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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 and Fn14-ΔEC proteins, but not the Fn14-ΔCT protein, have slower electrophoretic mobility when analyzed under non-reducing conditions due to intermolecular disulfide bond formation during cell lysis.(A) HEK293 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 subjected to SDS-PAGE under either reducing or non-reducing conditions. Western blot analysis was performed using either an anti-myc or anti-tubulin antibody. The positions of molecular size markers are shown on the left (in kDa). (B) HEK293 cells were transfected with vector (V) or the Fn14-FL-myc expression plasmid and 24 hr later the cells were harvested. Cells were lysed and an aliquot of the Fn14-FL lysate was incubated with iodoacetamide (IA). Equal amounts of protein were subjected to SDS-PAGE under non-reducing conditions and Western blot analysis was performed using either an anti-myc or anti-GAPDH antibody. The positions of molecular size markers are shown on the left (in kDa).
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pone-0065248-g007: The Fn14-FL and Fn14-ΔEC proteins, but not the Fn14-ΔCT protein, have slower electrophoretic mobility when analyzed under non-reducing conditions due to intermolecular disulfide bond formation during cell lysis.(A) HEK293 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 subjected to SDS-PAGE under either reducing or non-reducing conditions. Western blot analysis was performed using either an anti-myc or anti-tubulin antibody. The positions of molecular size markers are shown on the left (in kDa). (B) HEK293 cells were transfected with vector (V) or the Fn14-FL-myc expression plasmid and 24 hr later the cells were harvested. Cells were lysed and an aliquot of the Fn14-FL lysate was incubated with iodoacetamide (IA). Equal amounts of protein were subjected to SDS-PAGE under non-reducing conditions and Western blot analysis was performed using either an anti-myc or anti-GAPDH antibody. The positions of molecular size markers are shown on the left (in kDa).

Mentions: Fn14 contains a total of eight cysteine residues. Six of these residues are in the extracellular region, where they form the three intramolecular disulfide bonds that stabilize the cysteine-rich domain [41]–[43]. The other two cysteines, Cys104 and Cys122, are located in the cytoplasmic tail, and since disulfide bonds are formed in the lumen of the rough ER during protein synthesis, they are not expected to form intra- or intermolecular disulfide bonds in living cells. In order to determine which Fn14 cysteine residues were likely involved in the endogenous Fn14 dimerization noted above, HEK293 cells were transfected with vector or the Fn14-FL (contains all 8 cysteines), Fn14-ΔEC (contains only the 2 cysteines in the cytoplasmic tail), or Fn14-ΔCT (contains all 6 cysteines in the extracellular domain and Cys104 in the cytoplasmic tail) plasmids and 24 hr later the cells were harvested and lysed. Samples were subjected to SDS-PAGE under either reducing or non-reducing conditions and Western blot analysis was performed. Fn14-FL and Fn14-ΔEC dimers, but not Fn14-ΔCT dimers, were detected under non-reducing conditions (Fig. 7A). This result indicated that the most C-terminal Fn14 cysteine residue, Cys122, was critical for disulfide bond formation. Since this residue is within the cytoplasmic domain, this result also implied that disulfide bond formation was occurring after cell harvest. To confirm this, HEK293 cells were transfected with vector or the Fn14-FL protein and 24 hr later the cells were harvested. The Fn14-FL cells were then lysed in normal lysis buffer or lysis buffer supplemented with iodoacetamide, which reacts with free sulfhydryls on cysteine residues. Samples were subjected to SDS-PAGE under non-reducing conditions, and Western blot analysis was performed. Iodoacetamide treatment blocked Fn14-FL dimer formation (Fig. 7B), indicating that the Cys122 intermolecular disulfide bond was forming in the cell lysis buffer, a favorable redox environment for oxidation of SH groups.


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 and Fn14-ΔEC proteins, but not the Fn14-ΔCT protein, have slower electrophoretic mobility when analyzed under non-reducing conditions due to intermolecular disulfide bond formation during cell lysis.(A) HEK293 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 subjected to SDS-PAGE under either reducing or non-reducing conditions. Western blot analysis was performed using either an anti-myc or anti-tubulin antibody. The positions of molecular size markers are shown on the left (in kDa). (B) HEK293 cells were transfected with vector (V) or the Fn14-FL-myc expression plasmid and 24 hr later the cells were harvested. Cells were lysed and an aliquot of the Fn14-FL lysate was incubated with iodoacetamide (IA). Equal amounts of protein were subjected to SDS-PAGE under non-reducing conditions and Western blot analysis was performed using either an anti-myc or anti-GAPDH antibody. The positions of molecular size markers are shown on the left (in kDa).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3672086&req=5

pone-0065248-g007: The Fn14-FL and Fn14-ΔEC proteins, but not the Fn14-ΔCT protein, have slower electrophoretic mobility when analyzed under non-reducing conditions due to intermolecular disulfide bond formation during cell lysis.(A) HEK293 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 subjected to SDS-PAGE under either reducing or non-reducing conditions. Western blot analysis was performed using either an anti-myc or anti-tubulin antibody. The positions of molecular size markers are shown on the left (in kDa). (B) HEK293 cells were transfected with vector (V) or the Fn14-FL-myc expression plasmid and 24 hr later the cells were harvested. Cells were lysed and an aliquot of the Fn14-FL lysate was incubated with iodoacetamide (IA). Equal amounts of protein were subjected to SDS-PAGE under non-reducing conditions and Western blot analysis was performed using either an anti-myc or anti-GAPDH antibody. The positions of molecular size markers are shown on the left (in kDa).
Mentions: Fn14 contains a total of eight cysteine residues. Six of these residues are in the extracellular region, where they form the three intramolecular disulfide bonds that stabilize the cysteine-rich domain [41]–[43]. The other two cysteines, Cys104 and Cys122, are located in the cytoplasmic tail, and since disulfide bonds are formed in the lumen of the rough ER during protein synthesis, they are not expected to form intra- or intermolecular disulfide bonds in living cells. In order to determine which Fn14 cysteine residues were likely involved in the endogenous Fn14 dimerization noted above, HEK293 cells were transfected with vector or the Fn14-FL (contains all 8 cysteines), Fn14-ΔEC (contains only the 2 cysteines in the cytoplasmic tail), or Fn14-ΔCT (contains all 6 cysteines in the extracellular domain and Cys104 in the cytoplasmic tail) plasmids and 24 hr later the cells were harvested and lysed. Samples were subjected to SDS-PAGE under either reducing or non-reducing conditions and Western blot analysis was performed. Fn14-FL and Fn14-ΔEC dimers, but not Fn14-ΔCT dimers, were detected under non-reducing conditions (Fig. 7A). This result indicated that the most C-terminal Fn14 cysteine residue, Cys122, was critical for disulfide bond formation. Since this residue is within the cytoplasmic domain, this result also implied that disulfide bond formation was occurring after cell harvest. To confirm this, HEK293 cells were transfected with vector or the Fn14-FL protein and 24 hr later the cells were harvested. The Fn14-FL cells were then lysed in normal lysis buffer or lysis buffer supplemented with iodoacetamide, which reacts with free sulfhydryls on cysteine residues. Samples were subjected to SDS-PAGE under non-reducing conditions, and Western blot analysis was performed. Iodoacetamide treatment blocked Fn14-FL dimer formation (Fig. 7B), indicating that the Cys122 intermolecular disulfide bond was forming in the cell lysis buffer, a favorable redox environment for oxidation of SH groups.

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