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Previous Article | Next Article 
Molecular and Cellular Biology, May 2003, p. 3623-3635, Vol. 23, No. 10
0270-7306/03/$08.00+0 DOI: 10.1128/MCB.23.10.3623-3635.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Ruttenberg Cancer Center,1 Department of Molecular and Developmental Biology, Mount Sinai School of Medicine, New York, New York 10029,3 Department of Pathology, Columbia University, New York, New York 100322
Received 22 October 2002/ Returned for modification 11 December 2002/ Accepted 18 February 2003
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ABSTRACT |
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INTRODUCTION |
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FAP-1 is a 270-kDa protein containing an ezrin-like cytoskeleton binding domain, a leucine zipper motif, and six PSD95/Dlg/Z-1 homology (PDZ) domains which is widely expressed in almost all tissues (7, 29, 34, 36). FAP-1 has been implicated in the suppression of Fas-mediated apoptosis (28, 36), which requires functional interaction of either the third or the fifth PDZ domain of FAP-1 with the extreme C-terminal domain of Fas (35, 36, 47). However, the mechanisms by which FAP-1 inhibits Fas-mediated death signaling are still unknown.
Colocalization of FAP-1 with Fas protein within the Golgi apparatus has been observed in pancreatic cancer cells, implying that FAP-1 may be involved in regulating Fas processing (45). Interestingly, increased Fas translocation from the Golgi apparatus to the cell membrane can be induced upon cellular treatment, as seen in hepatocytes that were exposed to bile salts (41). An adenovirus infection that leads to forced degradation of Fas by the adenovirus E3 proteins also results in down-regulation of Fas surface expression (14, 43).
Elevated levels of FAP-1 expression have been reported in various types of human cancer, including colon, pancreatic, hepatocellular, hematological, and ovarian cancers (4, 13, 26, 27, 28, 30, 42, 45). Overall, the level of FAP-1 expression positively correlates with the resistance of human tumors to FasL-mediated apoptosis (28, 30).
As a protein tyrosine phosphatase, FAP-1 is expected to alter tyrosine phosphorylation of its associated substrates. Although such data are not yet available for human cells, the mouse homologue of FAP-1 (PTP-BL phosphatase) dephosphorylates phosphoephrin B ligands following their phosphorylation by Src kinases (46). In the case of Fas, information about its own phosphorylation is very limited, although several studies have implicated Fas phosphorylation as an important regulatory event for its activities. For example, tyrosine kinases, such as Lyn, were implicated in the generation of a Fas receptor-linked transmembrane death signal in eosinophils (40). A physical interaction between Fas and the tyrosine kinase Fyn also has been reported (5).
Here, we demonstrate that FAP-1 association with Fas inhibits its export to the cell surface. The implication of our findings for down-regulation of Fas death signaling in human tumors is discussed.
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MATERIALS AND METHODS |
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Plasmid constructs.
The original human Apo-1/Fas cDNA was recloned into a pEF-GFP plasmid. Mutations within this basal sequence were generated using the Quick Change kit (Stratagene, La Jolla, Calif.). In all cases, the integrity of modified or cloned constructs was confirmed via sequencing. The FAP-1 expression vector and its dominant-negative variants, FAP-1
CD (without the catalytic domain) and FAP-1PDZ3 (without the PDZ3 domain, which binds Fas), have been previously described (17, 28). Fas was mutated on Y275 to histidine or aspartic acid and on Y75 to histidine, using a Quick Change site-directed mutagenesis kit. All constructs were sequenced to assure the integrity of mutations.
Transfection, ß-galactosidase assay, and FACS analysis. Transient transfection of the Fas-green fluorescent protein (GFP) construct (0.2 µg) together with different expression vectors (0.7 µg) and pCMV-ßgal (0.1 µg) into 3 x 105 cells was performed using Lipofectamine (Invitrogen, Carlsbad, Calif.). Eight to 30 h after transfection, surface Fas expression in GFP-positive cells was determined by staining them with phycoerythrin (PE)-anti-Fas monoclonal antibodies (MAbs) and by flow cytometry. Two parameters, the percentage and number of Fas+ GFP+ cells and the mean fluorescence intensity (MFI) of surface Fas levels, were used to evaluate surface Fas-GFP translocation. The specific activity of ß-galactosidase was used for normalization of the efficiency of transfection in fluorescence-activated cell sorter (FACS) data.
Confocal immunofluorescence. Fas-GFP-transfected cells grown on coverslips were fixed in 2% para-formaldehyde in phosphate-buffered saline (PBS). The cells were then washed three times in PBS followed by permeabilization in 0.1% Triton X-100 in PBS for 10 min with three additional 5-min washes in PBS. The cells were incubated with primary antibodies (Abs) for 1 h at room temperature. After additional washes, the cells were incubated with the secondary Abs labeled with Texas Red. The cells were washed several times, and the coverslips were mounted on glass slides in Vectashield (Vector Laboratory).
Treatment and apoptosis studies. Surface expression of endogenous Fas was determined using anti-Fas-PE Abs (Pharmingen). Cells were permeabilized using a Cytofix/cytoperm kit (Pharmingen) for detection of internal Fas levels via FACS. The cells were exposed to FasL as previously described (19, 20). FasL (25 to 50 ng/ml) was used in combination with cycloheximide (10 µg/ml). For apoptosis studies, cells were stained with propidium iodide (50 µg/ml) and analyzed by flow cytometry using a FACS Calibur flow cytometer (Becton Dickinson) with the CellQuest program. Apoptosis was assessed by quantifying the percentage of hypodiploid nuclei undergoing DNA fragmentation (32).
Western blot analysis and immunoprecipitation. RIPA buffer (PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitor cocktail) was used for the preparation of total cell extracts. Alternatively, Triton X-100-soluble and -insoluble fractions were prepared. To this end, proteins that were soluble in 1% Triton X-100 were separated from the insoluble fraction, which was then solubilized with 1% SDS in the presence of DNase (5 µg/µl) for 30 min at 37°C. The cell lysates (50 to 100 µg of protein) were resolved by SDS- 8 or 10% polyacrylamide gel electrophoresis and processed according to the standard protocols. The primary Abs used were rabbit anti-FAP-1 or monoclonal anti-Fas (Pharmingen), anti-ß-actin (Sigma), and anti-c-Src (UBI) (1:1,000 to 1:5,000). The secondary Abs (anti-rabbit or anti-mouse) were conjugated to horseradish peroxidase (dilution, 1:5,000). Signals were detected using the ECL system (Amersham). Immunoprecipitation was carried out by standard methods (20).
RT-PCR analysis.
Reverse transcription (RT)-PCR was carried out on cDNA prepared from the RNA using primers designed to amplify 499 bp of Fas promoter and 250 bp of the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) cDNAs. Amplifications were carried out for 17 cycles in the presence of [
-32P]dCTP (1 min each at 94, 55, and 72°C), followed by separation on polyacrylamide gels and autoradiography.
FAP-1 suppression by RNAi. The p SUPER retro (pRS) RNA interference (RNAi) system was used for the production of small RNAi transcripts to suppress FAP-1 gene expression as previously described (10, 11); 19-mer FAP-1-based oligonucleotides in both sense and antisense orientations (FAP-1 RNAi, 653 GCCAGGCTATTCGAGATCG 671; control RNAi [of a different region from FAP-1], 1190 GTTCTTTGGACCGAATCCG 1208; based on FAP-1 mRNA [GenBank accession no. NM_080683]) were synthesized and cloned into pRS. A FAP-1 BLAST search confirmed the specificity of the selected oligonucleotides, and sequencing of cloned constructs confirmed their integrity. TIG3 cells (3 x 105) were cotransfected with 1 µg of pRS-FAP-1 RNAi and 0.2 µg of Fas-GFP or GFP, using Lipofectamine. Analysis of FAP-1 and Fas expression was carried out 48 h later as detailed in Results.
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RESULTS |
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Earlier reports pointed to the correlation between FAP-1 overexpression and decreased Fas-dependent death signaling (28, 30). Monitoring FAP-1 expression by RT-PCR (Fig. 1C) and Western blotting (Fig. 1D) revealed low levels of FAP-1 expression in WM9 cells (in which most Fas was found on the cell surface) and high levels of FAP-1 expression in OM431 and HHMSX cells (which exhibit internal and low cell surface Fas expression). FEMX cells (in which total Fas expression is low) were found to exhibit very low levels of FAP-1 expression. These observations point to an inverse correlation between surface Fas and FAP-1 expression.
A series of immunoprecipitation assays revealed an association of FAP-1 with Fas that coincides with the overall level of FAP-1 expressed (Fig. 1D) and confirms the findings of earlier studies (47). Only a small portion of the total FAP-1 expressed appears to be associated with Fas (Fig. 1D), suggesting that even low levels of FAP-1 expression may suffice to affect Fas. Subcellular fractionations were carried out to localize endogenous Fas and FAP-1 in melanoma cell lines. Whereas Fas protein was found in both 1% Triton X-100-soluble and -insoluble fractions of FEMX cells, a substantially higher level of Fas was found in the insoluble fraction of OM431 cells (Fig. 1E). FAP-1 was predominantly found in the insoluble fraction of OM431 cells but not in that of FEMX cells (Fig. 1E), where it was poorly expressed (Fig. 1D and E). These data confirm that FAP-1 colocalizes with Fas in OM431 cells.
Fas-GFP is accumulated within cytoplasmic pools and the Golgi apparatus in cells exhibiting impaired cell surface expression. To follow up possible effects of FAP-1 on Fas cell surface expression, we constructed a Fas-GFP expression vector that was first tested for cellular localization and for its ability to induce apoptosis following the addition of FasL. Transfection of Fas-GFP to FEMX melanoma cells that express overall low levels of Fas resulted in an approximately sixfold increase in the expression of Fas on the cell surface, detected by an increase in the MFI of surface Fas (Fig. 2A). Time lapse fluorescence analysis revealed mixed cytoplasmic and cell surface expression within 6 to 16 h, followed by primary cell surface expression after 28 h (Fig. 2B). These data demonstrate that Fas-GFP is subject to time-dependent trafficking. Total levels of Fas-GFP expression were monitored by Western analysis of the transfected cells with anti-GFP Ab (Fig. 2C). Two forms of Fas-GFP are likely to represent posttranslationally modified forms of Fas. These findings demonstrate that Fas-GFP is properly expressed and localized within the cell, similar to the endogenous form of Fas.
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We next monitored changes in the localization of Fas-GFP in FEMX and OM431 cells that exhibited a different subcellular distribution of Fas. Expression of Fas-GFP in the FEMX cells (which exhibit overall low endogenous Fas levels) revealed a partial colocalization of Fas-GFP with 
-adaptin, indicating localization within the Golgi compartment. Localization of Fas-GFP within the Golgi compartment decreased in a time-dependent manner, as greater amounts of Fas-GFP could be found on the cell surface at later time points (Fig. 3A). In contrast, OM431 cells, which mostly exhibit internal Fas expression, retained most of the Fas-GFP within the cytoplasm. Only a small portion of Fas-GFP was found within the Golgicompartment (Fig. 3B). Additional staining of OM431 cells using Abs to different subcellular compartments revealed localization of Fas within the cellular cytoskeleton based on its colocalization with the tubulin network but not the plasma membrane, monitored here by the use of Abs to ezrin (Fig. 3B). These data further establish changes in Fas localization among melanoma cells, as represented with the OM431 and FEMX cells.
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PDZ3) or that is mutated within the domain expected to enable its phosphatase activity (FAP-1CD). Since PDZ domain-containing proteins are known for their notorious homo- and heterodimerization capabilities (38), it is likely that the deleted forms of FAP-1 used in the present study efficiently heterodimerize with the endogenous wild-type (wt) FAP-1, which is expected to inhibit endogenous FAP-1 activities. Accordingly, we consider the forms of FAP-1 with deletions as dominant negatives. Expression of either form of mutant FAP-1 together with Fas-GFP in OM431 cells (Fig. 5A), which exhibit impaired Fas cell surface expression, resulted in a substantial increase in Fas surface expression (a three- to fourfold increase in the percentage of surface Fas-GFP-positive cells [Fig. 5B]). Similarly, expression of Fas-GFP in the presence of the FAP-1 dominant-negative form that lacks phosphatase activity resulted in a two- to threefold increase in the percentage of surface Fas-expressing cells in OM431 cells (Fig. 5B), as in HeLa cells (data not shown) and normal human TIG3 fibroblasts (Fig. 6C). Overexpression of wt FAP-1 did not affect Fas-GFP localization in OM431 cells (which already express high levels of endogenous FAP-1). Immunohistochemistry confirmed a decrease in the number of cells that contain intracellular pools of Fas-GFP upon exogenous expression of dominant-negative FAP-1 constructs (data not shown). In all cases, dominant-negative forms of FAP-1 affected the relative number of cells expressing Fas on their surfaces but not the number of Fas molecules expressed per cell (which is reflected in unchanged MFI values [Fig. 5B]). This is possibly due to the limited transfection efficiency of high-molecular-weight FAP-1 in these cultures. Equally plausible is the notion that additional factors play important roles in regulating Fas trafficking to the cell surface.
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Analysis of normal human fibroblasts that express low levels of Fas (Fig. 6A) was performed after forced expression of FAP-1 (Fig. 6B). Similar to what was observed in the OM431 melanoma cells, expression of FAP decreased the levels of Fas on the cell surface whereas dominant-negative forms of FAP-1 increased it (Fig. 6C). These observations suggest that FAP regulation of Fas surface expression is not limited to melanoma cells, as it is also seen in nontransformed fibroblasts. Given the higher efficiency of transfection in these cells compared with the melanoma cultures, we have generated and expressed RNAi constructs designed to inhibit the expression of endogenous FAP-1 (FAP-1 RNAi) and control sequences designed from a different region of the FAP-1 sequence which poorly affect FAP-1 expression (control RNAi). Forced expression of FAP-1 RNAi resulted in the inhibition of FAP-1 transcription, as determined by RT-PCR (not shown), and expression, as revealed in immunoblot analysis (Fig. 6D), and increased the level of endogenous Fas surface expression, as determined by immunohistochemistry (Fig. 6E) and FACS analysis (Fig. 6F). These results provide important support for the role of FAP-1 in the inhibition of Fas surface expression.
Overexpression of FAP-1 decreases Fas surface expression in melanoma cells that exhibit proper Fas trafficking.
We next assessed whether the expression of dominant-negative FAP-1 would enable further-increased cell surface expression of Fas in cells in which most of the Fas is already found on the cell surface. To this end, we used LU1205 melanoma cells, in which the amount of endogenous surface Fas is almost equal to that of total Fas (data not shown). Coexpression of mutant FAP-1 and Fas-GFP in LU1205 cells caused further increase (
3-fold) in the fraction of cells exhibiting surface Fas-GFP expression, with a noticeable increase also seen in the intensity of cell surface Fas, reflected in increased MFI (Fig. 7A). Such increases coincided with smaller amounts of Fas-GFP able to colocalize within the Golgi and cytosol fractions (Fig. 7B). In contrast, coexpression of wt FAP-1 and Fas-GFP did not affect surface Fas expression compared with control LU1205 cells (due to high basal levels of FAP-1 in LU1205 cells). Consistent with these observations, it was also observed that LU1205 cells stably expressing FAP-1PDZ3 (Fig. 7C) exhibited a noticeable increase in the level of endogenously expressed Fas on the cell surface compared to the control LU1205-neo cells (Fig. 7D). Up-regulation of surface Fas levels upon expression of the dominant-negative form of FAP-1 was also reflected in the proportion of LU1205 cells that underwent apoptosis in response to FasL and CHX stimuli (Fig. 7E). The ability of FAP-1 to further increase Fas surface expression even in the LU1205 cells suggests that it serves to limit the level of Fas surface expression even in cells where a noticeable fraction of Fas appears to be localized on the cell surface.
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To exclude the possible effects of endogenous Fas on Fas-GFP trafficking, we have generated mouse embryo fibroblasts, which were subsequently immortalized, from mice lacking the Fas gene (2) (Fig. 9A and B). The control for actual expression of wt and mutant Fas-GFPs that were transfected into Fas null cells is depicted in Fig. 9C. It is important to note that the mouse analogue of FAP-1 (PTB-BL) can interact with human Fas (46). Monitoring the pattern of Fas-GFP surface expression in the Fas null cells revealed a low basal level of wt Fas-GFP and the N-terminal Y75H mutant. In contrast, Fas mutated on Y275H exhibited a marked (>3-fold) increase in the level of surface expression (MFI) (Fig. 9D), similar to its elevated trafficking in other cell lines. Similar changes were also observed in the normal human fibroblasts. Expression of the C-terminal mutant R275H resulted in an increased number of Fas molecules on the cell surface, whereas expression of Y75H did not cause such an increase (Fig. 9E).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We observed that, of the total amount of FAP-1 expressed, only a small portion appears to be associated with Fas, thus providing further support for controlled Fas-FAP-1 association. While this implies the role of Fas modification as a prerequisite for association, it also suggests that FAP-1 may be involved in the regulation of trafficking of other proteins and that even a low expression of FAP-1 may be sufficient for eliciting its trafficking control. Candidates include the neurotrophin receptor p75NTR, a member of the tumor necrosis factor receptor superfamily shown to interact with FAP-1 (17). It is likely that FAP-1 activities could be regulated by posttranslational modification, which would render only a portion available for both association with and effect on Fas or other associated proteins.
The level of FAP-1 expression inversely correlates with cell surface Fas expression. However, at least one of the cell lines studied here, LU1205, exhibited marked cell surface expression and also expression of high levels of FAP-1. This suggests either that expression per se may not suffice to enable FAP-1 to attenuate Fas surface expression or that FAP-1 or Fas may be subject to changes at the genomic or posttranslational level, explaining the limited effect in the LU1205 cells.
The nature of FAP-1 as a protein phosphatase is intriguing, given that Fas phosphorylation remains elusive. Human Fas (Apo-1) contains three tyrosine residues, Y75, Y216, and Y275, the last two being positioned within the death domain. Using NetPhos software for sequence- and structure-based prediction of tyrosine phosphorylation sites (9), Y75 and Y275 were highlighted as the putative Tyr-phosphoryated sites. Although our data suggest that the binding of Fas to FAP-1 and to Fas requires intact tyrosine at position 275, additional studies are needed to assess the role of Fas phosphorylation in the context of its stability and trafficking.
Understanding the nature of Fas phosphorylation may also provide an explanation for the differences in the effect of FAP-1 on the number of cells expressing Fas versus the number of Fas surface molecules expressed per cell. Whereas overexpression of FAP-1 decreased both parameters, the expression of FAP-1 mutants increased the number of cells that expressed surface Fas but not the number of Fas molecules per cell. In contrast, the expression of a Fas mutant (Y275H or Y275D) resulted in increased intensity (number of molecules per cells) but not increased numbers of cells expressing surface Fas. The nature of these changes could be attributed to additional parameters that govern Fas trafficking, including altered phosphorylation or glycosylation, as well as changes in Fas stability and recycling. The role of such changes in FAP-1-dependent Fas export is being investigated.
Our data, using a naturally occurring mutant form of Fas at the 275 site (25), provide direct evidence for FAP-1- Fas association as a prerequisite for Fas trafficking inhibition. Our data are in agreement with the observation that the C-terminal peptide that harbors the last three amino acids of the Fas protein increases Fas surface expression (37, 45), probably due to interference with FAP-1 binding. It is interesting that the extreme C-terminal domain (i.e., the last three amino acids of the Fas protein) is not conserved in evolution (i.e., mouse Fas lacks these residues), further highlighting the requirement for the integrity of Fas conformation within the carboxyl end of the protein, as revealed in our analysis of the Fas mutants at amino acid 275.
Elevated FAP-1 expression in human tumors is often seen and has been linked with impaired Fas-mediated apoptosis (26, 28, 30, 44, 45). Our findings, which provide direct evidence for the effect of FAP-1 on Fas cell surface expression, point to the mechanism underlying the inhibition of Fas surface expression commonly seen in advanced human tumors. NF
B, which has been implicated in melanoma progression (12, 23), was also linked to the regulation of FAP-1 (18).
FAP-1 expression may also play a role in dictating the primary apoptosis cascade in normal untransformed cells, as shown here in normal TIG3 lung fibroblasts. This is in accord with the notions that pancreatic ß cells are triggered to express cell surface Fas following progressive insulitis (6) and that bile salts mediate hepatocyte apoptosis by increasing cell surface trafficking of Fas (41).
Taken together, our findings provide direct evidence for the role of FAP-1 in the regulation of Fas expression on the cell surface, thereby highlighting novel insights into the mechanisms underlying the regulation of Fas cell surface expression in normal and tumor cells. The use of melanoma cells with very low Fas expression (FEMX) allowed analysis in a model system (using Fas-GFP) resembling Fas null cells, albeit in a true melanoma setting. FAP-1 inhibition of Fas surface expression has also been observed in the context of different melanoma cultures, as well as in untransformed human fibroblasts, further validating our model and highlighting its physiological relevance.
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ACKNOWLEDGMENTS |
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Support by NCI grant CA51995 (to Z.R.) and from the Sharp foundation (to Z.R.) is gratefully acknowledged.
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FOOTNOTES |
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