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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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Fn14-FL dimerization during cell lysis can be abrogated by mutagenesis of cysteine residue 122.(A) The human Fn14-WT (FL), Fn14-ΔCT, Fn14-C104S, and Fn14-C122S cytoplasmic tail amino acid sequences are shown with cysteine residues in red and the serine residue substitutions in blue. (B) HEK293 cells were transfected with vector or the indicated Fn14-myc expression plasmids and 24 hr later the cells were harvested and lysed. Equal amounts of protein were subjected to SDS-PAGE under either reducing or non-reducing conditions and 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). (C) Schematic representation of predicted Fn14 cysteine residue status prior to and after cell harvesting and lysis. The Fn14 extracellular (EC) domain, transmembrane (TM) domain, and cytoplasmic tail (CT) are indicated and the cysteine residue distribution within the Fn14-FL protein is shown. Disulfide bonds are indicated with brackets. Non-covalent Fn14 association mediated by the most distal 18-aa residues of the Fn14 CT is illustrated with a double-headed arrow.
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pone-0065248-g008: Fn14-FL dimerization during cell lysis can be abrogated by mutagenesis of cysteine residue 122.(A) The human Fn14-WT (FL), Fn14-ΔCT, Fn14-C104S, and Fn14-C122S cytoplasmic tail amino acid sequences are shown with cysteine residues in red and the serine residue substitutions in blue. (B) HEK293 cells were transfected with vector or the indicated Fn14-myc expression plasmids and 24 hr later the cells were harvested and lysed. Equal amounts of protein were subjected to SDS-PAGE under either reducing or non-reducing conditions and 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). (C) Schematic representation of predicted Fn14 cysteine residue status prior to and after cell harvesting and lysis. The Fn14 extracellular (EC) domain, transmembrane (TM) domain, and cytoplasmic tail (CT) are indicated and the cysteine residue distribution within the Fn14-FL protein is shown. Disulfide bonds are indicated with brackets. Non-covalent Fn14 association mediated by the most distal 18-aa residues of the Fn14 CT is illustrated with a double-headed arrow.

Mentions: We next confirmed that Cys122 within the Fn14 cytoplasmic domain was indeed the residue participating in intermolecular disulfide bond formation by site-specific mutagenesis. Expression plasmids were constructed that encoded myc-tagged Fn14 proteins with a cysteine to serine substitution at position 104 or 122 (Fig. 8A). HEK293 cells were transiently transfected with each of these plasmids, as well as the Fn14-FL (denoted wild-type (WT) in this figure) and Fn14-ΔCT plasmids used earlier. Cells were harvested, lysed, and samples were subjected to SDS-PAGE under either reducing or non-reducing conditions. Western blot analysis was performed. All four of the Fn14 proteins were expressed in the transfected cells and were detected as single species when analyzed under reducing conditions (Fig. 8B). Fn14-WT and Fn14-C104S dimers, but not Fn14-ΔCT or Fn14-C122S dimers, were detected under non-reducing conditions, confirming the importance of the C-terminal region of the cytoplasmic tail and Cys122 in particular in intermolecular disulfide bond formation (Fig. 8B).


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)

Fn14-FL dimerization during cell lysis can be abrogated by mutagenesis of cysteine residue 122.(A) The human Fn14-WT (FL), Fn14-ΔCT, Fn14-C104S, and Fn14-C122S cytoplasmic tail amino acid sequences are shown with cysteine residues in red and the serine residue substitutions in blue. (B) HEK293 cells were transfected with vector or the indicated Fn14-myc expression plasmids and 24 hr later the cells were harvested and lysed. Equal amounts of protein were subjected to SDS-PAGE under either reducing or non-reducing conditions and 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). (C) Schematic representation of predicted Fn14 cysteine residue status prior to and after cell harvesting and lysis. The Fn14 extracellular (EC) domain, transmembrane (TM) domain, and cytoplasmic tail (CT) are indicated and the cysteine residue distribution within the Fn14-FL protein is shown. Disulfide bonds are indicated with brackets. Non-covalent Fn14 association mediated by the most distal 18-aa residues of the Fn14 CT is illustrated with a double-headed arrow.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3672086&req=5

pone-0065248-g008: Fn14-FL dimerization during cell lysis can be abrogated by mutagenesis of cysteine residue 122.(A) The human Fn14-WT (FL), Fn14-ΔCT, Fn14-C104S, and Fn14-C122S cytoplasmic tail amino acid sequences are shown with cysteine residues in red and the serine residue substitutions in blue. (B) HEK293 cells were transfected with vector or the indicated Fn14-myc expression plasmids and 24 hr later the cells were harvested and lysed. Equal amounts of protein were subjected to SDS-PAGE under either reducing or non-reducing conditions and 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). (C) Schematic representation of predicted Fn14 cysteine residue status prior to and after cell harvesting and lysis. The Fn14 extracellular (EC) domain, transmembrane (TM) domain, and cytoplasmic tail (CT) are indicated and the cysteine residue distribution within the Fn14-FL protein is shown. Disulfide bonds are indicated with brackets. Non-covalent Fn14 association mediated by the most distal 18-aa residues of the Fn14 CT is illustrated with a double-headed arrow.
Mentions: We next confirmed that Cys122 within the Fn14 cytoplasmic domain was indeed the residue participating in intermolecular disulfide bond formation by site-specific mutagenesis. Expression plasmids were constructed that encoded myc-tagged Fn14 proteins with a cysteine to serine substitution at position 104 or 122 (Fig. 8A). HEK293 cells were transiently transfected with each of these plasmids, as well as the Fn14-FL (denoted wild-type (WT) in this figure) and Fn14-ΔCT plasmids used earlier. Cells were harvested, lysed, and samples were subjected to SDS-PAGE under either reducing or non-reducing conditions. Western blot analysis was performed. All four of the Fn14 proteins were expressed in the transfected cells and were detected as single species when analyzed under reducing conditions (Fig. 8B). Fn14-WT and Fn14-C104S dimers, but not Fn14-ΔCT or Fn14-C122S dimers, were detected under non-reducing conditions, confirming the importance of the C-terminal region of the cytoplasmic tail and Cys122 in particular in intermolecular disulfide bond formation (Fig. 8B).

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