FAP-1 association with Fas (Apo-1) inhibits Fas expression on the cell surface

Abstract

As revealed by intracellular pools of nonactive Fas (Apo-1), export of Fas to the cell surface is often impaired in human tumors, thereby inactivating Fas ligand-mediated apoptosis. Here, we demonstrate that association with Fas-associated phosphatase 1 (FAP-1) attenuates Fas export to the cell surface. Forced expression of FAP-1 reduces cell surface Fas levels and increases the intracellular pool of Fas within the cytoskeleton network. Conversely, expression of dominant-negative forms of FAP-1, or inhibition of FAP-1 expression by short interfering RNA, efficiently up-regulates surface expression of Fas. Inhibition of Fas surface expression by FAP-1 depends on its association with the C terminus of Fas. Mutation within amino acid 275 results in decreased association with FAP-1 and greater export of Fas to the cell surface in melanomas, normal fibroblasts, or Fas null cells. Identifying the role of FAP-1 in binding to, and consequently inhibition of, Fas export to the cell surface provides novel insight into the mechanism underlying the regulation of Fas trafficking, which is commonly impaired in advanced tumors with FAP-1 overexpression.

FIG. 1.

Fas and FAP-1 expression in human melanoma cell lines. (A) Surface and total (after cell permeabilization) Fas levels were determined by staining with PE-conjugated anti-human Fas MAbs and flow cytometry of melanoma cells. (B) Immunostaining of endogenous Fas in WM9 and OM431 melanoma cells. Anti-Fas MAb and fluorescein isothiocyanate-labeled secondary Abs were used. DAPI was used for nuclear staining. The merged images were obtained by confocal microscopy. (C) RT-PCR analysis of FAP-1 mRNA levels in human melanomas. GAPDH mRNA levels were used as an internal control. (D) Western blot analysis (W) of FAP-1 and Fas levels in melanomas. The positions of FAP-1 and of a nonspecific form (ns) recognized by these Abs are indicated by arrowheads. The β-actin level was used as a loading control. Fas and FAP-1 proteins prepared from the indicated melanoma cell lines using RIPA buffer were subjected to coimmunoprecipitation (IP) with Abs to FAP-1 followed by immunoblot analysis with Abs to Fas and FAP-1. Apparent molecular masses (kilodaltons) are in parentheses. (E) Western blot analyses of 1% Triton X-100-soluble and -insoluble fractions from FEMX and OM431 cells were performed using Abs against Fas, FAP1, vinculin, RIP-1, and Src.

FIG. 2.

Fas-GFP is a functional chimeric protein that exhibits time-dependent localization on the cell surface and triggers FasL-mediated apoptosis. (A) FEMX cells were transiently transfected with a pEF-FAS- GFP expression construct or with a pEF-GFP control plasmid. Surface Fas levels were determined by staining the cells with anti-N-terminal Fas-PE MAbs and by FACS analysis. The MFIs of Fas and the percentages of double-positive Fas⁺ GFP⁺ cells are indicated. (B) Intracellular localization of Fas-GFP fused protein 6 to 28 h after transfection; GFP expression vector was used as a control. Phase reflects phase-contrast microscopy. (C) Expression pattern of total Fas-GFP was determined by Western blot analysis 16 to 28 h after transfection; nt, nontransfected cells. The Fas-GFP fused protein has an apparent mass of 75 kDa; an additional band, probably reflecting a posttranslationally modified form of Fas, was designated Fas-GFP*. The β-actin level was used as a loading control. (D) Apoptosis analysis of Fas-GFP-transfected FEMX cells. Apoptosis was induced by FasL (10 ng/ml)-CHX (10 μg/ml) treatment.

FIG. 3.

Time-dependent localization of Fas-GFP in melanoma cell lines. (A) Confocal analysis of Fas-GFP subcellular location in FEMX. Localization of Fas-GFP (green) and a Golgi marker, γ-adaptin (red; the secondary Ab labeled with Texas Red), was performed at the indicated time points. Yellow represents the overlay of green and red. (B) Confocal analysis of Fas-GFP (green) and γ-adaptin, β-tubulin, and ezrin (red) in OM431 cells.

FIG. 4.

FAP-1 expression attenuates Fas-GFP cell surface expression. (A) Western blot analysis of FAP-1 levels in 1% Triton X-100-soluble and -insoluble fractions in control (empty vector) or FAP-1-tranfected FEMX cells. ns, a nonspecific form recognized by the Abs. (B) Western blot analysis (W) of FAP-1 levels (RIPA buffer) in control (empty vector) or FAP-1-transfected FEMX cells. Both cell lines were cotransfected with Fas-GFP. IP, immunoprecipitation. (C) Surface Fas levels were determined in transfected FEMX cells by staining them with anti-N-terminal Fas-PE MAb and by FACS analysis. The MFIs of Fas and the percentages of double-positive Fas⁺ GFP⁺ cells are indicated. (D) Confocal images of FEMX cells transfected with Fas-GFP plus empty vector or Fas-GFP plus FAP-1 expression construct. γ-Adaptin was used as a Golgi marker.

FIG. 5.

Mutated forms of FAP-1 increase cell surface expression of Fas. (A) Expression levels of mutated forms of transfected FAP-1 in OM431 cells were determined by Western blot analysis (W). IP, immunoprecipitation. Apparent molecular masses (kilodaltons) are in parentheses. (B) FACS analysis of Fas-GFP surface expression was performed in OM431 cells after cotransfection with vector, FAP-1, or mutated forms of FAP-1 using anti-N-terminal Fas-PE MAbs and C-terminally tagged GFP. The MFIs and the percentages of double-positive cells (or numbers of double-positive cells of a total of 40,000 cells analyzed) are indicated in the upper right quadrants. (C) Effect of FAP-1 or FAP-1ΔPGZ3 on endogenous Fas expression in OM431 cells. The indicated constructs were cotransfected together with GFP, and 24 h later, FACS analysis allowed the analysis of endogenous Fas levels in GFP-positive cells. The ratios of Fas⁺ GFP⁺ cells to the total number of GFP⁺ cells are indicated.

FIG. 6.

Mutated forms of FAP-1 and RNAi of FAP-1 increase cell surface expression of Fas. (A) FACS analysis of surface and total Fas levels in TIG3 cells. (B) Western blot analysis (W) of FAS-GFP and FAP-1 levels in TIG3 cells. IP, immunoprecipitation; ns, a nonspecific form recognized by the Abs. (C) FACS analysis of Fas-GFP surface expression was performed in TIG3 cells after cotransfection with vector, FAP-1, or dominant-negative forms of FAP-1 using anti-N-terminal Fas-PE MAbs and C-terminally tagged GFP. (D) Western blot analysis of FAP-1 levels in TIG3 cells transfected with pRS, FAP-1 RNAi, or control RNAi 60 h after transfection. (E) Immunohistochemistry using confocal microscopy follows changes in surface expression of endogenous Fas in TIG3 cells that were transfected with FAP-1 RNAi or control RNAi. (F) Changes in endogenous levels of surface Fas were determined in TIG3 cells after cotransfection of red fluorescent protein (RFP) expression vector together with either pSR or FAP-1 RNAi and control RNAi. FACS analysis was performed for the determination of the percentage of Fas-positive cells and Fas MFI in the population of GFP-positive cells. The error bars indicate standard deviations.

FIG. 7.

Dominant-negative forms of FAP-1 further increase cell surface expression of Fas in LU1205 melanoma cells. (A) FACS analysis of Fas-GFP expression in cells expressing wt and mutant forms of FAP-1 was performed as indicated in Materials and Methods. (B) Corresponding confocal images of merged GFP-Fas and Golgi marker (γ-adaptin) are shown for the experiment depicted in panel A. (C) Western blotting of FAP-1 levels in LU1205 cells that stably express control or FAP-1ΔPDZ3 constructs. ns, a nonspecific form recognized by the Abs. (D) FACS analysis of Fas levels in LU1205 cells that stably express pcDNA3 (neo) or FAP-1ΔPDZ3. (E) Apoptosis analysis of control or FAP-1ΔPDZ3-transfected LU1205 cells after treatment with FasL (50 ng/ml) plus CHX (10 μg/ml). The percentages of apoptotic cells are indicated.

FIG. 8.

Mutation within C-terminal Fas abolishes FAP-1 association and inhibition of Fas trafficking. (A) Effect of N- and/or C-terminal mutations of Fas on association of Fas-GFP with FAP-1. The corresponding constructs were expressed in OM431 cells following their coimmunoprecipitation (IP) and Western analysis (W) with the indicated Abs. The positions of Fas-GFP and the imunoglobulin heavy chain (H, Ig) are indicated. (B) Immunohistochemical analysis of Fas-GFP expression in OM431 cells that were transfected with the wt or mutant forms of Fas-GFP.

FIG. 9.

Effects of N- and or C-terminal mutation of Fas-GFP on its trafficking in Fas knockout (KO) cells. (A) Mouse embryo fibroblasts (MEF) from Fas null (KO) mice have been established in culture. FACS analysis was used for monitoring Fas surface levels in KO fibroblasts before and after transfection by Fas expression vector. ns, nonstained cells. (B) Western analysis of total Fas levels in wt and Fas KO fibroblasts. (C) Analysis of wt and mutant Fas-GFP fusion protein expression after transfection intot Fas KO fibroblasts was done by immunoprecipitation (IP) with anti-Fas MAb followed by Western analysis (W). (D and E) FACS analysis of surface Fas-GFP expression was performed on FAS KO (D) or TIG3 (E) cells as indicated in Materials and Methods.