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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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TWEAK cannot bind to the Fn14-ΔEC protein.(A) Schematic representation of the expression constructs encoding the Fn14-FL or Fn14-ΔEC protein. The Fn14 signal peptide (SP) region, extracellular (EC) domain, transmembrane (TM) domain, and cytoplasmic tail (CT) are indicated, the size of each EC domain is provided, and the positions of the signal peptide cleavage site (scissors) and the myc epitope tag are shown for both constructs. (B) HEK293 cells were transfected with vector or the indicated Fn14-myc expression plasmids. Cells were harvested 24 hr later, lysed, and equal amounts of protein were subjected to ligand blot analysis and Western blot analysis using either the anti-Fn14 #3600 antibody or an anti-tubulin antibody. The positions of molecular size markers are shown on the right (in kDa).
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pone-0065248-g002: TWEAK cannot bind to the Fn14-ΔEC protein.(A) Schematic representation of the expression constructs encoding the Fn14-FL or Fn14-ΔEC protein. The Fn14 signal peptide (SP) region, extracellular (EC) domain, transmembrane (TM) domain, and cytoplasmic tail (CT) are indicated, the size of each EC domain is provided, and the positions of the signal peptide cleavage site (scissors) and the myc epitope tag are shown for both constructs. (B) HEK293 cells were transfected with vector or the indicated Fn14-myc expression plasmids. Cells were harvested 24 hr later, lysed, and equal amounts of protein were subjected to ligand blot analysis and Western blot analysis using either the anti-Fn14 #3600 antibody or an anti-tubulin antibody. The positions of molecular size markers are shown on the right (in kDa).

Mentions: The Fn14-ΔEC isoform should not be able to bind TWEAK since it is missing most of the extracellular domain, and in particular, it does not have the cysteine-rich region within this domain that has been previously shown to contain the three disulfide bonds critical for proper Fn14 folding and TWEAK:Fn14 interaction [41]–[43]. We confirmed that TWEAK could not bind to Fn14-ΔEC using a ligand blot assay. Expression plasmids encoding either myc epitope-tagged Fn14-FL or Fn14-ΔEC (Fig. 2A), as well as the vector itself, were transfected into HEK293 cells. Cell lysates were prepared and protein was subjected to SDS-PAGE under non-reducing conditions (for ligand blot analysis) or reducing conditions (for Western blot analysis). TWEAK interaction with the Fn14-FL protein, but not the Fn14-ΔEC protein, was detected using the ligand blot assay, although Western blot analysis indicated that both proteins were expressed to a similar degree in the transfected cells (Fig. 2B).


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)

TWEAK cannot bind to the Fn14-ΔEC protein.(A) Schematic representation of the expression constructs encoding the Fn14-FL or Fn14-ΔEC protein. The Fn14 signal peptide (SP) region, extracellular (EC) domain, transmembrane (TM) domain, and cytoplasmic tail (CT) are indicated, the size of each EC domain is provided, and the positions of the signal peptide cleavage site (scissors) and the myc epitope tag are shown for both constructs. (B) HEK293 cells were transfected with vector or the indicated Fn14-myc expression plasmids. Cells were harvested 24 hr later, lysed, and equal amounts of protein were subjected to ligand blot analysis and Western blot analysis using either the anti-Fn14 #3600 antibody or an anti-tubulin antibody. The positions of molecular size markers are shown on the right (in kDa).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3672086&req=5

pone-0065248-g002: TWEAK cannot bind to the Fn14-ΔEC protein.(A) Schematic representation of the expression constructs encoding the Fn14-FL or Fn14-ΔEC protein. The Fn14 signal peptide (SP) region, extracellular (EC) domain, transmembrane (TM) domain, and cytoplasmic tail (CT) are indicated, the size of each EC domain is provided, and the positions of the signal peptide cleavage site (scissors) and the myc epitope tag are shown for both constructs. (B) HEK293 cells were transfected with vector or the indicated Fn14-myc expression plasmids. Cells were harvested 24 hr later, lysed, and equal amounts of protein were subjected to ligand blot analysis and Western blot analysis using either the anti-Fn14 #3600 antibody or an anti-tubulin antibody. The positions of molecular size markers are shown on the right (in kDa).
Mentions: The Fn14-ΔEC isoform should not be able to bind TWEAK since it is missing most of the extracellular domain, and in particular, it does not have the cysteine-rich region within this domain that has been previously shown to contain the three disulfide bonds critical for proper Fn14 folding and TWEAK:Fn14 interaction [41]–[43]. We confirmed that TWEAK could not bind to Fn14-ΔEC using a ligand blot assay. Expression plasmids encoding either myc epitope-tagged Fn14-FL or Fn14-ΔEC (Fig. 2A), as well as the vector itself, were transfected into HEK293 cells. Cell lysates were prepared and protein was subjected to SDS-PAGE under non-reducing conditions (for ligand blot analysis) or reducing conditions (for Western blot analysis). TWEAK interaction with the Fn14-FL protein, but not the Fn14-ΔEC protein, was detected using the ligand blot assay, although Western blot analysis indicated that both proteins were expressed to a similar degree in the transfected cells (Fig. 2B).

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