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The PAAD/PYRIN-family protein ASC is a dual regulator of a conserved step in nuclear factor kappaB activation pathways.

Stehlik C, Fiorentino L, Dorfleutner A, Bruey JM, Ariza EM, Sagara J, Reed JC - J. Exp. Med. (2002)

Bottom Line: Apoptosis-associated speck-like protein containing a Caspase recruitment domain (ASC) belongs to a large family of proteins that contain a Pyrin, AIM, ASC, and death domain-like (PAAD) domain (also known as PYRIN, DAPIN, Pyk).Conversely, reducing endogenous levels of ASC using siRNA enhanced TNF- and LPS-induced degradation of the IKK substrate, IkappaBalpha.Our findings suggest that ASC modulates diverse NF-kappaB induction pathways by acting upon the IKK complex, implying a broad role for this and similar proteins containing PAAD domains in regulation of inflammatory responses.

View Article: PubMed Central - PubMed

Affiliation: The Burnham Institute, The Scripps Research Institute, La Jolla, CA 92037, USA.

ABSTRACT
Apoptosis-associated speck-like protein containing a Caspase recruitment domain (ASC) belongs to a large family of proteins that contain a Pyrin, AIM, ASC, and death domain-like (PAAD) domain (also known as PYRIN, DAPIN, Pyk). Recent data have suggested that ASC functions as an adaptor protein linking various PAAD-family proteins to pathways involved in nuclear factor (NF)-kappaB and pro-Caspase-1 activation. We present evidence here that the role of ASC in modulating NF-kappaB activation pathways is much broader than previously suspected, as it can either inhibit or activate NF-kappaB, depending on cellular context. While coexpression of ASC with certain PAAD-family proteins such as Pyrin and Cryopyrin increases NF-kappaB activity, ASC has an inhibitory influence on NF-kappaB activation by various proinflammatory stimuli, including tumor necrosis factor (TNF)alpha, interleukin 1beta, and lipopolysaccharide (LPS). Elevations in ASC protein levels or of the PAAD domain of ASC suppressed activation of IkappaB kinases in cells exposed to pro-inflammatory stimuli. Conversely, reducing endogenous levels of ASC using siRNA enhanced TNF- and LPS-induced degradation of the IKK substrate, IkappaBalpha. Our findings suggest that ASC modulates diverse NF-kappaB induction pathways by acting upon the IKK complex, implying a broad role for this and similar proteins containing PAAD domains in regulation of inflammatory responses.

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ASC associates with IKKα and IKKβ. (A and B) CoIP assays were performed using THP-1 cells that had been treated with LPS for 30 min. Cleared lysates were subject to coIP using either IgG, anti-IKKα, or anti-ASC antibodies and the resulting immune-complexes were analyzed by WB using various antibodies, as indicated. (B) Cleared lysates from stable HEK293N-Neo and HEK293N-ASC-PAAD cells were subjected to coIP using either IgG, anti-IKKα, anti-IKKβ, or anti-Myc antibodies and the resulting immune-complexes were analyzed by WB. (C and D) Immunofluorescence analysis of stably transfected HEK293N-Neo and HEK293N-ASC-PAAD cells was performed, using either anti-IKKα (C) or anti-IKKβ (D) polyclonal rabbit and goat antibodies, respectively, in combination with mouse monoclonal anti-Myc epitope antibody for detection of Myc-ASC-PAAD protein. From left to right: Localization of FITC-labeled IKKα or IKKβ is shown for Neo cells and ASC-PAAD–expressing cells, followed by localization of TRITC-labeled Myc-ASC-PAAD, and then two-color fluorescence analysis (merge).
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fig4: ASC associates with IKKα and IKKβ. (A and B) CoIP assays were performed using THP-1 cells that had been treated with LPS for 30 min. Cleared lysates were subject to coIP using either IgG, anti-IKKα, or anti-ASC antibodies and the resulting immune-complexes were analyzed by WB using various antibodies, as indicated. (B) Cleared lysates from stable HEK293N-Neo and HEK293N-ASC-PAAD cells were subjected to coIP using either IgG, anti-IKKα, anti-IKKβ, or anti-Myc antibodies and the resulting immune-complexes were analyzed by WB. (C and D) Immunofluorescence analysis of stably transfected HEK293N-Neo and HEK293N-ASC-PAAD cells was performed, using either anti-IKKα (C) or anti-IKKβ (D) polyclonal rabbit and goat antibodies, respectively, in combination with mouse monoclonal anti-Myc epitope antibody for detection of Myc-ASC-PAAD protein. From left to right: Localization of FITC-labeled IKKα or IKKβ is shown for Neo cells and ASC-PAAD–expressing cells, followed by localization of TRITC-labeled Myc-ASC-PAAD, and then two-color fluorescence analysis (merge).

Mentions: Having mapped the site of action of ASC to the IKK complex, we performed experiments to explore whether ASC associated with these protein kinases. In the course of our studies of ASC, we observed that expression of ASC is induced in myeloid-lineage hematopoietic cells such as THP-1 monocytic or HL-60 monomyelocytic leukemia cell lines by LPS and TNFα (Fig. 2 E, and unpublished data). We therefore asked whether endogenous ASC protein could be found associated with endogenous IKK complex components after LPS- or TNFα-stimulation in these cells. Co-IP experiments provided evidence of association of ASC with both IKKα and IKKβ in LPS-stimulated THP-1 and TNFα-treated HL-60 cells (Fig. 4 A, and unpublished data). These protein interactions were reciprocally demonstrable, regardless of whether immune-complexes were prepared using anti-IKKα and anti-IKKβ antibodies (followed by immunoblotting with anti-ASC antiserum) or using anti-ASC antiserum (followed by immunoblotting with anti-IKKα or anti-IKKβ antibodies; Fig. 4 A, and unpublished data). We also analyzed the HEK293N-ASC-PAAD stable transfectants to determine whether the PAAD is sufficient for association with the endogenous IKK complex. Again, coimmunoprecipitation (coIP) experiments demonstrated specific interaction of ASC-PAAD with the endogenous IKK complex, as indicated by the association of ASC with IKKα, IKKβ, as well as IKKγ (Fig. 4 B).


The PAAD/PYRIN-family protein ASC is a dual regulator of a conserved step in nuclear factor kappaB activation pathways.

Stehlik C, Fiorentino L, Dorfleutner A, Bruey JM, Ariza EM, Sagara J, Reed JC - J. Exp. Med. (2002)

ASC associates with IKKα and IKKβ. (A and B) CoIP assays were performed using THP-1 cells that had been treated with LPS for 30 min. Cleared lysates were subject to coIP using either IgG, anti-IKKα, or anti-ASC antibodies and the resulting immune-complexes were analyzed by WB using various antibodies, as indicated. (B) Cleared lysates from stable HEK293N-Neo and HEK293N-ASC-PAAD cells were subjected to coIP using either IgG, anti-IKKα, anti-IKKβ, or anti-Myc antibodies and the resulting immune-complexes were analyzed by WB. (C and D) Immunofluorescence analysis of stably transfected HEK293N-Neo and HEK293N-ASC-PAAD cells was performed, using either anti-IKKα (C) or anti-IKKβ (D) polyclonal rabbit and goat antibodies, respectively, in combination with mouse monoclonal anti-Myc epitope antibody for detection of Myc-ASC-PAAD protein. From left to right: Localization of FITC-labeled IKKα or IKKβ is shown for Neo cells and ASC-PAAD–expressing cells, followed by localization of TRITC-labeled Myc-ASC-PAAD, and then two-color fluorescence analysis (merge).
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fig4: ASC associates with IKKα and IKKβ. (A and B) CoIP assays were performed using THP-1 cells that had been treated with LPS for 30 min. Cleared lysates were subject to coIP using either IgG, anti-IKKα, or anti-ASC antibodies and the resulting immune-complexes were analyzed by WB using various antibodies, as indicated. (B) Cleared lysates from stable HEK293N-Neo and HEK293N-ASC-PAAD cells were subjected to coIP using either IgG, anti-IKKα, anti-IKKβ, or anti-Myc antibodies and the resulting immune-complexes were analyzed by WB. (C and D) Immunofluorescence analysis of stably transfected HEK293N-Neo and HEK293N-ASC-PAAD cells was performed, using either anti-IKKα (C) or anti-IKKβ (D) polyclonal rabbit and goat antibodies, respectively, in combination with mouse monoclonal anti-Myc epitope antibody for detection of Myc-ASC-PAAD protein. From left to right: Localization of FITC-labeled IKKα or IKKβ is shown for Neo cells and ASC-PAAD–expressing cells, followed by localization of TRITC-labeled Myc-ASC-PAAD, and then two-color fluorescence analysis (merge).
Mentions: Having mapped the site of action of ASC to the IKK complex, we performed experiments to explore whether ASC associated with these protein kinases. In the course of our studies of ASC, we observed that expression of ASC is induced in myeloid-lineage hematopoietic cells such as THP-1 monocytic or HL-60 monomyelocytic leukemia cell lines by LPS and TNFα (Fig. 2 E, and unpublished data). We therefore asked whether endogenous ASC protein could be found associated with endogenous IKK complex components after LPS- or TNFα-stimulation in these cells. Co-IP experiments provided evidence of association of ASC with both IKKα and IKKβ in LPS-stimulated THP-1 and TNFα-treated HL-60 cells (Fig. 4 A, and unpublished data). These protein interactions were reciprocally demonstrable, regardless of whether immune-complexes were prepared using anti-IKKα and anti-IKKβ antibodies (followed by immunoblotting with anti-ASC antiserum) or using anti-ASC antiserum (followed by immunoblotting with anti-IKKα or anti-IKKβ antibodies; Fig. 4 A, and unpublished data). We also analyzed the HEK293N-ASC-PAAD stable transfectants to determine whether the PAAD is sufficient for association with the endogenous IKK complex. Again, coimmunoprecipitation (coIP) experiments demonstrated specific interaction of ASC-PAAD with the endogenous IKK complex, as indicated by the association of ASC with IKKα, IKKβ, as well as IKKγ (Fig. 4 B).

Bottom Line: Apoptosis-associated speck-like protein containing a Caspase recruitment domain (ASC) belongs to a large family of proteins that contain a Pyrin, AIM, ASC, and death domain-like (PAAD) domain (also known as PYRIN, DAPIN, Pyk).Conversely, reducing endogenous levels of ASC using siRNA enhanced TNF- and LPS-induced degradation of the IKK substrate, IkappaBalpha.Our findings suggest that ASC modulates diverse NF-kappaB induction pathways by acting upon the IKK complex, implying a broad role for this and similar proteins containing PAAD domains in regulation of inflammatory responses.

View Article: PubMed Central - PubMed

Affiliation: The Burnham Institute, The Scripps Research Institute, La Jolla, CA 92037, USA.

ABSTRACT
Apoptosis-associated speck-like protein containing a Caspase recruitment domain (ASC) belongs to a large family of proteins that contain a Pyrin, AIM, ASC, and death domain-like (PAAD) domain (also known as PYRIN, DAPIN, Pyk). Recent data have suggested that ASC functions as an adaptor protein linking various PAAD-family proteins to pathways involved in nuclear factor (NF)-kappaB and pro-Caspase-1 activation. We present evidence here that the role of ASC in modulating NF-kappaB activation pathways is much broader than previously suspected, as it can either inhibit or activate NF-kappaB, depending on cellular context. While coexpression of ASC with certain PAAD-family proteins such as Pyrin and Cryopyrin increases NF-kappaB activity, ASC has an inhibitory influence on NF-kappaB activation by various proinflammatory stimuli, including tumor necrosis factor (TNF)alpha, interleukin 1beta, and lipopolysaccharide (LPS). Elevations in ASC protein levels or of the PAAD domain of ASC suppressed activation of IkappaB kinases in cells exposed to pro-inflammatory stimuli. Conversely, reducing endogenous levels of ASC using siRNA enhanced TNF- and LPS-induced degradation of the IKK substrate, IkappaBalpha. Our findings suggest that ASC modulates diverse NF-kappaB induction pathways by acting upon the IKK complex, implying a broad role for this and similar proteins containing PAAD domains in regulation of inflammatory responses.

Show MeSH
Related in: MedlinePlus