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AIP augments CARMA1-BCL10-MALT1 complex formation to facilitate NF-κB signaling upon T cell activation.

Schimmack G, Eitelhuber AC, Vincendeau M, Demski K, Shinohara H, Kurosaki T, Krappmann D - Cell Commun. Signal (2014)

Bottom Line: In T cells, transient binding of CARMA1 and AIP enhanced formation of the CBM complex.Thereby, AIP promoted optimal IKK/NF-κB signaling and IL-2 production in response to TCR/CD28 co-stimulation.Our data demonstrate that AIP acts as a positive regulator of NF-κB signaling upon T cell activation.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: The CARMA1-BCL10-MALT1 (CBM) complex bridges T cell receptor (TCR) signaling to the canonical IκB kinase (IKK)/NF-κB pathway. The CBM complex constitutes a signaling cluster of more than 1 Mio Dalton. Little is known about factors that facilitate the rapid assembly and maintenance of this dynamic higher order complex.

Findings: Here, we report the novel interaction of the aryl hydrocarbon receptor (AHR) interacting protein (AIP) and the molecular scaffold protein CARMA1. In T cells, transient binding of CARMA1 and AIP enhanced formation of the CBM complex. Thereby, AIP promoted optimal IKK/NF-κB signaling and IL-2 production in response to TCR/CD28 co-stimulation.

Conclusions: Our data demonstrate that AIP acts as a positive regulator of NF-κB signaling upon T cell activation.

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Related in: MedlinePlus

AIP regulates optimal T cell signaling responses. (A) IL-2 mRNA levels are decreased in AIP knockdown cells. Jurkat T cells were transfected with siGFP or siAIP1 and 3 and stimulated with P/I or CD3/CD28 for 3 h. RNA was isolated and IL-2 transcript levels were analyzed by quantitative RT-PCR. Bars show average and standard deviation of three independent experiments. (B) Downregulation of AIP reduces IL-2 production. Jurkat T cells were transfected with siRNAs (siAIP1, 2 and 3) as in (A) and stimulated with P/I. Secreted IL-2 was measured by ELISA. In A and B bars show average and standard deviation of three independent experiments. Significance was evaluated using Student t-test (one star: p < 0,05; three stars p < 0,001). (C) AIP knockdown diminishes NF-κB and NF-AT activation, while AP-1 activation is only slightly affected. Jurkat T cells were transfected with siRNAs against GFP and AIP (siAIP1 and 3), respectively, and stimulated with P/I or CD3/CD28 for 3 hours. NF-κB, NF-AT and AP-1 DNA binding was assessed by EMSA. (D) Working model how AIP affects CARMA1. Upon CARMA1 linker phosphorylation by PKCθ, AIP binding to PDZ-SH3 can compete for intramolecular GUK-SH3 interaction, which may facilitate structural rearrangements to transfer the double-closed conformation of CARMA1 into an active open state.
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Figure 4: AIP regulates optimal T cell signaling responses. (A) IL-2 mRNA levels are decreased in AIP knockdown cells. Jurkat T cells were transfected with siGFP or siAIP1 and 3 and stimulated with P/I or CD3/CD28 for 3 h. RNA was isolated and IL-2 transcript levels were analyzed by quantitative RT-PCR. Bars show average and standard deviation of three independent experiments. (B) Downregulation of AIP reduces IL-2 production. Jurkat T cells were transfected with siRNAs (siAIP1, 2 and 3) as in (A) and stimulated with P/I. Secreted IL-2 was measured by ELISA. In A and B bars show average and standard deviation of three independent experiments. Significance was evaluated using Student t-test (one star: p < 0,05; three stars p < 0,001). (C) AIP knockdown diminishes NF-κB and NF-AT activation, while AP-1 activation is only slightly affected. Jurkat T cells were transfected with siRNAs against GFP and AIP (siAIP1 and 3), respectively, and stimulated with P/I or CD3/CD28 for 3 hours. NF-κB, NF-AT and AP-1 DNA binding was assessed by EMSA. (D) Working model how AIP affects CARMA1. Upon CARMA1 linker phosphorylation by PKCθ, AIP binding to PDZ-SH3 can compete for intramolecular GUK-SH3 interaction, which may facilitate structural rearrangements to transfer the double-closed conformation of CARMA1 into an active open state.

Mentions: To assess the downstream consequences of diminished TCR signaling, we determined interleukin-2 (IL-2) production as a hallmark of T cell activation after siRNA mediated AIP downregulation. As measured by quantitative RT-PCR, upregulation of IL-2 mRNA in response to T cell stimulation by P/I or CD3/CD28 stimulation was significantly impaired by AIP knockdown (Figure 4A). Congruently, P/I induced IL-2 production and secretion was also decreased in AIP depleted Jurkat T cells as determined by ELISA (Figure 4B). IL-2 induction in T cells does not only rely on IKK/NF-κB signaling, but also on activation of the transcription factors NF-AT and AP-1 [[22]]. Therefore, we also assessed NF-AT and AP-1 DNA binding in nuclear extracts of Jurkat T cells after AIP knockdown (Figure 4C). NF-AT was severely reduced and AP-1 was slightly diminished in AIP downregulated Jurkat T cells, highlighting that AIP augments TCR/CD28 downstream pathways that contribute to optimal IL-2 induction. Effects on AP-1 may be downstream of NF-κB, because NF-κB activation can contribute to expression of Jun/AP-1 transcription factors such as JUNB and JUND [[23]]. Intriguingly, the MAGUK family member DLGH1 was shown to be an essential factor for TCR-triggered NF-AT activation, which may indicate that AIP could be a general regulatory factor for MAGUK dependent signaling events [[24]].


AIP augments CARMA1-BCL10-MALT1 complex formation to facilitate NF-κB signaling upon T cell activation.

Schimmack G, Eitelhuber AC, Vincendeau M, Demski K, Shinohara H, Kurosaki T, Krappmann D - Cell Commun. Signal (2014)

AIP regulates optimal T cell signaling responses. (A) IL-2 mRNA levels are decreased in AIP knockdown cells. Jurkat T cells were transfected with siGFP or siAIP1 and 3 and stimulated with P/I or CD3/CD28 for 3 h. RNA was isolated and IL-2 transcript levels were analyzed by quantitative RT-PCR. Bars show average and standard deviation of three independent experiments. (B) Downregulation of AIP reduces IL-2 production. Jurkat T cells were transfected with siRNAs (siAIP1, 2 and 3) as in (A) and stimulated with P/I. Secreted IL-2 was measured by ELISA. In A and B bars show average and standard deviation of three independent experiments. Significance was evaluated using Student t-test (one star: p < 0,05; three stars p < 0,001). (C) AIP knockdown diminishes NF-κB and NF-AT activation, while AP-1 activation is only slightly affected. Jurkat T cells were transfected with siRNAs against GFP and AIP (siAIP1 and 3), respectively, and stimulated with P/I or CD3/CD28 for 3 hours. NF-κB, NF-AT and AP-1 DNA binding was assessed by EMSA. (D) Working model how AIP affects CARMA1. Upon CARMA1 linker phosphorylation by PKCθ, AIP binding to PDZ-SH3 can compete for intramolecular GUK-SH3 interaction, which may facilitate structural rearrangements to transfer the double-closed conformation of CARMA1 into an active open state.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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Figure 4: AIP regulates optimal T cell signaling responses. (A) IL-2 mRNA levels are decreased in AIP knockdown cells. Jurkat T cells were transfected with siGFP or siAIP1 and 3 and stimulated with P/I or CD3/CD28 for 3 h. RNA was isolated and IL-2 transcript levels were analyzed by quantitative RT-PCR. Bars show average and standard deviation of three independent experiments. (B) Downregulation of AIP reduces IL-2 production. Jurkat T cells were transfected with siRNAs (siAIP1, 2 and 3) as in (A) and stimulated with P/I. Secreted IL-2 was measured by ELISA. In A and B bars show average and standard deviation of three independent experiments. Significance was evaluated using Student t-test (one star: p < 0,05; three stars p < 0,001). (C) AIP knockdown diminishes NF-κB and NF-AT activation, while AP-1 activation is only slightly affected. Jurkat T cells were transfected with siRNAs against GFP and AIP (siAIP1 and 3), respectively, and stimulated with P/I or CD3/CD28 for 3 hours. NF-κB, NF-AT and AP-1 DNA binding was assessed by EMSA. (D) Working model how AIP affects CARMA1. Upon CARMA1 linker phosphorylation by PKCθ, AIP binding to PDZ-SH3 can compete for intramolecular GUK-SH3 interaction, which may facilitate structural rearrangements to transfer the double-closed conformation of CARMA1 into an active open state.
Mentions: To assess the downstream consequences of diminished TCR signaling, we determined interleukin-2 (IL-2) production as a hallmark of T cell activation after siRNA mediated AIP downregulation. As measured by quantitative RT-PCR, upregulation of IL-2 mRNA in response to T cell stimulation by P/I or CD3/CD28 stimulation was significantly impaired by AIP knockdown (Figure 4A). Congruently, P/I induced IL-2 production and secretion was also decreased in AIP depleted Jurkat T cells as determined by ELISA (Figure 4B). IL-2 induction in T cells does not only rely on IKK/NF-κB signaling, but also on activation of the transcription factors NF-AT and AP-1 [[22]]. Therefore, we also assessed NF-AT and AP-1 DNA binding in nuclear extracts of Jurkat T cells after AIP knockdown (Figure 4C). NF-AT was severely reduced and AP-1 was slightly diminished in AIP downregulated Jurkat T cells, highlighting that AIP augments TCR/CD28 downstream pathways that contribute to optimal IL-2 induction. Effects on AP-1 may be downstream of NF-κB, because NF-κB activation can contribute to expression of Jun/AP-1 transcription factors such as JUNB and JUND [[23]]. Intriguingly, the MAGUK family member DLGH1 was shown to be an essential factor for TCR-triggered NF-AT activation, which may indicate that AIP could be a general regulatory factor for MAGUK dependent signaling events [[24]].

Bottom Line: In T cells, transient binding of CARMA1 and AIP enhanced formation of the CBM complex.Thereby, AIP promoted optimal IKK/NF-κB signaling and IL-2 production in response to TCR/CD28 co-stimulation.Our data demonstrate that AIP acts as a positive regulator of NF-κB signaling upon T cell activation.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: The CARMA1-BCL10-MALT1 (CBM) complex bridges T cell receptor (TCR) signaling to the canonical IκB kinase (IKK)/NF-κB pathway. The CBM complex constitutes a signaling cluster of more than 1 Mio Dalton. Little is known about factors that facilitate the rapid assembly and maintenance of this dynamic higher order complex.

Findings: Here, we report the novel interaction of the aryl hydrocarbon receptor (AHR) interacting protein (AIP) and the molecular scaffold protein CARMA1. In T cells, transient binding of CARMA1 and AIP enhanced formation of the CBM complex. Thereby, AIP promoted optimal IKK/NF-κB signaling and IL-2 production in response to TCR/CD28 co-stimulation.

Conclusions: Our data demonstrate that AIP acts as a positive regulator of NF-κB signaling upon T cell activation.

Show MeSH
Related in: MedlinePlus