mutation Destabilizes Filamin A by Inhibiting the Hsp90 Chaperoning Pathway and Suppresses Cell Migration

Abstract

Filamin A, the first discovered non-muscle actin filament cross-linking protein, plays a crucial role in regulating cell migration that participates in diverse cellular and developmental processes. However, the regulatory mechanism of filamin A stability remains unclear. Here, we find that nuclear distribution gene C (NudC), a cochaperone of heat shock protein 90 (Hsp90), is required to stabilize filamin A in mammalian cells. Immunoprecipitation-mass spectrometry and western blotting analyses reveal that NudC interacts with filamin A. Overexpression of human NudC-L279P (an evolutionarily conserved mutation in NudC that impairs its chaperone activity) not only decreases the protein level of filamin A but also results in actin disorganization and the suppression of cell migration. Ectopic expression of filamin A is able to reverse these defects induced by the overexpression of NudC-L279P. Furthermore, Hsp90 forms a complex with filamin A. The inhibition of Hsp90 ATPase activity by either geldanamycin or radicicol decreases the protein stability of filamin A. In addition, ectopic expression of Hsp90 efficiently restores NudC-L279P overexpression-induced protein stability and functional defects of filamin A. Taken together, these data suggest NudC L279P mutation destabilizes filamin A by inhibiting the Hsp90 chaperoning pathway and suppresses cell migration.

Keywords: Hsp90; NudC-L279P; cell migration; filamin A; protein stability.

FIGURE 1

L279P mutation of NudC destabilizes filamin A. (A) Venn diagram showing the overlap between two sets of interactors identified by IP (with anti-NudC or anti-Flag antibodies) coupled with mass spectrometry in HeLa cells. (B,C) RPE-1 cells transfected with either Flag-NudC or Myc-Filamin A were subjected to IP with anti-Flag or anti-Myc antibodies and western blotting analysis, respectively. 3% of total input is shown. (D,E) Total lysates of RPE-1 cells were immunoprecipitated with the indicated antibodies or IgGs and processed for western blotting. 3% of total input is shown. (F) Purified GST or GST-NudC protein was incubated with RPE-1 cell lysates and subjected to immunoblotting with anti-filaminA antibody. 3% of total input is shown. GST and GST-NudC input were stained with Coomassie brilliant blue. (G) Summary of filamin A truncation mutants. (H) The indicated fragments of GST-tagged filamin A and His-NudC proteins were purified. Fragments were incubated with His-NudC and subjected to immunoblotting with the indicated antibodies. (I) Cells transfected with GFP-NudC were fixed and stained with anti-filamin A antibody. DNA was visualized with DAPI. Bar, 10 μm. (J) RPE-1 cells transfected with NudC RNAi-1 or -2 were harvested at various times and subjected to western blotting with anti-NudC and anti-filamin A antibodies. GAPDH, a loading control. (K) RPE-1 cells stably expressing GFP, GFP-NudC, or GFP-NudC-L279P were subjected to western blotting analysis by using anti-GFP, -NudC and -filamin A antibodies. GAPDH, a loading control. (L) Relative protein levels of filamin A compared to GAPDH in panels (G) were measured using ImageJ software. (M) Quantitative RT-PCR analysis of filamin A mRNA in RPE-1 cells transfected with the indicated vectors. GAPDH, an internal control. (N) Cells transfected with either GFP or GFP-NudC-L279P were treated with 100 μg/ml cycloheximide for different times, harvested, and subjected to western blotting analysis using indicated antibodies. GAPDH, a loading control. (O) Cells transfected with either GFP or GFP-NudC-L279P were treated with either 5 μM MG132 or DMSO, lysed, and subjected to western blotting using the indicated antibodies. GAPDH, a loading control. Quantitative data are presented as the means ± SD (at least three independent experiments). *P < 0.05 and ns, not significant (P > 0.05). Student’s t-test.

FIGURE 2

Overexpression of NudC-L279P inhibits cell migration in RPE-1 cells. RPE-1 cells stably expressing GFP, GFP-NudC, or GFP-NudC-L279P were subjected to the following analyses. (A) Western blotting analysis of the expression of GFP, GFP-NudC, or GFP-NudC-L279P. GAPDH, a loading control. (B,C) Scratch wound assays displayed cell migration at the different time points. Cells expressing GFP signals were monitored with fluorescence microscopy. Dashed lines indicate the approximate line of wound edges. The distance between the two edge lines was measured by ImageJ software. Scale bar, 100 μm. (D,E) Transwell migration assays were performed to detect cell motility. Cells that migrated to the undersides of the filters were stained with 0.2% crystal violet and monitored with DIC (differential interference contrast) microscopy. The number of migrated cells per transwell was counted. (F–H) The migration track of individual cells was traced using Imaris 9.1.2 software. Representative cell migration tracks are shown. Euclidean distance and migration velocity were analyzed with Imaris 9.1.2 software. Scale bar, 100 μm. Quantitative data are presented as the means ± SD (at least three independent experiments). n, the sample size. *P < 0.05; **P < 0.01; and ns, not significant (P > 0.05). Student’s t-test.

FIGURE 3

Overexpression of NudC-L279P impairs actin dynamics. RPE-1 cells infected with lentiviruses expressing GFP, GFP-NudC, or GFP-NudC-L279P were subjected to the following analyses. (A) Western blotting analysis of the expression of the indicated proteins. GAPDH, a loading control. (B) Cells were fixed and stained with phalloidin. DNA was visualized with DAPI. Images were captured using an immunofluorescence microscope. Scale bar, 10 μm. Higher magnifications of the boxed regions are displayed. (C) Structural organization of lamellipodia is shown with scanning electron microscopy. Scale bar, 5 μm. Higher magnifications of the boxed regions are displayed. (D,E) Cells stably expressing the indicated proteins were fixed and stained with phalloidin after 3 h of scratching. DNA was visualized by DAPI. Scale bar, 20 μm. The lamellipodia at the leading edge of cells are indicated by arrowheads. Cells with lamellipodia were calculated. (F–H) A sequence of phase-contrast time-lapse images of the cells was obtained with an LSM880 confocal microscope. Kymographs were analyzed using MetaMorph software. The minimum intensity projection of a 250-frame movie (3 s per frame) is presented on the left. Pixel intensities along a one-pixel-wide line (white) were used to generate the kymograph presented on the right. Cells are outlined with dashed lines. (I,J) Cell spreading was detected using a phase contrast microscope. The areas of cell spreading are outlined by dashed lines and measured by ImageJ software (NIH). Scale bar, 10 μm. Quantitative data are presented as the means ± SD (at least three independent experiments). n, the sample size. *P < 0.05; **P < 0.01; ***P < 0.001; and ns, not significant (P > 0.05). Student’s t-test.

FIGURE 4

Enforced expression of filamin A reverses the defects caused by NudC-L279P overexpression. RPE-1 cells stably overexpressing GFP or GFP-NudC-L279P were transfected with Myc or Myc-filamin A and then subjected to the following analyses. (A) Western blotting analysis of the expression of the indicated proteins. GAPDH, a loading control. (B) Scratch wound assays detected cell migration at the different time points. The scratch closure was monitored with fluorescence microscopy. The distance between the two edge lines was measured using ImageJ software. (C,D) Transwell migration assays were performed to detect cell migration. Cells that migrated to the undersides of the filters were stained with 0.2% crystal violet and monitored with DIC microscopy. The number of migrated cells per transwell was counted. Scale bar, 100 μm. (E,F) The migration tracks of individual cells were traced by Imaris 9.1.2 software. Euclidean distance and migration velocity were analyzed with Imaris 9.1.2 software. (G) Cells were fixed and stained with phalloidin. DNA was visualized with DAPI. Images were captured by immunofluorescence microscopy. Scale bar, 10 μm. Higher magnifications of the boxed regions are displayed. (H,I) Cells were fixed and stained with phalloidin after 3 h of scratching. DNA was visualized by DAPI. Scale bar, 5 μm. The lamellipodia at the leading edge of cells are pointed by arrowheads. Cells with lamellipodia were counted. Quantitative data are presented as the means ± SD (at least three independent experiments). n, the sample size. *P < 0.05 and **P < 0.01. Student’s t-test.

FIGURE 5

Hsp90 binds to and stabilizes filamin A. (A,B) RPE-1 cells transfected with either Myc-Hsp90 or -Filamin A were subjected to IP and western blotting using the indicated antibodies. 3% of total input is shown. (C,D) Total lysates of RPE-1 cells were immunoprecipitated with anti-Hsp90, -filamin A, or -IgG antibodies. Then, the samples were subjected to western blotting analysis with the indicated antibodies. 3% of total input is shown. (E,F) RPE-1 cells were treated with different concentrations of GA or RA for 48 h and subjected to western blotting analyses with the indicated antibodies as shown. Relative protein levels of filamin A compared to GAPDH were measured using ImageJ software and are shown at the bottom. (G,H) RPE-1 cells were treated with 1.78 μM GA or RA for different times and then processed for western blotting analyses with the indicated antibodies. The relative abundances of filamin A compared to GAPDH were measured using ImageJ software and are shown at the bottom. Quantitative data are presented as the means ± SD (at least three independent experiments). *P < 0.05 and **P < 0.01. Student’s t-test.

FIGURE 6

Ectopic expression of Hsp90 reverses the defects induced by NudC-L279P overexpression. RPE-1 cells stably overexpressing GFP or GFP-NudC-L279P were transfected with Myc or Myc-Hsp90 and then subjected to the following analyses. (A) Western blotting analysis of the expression of the indicated proteins. GAPDH, a loading control. (B) Cells were fixed and stained with phalloidin. DNA was visualized with DAPI. Images were captured by immunofluorescence microscopy. Scale bar, 10 μm. Higher magnifications of the boxed regions are displayed. (C) Cells were fixed and stained with phalloidin after 3 h of scratching. Cells with lamellipodia were counted. (D) Scratch wound assays detected cell motility. The distance of scratch closure was measured by ImageJ software. (E,F) Transwell migration assays were performed to detect cell motility. Cells that migrated to the undersides of the filters were stained with 0.2% crystal violet and monitored with DIC microscopy. The number of migrated cells per transwell was calculated. Scale bar, 100 μm. (G,H) The migration tracks of individual cells were traced by Imaris 9.1.2 software. Euclidean distance and migration velocity were analyzed with Imaris 9.1.2 software. Quantitative data are presented as the means ± SD (at least three independent experiments). n, the sample size. *P < 0.05 and **P < 0.01. Student’s t-test.