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TWEAK-independent Fn14 self-association and NF-κB activation is mediated by the C-terminal region of the Fn14 cytoplasmic domain.

Brown SA, Cheng E, Williams MS, Winkles JA - PLoS ONE (2013)

Bottom Line: Endogenously-expressed Fn14 as well as ectopically-overexpressed Fn14 could also be detected in dimeric form when cell lysates were subjected to SDS-PAGE under non-reducing conditions.Additional experiments revealed that Fn14 dimerization occurs during cell lysis via formation of an intermolecular disulfide bond at cysteine residue 122.These findings provide insight into the Fn14 signaling mechanism and may aid current studies to develop therapeutic agents targeting this small cell surface receptor.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Center for Vascular and Inflammatory Diseases, University of Maryland School of Medicine, Baltimore, Maryland, USA.

ABSTRACT
The tumor necrosis factor (TNF) superfamily member TNF-like weak inducer of apoptosis (TWEAK) is a pro-inflammatory and pro-angiogenic cytokine implicated in physiological tissue regeneration and wound repair. TWEAK binds to a 102-amino acid type I transmembrane cell surface receptor named fibroblast growth factor-inducible 14 (Fn14). TWEAK:Fn14 engagement activates several intracellular signaling cascades, including the NF-κB pathway, and sustained Fn14 signaling has been implicated in the pathogenesis of chronic inflammatory diseases and cancer. Although several groups are developing TWEAK- or Fn14-targeted agents for therapeutic use, much more basic science research is required before we fully understand the TWEAK/Fn14 signaling axis. For example, we and others have proposed that TWEAK-independent Fn14 signaling may occur in cells when Fn14 levels are highly elevated, but this idea has never been tested directly. In this report, we first demonstrate TWEAK-independent Fn14 signaling by showing that an Fn14 deletion mutant that is unable to bind TWEAK can activate the NF-κB pathway in transfected cells. We then show that ectopically-expressed, cell surface-localized Fn14 can self-associate into Fn14 dimers, and we show that Fn14 self-association is mediated by an 18-aa region within the Fn14 cytoplasmic domain. Endogenously-expressed Fn14 as well as ectopically-overexpressed Fn14 could also be detected in dimeric form when cell lysates were subjected to SDS-PAGE under non-reducing conditions. Additional experiments revealed that Fn14 dimerization occurs during cell lysis via formation of an intermolecular disulfide bond at cysteine residue 122. These findings provide insight into the Fn14 signaling mechanism and may aid current studies to develop therapeutic agents targeting this small cell surface receptor.

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The Fn14-FL, Fn14-ΔEC, and Fn14-ΔCT proteins can all associate with the Fn14-FL protein.(A) Schematic representation of the expression construct encoding the HA-tagged Fn14-FL protein. The Fn14 signal peptide (SP) region, extracellular (EC) domain, transmembrane (TM) domain, and cytoplasmic tail (CT) are indicated and the positions of the signal peptide cleavage site (scissors) and the HA epitope tag are shown. (B) HEK293/NF-κB-luc/Fn14-HA cells were transfected with vector or the indicated Fn14-myc expression plasmids and 24 hr later the cells were harvested, lysed, and equal amounts of protein were used for Western blot analysis using either an anti-HA, anti-myc or anti-tubulin antibody (lower three panels). Lysates were also subjected to immunoprecipitation (IP) analysis using an immobilized anti-myc antibody and Fn14-HA:Fn14-myc association analyzed by Western blot analysis with an anti-HA antibody (top two panels). The positions of molecular size markers are shown on the left (in kDa).
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pone-0065248-g005: The Fn14-FL, Fn14-ΔEC, and Fn14-ΔCT proteins can all associate with the Fn14-FL protein.(A) Schematic representation of the expression construct encoding the HA-tagged Fn14-FL protein. The Fn14 signal peptide (SP) region, extracellular (EC) domain, transmembrane (TM) domain, and cytoplasmic tail (CT) are indicated and the positions of the signal peptide cleavage site (scissors) and the HA epitope tag are shown. (B) HEK293/NF-κB-luc/Fn14-HA cells were transfected with vector or the indicated Fn14-myc expression plasmids and 24 hr later the cells were harvested, lysed, and equal amounts of protein were used for Western blot analysis using either an anti-HA, anti-myc or anti-tubulin antibody (lower three panels). Lysates were also subjected to immunoprecipitation (IP) analysis using an immobilized anti-myc antibody and Fn14-HA:Fn14-myc association analyzed by Western blot analysis with an anti-HA antibody (top two panels). The positions of molecular size markers are shown on the left (in kDa).

Mentions: It has been reported that when CD40 [54] and CD30 [52] deletion mutants lacking TRAF binding sites in the cytoplasmic domain are overexpressed in cells they can act as dominant-negative inhibitors of ligand-stimulated signaling, presumably by associating with endogenous receptors and thereby forming non-functional receptor complexes. In a previous study, we reported that MDA-MB-231 breast cancer cell invasion capacity could be inhibited by forced Fn14-ΔCT expression [32], suggesting that Fn14-ΔCT may also be a dominant-negative inhibitor. Therefore, we wanted to determine whether Fn14-ΔCT could associate with the Fn14-FL protein even though it cannot associate with itself. For these studies, we used a co-immunoprecipitation approach. HEK293 cells engineered to stably overexpress HA epitope-tagged Fn14-FL (Fig. 5A) were transfected with vector or the Fn14-FL, Fn14-ΔEC, or Fn14-ΔCT expression plasmids (all proteins myc-tagged) and 24 hr later the cells were harvested. Western blot analysis was performed on an aliquot of each lysate using either an anti-HA or anti-myc antibody to confirm expression of all Fn14 proteins (Fig. 5B). Lysates were also subjected to immunoprecipitation analysis using an immobilized anti-myc antibody and Fn14-HA:Fn14-myc association evaluated by Western blot analysis with an anti-HA antibody. We found that all three myc-tagged Fn14 proteins were effectively immunoprecipitated with the anti-myc antibody and could interact with HA-tagged Fn14-FL (Fig. 5B).


TWEAK-independent Fn14 self-association and NF-κB activation is mediated by the C-terminal region of the Fn14 cytoplasmic domain.

Brown SA, Cheng E, Williams MS, Winkles JA - PLoS ONE (2013)

The Fn14-FL, Fn14-ΔEC, and Fn14-ΔCT proteins can all associate with the Fn14-FL protein.(A) Schematic representation of the expression construct encoding the HA-tagged Fn14-FL protein. The Fn14 signal peptide (SP) region, extracellular (EC) domain, transmembrane (TM) domain, and cytoplasmic tail (CT) are indicated and the positions of the signal peptide cleavage site (scissors) and the HA epitope tag are shown. (B) HEK293/NF-κB-luc/Fn14-HA cells were transfected with vector or the indicated Fn14-myc expression plasmids and 24 hr later the cells were harvested, lysed, and equal amounts of protein were used for Western blot analysis using either an anti-HA, anti-myc or anti-tubulin antibody (lower three panels). Lysates were also subjected to immunoprecipitation (IP) analysis using an immobilized anti-myc antibody and Fn14-HA:Fn14-myc association analyzed by Western blot analysis with an anti-HA antibody (top two panels). The positions of molecular size markers are shown on the left (in kDa).
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pone-0065248-g005: The Fn14-FL, Fn14-ΔEC, and Fn14-ΔCT proteins can all associate with the Fn14-FL protein.(A) Schematic representation of the expression construct encoding the HA-tagged Fn14-FL protein. The Fn14 signal peptide (SP) region, extracellular (EC) domain, transmembrane (TM) domain, and cytoplasmic tail (CT) are indicated and the positions of the signal peptide cleavage site (scissors) and the HA epitope tag are shown. (B) HEK293/NF-κB-luc/Fn14-HA cells were transfected with vector or the indicated Fn14-myc expression plasmids and 24 hr later the cells were harvested, lysed, and equal amounts of protein were used for Western blot analysis using either an anti-HA, anti-myc or anti-tubulin antibody (lower three panels). Lysates were also subjected to immunoprecipitation (IP) analysis using an immobilized anti-myc antibody and Fn14-HA:Fn14-myc association analyzed by Western blot analysis with an anti-HA antibody (top two panels). The positions of molecular size markers are shown on the left (in kDa).
Mentions: It has been reported that when CD40 [54] and CD30 [52] deletion mutants lacking TRAF binding sites in the cytoplasmic domain are overexpressed in cells they can act as dominant-negative inhibitors of ligand-stimulated signaling, presumably by associating with endogenous receptors and thereby forming non-functional receptor complexes. In a previous study, we reported that MDA-MB-231 breast cancer cell invasion capacity could be inhibited by forced Fn14-ΔCT expression [32], suggesting that Fn14-ΔCT may also be a dominant-negative inhibitor. Therefore, we wanted to determine whether Fn14-ΔCT could associate with the Fn14-FL protein even though it cannot associate with itself. For these studies, we used a co-immunoprecipitation approach. HEK293 cells engineered to stably overexpress HA epitope-tagged Fn14-FL (Fig. 5A) were transfected with vector or the Fn14-FL, Fn14-ΔEC, or Fn14-ΔCT expression plasmids (all proteins myc-tagged) and 24 hr later the cells were harvested. Western blot analysis was performed on an aliquot of each lysate using either an anti-HA or anti-myc antibody to confirm expression of all Fn14 proteins (Fig. 5B). Lysates were also subjected to immunoprecipitation analysis using an immobilized anti-myc antibody and Fn14-HA:Fn14-myc association evaluated by Western blot analysis with an anti-HA antibody. We found that all three myc-tagged Fn14 proteins were effectively immunoprecipitated with the anti-myc antibody and could interact with HA-tagged Fn14-FL (Fig. 5B).

Bottom Line: Endogenously-expressed Fn14 as well as ectopically-overexpressed Fn14 could also be detected in dimeric form when cell lysates were subjected to SDS-PAGE under non-reducing conditions.Additional experiments revealed that Fn14 dimerization occurs during cell lysis via formation of an intermolecular disulfide bond at cysteine residue 122.These findings provide insight into the Fn14 signaling mechanism and may aid current studies to develop therapeutic agents targeting this small cell surface receptor.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Center for Vascular and Inflammatory Diseases, University of Maryland School of Medicine, Baltimore, Maryland, USA.

ABSTRACT
The tumor necrosis factor (TNF) superfamily member TNF-like weak inducer of apoptosis (TWEAK) is a pro-inflammatory and pro-angiogenic cytokine implicated in physiological tissue regeneration and wound repair. TWEAK binds to a 102-amino acid type I transmembrane cell surface receptor named fibroblast growth factor-inducible 14 (Fn14). TWEAK:Fn14 engagement activates several intracellular signaling cascades, including the NF-κB pathway, and sustained Fn14 signaling has been implicated in the pathogenesis of chronic inflammatory diseases and cancer. Although several groups are developing TWEAK- or Fn14-targeted agents for therapeutic use, much more basic science research is required before we fully understand the TWEAK/Fn14 signaling axis. For example, we and others have proposed that TWEAK-independent Fn14 signaling may occur in cells when Fn14 levels are highly elevated, but this idea has never been tested directly. In this report, we first demonstrate TWEAK-independent Fn14 signaling by showing that an Fn14 deletion mutant that is unable to bind TWEAK can activate the NF-κB pathway in transfected cells. We then show that ectopically-expressed, cell surface-localized Fn14 can self-associate into Fn14 dimers, and we show that Fn14 self-association is mediated by an 18-aa region within the Fn14 cytoplasmic domain. Endogenously-expressed Fn14 as well as ectopically-overexpressed Fn14 could also be detected in dimeric form when cell lysates were subjected to SDS-PAGE under non-reducing conditions. Additional experiments revealed that Fn14 dimerization occurs during cell lysis via formation of an intermolecular disulfide bond at cysteine residue 122. These findings provide insight into the Fn14 signaling mechanism and may aid current studies to develop therapeutic agents targeting this small cell surface receptor.

Show MeSH
Related in: MedlinePlus