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. Author manuscript; available in PMC: 2014 May 15.
Published in final edited form as: J Immunol. 2013 Apr 19;190(10):4937–4945. doi: 10.4049/jimmunol.1202646

Pin1-FADD interactions regulate Fas-mediated apoptosis in activated eosinophils#

Jiyoung Oh 1, James S Malter 1
PMCID: PMC3652414  NIHMSID: NIHMS459379  PMID: 23606538

Abstract

Abnormally long-lived eosinophils (Eos) are the major inflammatory component of allergic responses in the lungs of active asthmatics. Eos recruited to the airways after allergen exposure produce and respond to IL-5 and GM-CSF, enhancing their survival. Pro-survival signaling activates Pin1, a cis-trans peptidyl isomerase (PPIase) that binds to Bax and prevents it activation. How long-lived Eos, despite the continued presence of GM-CSF or IL-5, eventually undergo apoptosis to end allergic inflammation remains unclear. Here we show that Pin1 location, activity and protein interactions are jointly influenced by Fas and pro-survival cytokine IL-5. Fas signaling strongly induced the phosphorylation of FADD at Ser194 and Pin1 at Ser16 as well as their nuclear accumulation. Phospho-mimic Ser194Glu FADD mutants accelerated Eos apoptosis compared to WT or Ser194Ala mutants. Downstream of FADD phosphorylation, Caspase 8, 9 and 3 cleavage as well as Eos apoptosis induced by Fas were reduced by constitutively active Pin1 and enhanced by Pin1 inhibition. Pin1 was activated by IL-5 while simultaneous IL-5 and anti-Fas treatment modestly reduced PPIase activity but induced Pin1 to associate with FADD after its phosphorylation at Ser194. Mechanistically, Pin1 mediated isomerization facilitated the subsequent dephosphorylation of Ser194 FADD and maintenance of cytoplasmic location. In vivo activated bronchoalvelolar (BAL) Eos obtained after allergen challenge showed elevated survival and Pin1 activity that could be reversed by anti-Fas. Therefore, our data suggest that Pin1 is a critical link between FADD mediated cell death and IL-5 mediated pro-survival signaling.

Keywords: IL-5, Pin1, FADD, Apoptosis, Asthma, eosinophils

Introduction

After maturation in the bone marrow, eosinophils (Eos) circulate briefly (~3 days) in the peripheral blood before undergoing programmed cell death (apoptosis). Despite this short life-span, Eos can be found in a variety of tissues such as spleen, uterus and lymph nodes but typically not the lung or skin. (1, 2). However, asthmatics exposed to allergen as well as other patients with parasitic infection, cancer, adrenal insufficiency, esophagitis, or collagen vascular disease may show elevated levels of circulating Eos (2-5). This often reflects increased serum concentrations of IL-5, GM-CSF or IL-3 that enhance the differentiation, release and longevity of circulating or tissue-based Eos (4). In allergen challenged asthmatics, activated, long-lived Eos accumulate in the bronchial and nasal mucosa (6-9). These biological properties provided the rationale for the development and clinical testing of anti-IL-5 monoclonal antibodies such as mepoluzimab and reslizumab for asthma(10-12).

Opposing the pro-survival effects of GM-CSF or IL-5 is FasL. This pro-apoptotic agonist is produced and secreted by Mast cells, T and B cells and macrophages in the inflamed bronchi (13) and could contribute to the eradication of pulmonary eosinophilia and resolution of allergic inflammation. FasL interacst with Fas, a TNF-R family member that transduces death signals (14) by recruiting FADD, leading to the formation of the receptor-associated death-inducing signaling complex (DISC) (15). The DISC complex triggers caspase 8 which then cleaves Bid, providing a link to mitochondrial-dependent death pathways (16). These events are likely cytoplasmic. However, FADD can also be anti-apoptotic, induce cell cycle progression and proliferation presumably by trans-locating into the nucleus (16-18). The latter absolutely requires phosphorylation of human FADD at Ser194 although how this modification triggers nuclear localization is unknown.

Pin1 is peptidyl-prolyl cis-trans isomerase (PPIase) that plays a key role in cell cycle progression, the regulation of innate immunity, the expression and signaling of cytokines and Eos cell death (19-22). Pin1 consists of an N-terminal WW domain that mediates binding to p-Ser-Pro or p-Thr-Pro dipeptide motifs and a C-terminal PPIase domain. Pin1 activity is reduced by inhibitory phosphorylation by Protein Kinase A (PKA), Mitogen-Activated Protein Kinase (MAPK), Death Associated Protein Kinase 1 (DAPK1) or Mixed Lineage Kinase 3 (MLK3) at Ser16, Ser65, Ser71 or Ser138, respectively, that block target interactions (Ser16) or modulate PPIase activity (Ser65, −71 or −138) or subcellular localization (22-24). Pin1 mediates IL-5 or GM-CSF induced survival of Eos by suppressing Bax activation (22). In the absence of Pin1, pro-survival signaling is ablated and Eos undergo accelerated apoptosis. Pin1 also binds to caspase 8, suggesting a possible role in the extrinsic apoptotic pathway as well (22, 25).

Here we show that Fas activation triggers Eos apoptosis through the extrinsic pathway. Critical to this process is the phosphorylation of FADD at Ser194 and it’s subsequent nuclear translocation, both of which were regulated by interactions with Pin1. Therefore, Pin1 plays a critical role in balancing Fas induced Eos apoptosis by interacting with FADD versus maintaining eosinophilic allergic inflammation driven by IL-5 or GM-CSF.

Materials and Methods

Reagents and antibodies

Anti-Fas was purchased from Millipore (clone CH-11, Billerica, MA, USA). IL-5 was obtained from R&D Systems (Minneapolis, MN, USA). Anti-caspase 9, anti-Bid, anti-procaspase 8, anti-cleaved caspase 8, anti-poly(ADP-ribose) polymerase, anti-hemagglutinin (HA) and anti-phospho-Pin1 Abs were purchased from Cell Signaling Technologies (Beverly, MA). Anti-caspase 3, anti-Pin1, anti-phospho-FADD, anti-α tubulin and anti-hnRNP C1/C2 were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-His and anti-β-actin were from Sigma-Aldrich (St.Louis, MO). Anti-FADD was from BD Bioscience (San Jose, CA)

Eosinophil preparation

Human peripheral blood was obtained from healthy donors or atopic donors. All participants have a clinical record at the University of Wisconsin Hospital and consent was obtained according to an apporoved University of Wisconsin Hospital Institutial Review Broad protocol. The characteristics of donor is described in Supplementary Materials. Eosinophils were purified from peripheral blood or BAL fluid by a negative immunomagnetic selection as described previously (22, 25). The purity of eosinophils was > 99% and cells were cultured as described (22, 25).

Plasmids and constructs

Human Pin1 WT and various mutants cDNAs were amplified by PCR and the products cloned into NotI and XhoI site of pTYB1 (New England Biolabs, Ipswich, MA). Pin1 mutants, S16A, S16D, S71A, and S71D were constructed with N-terminal TAT-HA tags in pTYB1 using the Phusion site-directed mutagenesis Kit (New England Biolabs). To generate human wild type (WT) and mutant FADD, cDNA was amplified and S194A, S194D and S194E were generated using the Phusion site directed mutagenesis kit prior to sequencing (Applied Biosystems, Foster, City, CA).

Protein purification and in vitro binding

Recombinant WT FADD, Pin1 and FADD mutant proteins were purified using ImpactTM (Intein Mediated Purification with an Affinity Chitin-binding Tag, New England Biolabs) protein purification system. For in vitro binding assays, 10 pmol GST, GST-WW, GST-S16E, WT-FADD, or FADD mutant proteins were incubated with specific antibodies in binding buffer (20 mM HEPES (pH 7.6), 15 mM KCl, 5 mM MgCl2, 10% glycerol and 0.1% NP40) for 4 h at 4 °C followed by Protein A/G-agarose beads (Sigma Aldrich, St. Louis, MO). After elution, proteins were analyzed by immunoblot.

Cell viability analysis by flow cytometry or trypan blue staining

Eos (106 cells/ml) were cultured in 96 well culture plates (BD Bioscience). Cell death was determined by flow cytometry (BD Biosciences FACSCalibur system) using an apoptosis detection kit (BD Bioscience) or assessed by trypan blue exclusion on a hemacytometer.

TAT fusion protein transduction and subcellular fractionation

TAT-proteins were produced in E. coli as described previously (22, 25). Eosinophils were treated with 100nM of various TAT proteins (TAT-GFP, TAT-Pin1 WT, TAT-S16D Pin1, TAT-S16A Pin1, TAT-FADD, TAT-S194A-FADD, TAT-S194D/E-FADD). For nuclear extracts, cells were lysed in cytoplasmic and nuclear extraction k buffer (NE-PER, Pierce) according to the manufacturer’s protocol. 10 μg of cytosolic and nuclear fraction was analyzed by western blot for cytosolic (α-tubulin) or nuclear markers (hnRNP C1/C2).

Immunoblotting and Immunoprecipitation

Immunoblot (IB) analysis and densitometry of protein expression was determined by Li-COR software as described (22, 26). Protein extracts were prepared as described previously (22). Cells were lysed in buffer containing 20 mM HEPES (pH 7.3), 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM PMSF, 1 mM Na3VO4, 10 mM NaF and protease inhibitor. Protein concentration was measured by Lowry protein assay (Bio-Rad). Proteins were separated by SDS-PAGE and transferred to Immobilon FL membranes (Millipore) prior to incubation with primary antibodies and then IR-Dye infrared secondary antibodies at room temperature (Li-Cor Biosciences, Lincoln, NE). Membranes were scanned and quantified with Odyssey infrared Imaging System (Li-Cor). For immunoprecipitation (IP), cell extracts were lysed, precipitated and analyzed as described (22).

Immunofluorescence staining

Cells were treated as described in the figure legends, cytospun onto slides, fixed with 4% paraformaldehyde, and permeabilized with 0.2% Triton-X100. The cells were stained with antibodies vs. FADD, phospho-FADD, Pin1, phospho-Ser16-Pin1 and β-actin and incubated with Alexa 488-congugated goat anti-rabbit or 586-congugated goat anti-mouse antibodies (Jackson Immunoresearch, West Grove, PA). Cells were mounted with ProLong Gold antifade reagent including DAPI (Invitrogen). Images were gathered by confocal laser microscopy (C1 Laser Scanning Confocal, Nikon Instruments).

Pin1 activity assay

Pin1 activity was determined as described previously (22).

Statistical analysis

Statistical differences beween control and treated group were determined by Student’s t-Test. All experiments were performed at least three times with different donors. The values are presented as mean ± s.d. p <0.05 was considered significant.

Results

Fas activation overcomes pro-survival signaling

Airway Eos, activated in vivo by IL-5 and GM-CSF, show significantly prolonged survival (1, 2, 4, 5). Peripheral blood Eos show similar phenotypic changes and thus are a good model to study the underlying mechanisms in vitro. FasL delivered systemically increased pulmonary eosinophilia after allergen challenge (27) while anti-Fas induced Eos apoptosis but also pro-inflammatory, Eos necrosis (28). Thus the end-points and mechanism of action of Fas-FasL in Eos airway inflammation remains murky. We modeled these events by assessing the response of peripheral blood Eos to IL-5, Pin1 inhibitors and Fas activation. As expected, untreated Eos showed significant apoptosis (~55% by 48 h in culture, Fig. 1A) that could be antagonized by IL-5 (Fig. 1B, 3D and S1). Co-treatment with anti-Fas reduced but did not completely attenuate the effects of IL-5 as assessed by Eos survival, and the cleavage of PARP, caspase 3 and Bid (Fig. 1B, C and S1). Fas activation also accelerated apoptosis by otherwise untreated Eos (Fig. 1A). Therefore, Fas activation can enhance Eos death and overcome the pro-survival program initiated by IL-5 through the induction of the extrinsic apoptosis pathway.

Figure 1.

Figure 1

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Fas activation or Pin1 inhibition accelerates Eos apoptosis despite pro-survival signaling.

Purified Eos (>99% pure) from 3 different donors were incubated for 0 or 48 hours in the absence or presence of IL-5 (0.1 ng/ml), various concentrations of anti-Fas (as shown) or Pin1 inhibitors (Juglone (J) or TAT-WW Pin1 (WW) at 100 nM). A) In vitro Eos viability at 0 h or 48 h was determined by trypan blue dye exclusion after treatments shown. The results shown are representative of three independent experiments with 3 different donors. Mean values are shown with s.d. * = p<0.05 B) Eos were incubated as shown for 0 or 24 h. Whole cell lysates were assessed by western blot analysis using antibodies shown. C) Eos were incubated in IL-5 (0.1 ng/ml) with or without anti-Fas (50 ng/ml), TAT-GFP, TAT-WW-Pin1 or juglone (0.5 μM) for 24 h and whole cell lysates analyzed by western blot. D) Eos were pretreated with TAT-β-gal, TAT-WW or juglone (0.5 μM) for 24 h prior to flow analysis for Annexin V. Control fresh Eos were stained for Annexin V immediately after isolation from donors. E) Eos viability after 72 h for the treatment shown, determined by trypan blue exclusion. * = p<0.05 in all panels. All experiments were done in triplicate with different donors.

Figure 3.

Figure 3

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Anti-Fas-mediated apoptosis is blocked by S16A Pin1 but not Ser71Ala Pin1.

A) Schematic representation of TAT-HA-WT and Pin1 mutants. B) Eos were pretreated with IL-5 (0.1 ng/ml) alone or pretreated with TAT-HA-GFP, TAT-HA-WT Pin1, TAT-HA-S16A Pin1, TAT-HA-S16D Pin1, TAT-HA-S71A Pin1 or TAT-HA-S71D Pin1 (100 nM) for 30 min prior to anti-Fas for 24 h and western blot analysis as shown. C) Eos were pretreated with TAT-HA-GFP or TAT-HA-S16A for 30 min prior to anti-Fas, IL-5 or anti-Fas plus IL-5 for 24 h. Whole cell lysates were assessed by Western blot analysis using antibodies shown. D) Eos viability was determined by trypan blue exclusion after no treatment (-) or after 48 h of the treatments in (B). * = p<0.05. All experiments were done in triplicate with different donors.

Pin1 participates in Eos pro-survival signaling by suppressing Bax and the activation of the intrinsic pathway (22, 25). To explore the role of Pin1 in anti-Fas signaling, we transduced Eos with dominant negative WW domain (WW-Pin1) or control (GFP) protein fused to a TAT penetratin tag (22, 25) or treated cells with the Pin1 inhibitor, juglone. Treatment of Eos with TAT-WW-Pin1 or juglone synergized with anti-Fas, irrespective of the presence of IL-5, to significantly increase Bid cleavage and Eos apoptosis (Fig. 1C, D and E). These data suggest that Pin1, in addition to regulating Bax, also participates with Fas in the control of the extrinsic pathway.

Fas-induced phosphorylation of Pin1 at Ser16 is blocked by IL-5

The ability of anti-Fas to both accelerate the death of resting, untreated Eos as well as antagonize IL-5 suggested Fas signaling regulated Pin1. Pin1 activity is controlled by reversible phosphorylation at Ser16 within the WW domain, or at Ser71 or Ser138 within the PPIase domain (22-24, 29). Ser16 phosphorylation prevents target protein binding while Ser71 or Ser138 directly interfere with PPIase action. Thus, freshly isolated Eos were left untreated (control) or briefly exposed to IL-5 and/or anti-Fas prior to lysis and PPIase assay. As seen previously (22, 25) endogenous Pin1 activity was rapidly induced by IL-5 (Fig. 2A) that could be modestly antagonized by either simultaneous or post-hoc anti-Fas treatment (Fig. 2A). In resting, freshly isolated Eos, Pin1 was largely dephosphorylated at Ser16 and diffusely cytoplasmic. Fas activation enhanced the phosphorylation of Pin1 at Ser16 that was reversed by IL-5 (Fig. 2B) and dependent on PP2A (Fig. 2D). The subcellular localization of Pin1 was also dependent on Fas or IL-5 mediated signaling (Fig. 2C). IL-5 treatment had little effect on Ser16 phosphorylation but caused Pin1 to concentrate along the plasma membrane. Conversely, anti-Fas alone markedly increased nuclear phospho-Ser16 Pin1. The combined effects of IL-5 and anti-Fas were intermediate between these phenotypes (Fig. 2C). These data indicate that Fas signaling has profound effects on Pin1 phosphorylation and location, possibly by inhibiting PP2A action and partially antagonizes IL-5 mediated events.

Figure 2.

Figure 2

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IL-5 and Fas signaling affect Pin1 activity and location through Ser16 phosphorylation.

A) Eos were untreated or treated for 10 min with IL-5 (0.1 ng/ml), anti-Fas (50 ng/ml), IL-5 (for 10 min prior) plus anti-Fas or IL-5 and anti-Fas together. PPIase activity was then measured on whole cell lysates. B) Eos were treated as shown for 30 min (same concentrations as in A above) and lysates analyzed by western blotting as shown. Below, quantification of a representative blot. C) Cells were treated for 30 min as shown along the top (same concentrations as in A above) and stained with antibodies as shown. D) Eos were pretreated with DMSO or 100 nM calyculin A for 30 min before treatments as shown for 30 min. Cell lysates were prepared and analyzed by western blotting as shown. All experiments were done in triplicate with different donors.

Phosphorylation of Pin1 at Ser16 determines Eos survival

The above results suggested that Pin1 phospho-status, activity and/or location were critical for Eos apoptotic decisions induced by Fas. To clarify these hypotheses, we generated TAT-HA-Pin1 mutants where Ser16 or Ser71 were mutated to alanine (A; phospho-null) or aspartic acid (D; phospho-mimetic) (Fig. 3A). Eos were incubated with 100 nM recombinant protein and the effects on Fas-mediated apoptosis analyzed. Only Ser16A mutants suppressed caspase 8 activation and Eos apoptosis (Fig. 3B and D). These results did not reflect differences in transduction efficiency (Fig. 3B). Interestingly, neither Ser71A nor Ser71D mutants affected viability or caspase 8 cleavage in Fas activated Eos (Fig. 3B and D). These data suggest that Pin1 phosphorylation at Ser71 by DAPK1 (23) is not relevant for Fas-mediated signal transduction. Instead, the data point to the phosphorylation of Ser16 as an essential step for Fas-mediated apoptosis.

The role of Pin1 in mediating pro-survival signaling was assessed biochemically and morphologically after adding IL-5 to Eos transduced with S16A or other Pin1 mutants. Only the S16A mutant blocked procaspase 8, 9 or 3 cleavage (Fig. 3B and C) and significantly increased Eos survival (Fig. 3D) despite treatment wth anti-Fas and did so to an equivalent level as IL-5 itself (Fig. 3D). As the S16A mutation facilitates Pin1 interactions with targets (22, 24), these results suggest that Pin1 binding and isomerization activity are required to prevent Fas-induced, Eos apoptosis. Furthermore, these data suggest that the maintenance of Ser16 dephosphorylation by IL-5 mediated signaling is both necessary and sufficient for full prosurvival signaling.

Fas activation induced phosphorylation and nuclear localization of FADD

In T cells, Fas activation is accompanied by recruitment and oligomerization of FADD to the death receptor through its death domain (DD). FADD then binds to and activates caspase 8, leading to Bid cleavage (17, 30). However, after phosphorylation at Ser194 by Casein Kinase 1α or Polo-like Kinase 1 (Plk 1) (30, 31), p-FADD can traffic into the nucleus where it influences other processes including cell proliferation and cell cycle progression (30-32). As Ser194-Pro195 is a possible Pin1 binding site, we investigated if this site was phosphorylated and supported interactions between FADD and Pin1. Eos were incubated with media, anti-Fas, IL-5 or both for 30 min. FADD was diffusely expressed in both the cytosol as well as nucleus irrespective of anti-Fas (S3) or IL-5 (data not shown) treatment. Western blot and immunofluorescence revealed anti-Fas rapidly induced the phosphorylation of FADD at Ser194 that was antagonized by IL-5 (Fig. 4A, B and S3).As seen in other cells, p-Ser194 FADD transited to the nucleus that was largely, but not completely blocked by co-treatment with IL-5 (Fig. 4B). Therefore, Fas signaling induced FADD phosphorylation on Ser194 that can be markedly attenuated by IL-5.

Figure 4.

Figure 4

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Anti-Fas triggers FADD phosphorylation and nuclear localization.

A) Eos were incubated with media alone (-), IL-5 and/or anti-Fas as shown for 30 min prior to lysis and western blotting as shown. Ratio of p-FADD to FADD for a representative blot is shown below. B) Eos treated as shown along the left (IL-5: 0.1 ng/ml; anti-Fas: 50 ng/ml) were analyzed by confocal microscopy as shown along the top. C) Schematic representation of TAT-HA-FADD WT and TAT-HA-Ser194 FADD mutants. D) Eos were incubated with 100 nM TAT-HA-β-gal, TAT-HA-FADD WT, TAT-HA-S194A FADD, TAT-HA-S194D FADD and TAT-HA-S194E FADD for 24 h prior to analysis of AnnexinV staining by flow cytometry. All experiments were done in triplicate with different donors.

In order to establish if phosphorylation of Ser194 FADD was required for Fas-mediated, Eos apoptosis, we transduced Eos with recombinant TAT-WT FADD or versions with Ser194 mutated to Ala (phospho-null), Glu or Asp (phospho-mimetics) (Fig. 4C). Otherwise untreated Eos transduced with phospho-mimic FADD showed significantly greater cell death as compared to phospho-null mutants or WT (Fig. 4D). These results suggest that Fas induced, FADD phosphorylation on Ser194 accelerates Eos apoptosis and that this signaling can be blocked by IL-5.

Pin1 regulates FADD phosphorylation

Pin1 can influence the phospho-status of its binding sites, presumably by modulating phosphatase or kinase access (21, 33). To dermine whether the anti-apoptotic effect of Pin1 in Fas-mediated signaling is upstream or downstream of FADD phosphorylation, we examined FADD phosphorylation after Pin1 blockade with juglone. As shown in Fig 5A, Fas-mediated FADD phosphorylation was decreased by IL-5 and was substantially increased by juglone (~2.5 fold), indicating that FADD phosphorylation is negatively regulated by Pin1. Thus we asked if FADD Ser194 phosphorylation and apoptotic action were similarly Pin1 dependent. Eos were incubated with FADD WT or mutants for 24 h prior to western blot analysis for activation of the extrinsic pathway and for 48 h for determination of cell viability. Eos transduced with phospho-mimic FADD showed significantly greater cleavage of Bid, caspase 3 and caspase 9 as well as accelerated apoptosis than control Eos or those transduced with control TAT-GFP (Fig. 4D, 5B and 5C). Consistent with the above data, IL-5 prevented Bid and caspase cleavage as well as cell death despite the transduction of phospho-mimic FADD (Fig. 5B and C). However, the pro-survival effects of IL-5 were completely abolished in Eos simultaneously treated with the Pin1 inhibitor juglone (Fig. 5A and B).

Figure 5.

Figure 5

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Pin1 suppresses Fas-mediated FADD function and translocation.

A) Eos were incubated for 30 min in the absence or presence of IL-5 (0.1 ng/ml) and/or anti-Fas (50 ng/ml) or juglone (0.5 μM). Whole cell lysates were then analyzed by western blot for phospho-FADD, FADD and β-actin. Quantitation of the western blot is shown below. B) Eos were untreated (-), pretreated with TAT-GFP or TAT-S194D FADD for 30min prior to IL-5, anti-Fas and/or juglone (as in A above) for 24 h, after which the cell lysates were analyzed by western blot. C) Eos were untreated or treated as shown along the bottom for 48 h prior to collection. Cell death determined by trypan blue exclusion. * = p<0.05, **= p< 0.05 vs untreated cells(-). D) Cells were pretreated with TAT-GFP, TAT-S16D Pin1 or TAT-S16A Pin1 for 30 min and then incubated with anti-Fas (50 ng/ml) for 30 min or 24 h prior to lysis and western blotting. E) Eos were treated with anti-Fas (50 ng/ml) for 30 min prior to fixation and confocal analysis with antibodies shown along the top. F) Eos were pretreated with TAT-GFP, TAT-S16A or TAT-S16D for 30 min prior to anti-Fas (50 ng/ml) for 30 min. Cytosolic or nuclear extracts were prepared and then analyzed by western blot.

These results suggested that the anti-apoptotic effect of IL-5 were fully mediated by Pin1 and involved suppression of FADD phosphorylation. To explore these hypotheses, FADD phosphorylation was measured in Eos transduced with S16A (constitutively active) Pin1 or S16D (constitutively inactive) Pin1 prior to Fas activation. Western blot revealed strong, anti-Fas induced FADD phosphorylation on Ser194 in control (TAT-GFP) or S16D Pin1 transduced Eos but markedly less so in Eos treated with S16A Pin1 (Fig. 5D and E). Consistent with this observation, cleaved caspase 3, 9, 8 were also markedly reduced in S16A Pin1 transduced cells (Fig. 5D). Immunofluorescence showed typical nuclear localization of p-Ser194 FADD after anti-Fas that was unaffected by transduction with TAT-constitutively inactive Pin1 or TAT-GFP (Fig. 5E) However, nuclear p-Ser194 FADD levels were barely detectable in anti-Fas treated Eos previously transduced with TAT-constitutively active Pin1. These data imply that the maintenance of Pin1 activity by IL-5, likely by preventing its phosphorylation at Ser16, is required to block FADD phosphorylation at Ser194 and subsequent FADD translocation and cell death.

In order to better quantitate pFADD intracellular distribution and relationship to Pin1 activity, Eos were transduced with S16A or S16D, treated with anti-Fas and cytoplasmic and nuclear fractions analyzed by western blotting (Fig. 5F). In all cells, pFADD and pSer16 Pin1 were exclusively nuclear. Treatment with anti-Fas increased nuclear pFADD and pSer16 Pin1 in Eos transduced with GFP or S16D Pin1. However, despite anti-Fas treatment, Eos transduced with S16A Pin1 showed reduced nuclear pFADD and pSer16 Pin1. As pSer16 Pin1 is unable to bind to targets, we propose anti-Fas induces the phosphorylation of nuclear Pin1, presumably attenuating its function in that compartment. Irrespectively, the data show that Pin1 likely plays a critical role in suppressing Fas-mediated apoptosis, presumably by influencing FADD nuclear localization and phosphorylation.

Pin1 binds to p-Ser194-Pro195 of FADD

Pin1 interacts with a subset of target proteins containing phosphorylated serine-proline or threonine-proline (S/T-P) dipeptide motifs and the phosphorylation of Ser194 FADD was Pin1 dependent (Fig. 5D and E), we examined if Pin1 interacted with FADD through Ser194-Pro195. Thus recombinant GST, GST-WW-Pin1, FADD WT and mutant (S194A, S194D or S194E) proteins were incubated with anti-FADD and the pellets immuno-blotted. Phospho-mimic FADD interacted strongly with the WW domain of Pin1 while interactions with WT or Ser194Ala FADD, while much weaker, were detectable (Fig. 6A). Conversely, Pin1 binding to FADD was abolished when Ser16 of Pin1 was mutated to phospho-mimetic Glu or Asp (Fig. 6B). These results are consistent with the data in Fig 5D-F, demonstrating an inability of phospho-mimic Pin1 to modulate FADD phosphorylation, location or pro-apoptotic activity. Thus, Pin1 must remain active (dephosphorylated at Ser16) to interact with FADD at p-Ser194-Pro195 and influence its functionality.

Figure 6.

Figure 6

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Pin1 interacts with FADD after IL-5 and anti-Fas.

A) GST or GST-WW-Pin1 was incubated with FADD, S194A FADD, S194E FADD or S194D FADD for 8 h at 4°C prior to anti-FADD pull-down and analysis by western blotting. B) GST-WW-Pin1 or GST-S16E-Pin1 were mixed with S194D FADD or S194E FADD for 8 h prior to immuno-precipitation with anti-GST antibodies and western blotting. C) Eos were incubated with media (-), anti-Fas (50 ng/ml), or anti-Fas plus IL-5 (0.1 ng/ml) for time shown, prior to cell lysis and immuno-precipitation with anti-Pin1 (Top Panel) or anti-FADD antibodies (Bottom Panel) and western blotting as shown. N denotes IP with nonimmune IgG. D) Anti-Fas treated cells were incubated with TAT-HA-GFP, TAT-HA-S16A-Pin1 or TAT-HA-S16D-Pin1 for 30 min prior to lysis and pull-down with anti-HA and western blotting as shown along the left.

As Fas activation triggers FADD phosphorylation at Ser194, we investigated if that was sufficient to induce an interaction with Pin1. Resting Eos were stimulated with either IL-5 and/or anti-Fas prior to IP/IB. Bidirectional IP’s revealed FADD and Pin1 interacted but only after brief (30 min) co-treatment with both anti-Fas and IL-5 (Fig. 6C). Prosurvival signaling initiated up to 24 h prior to anti-Fas (Fig. 7A and B) was sufficient to promote FADD-Pin1 interactions, suggesting IL-5 prevents Pin1 phosphorylation and maintains activity for extended periods of time (Fig. 7A). In order to determine if active Pin1 was necessary and sufficient to mediate IL-5 signaling, we performed pull-downs with lysates from anti-Fas treated Eos. Control GFP or S16D Pin1 (phospho-mimetic and constitutively inactive) failed to pull-down appreciable amounts of pSer194 FADD in contrast to S16A Pin1 (Fig. 6D). Thus, a dual signal provided by IL-5 and Fas signaling induce FADD and Pin1 to interact. Dominant active Pin1 (S16A) can replace the IL-5 signal suggesting that Pin1 is the critical, final effector for the modulation of Fas signaling by pro-survival cytokines. Mechanistically, the data support a model (Fig. 8) whereby Pin1 is activated by IL-5, binds to p-Ser194 that is induced by Fas ligature and isomerizes the Ser194-Pro195 bond from cis to trans, permitting de-phosphorylation of FADD and its retention in the cytoplasm.

Figure 7.

Figure 7

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Anti-Fas partially antagonizes IL-5 induced Pin1 activity and Eos viability.

A) Eos were treated for 24 h as shown (IL-5: 0.1 ng/ml; Anti-Fas: 50 ng/ml) prior to lysis and Pin1 PPIase assay. B) Eos were treated as shown prior to immuno-precipitation with anti-Pin1 (Top Panel) or anti-FADD (Bottom Panel) and western blotting as shown. N is IP with nonimmune IgG. C) Bronchoalveolar lavage (BAL) Eos were untreated (BAL Eos), or treated for 30 min with anti-Fas prior to lysis and Pin1 PPIase assay. Control Eos unstimulated and from normal donors. D) Eos from control donors, BAL Eos obtained 48 h after allergen challenge or Eos from the blood of allergen challenged donors (48 h after challenge) and treated as shown wih concentrations as in A) above. IL+α-F denotes cotreatment with IL-5 and anti-Fas.

Figure 8.

Figure 8

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Proposed model of Pin1 function in Fas-mediated apoptosis in activated eosinophils.

Finally, in order to establish the in vivo relevance of our observations, we asked if broncho-alveolar lavage (BAL) derived Eos, derived from asthmatics after segmental allergen challenge, were sensitive to Fas signaling. BAL Eos or peripheral blood Eos derived from these subjects have been activated through cytokine exposure in vivo and display prolonged lifespan (Fig. 7D) (25). Incubation of these cells with anti-Fas accelerated apoptosis (Fig. 7D). Consistent with in vivo activation, lysates from otherwise untreated BAL Eos showed elevated Pin1 activity that was modestly reduced by anti-Fas (Fig. 7C).

Discussion

In this study, we demonstrate that Pin1 is a critical mediator of IL-5 prosurvival signaling and FADD mediated apoptosis initiated by Fas activation. Pin1 interacts with FADD after its phosphorylation at Ser194-Pro195 but does so only if Ser16 of Pin1 remains dephosphorylated. Upon interaction with Pin1, FADD phosphorylation, nuclear translocation and pro-apoptotic signaling are largely suppressed. Therefore Pin1 can attenuate cell death initiated through the extrinsic pathway only in the presence of simultaneous pro-survival signaling; leading to the pulmonary persistence of in vivo activated Eos, despite ongoing Fas mediated death signaling. As this is likely the case in allergically inflamed airways, Pin1 is an important determinant of whether activated Eos will be cleared or accumulated in the lungs of asthmatics.

The balance between Eos survival and death is crucial for the development, maintenance or resolution of asthma (7). In chronic asthma, airway eosinophilia continues and predominantly reflects defective or reduced Eos apoptosis (8). FasL is expressed in the lung on some activated T and B cells, macrophages and epithelia and thus is a likely candidate to oppose pro-survival mediated signaling by GM-CSF or IL-5 (34). In vivo manipulation of Fas or FasL have generated data that largely, but not entirely reinforces this hypothesis (27, 28, 35, 36). Here we confirm that Fas activation accelerated Eos apoptosis through the extrinsic pathway in both the absence and presence of IL-5 mediated signaling. Consistent with this conclusion, allergen challenged, Bid knock-out mice showed elevated numbers of BAL Eos in vivo and reduced apoptosis and caspase 3 cleavage after in vitro Fas activation (36). Thus Fas activation of the extrinsic pathway is likely one mechanism by which Eos apoptosis is enhanced despite pro-survival signaling after allergen challenge.

Phosphorylation at Ser194 in human cells or Ser191 in mouse cells is a critical determinant of FADD intracellular trafficking and function (30, 37). Phospho-null Ser194Ala FADD mutants were unable to interact with exportin 5, leading to diffuse distribution in both the cytoplasm and nucleus (32). Phosphorylation is normally mediated by Casein Kinase 1 or Plk1 after death receptor activation (38, 39). Nuclear p-FADD has been associated with reduced cell death, possibly by up-regulating survival gene expression, modifying Methyl-CpG BP4 function or by reducing the interactions between FADD and caspase 8 in the cytoplasm (32). Resting Eos showed low levels of p-FADD by western blot and very little on IF analysis. Fas activation triggered very rapid phosphorylation on Ser194, localization into the nucleus and the acceleration of caspase 8 and Bid cleavage. Either IL-5 or constitutively active (S16A) Pin1 antagonized FADD phosphorylation, nuclear translocation and apoptotic activity, irrespective of Fas signaling. Phospho-mimic FADD mutants had similar effects that could also be antagonized by IL-5 or enhanced by Pin1 inhibitors. Thus, FADD phospho-mutants were functionally similar to wild type, p-FADD. These data suggest that in Eos, FADD mediated apoptosis depends on both Ser194 phosphorylation as well as nuclear translocation, processes that are antagonized by IL-5 and Pin1. As constitutively active Pin1 was able to reduce FADD phosphorylation, translocation and apoptosis, all in the absence of IL-5, Pin1 is likely the downstream effector for IL-5. It remains unclear how nuclear p-FADD activates caspase 8 to drive Eos apoptosis. Caspase 8 is expressed exclusively in the cytoplasm of T cells treated with anti-CD3 or anti-Fas (36). Thus, caspase 8 is likely cleaved in the cytoplasm, presumably by the modest amount of residual p-FADD present there.

Overexpression of constitutively active Pin1 attenuates Fas mediated phosphorylation of FADD as well as its nuclear translocation. However, IP/WB revealed that endogenous Pin1 or recombinant WW domain preferentially interacts with p-FADD or phospho-mimic FADD mutants. These observations suggest active Pin1 initially interacts with p-FADD but that once bound, Pin1 isomerizes the target, converting the p-Ser194-Pro195 site from cis to trans. Due to increased availability, the trans phosphate is then cleaved as has been described for trans p-tau (40). As Pin1 has ~1000 fold lower activity towards un-phosphorylated targets (21), FADD in the trans, nonphosphorylated conformation would accumulate. While we have not yet examined the issue, trans FADD might also be subject to accelerated degradation.

Pin1 is regulated by reversible, multi-site phosphorylation. Recent work has shown phosphorylation of Ser16 by PKA (22, 24) or PKMζ (41), Ser71 by DAPK1 (23) or Ser138 by MLK3 (29) can modulate Pin1 activity and/or location. We were unable to detect any changes in Ser71 after Fas or IL-5 mediated activation. Our data suggest that modulation of Pin1 activity in Eos by pro-survival (IL-5) and pro-apoptotic (Fas) signaling focuses on reversible events at Ser16. Fas activation rapidly reduced PPIase activity in resting Eos that was associated with increased phosphorylation of Pin1 at Ser16. Calyculin increased Ser16 phosphorylation and prevented any change after agonists suggesting PP2A or PP1 mediated Fas signaling. In addition, Eos transduced with S16A mutants were largely but not completely resistant to the effects of anti-Fas. PP1 and PP2A were required for Fas mediated apoptosis of tumor cell lines (42) and FADD has recently been shown to interact with and regulate PP2A activity (43). As Pin1 interacts with and is regulated by PP2A (44), these results suggest FADD, Pin1 and PP2A form a complex with FADD signaling altering both Pin1 and PP2A activity and function.

In conclusion, our data collectively show that Pin1 prevents Fas-mediated apoptosis in activated Eos via interactions with p-FADD (Fig. 8). Our results also support the hypothesis that Fas signaling can overcome IL-5 signaling and accelerate apoptosis to facilitate the elimination of Eos after allergen challenge.

Supplementary Material

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Acknowledgements

We would like to thank the members of the lab as well as the UW-asthma group for thoughtful suggestions, cells and reagents.

Abbreviations

Eos

Eosinophils

FADD

Fas-associated protein with death domain

PARP

Poly (ADP-ribose) polymerase

IB

immunoblot

IP

immunoprecipitation

Footnotes

#

Supported by NIH (P01HL88594 and R01HL87950) to J.S.M.

References

  • 1.Rosenberg HF, Phipps S, Foster PS. Eosinophil trafficking in allergy and asthma. J. Allergy. Clin. Immunol. 2007;119(6):1303–1310. doi: 10.1016/j.jaci.2007.03.048. quiz 1311-1302. [DOI] [PubMed] [Google Scholar]
  • 2.Radinger M, Lotvall J. Eosinophil progenitors in allergy and asthma - do they matter? Pharmacol. Ther. 2009;121(2):174–184. doi: 10.1016/j.pharmthera.2008.10.008. [DOI] [PubMed] [Google Scholar]
  • 3.Laitinen LA, Laitinen A, Heino M, Haahtela T. Eosinophilic airway inflammation during exacerbation of asthma and its treatment with inhaled corticosteroid. Am. Rev. Respir. Dis. 1991;143(2):423–427. doi: 10.1164/ajrccm/143.2.423. [DOI] [PubMed] [Google Scholar]
  • 4.Kankaanranta H, Moilanen E, Zhang X. Pharmacological regulation of human eosinophil apoptosis. Curr. Drug. Targets. Inflamm. Allergy. 2005;4(4):433–445. doi: 10.2174/1568010054526395. [DOI] [PubMed] [Google Scholar]
  • 5.Kroegel C, Warner JA, Virchow JC, Jr., Matthys H. Pulmonary immune cells in health and disease: the eosinophil leucocyte (Part II) Eur. Respir. J. 1994;7(4):743–760. doi: 10.1183/09031936.94.07040743. [DOI] [PubMed] [Google Scholar]
  • 6.Saita N, Yamanaka T, Kohrogi H, Ando M, Hirashima M. Apoptotic response of eosinophils in chronic eosinophilic pneumonia. Eur. Respir. J. 2001;17(2):190–194. doi: 10.1183/09031936.01.17201900. [DOI] [PubMed] [Google Scholar]
  • 7.Bousquet J, Jeffery PK, Busse WW, Johnson M, Vignola AM. Asthma. From bronchoconstriction to airways inflammation and remodeling. Am. J. Respir. Crit. Care. Med. 2000;161(5):1720–1745. doi: 10.1164/ajrccm.161.5.9903102. [DOI] [PubMed] [Google Scholar]
  • 8.Haslett C. Granulocyte apoptosis and its role in the resolution and control of lung inflammation. Am. J. Respir. Crit. Care. Med. 1999;160(5 Pt 2):S5–11. doi: 10.1164/ajrccm.160.supplement_1.4. [DOI] [PubMed] [Google Scholar]
  • 9.Maret M, Ruffie C, Letuve S, Phelep A, Thibaudeau O, Marchal J, Pretolani M, Druilhe A. A role for Bid in eosinophil apoptosis and in allergic airway reaction. J. Immunol. 2009;182(9):5740–5747. doi: 10.4049/jimmunol.0800864. [DOI] [PubMed] [Google Scholar]
  • 10.Gleich GJ. Anti-interleukin-5 therapy and severe asthma. N. Engl. J. Med. 2009;360(24):2577. author reply 2578. [PubMed] [Google Scholar]
  • 11.Kouro T, Takatsu K. IL-5- and eosinophil-mediated inflammation: from discovery to therapy. Int. Immunol. 2009;21(12):1303–1309. doi: 10.1093/intimm/dxp102. [DOI] [PubMed] [Google Scholar]
  • 12.Heasman SJ, Giles KM, Ward C, Rossi AG, Haslett C, Dransfield I. Glucocorticoid-mediated regulation of granulocyte apoptosis and macrophage phagocytosis of apoptotic cells: implications for the resolution of inflammation. J. Endocrinol. 2003;178(1):29–36. doi: 10.1677/joe.0.1780029. [DOI] [PubMed] [Google Scholar]
  • 13.Robinson DS, Hamid Q, Ying S, Tsicopoulos A, Barkans J, Bentley AM, Corrigan C, Durham SR, Kay AB. Predominant TH2-like bronchoalveolar T-lymphocyte population in atopic asthma. N. Engl. J. Med. 1992;326(5):298–304. doi: 10.1056/NEJM199201303260504. [DOI] [PubMed] [Google Scholar]
  • 14.Nagata S. Apoptosis by death factor. Cell. 1997;88(3):355–365. doi: 10.1016/s0092-8674(00)81874-7. [DOI] [PubMed] [Google Scholar]
  • 15.Kischkel FC, Hellbardt S, Behrmann I, Germer M, Pawlita M, Krammer PH, Peter ME. Cytotoxicity-dependent APO-1 (Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC) with the receptor. EMBO. J. 1995;14(22):5579–5588. doi: 10.1002/j.1460-2075.1995.tb00245.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Tourneur L, Chiocchia G. FADD: a regulator of life and death. Trends Immunol. 2010;31(7):260–269. doi: 10.1016/j.it.2010.05.005. [DOI] [PubMed] [Google Scholar]
  • 17.Scott FL, Stec B, Pop C, Dobaczewska MK, Lee JJ, Monosov E, Robinson H, Salvesen GS, Schwarzenbacher R, Riedl SJ. The Fas-FADD death domain complex structure unravels signalling by receptor clustering. Nature. 2009;457(7232):1019–1022. doi: 10.1038/nature07606. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Gomez-Angelats M, Cidlowski JA. Molecular evidence for the nuclear localization of FADD. Cell Death Differ. 2003;10(7):791–797. doi: 10.1038/sj.cdd.4401237. [DOI] [PubMed] [Google Scholar]
  • 19.Anderson P. Pin1: a proline isomerase that makes you wheeze? Nat. Immunol. 2005;6(12):1211–1212. doi: 10.1038/ni1205-1211. [DOI] [PubMed] [Google Scholar]
  • 20.Lu KP, Zhou XZ. The prolyl isomerase PIN1: a pivotal new twist in phosphorylation signalling and disease. Nat. Rev. Mol. Cell Biol. 2007;8(11):904–916. doi: 10.1038/nrm2261. [DOI] [PubMed] [Google Scholar]
  • 21.Liou YC, Zhou XZ, Lu KP. Prolyl isomerase Pin1 as a molecular switch to determine the fate of phosphoproteins. Trends Biochem. Sci. 2011;36(10):501–514. doi: 10.1016/j.tibs.2011.07.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Shen ZJ, Esnault S, Schinzel A, Borner C, Malter JS. The peptidyl-prolyl isomerase Pin1 facilitates cytokine-induced survival of eosinophils by suppressing Bax activation. Nat. Immunol. 2009;10(3):257–265. doi: 10.1038/ni.1697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Lee TH, Chen CH, Suizu F, Huang P, Schiene-Fischer C, Daum S, Zhang YJ, Goate A, Chen RH, Zhou XZ, Lu KP. Death-associated protein kinase 1 phosphorylates Pin1 and inhibits its prolyl isomerase activity and cellular function. Mol. Cell. 2011;42(2):147–159. doi: 10.1016/j.molcel.2011.03.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Lu PJ, Zhou XZ, Liou YC, Noel JP, Lu KP. Critical role of WW domain phosphorylation in regulating phosphoserine binding activity and Pin1 function. J. Biol. Chem. 2002;277(4):2381–2384. doi: 10.1074/jbc.C100228200. [DOI] [PubMed] [Google Scholar]
  • 25.Shen ZJ, Esnault S, Malter JS. The peptidyl-prolyl isomerase Pin1 regulates the stability of granulocyte-macrophage colony-stimulating factor mRNA in activated eosinophils. Nat. Immunol. 2005;6(12):1280–1287. doi: 10.1038/ni1266. [DOI] [PubMed] [Google Scholar]
  • 26.Gong H, Kovar J, Little G, Chen H, Olive DM. In vivo imaging of xenograft tumors using an epidermal growth factor receptor-specific affibody molecule labeled with a near-infrared fluorophore. Neoplasia. 2010;12(2):139–149. doi: 10.1593/neo.91446. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Sharma SK, Almeida FA, Kierstein S, Hortobagyi L, Lin T, Larkin A, Peterson J, Yagita H, Zangrilli JG, Haczku A. Systemic FasL neutralization increases eosinophilic inflammation in a mouse model of asthma. Allergy. 2012;67(3):328–335. doi: 10.1111/j.1398-9995.2011.02763.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Uller L, Rydell-Tormanen K, Persson CG, Erjefalt JS. Anti-Fas mAb-induced apoptosis and cytolysis of airway tissue eosinophils aggravates rather than resolves established inflammation. Respir. Res. 2005;6:90. doi: 10.1186/1465-9921-6-90. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Rangasamy V, Mishra R, Sondarva G, Das S, Lee TH, Bakowska JC, Tzivion G, Malter JS, Rana B, Lu KP, Kanthasamy A, Rana A. Mixed-lineage kinase 3 phosphorylates prolyl-isomerase Pin1 to regulate its nuclear translocation and cellular function. Proc. Natl. Acad. Sci. U S A. 2012;109(21):8149–8154. doi: 10.1073/pnas.1200804109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Jang MS, Lee SJ, Kim CJ, Lee CW, Kim E. Phosphorylation by polo-like kinase 1 induces the tumor-suppressing activity of FADD. Oncogene. 2011;30(4):471–481. doi: 10.1038/onc.2010.423. [DOI] [PubMed] [Google Scholar]
  • 31.Alappat EC, Feig C, Boyerinas B, Volkland J, Samuels M, Murmann AE, Thorburn A, Kidd VJ, Slaughter CA, Osborn SL, Winoto A, Tang WJ, Peter ME. Phosphorylation of FADD at serine 194 by CKIalpha regulates its nonapoptotic activities. Mol. Cell. 2005;19(3):321–332. doi: 10.1016/j.molcel.2005.06.024. [DOI] [PubMed] [Google Scholar]
  • 32.Screaton RA, Kiessling S, Sansom OJ, Millar CB, Maddison K, Bird A, Clarke AR, Frisch SM. Fas-associated death domain protein interacts with methyl-CpG binding domain protein 4: a potential link between genome surveillance and apoptosis. Proc. Natl. Acad. Sci. U S A. 2003;100(9):5211–5216. doi: 10.1073/pnas.0431215100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Ranganathan R, Lu KP, Hunter T, Noel JP. Structural and functional analysis of the mitotic rotamase Pin1 suggests substrate recognition is phosphorylation dependent. Cell. 1997;89(6):875–886. doi: 10.1016/s0092-8674(00)80273-1. [DOI] [PubMed] [Google Scholar]
  • 34.Krug N, Tschernig T, Balke K, Erpenbeck VJ, Meyer L, Bode U, Hohlfeld JM, Pabst R, Fabel H. Enhanced expression of fas ligand (CD95L) on T cells after segmental allergen provocation in asthma. J. Allergy. Clin. Immunol. 1999;103(4):649–655. doi: 10.1016/s0091-6749(99)70238-1. [DOI] [PubMed] [Google Scholar]
  • 35.Tsuyuki S, Bertrand C, Erard F, Trifilieff A, Tsuyuki J, Wesp M, Anderson GP, Coyle AJ. Activation of the Fas receptor on lung eosinophils leads to apoptosis and the resolution of eosinophilic inflammation of the airways. J. Clin. Invest. 1995;96(6):2924–2931. doi: 10.1172/JCI118364. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Koenig A, Russell JQ, Rodgers WA, Budd RC. Spatial differences in active caspase-8 defines its role in T-cell activation versus cell death. Cell Death Differ. 2008;15(11):1701–1711. doi: 10.1038/cdd.2008.100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Drakos E, Leventaki V, Atsaves V, Schlette EJ, Lin P, Vega F, Miranda RN, Claret FX, Medeiros LJ, Rassidakis GZ. Expression of serine 194-phosphorylated Fas-associated death domain protein correlates with proliferation in B-cell non-Hodgkin lymphomas. Hum. Pathol. 2011;42(8):1117–1124. doi: 10.1016/j.humpath.2010.11.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Desagher S, Osen-Sand A, Montessuit S, Magnenat E, Vilbois F, Hochmann A, Journot L, Antonsson B, Martinou JC. Phosphorylation of bid by casein kinases I and II regulates its cleavage by caspase 8. Mol. Cell. 2001;8(3):601–611. doi: 10.1016/s1097-2765(01)00335-5. [DOI] [PubMed] [Google Scholar]
  • 39.Jang MS, Lee SJ, Kang NS, Kim E. Cooperative phosphorylation of FADD by Aur-A and Plk1 in response to taxol triggers both apoptotic and necrotic cell death. Cancer Res. 2011;71(23):7207–7215. doi: 10.1158/0008-5472.CAN-11-0760. [DOI] [PubMed] [Google Scholar]
  • 40.Lu KP, Liou YC, Zhou XZ. Pinning down proline-directed phosphorylation signaling. Trends Cell Biol. 2002;12(4):164–172. doi: 10.1016/s0962-8924(02)02253-5. [DOI] [PubMed] [Google Scholar]
  • 41.Westmark PR, Westmark CJ, Wang S, Levenson J, O’Riordan KJ, Burger C, Malter JS. Pin1 and PKMzeta sequentially control dendritic protein synthesis. Sci. Signal. 2010;3(112):ra18. doi: 10.1126/scisignal.2000451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Chatfield K, Eastman A. Inhibitors of protein phosphatases 1 and 2A differentially prevent intrinsic and extrinsic apoptosis pathways. Biochem. Biophys. Res. Commun. 2004;323(4):1313–1320. doi: 10.1016/j.bbrc.2004.09.003. [DOI] [PubMed] [Google Scholar]
  • 43.Cheng W, Wang L, Zhang R, Du P, Yang B, Zhuang H, Tang B, Yao C, Yu M, Wang Y, Zhang J, Yin W, Li J, Zheng W, Lu M, Hua Z. Regulation of PKC Inactivation by FADD. J. Biol. Chem. 2012 doi: 10.1074/jbc.M112.342170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Shen ZJ, Esnault S, Rosenthal LA, Szakaly RJ, Sorkness RL, Westmark PR, Sandor M, Malter JS. Pin1 regulates TGF-beta1 production by activated human and murine eosinophils and contributes to allergic lung fibrosis. J. Clin. Invest. 2008;118(2):479–490. doi: 10.1172/JCI32789. [DOI] [PMC free article] [PubMed] [Google Scholar]

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Supplementary Materials

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NIHMS459379-supplement-1.docx (45.4KB, docx)
2
NIHMS459379-supplement-2.doc (71KB, doc)
3
NIHMS459379-supplement-3.pdf (195.2KB, pdf)
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