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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 inhibits NF-κB induction at the level of the IKK complex. (A) HEK293N cells were transfected with 200 ng of plasmids encoding various NF-κB–inducing proteins, as indicated, and either 200 or 400 ng of ASC-encoding plasmid. NF-κB activity was measured 1 d later by reporter gene assays as described above, and data presented as fold-induction relative to cells not transfected with effector plasmids (mean ± SD; n = 3). The IκBαM (S32, 36A) mutant served as a negative control. (B) IKKα in vitro kinase activity toward GST-IκBα in transient transfected HEK293N cells in response to TNFα. Kinase dead IKKα (K44M) was used as a control for blocking TNFα induction of IKK activity. Shown are phosphorylated GST-IκBα, as well as auto-phosphorylated IKKα and phosphorylated endogenous IκBα, which associates with the IKK complex. (C) In vitro kinase assays as above were performed using HA-IKKβ and HA-IKKβ (K44A). (D) HEK293N cells were transiently transfected with plasmids encoding effectors of the TNFα (TRAF2) and IL-1β (TRAF6) pathways and in vitro kinase assays were performed, measuring IKKα kinase activity on GST-IκBα. (E) Endogenous IKKα kinase activity was measured by in vitro kinase assay in HEK293N-ASC-PAAD or -Neo stable transfectants. The IKKα kinase assay data from five experiments were quantified by scanning-densitometry analysis of the autoradiograms, and are presented below the gel as a bar graph showing fold-activity compared with uninduced HEK293N-Neo cells, normalized to IKKα expression as determined by immunoblotting (mean ± SD). KA, kinase assay; WB, Western blot.
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fig3: ASC inhibits NF-κB induction at the level of the IKK complex. (A) HEK293N cells were transfected with 200 ng of plasmids encoding various NF-κB–inducing proteins, as indicated, and either 200 or 400 ng of ASC-encoding plasmid. NF-κB activity was measured 1 d later by reporter gene assays as described above, and data presented as fold-induction relative to cells not transfected with effector plasmids (mean ± SD; n = 3). The IκBαM (S32, 36A) mutant served as a negative control. (B) IKKα in vitro kinase activity toward GST-IκBα in transient transfected HEK293N cells in response to TNFα. Kinase dead IKKα (K44M) was used as a control for blocking TNFα induction of IKK activity. Shown are phosphorylated GST-IκBα, as well as auto-phosphorylated IKKα and phosphorylated endogenous IκBα, which associates with the IKK complex. (C) In vitro kinase assays as above were performed using HA-IKKβ and HA-IKKβ (K44A). (D) HEK293N cells were transiently transfected with plasmids encoding effectors of the TNFα (TRAF2) and IL-1β (TRAF6) pathways and in vitro kinase assays were performed, measuring IKKα kinase activity on GST-IκBα. (E) Endogenous IKKα kinase activity was measured by in vitro kinase assay in HEK293N-ASC-PAAD or -Neo stable transfectants. The IKKα kinase assay data from five experiments were quantified by scanning-densitometry analysis of the autoradiograms, and are presented below the gel as a bar graph showing fold-activity compared with uninduced HEK293N-Neo cells, normalized to IKKα expression as determined by immunoblotting (mean ± SD). KA, kinase assay; WB, Western blot.

Mentions: To map where the PAAD of ASC affects the NF-κB activation pathway, NF-κB activity was induced by transient transfection of plasmids encoding various intracellular signal-transducers that operate within cytokine receptor pathways leading to phosphorylation of IκB, a key event required for NF-κB release. Coexpression of ASC blocked induction of NF-κB activity by the adaptor proteins TRAF2 and TRAF6, the TRAF-binding kinases TBK1 and NIK, the IKK complex constituents IKKα and IKKβ, and the related kinase IKKi (Fig. 3 A). In contrast, coexpression of ASC did not suppress reporter gene activation induced by NF-κB-p65. Thus, ASC blocks upstream of NF-κB, apparently at the level of the IKK complex.


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 inhibits NF-κB induction at the level of the IKK complex. (A) HEK293N cells were transfected with 200 ng of plasmids encoding various NF-κB–inducing proteins, as indicated, and either 200 or 400 ng of ASC-encoding plasmid. NF-κB activity was measured 1 d later by reporter gene assays as described above, and data presented as fold-induction relative to cells not transfected with effector plasmids (mean ± SD; n = 3). The IκBαM (S32, 36A) mutant served as a negative control. (B) IKKα in vitro kinase activity toward GST-IκBα in transient transfected HEK293N cells in response to TNFα. Kinase dead IKKα (K44M) was used as a control for blocking TNFα induction of IKK activity. Shown are phosphorylated GST-IκBα, as well as auto-phosphorylated IKKα and phosphorylated endogenous IκBα, which associates with the IKK complex. (C) In vitro kinase assays as above were performed using HA-IKKβ and HA-IKKβ (K44A). (D) HEK293N cells were transiently transfected with plasmids encoding effectors of the TNFα (TRAF2) and IL-1β (TRAF6) pathways and in vitro kinase assays were performed, measuring IKKα kinase activity on GST-IκBα. (E) Endogenous IKKα kinase activity was measured by in vitro kinase assay in HEK293N-ASC-PAAD or -Neo stable transfectants. The IKKα kinase assay data from five experiments were quantified by scanning-densitometry analysis of the autoradiograms, and are presented below the gel as a bar graph showing fold-activity compared with uninduced HEK293N-Neo cells, normalized to IKKα expression as determined by immunoblotting (mean ± SD). KA, kinase assay; WB, Western blot.
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fig3: ASC inhibits NF-κB induction at the level of the IKK complex. (A) HEK293N cells were transfected with 200 ng of plasmids encoding various NF-κB–inducing proteins, as indicated, and either 200 or 400 ng of ASC-encoding plasmid. NF-κB activity was measured 1 d later by reporter gene assays as described above, and data presented as fold-induction relative to cells not transfected with effector plasmids (mean ± SD; n = 3). The IκBαM (S32, 36A) mutant served as a negative control. (B) IKKα in vitro kinase activity toward GST-IκBα in transient transfected HEK293N cells in response to TNFα. Kinase dead IKKα (K44M) was used as a control for blocking TNFα induction of IKK activity. Shown are phosphorylated GST-IκBα, as well as auto-phosphorylated IKKα and phosphorylated endogenous IκBα, which associates with the IKK complex. (C) In vitro kinase assays as above were performed using HA-IKKβ and HA-IKKβ (K44A). (D) HEK293N cells were transiently transfected with plasmids encoding effectors of the TNFα (TRAF2) and IL-1β (TRAF6) pathways and in vitro kinase assays were performed, measuring IKKα kinase activity on GST-IκBα. (E) Endogenous IKKα kinase activity was measured by in vitro kinase assay in HEK293N-ASC-PAAD or -Neo stable transfectants. The IKKα kinase assay data from five experiments were quantified by scanning-densitometry analysis of the autoradiograms, and are presented below the gel as a bar graph showing fold-activity compared with uninduced HEK293N-Neo cells, normalized to IKKα expression as determined by immunoblotting (mean ± SD). KA, kinase assay; WB, Western blot.
Mentions: To map where the PAAD of ASC affects the NF-κB activation pathway, NF-κB activity was induced by transient transfection of plasmids encoding various intracellular signal-transducers that operate within cytokine receptor pathways leading to phosphorylation of IκB, a key event required for NF-κB release. Coexpression of ASC blocked induction of NF-κB activity by the adaptor proteins TRAF2 and TRAF6, the TRAF-binding kinases TBK1 and NIK, the IKK complex constituents IKKα and IKKβ, and the related kinase IKKi (Fig. 3 A). In contrast, coexpression of ASC did not suppress reporter gene activation induced by NF-κB-p65. Thus, ASC blocks upstream of NF-κB, apparently at the level of the IKK complex.

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