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. 2014 May 13;7(325):ra45.
doi: 10.1126/scisignal.2004882.

T cell receptor signals to NF-κB are transmitted by a cytosolic p62-Bcl10-Malt1-IKK signalosome

Suman Paul  1 , Maria K Traver  1 , Anuj K Kashyap  1 , Michael A Washington  1 , Joseph R Latoche  1 , Brian C Schaefer  2
Affiliations

T cell receptor signals to NF-κB are transmitted by a cytosolic p62-Bcl10-Malt1-IKK signalosome

Suman Paul et al. Sci Signal. .

Abstract

Antigen-mediated stimulation of the T cell receptor (TCR) triggers activation of nuclear factor κB (NF-κB), a key transcriptional regulator of T cell proliferation and effector cell differentiation. TCR signaling to NF-κB requires both the Carma1-Bcl10-Malt1 (CBM) complex and the inhibitor of κB (IκB) kinase (IKK) complex; however, the molecular mechanisms connecting the CBM complex to activation of IKK are incompletely defined. We found that the active IKK complex is a component of a TCR-dependent cytosolic Bcl10-Malt1 signalosome containing the adaptor protein p62, which forms in effector T cells. Phosphorylated IκBα and NF-κB were transiently recruited to this signalosome before NF-κB translocated to the nucleus. Inhibiting the activity of the kinase TAK1 or IKK blocked the phosphorylation of IKK, but not the formation of p62-Bcl10-Malt1 clusters, suggesting that activation of IKK occurs after signalosome assembly. Furthermore, analysis of T cells from p62-deficient mice demonstrated that the p62-dependent clustering of signaling components stimulated activation of NF-κB in effector T cells. Thus, TCR-stimulated activation of NF-κB requires the assembly of cytosolic p62-Bcl10-Malt1-IKK signalosomes, which may ensure highly regulated activation of NF-κB in response to TCR engagement.

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Figures

Fig. 1.
Fig. 1.. Colocalization of pIKKα/β and TRAF6 with cytosolic Bcl10-p62 clusters after TCR stimulation.
(A to C) D10 cells transduced with retroviruses encoding PKCθ-CFP and Bcl10-YFP were stimulated for the indicated times by CH12 cells that were either not loaded (No Antigen) or were loaded with conalbumin (+ Antigen). Cells were then analyzed by confocal microscopy. Fluorescent proteins and antibody stains are indicated above each image. (D and E) D10 cells transduced with retroviruses encoding Bcl10-CFP and Malt1-YFP were left untreated or were stimulated for the indicated times with (D) CH12 cells that were either not loaded (No Antigen) or were loaded with conalbumin (+ Antigen) or (E) anti-CD3 antibody. Cells were then stained with antibodies against the indicated proteins before being analyzed by confocal microscopy. (F) Primary mouse naïve CD4+ T cells were differentiated in vitro into TH2 cells and then were incubated on coverslips coated with anti-CD3 and anti-CD28 antibodies. Cells were then stained with antibodies against the indicated proteins and analyzed by confocal microscopy. All images are representative of three or four independent experiments. Scale bar: 10 μm.
Fig. 2.
Fig. 2.. pIκBα and RelA exhibit transient colocalization with cytosolic Bcl10-containing signalosomes before translocating to the nucleus.
(A) D10 cells transduced with retroviruses encoding Bcl10-CFP and Malt1-YFP were left untreated or were stimulated with anti-CD3 antibodies. Cells were then stained with antibodies against the indicated proteins, and analyzed by confocal microscopy. (B) D10 cells transduced with retroviruses encoding PKCθ-CFP and Bcl10-YFP were stimulated for the indicated times with CH12 cells that were either unloaded (No Antigen) or were loaded with conalbumin (+ Antigen). Cells were then stained with anti-RelA and analyzed by confocal microscopy. (C) D10 cells transduced with retroviruses encoding Bcl10-CFP and Malt1-YFP were left untreated or were stimulated with anti-CD3 antibodies. Cells were then stained with anti-RelA and analyzed by confocal microscopy. Inset images below the 10-min time points are magnified views of the areas marked by white rectangles. All of the images are representative of at least four independent experiments. Scale bar: 10 μm.
Fig. 3.
Fig. 3.. Inhibition of TAK1 or IKK blocks IKK phosphorylation and RelA activation, but not Bcl10 clustering.
(A) D10 cells were treated with DMSO (control), BAY 11-7082 (BAY 11), or 5Z-7-oxozeaenol (5 oxo) before being stimulated with anti-CD3 antibody for 20 min. Cells were then lysed and analyzed by Western blotting with antibodies against the indicated proteins. Migration of molecular mass markers (kD) is indicated to left. Data are representative of three independent experiments. (B to D) D10 cells transduced with retroviruses encoding PKCθ-CFP and Bcl10-YFP were treated with DMSO (control), BAY 11, or 5 oxo, and were stimulated with (B and C) antigen-loaded CH12 cells (APCs) or (D) anti-CD3 antibody. Cells were then stained with antibodies against the indicated proteins and were analyzed by confocal microscopy. See fig. S2 for control images. DRAQ5 was used to stain nuclear DNA in (D). (E) The ratios of the abundances of nuclear and cytosolic RelA protein were quantified in cells from the experiments shown in (C) and (D). At least 20 cells were quantified in each group. Data are means ± SEM. *P < 0.05. Images in (B to D) are representative of three independent experiments. Scale bar: 10 μm.
Fig. 4.
Fig. 4.. p62 is required for efficient cytosolic Bcl10 clustering, IKK phosphorylation, and RelA nuclear translocation in primary effector T cells.
(A) Naïve CD4+ T cells isolated from WT C57BL/6 mice were stimulated with plate-bound anti-CD3 and anti-CD28 antibodies, lysed, and then analyzed by Western blotting with antibodies against the indicated proteins. (B) Lymph node cells isolated from WT or p62−/− mice were incubated on ice with biotinylated anti-CD3 antibody, which was followed by streptavidin-induced crosslinking on ice (control) or at 37°C (for 20 min). The cells were then fixed and stained with antibodies against the indicated proteins. (C) Primary TH2 cells derived from WT or p62−/− mice were stimulated with plate-bound anti-CD3 and anti-CD28 antibodies, lysed, and analyzed by Western blotting with antibodies against the indicated proteins. (A and C) Migration of molecular mass markers (kD) is indicated to left. Blots are representative of two independent experiments. (D and E) TH2 cells derived from WT or p62−/− mice were stimulated with SEB-loaded CHb cells (APCs), stained with the antibodies against the indicated proteins, and then analyzed by confocal microscopy. Note that the p62 speckles in (D) are present only in the CHb cells. (F) TH2 cells derived from WT or p62−/− mice were stimulated on coverslips coated with anti-CD3 and anti-CD28 antibodies for 20 min, and stained with antibodies against the indicated proteins and DAPI (to visualize nuclei). Cells were then analyzed by confocal microscopy. (G to I) Data from the experiment in (F) were quantified. Graphs show the percentages of cells that exhibited (G) cytosolic Bcl10 clusters (POLKADOTS) alone, (H) nuclear RelA alone, or (I) both. (J) Graph showing the ratios of the staining intensities of nuclear and cytosolic RelA in anti-CD3– and anti-CD28–stimulated WT and p62−/− TH2 cells that did or did not exhibit cytosolic POLKADOTS. At least 20 cells were quantified in each group. Data in bar graphs are means ± SEM. *P <0.05. Data in each panel are representative of two independent experiments. Scale bar: 5 μm (for B) or 10 μm (for D to F).
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