Cellular and Mathematical Analyses of LUBAC Involvement in T Cell Receptor-Mediated NF-κB Activation Pathway

Abstract

The LUBAC ubiquitin ligase complex, composed of the HOIP, HOIL-1L, and SHARPIN subunits, stimulates the canonical nuclear factor-κB (NF-κB) activation pathways through its Met1-linked linear ubiquitination activity. Here we performed cellular and mathematical modeling analyses of the LUBAC involvement in the T cell receptor (TCR)-mediated NF-κB activation pathway, using the Jurkat human T cell line. LUBAC is indispensable for TCR-induced NF-κB and T cell activation, and transiently associates with and linearly ubiquitinates the CARMA1-BCL10-MALT1 (CBM) complex, through the catalytic HOIP subunit. In contrast, the linear ubiquitination of NEMO, a substrate of the TNF-α-induced canonical NF-κB activation pathway, was limited during the TCR pathway. Among deubiquitinases, OTULIN, but not CYLD, plays a major role in downregulating LUBAC-mediated TCR signaling. Mathematical modeling indicated that linear ubiquitination of the CBM complex accelerates the activation of IκB kinase (IKK), as compared with the activity induced by linear ubiquitination of NEMO alone. Moreover, simulations of the sequential linear ubiquitination of the CBM complex suggested that the allosteric regulation of linear (de)ubiquitination of CBM subunits is controlled by the ubiquitin-linkage lengths. These results indicated that, unlike the TNF-α-induced NF-κB activation pathway, the TCR-mediated NF-κB activation in T lymphocytes has a characteristic mechanism to induce LUBAC-mediated NF-κB activation.

Keywords: CBM complex; LUBAC; NF-κB; T cell receptor; linear ubiquitin; mathematical model.

Figure 1

LUBAC is necessary for the TCR-mediated NF-κB activation pathway. (A) Parental Jurkat cells, HOIP-deficient cells (RNF31-KO), or WT-HOIP-restored RNF31-KO cells (RNF31-KO+HOIP-WT) were stimulated with 1 μg/ml each of anti-CD3 and anti-CD28 antibodies for the indicated periods of time. Cell lysates and nuclear fractions were immunoblotted with the indicated antibodies. (B) Impaired IKK activation in RNF31-KO cells. WT or RNF31-deficient Jurkat cells were stimulated and analyzed as in (A), using the indicated antibodies. *nonspecific signal. (C) Reduced expression of TCR-mediated NF-κB target genes in RNF31-KO cells. Cells were stimulated with anti-CD3 and anti-CD28 antibodies as in (A) for 1 h, and qPCR analyses were performed. (D) LUBAC activity is required for efficient IL-2 secretion. Cells were stimulated with 5 μg/ml each of anti-CD3 and anti-CD28 antibodies for 18 h, and secreted IL-2 was quantified by ELISA. (E) The expression of T cell activation marker CD69 was suppressed in RNF31-KO Jurkat cells. WT or RNF31-deficient Jurkat cells were stimulated with 3 μg/ml each of anti-CD3 and anti-CD28 antibodies for 20 h. The expression of CD69 was analyzed by a flow cytometer. (C, D) Data are shown as Means ±SD (n = 3). ***P<0.001, ****P<0.0001, NS, not significant.

Figure 2

The N-terminal domains in HOIP associate with CARMA1 and MALT1. (A) Endogenous association of LUBAC and the CBM complex upon TCR-stimulation. Jurkat cells were stimulated with 20 ng/ml PMA and 150 ng/ml ionomycin for the indicated time periods. The cells lysates and anti-HOIL-1L immunoprecipitates were subjected to immunoblotting with the indicated antibodies. (B) HOIP binds CARMA1 and MALT1. Myc-tagged LUBAC subunits and FLAG-tagged CBM components were co-expressed in HEK293T cells, as indicated. The cell lysates and anti-Myc immunoprecipitates were immunoblotted with the depicted antibodies. (C) Domain structure and mutants of HOIP. PUB: peptide:N-glycanase/UBA or UBX-containing proteins; ZF: zinc finger; NZF: Npl4-type zinc finger; UBA: ubiquitin-associated; RING: really interesting new gene; IBR: in-between RING; and LDD: linear ubiquitin determining. (D) The B box domain of HOIP is crucial for CARMA1-binding. WT and various mutants of Myc-tagged HOIP were co-expressed with FLAG-CARMA1 in HEK293T cells, and immunoprecipitations followed by immunoblotting analyses were performed as indicated. (E) The PUB domain of HOIP is responsible for MALT1-binding. A similar analysis to that in Figure 2D was performed, using FLAG-MALT1. (F) Domain structure and mutants of CARMA1. CARD, caspase-recruitment domain; BAR, Bin/Amphiphysin/Rvs; GBP-C, guanylate-binding protein C‐terminal; PDZ, post synaptic density protein (PSD95)-Drosophila disc large tumor suppressor (Dlg1)-Zonula occludens-1 protein (ZO-1); SH3, Src homology 3; and GUK, guanylate kinase. (G) The PDZ-SH3 region of CARMA1 binds HOIP. A similar analysis to that in Figure 2D was performed, using WT and various mutants of FLAG-CARMA1 and Myc-HOIP. (H) Domain structure and mutants of MALT1. Ig, immunoglobulin-like. (I) The death domain in MALT1 is the HOIP binding site. A similar analysis to that in Figure 2D was performed, using WT and various mutants of FLAG-MALT1 and Myc-HOIP.

Figure 3

OTULIN and MALT1 simultaneously bind to the PUB domain of HOIP. (A) WT and various mutants of Myc-HOIP were co-expressed with FLAG-MALT1 and/or FLAG-OTULIN in HEK293T cells, as indicated. Cell lysates and anti-Myc immunoprecipitates were immunoblotted with the indicated antibodies. (B) The endogenous association of CYLD, OTULIN, LUBAC, and the CBM complex. Jurkat cell lysates were immunoprecipitated with normal mouse IgG or anti-MALT1 antibody, and subjected to immunoblotting by the depicted antibodies. *; nonspecific signal. (C) Schematic illustration of the LUBAC/DUBs-CBM complex interaction.

Figure 4

The CBM complex is linearly ubiquitinated upon TCR stimulation. (A) Parental Jurkat cells were stimulated with 20 ng/ml PMA and 150 ng/ml ionomycin for the indicated time periods. The heat-denatured cell lysates were immunoprecipitated and immunoblotted with the indicated antibodies. Taking the maximum intensities of linear ubiquitination as 100%, the relative intensities of the linear ubiquitinated CBM complex are indicated. Means ±SD (n = 3). (B) HOIP is required for linear ubiquitination of MALT1. Parental and RNF31-KO Jurkat cells were stimulated with PMA/ionomycin as in (A), immunoprecipitated with anti-MALT1, and immunoblotted with the indicated antibodies. (C) Suppressed linear ubiquitination of NEMO in the TCR-mediated NF-κB activation pathway. Jurkat cells were stimulated with 20 ng/ml PMA and 150 ng/ml ionomycin or 1 µg/ml TNF-α for the indicated time periods, and cell lysates were immunoprecipitated with an anti-NEMO antibody and then immunoblotted with the depicted antibodies. (D) MALT1 is exclusively linearly ubiquitinated upon TCR stimulation. A similar analysis to that in Figure 4C was performed after anti-MALT1 immunoprecipitation. (E) Linear ubiquitination of MALT1 after stimulation with CD3 and CD28. Jurkat cells were stimulated with 5 µg/ml each of anti-CD3 and anti-CD28 antibodies as indicated. The cell lysates and anti-MALT1 immunoprecipitates were immunoblotted by the indicated antibodies. (F) Transient activation of canonical IKK. Jurkat cells were stimulated with 20 ng/ml PMA and 150 ng/ml ionomycin for the indicated time periods. After immunoprecipitation with an anti-NEMO antibody, an in vitro canonical IKK assay was performed using GST-IκBα^1–54 as the substrate. Samples were immunoblotted with the indicated antibodies, and taking the maximum intensities of P-IκBα as 100%, the relative intensities are indicated. Means ±SD (n = 3). (A, B, E) *; nonspecific signal.

Figure 5

OTULIN predominantly downregulates TCR-mediated NF-κB activation in Jurkat cells. (A) Parental, CYLD-deficient (CYLD-KO), and OTULIN-deficient (OTULIN-KO) Jurkat cells were stimulated with 4 μg/ml each of anti-CD3 and anti-CD28 antibodies for the indicated time periods, and cell lysates were immunoblotted with the depicted antibodies. (B) Augmented linear ubiquitination of MALT1 in OTULIN-deficient cells. Parental and OTULIN-KO Jurkat cells were stimulated with 20 ng/ml PMA and 150 ng/ml ionomycin for the indicated time periods, and the linear ubiquitination of MALT1 was compared as in Figure 4A . (C) Semi-quantification of MALT1 linear ubiquitination in OTULIN-KO cells. OTULIN-KO cells were stimulated with PMA and ionomycin, and analyzed as in Figure 4A . Taking the maximum intensities of the linear ubiquitination of MALT1 in parental Jurkat cells as 100% (closed circles), the relative intensities of linearly ubiquitinated MALT1 in OTULIN-KO cells are indicated (open circles). Means ±SD (n = 3). (D) Transient activation of canonical IKK in OTULIN-KO cells. OTULIN-KO cells were stimulated with PMA and ionomycin, and analyzed as in Figure 4E . (E) Enhanced linear ubiquitination of NEMO in OTULIN-KO cells upon TNFα treatment. Jurkat and OTULIN-KO cells were stimulated with 1 µg/ml TNF-α for the indicated time periods, and analyzed as in Figure 4C . (A–C) *; nonspecific signal.

Figure 6

Mathematical simulation of the effects of linear ubiquitination of the CBM complex and NEMO on the TCR-mediated IKK activation. (A) LUBAC ubiquitinates NEMO and the CBM complex. In this model, ubiquitination is assumed to be a first-order reaction. (B) Fitting result by GA. Solid lines represent simulation results, and dots represent experimental data. (C) The simulation results of IKK activation, changing k_C and k_N. From the blue line to the red line, k_C increases under condition (i), and k_N increases under condition (ii). Condition (iii) is a simulation result when θ = π/4. From the blue line to the red line, k_C increases and k_N decreases. Condition (iv) is a simulation result when θ = 3π/4. From the blue line to the red line, k_C decreases and k_N increases.

Figure 7

Mathematical model for different linear ubiquitinations of the CBM complex. (A) Detailed reaction pathway of the ubiquitination of the CBM complex. The red lines show parameters that are larger than the average value. The blue lines show parameters that are smaller than the average value. (B) Fitting results by GA in the CBM_DM. (C) Fitting results by GA. The ubiquitination reaction is assumed to be a Hill function.