CARD14 signalosome formation is associated with its endosomal relocation and mTORC1-induced keratinocyte proliferation

Abstract

Rare mutations in CARD14 promote psoriasis by inducing CARD14-BCL10-MALT1 complexes that activate NF-κB and MAP kinases. Here, the downstream signalling mechanism of the highly penetrant CARD14E138A alteration is described. In addition to BCL10 and MALT1, CARD14E138A associated with several proteins important in innate immune signalling. Interactions with M1-specific ubiquitin E3 ligase HOIP, and K63-specific ubiquitin E3 ligase TRAF6 promoted BCL10 ubiquitination and were essential for NF-κB and MAP kinase activation. In contrast, the ubiquitin binding proteins A20 and ABIN1, both genetically associated with psoriasis development, negatively regulated signalling by inducing CARD14E138A turnover. CARD14E138A localized to early endosomes and was associated with the AP2 adaptor complex. AP2 function was required for CARD14E138A activation of mTOR complex 1 (mTORC1), which stimulated keratinocyte metabolism, but not for NF-κB nor MAP kinase activation. Furthermore, rapamycin ameliorated CARD14E138A-induced keratinocyte proliferation and epidermal acanthosis in mice, suggesting that blocking mTORC1 may be therapeutically beneficial in CARD14-dependent psoriasis.

Keywords: ABIN1; CARD14; LUBAC; NF-κB; a20; keratinocytes; mTOR; psoriasis.

Figure 1.. CARD14^E138A activates a transcriptional response similar to TNF stimulation in HaCaT-TR keratinocytes.

(A) CARD14^WT-3xFLAG or CARD14^E138A-3xFLAG expression was induced in HaCaT-TR cells with tetracycline (Tet; 3 h). Cytoplasmic and nuclear fractions were immunoblotted for the indicated antigens. (B) CARD14E138A-3xFLAG HaCaT-TR cells, incubated with siRNAs targeting IKK2 or NEMO or control non-targeting siRNAs, were cultured with tetracycline (Tet; 3 h). RelA nuclear translocation was determined by confocal microscopy assay, with positive values indicating increased nuclear RelA and negative values indicating cytoplasmic RelA (mean ± SD). Two-tailed unpaired t-test. Representative of two independent experiments. Anti-FLAG staining demonstrated that CARD14E138A-3xFLAG expression was not altered by IKK2 or NEMO siRNA knockdown. (C) Lysates of CARD14^WT/E138A-3xFLAG HaCaT-TR cells, incubated with tetracycline (Tet) for the times shown, were immunoblotted for the indicated antigens. (D) Volcano plot of gene expression in CARD14^WT-3xFLAG versus CARD14^E138A-3xFLAG HaCaT-TR cells, as determined by RNA sequencing. Data from 6 h timepoint (+ tetracycline), with biologically significant and strongly up-regulated genes are highlighted. Red circles indicate genes shared with the hallmark TNF/NF-κB response. (E) −Log₁₀(FDR) comparison of CARD14^E138A-3xFLAG-induced differential gene expression with indicated hallmark and pathway genesets.

Figure 2.. Characterization of the CARD14^E138A signalosome.

(A) CARD14^E138A-3xFLAG HaCaT-TR cells were incubated for the indicated times with tetracycline (Tet). RIPA cell lysates were separated into soluble and insoluble fractions, resolved by SDS–PAGE and immunoblotted for 3xFLAG and HSP90 (loading control). (B) CARD14^E138A-3xFLAG HaCaT-TR cell lysates were mixed with lysates of HaCaT-TR parental control cell line to create forward and reverse SILAC conditions. Eluates of 3xFLAG pull downs from these mixtures were analyzed by MS. Scatter plot of Log₂ ratio of heavy (H) versus light (L) normalized peptide intensities for forward and reverse SILAC conditions. CARD14^E138A-3xFLAG-associated proteins found in both experimental conditions are located in upper left quadrant. Positions of CARD14 and signalosome components are indicated. Log₂ ratio 0.5 cut-off is shown with a red line. (C) Known and computationally derived STRING protein-protein association network for CARD14^E138A-3xFLAG [33]. (D) Lysates of CARD14^WT-3xFLAG HaCaT-TR (WT) and CARD14^E138A-3xFLAG (E138A) HaCaT-TR cells, cultured for tetracycline (Tet; 3 h), were immunoprecipitated with anti-FLAG beads. Immunoprecipitates (IP) and input lysates were immunoblotted for the indicated antigens. Molecular mass (kDa) are shown. (E) Two monoclonal CARD14^E138A-3xFLAG HaCaT-TR (Cas9) cell lines deficient in HOIP (HOIP Def. 1, 2) and control non-targeted cells (Con) were cultured with tetracycline (Tet; 3 h). Cell lysates were immunoprecipitated with anti-FLAG. Immunoprecipitates (IP) and input lysates were immunoblotted for the indicated antigens. (F) TRAF6-deficient CARD14^E138A-3xFLAG HaCaT-TR (Cas9) cell lines (TRAF6 Def. 1, 2) and control non-targeted cells (Con) were analyzed as in Figure 2E. (G) CARD14^E138A-3xFLAG HaCaT-TR (Cas9) cell lines deficient in HOIP (HOIP Def. 1, 2) and control non-targeted cells (Con) were cultured ± tetracycline (Tet; 3 h). Lysates were immunoblotted for the indicated antigens. (H) Cytoplasmic and nuclear fractions from control (Con) and HOIP-deficient (HOIP Def.) CARD14^E138A-3xFLAG HaCaT-TR (Cas9) cells ± tetracycline (Tet; 3 h) were immunoblotted. Lamin (nuclear) and tubulin (cytoplasm) blotting confirmed effective fractionation. (I) RelA nuclear localization in control non-targeted (Con) and HOIP-deficient (HOIP Def.) CARD14^E138A-3xFLAG HaCaT-TR (Cas9) cells cultured ± tetracycline (Tet; 3 h) was determined by confocal microscopy. Positive values indicate increased nuclear RelA and negative values indicating cytoplasmic RelA (mean ± SD). Two-tailed unpaired t-test. (J) CARD14^E138A-3xFLAG HaCaT-TR (Cas9) cell lines deficient in HOIP (HOIP Def.) and control non-targeted cells (Con) were cultured ± tetracycline (Tet; 6 h). IL36G and CCL20 mRNA expression was determined by qRT-PCR (mean ± SD). Two-tailed unpaired t-test. (K) TRAF6-deficient CARD14^E138A-3xFLAG HaCaT-TR (Cas9) cells (TRAF6 Def.) and control non-targeted cells (Con) were analyzed as in Figure 2G. (L) TRAF6-deficient CARD14^E138A-3xFLAG HaCaT-TR (Cas9) cells (TRAF6 Def.) and control non-targeted cells (Con) were analyzed as in Figure 2H. (M) TRAF6-deficient CARD14^E138A-3xFLAG HaCaT-TR (Cas9) cells (TRAF6 Def.) and control non-targeted cells (Con) were analyzed as in Figure 2I. (N) TRAF6-deficient CARD14^E138A-3xFLAG HaCaT-TR (Cas9) cells (TRAF6 Def.) and control non-targeted cells (Con) were analyzed as in Figure 2J. (O) CARD14^E138A-3xFLAG HaCaT-TR (Cas9) cell lines deficient in HOIP (HOIP Def.) and control non-targeted cells (Con) were cultured ± tetracycline (Tet; 3 h). Lysates were immunoblotted for RelB. Arrowhead shows position of proteolysed RelB band. (P) TRAF6-deficient CARD14^E138A-3xFLAG HaCaT-TR (Cas9) cells (TRAF6 Def.) and control non-targeted cells (Con) were analyzed as in Figure 2O.

Figure 3.. CARD14^E138A induces HOIP- and TRAF6-dependent ubiquitination of BCL10.

(A) HALO-NEMO pull downs were performed with lysates from CARD14^E138A 3xFLAG HaCaT-TR cells (E138A) incubated ± tetracycline (Tet; 3 h). HALO-NEMO resin was then treated with the M1-Ub and/or K63-Ub specific DUBs, OTULIN (1 μM) and AMSH-LP (0.2 μM), respectively. Control pulldowns (-) were left untreated. Pulldowns and input lysates were immunoblotted for the indicated antigens. (B) HALO-NEMO pull downs were carried out with HOIP-deficient (HOIP Def.) and control non-targeted (Con) CARD14^E138A 3xFLAG HaCaT-TR (Cas9) cells as in E. Pull downs and input lysates were immunoblotted. (C) HALO-NEMO pull downs were carried out with TRAF6-deficient (TRAF6 Def.) and control non-targeted (Con) CARD14^E138A 3xFLAG HaCaT-TR (Cas9) cells as in E. Pull downs and input lysates were immunoblotted. (D) HALO-TAB2 (K63-Ub) pull downs were performed with lysates from CARD14^E138A 3xFLAG HaCaT-TR cells cultured ± tetracycline (Tet; 3 h). HALO-NEMO resin was then treated with the K63-Ub specific DUB AMSH-LP (+) or left untreated (−). Pulldowns and input lysates were immunoblotted. (E) Two monoclonal CARD14^E138A-3xFLAG HaCaT-TR (Cas9) cell lines deficient in HOIP (1, 2) and control non-targeted cells (C) were cultured with tetracycline (3 h). Lysates were immunoprecipitated with anti-FLAG and immunoprecipitates (IP) immunoblotted for the indicated antigens. (F) Two monoclonal CARD14^E138A-3xFLAG HaCaT-TR (Cas9) cell lines deficient in TRAF6 (1, 2) and control non-targeted cells (C) were cultured with tetracycline (3 h). Lysates were immunoprecipitated with anti-FLAG and immunoprecipitates (IP) immunoblotted for the indicated antigens. (G) Lysates from control (Con), TRAF6-deficient (TRAF6 Def.) and HOIP-deficient (HOIP Def.) CARD14^E138A 3xFLAG HaCaT-TR (Cas9) cells were immunoblotted for the indicated antigens. Molecular mass (kDa) are shown. All data in this figure are representative of at least two similar independent experiments.

Figure 4.. CARD14^E138A is associated with early endosomes.

(A) Lysates of CARD14^WT-3xFLAG HaCaT-TR (WT) and CARD14^E138A-3xFLAG HaCaT-TR cells (E138A), cultured for 3 h with tetracycline, were subjected to immunoprecipitation with anti-FLAG beads. Immunoprecipitates (IP) and input lysates were immunoblotted for the indicated antigens. Molecular mass (kDa) are shown. (B) Confocal microscopy imaging of anti-FLAG and anti-MALT1 localization in CARD14^WT-3xFLAG HaCaT-TR (WT) and CARD14^E138A-3xFLAG (E138A) HaCaT-TR cells ± tetracycline induction (Tet; 3 h). Nuclei were stained with DAPI. (C) Proximity ligations assays determined whether CARD14^WT-3xFLAG (WT) and CARD14^E138A-3xFLAG (E138A) co-localized with MALT1 (by anti-FLAG and anti-MALT1 staining) in HaCaT-TR cells ± tetracyline induction (Tet; 3 h). Overlay box (E138A + Tet) shows blow up of indicated region. (D) Confocal microscopy imaging of anti-FLAG and anti-EEA1 localization in CARD14^WT-3xFLAG HaCaT-TR (WT) and CARD14^E138A-3xFLAG (E138A) HaCaT-TR cells ± tetracycline induction (Tet; 3 h). Nuclei were stained with DAPI. Arrowheads show regions of coincident staining. Overlay box (E138A + Tet) shows blow up of indicated region. (E) Proximity ligations assays determined whether CARD14^WT-3xFLAG (WT) and CARD14^E138A-3xFLAG (E138A) were in close proximity with EEA1 (by anti-FLAG and anti-EEA1 staining) in HaCaT-TR cells ± tetracycline induction (Tet; 3 h). (F) Confocal microscopy localization of CARD14^E138A-EGFP (EGFP) induced by tetracycline (3 h) in live HaCaT-TR cells was determined. Microtubules were fluorescently labelled with SiR-Tubulin (Tubulin). Lines tracking movement of CARD14^E138A-EGFP foci were created post-acquisition using TrackMate (ImageJ). Time stamp = mm:ss, scale bar = 5 μm. Images are representative of several acquisitions across separate cells and those shown are obtained from Supplementary Video S1. Bottom row of images show time series from region highlighted with box in overlay panel. (G) CARD14^E138A 3xFLAG HaCaT-TR cells were pre-treated with AP2M1 or control (con) siRNA. Cell lysates were prepared 3 h after tetracycline induction (Tet; 3 h) and immunoblotted for the indicated antigens. All data in this figure are representative of at least two replicate experiments.

Figure 5.. CARD14^E138A activates mTOR complex 1 independently of NF-κB activation.

(A) Parental HaCaT-TR cells or CARD14^E138A 3xFLAG HaCaT-TR cells were cultured in heavy or light amino acid SILAC media. Cell lysates were mixed according to Supplementary Figure S1E table and analysed by MS. Scatter plot shows Log₂ ratio of phosphosite intensities, heavy versus light, normalized from forward and reverse SILAC conditions. Red lines = threshold of Log₂ ratio (0.5). Hollow grey circle = detected phosphosite. Annotated phosphosites of interest are highlighted by red squares. (B) CARD14^WT-3xFLAG (WT) and CARD14^E138A-3xFLAG (E138A) were induced with tetracycline (Tet; 3 h) in HaCaT-TR cells. Cell lysates were immunoblotted for the indicated antigens. (C) Expression of CARD14^E138A-3xFLAG was induced with tetracycline (Tet; 3 h) in HaCaT-TR cells (E138A) ± BI605906 (BI6) treatment to block IKK2. Control cells were treated with DMSO vehicle (Con). Cell lysates were immunoblotted. (D) Expression of CARD14^E138A-3xFLAG was induced with tetracycline (Tet; 3 h) in HaCaT-TR cells (E138A) ± AZD8055 (AZD) treatment to block mTOR. Control cells were treated with DMSO vehicle (Con). Cell lysates were immunoblotted. (E) HOIP-deficient (HOIP Def.) and control non-targeted (Con) CARD14^E138A 3xFLAG HaCaT-TR (Cas9) cells were induced with tetracycline (Tet; 3 h). Cell lysates were immunoblotted. (F) TRAF6-deficient (TRAF6 Def.) and control non-targeted (Con) CARD14^E138A 3xFLAG HaCaT-TR (Cas9) cells were induced with tetracycline (Tet; 3 h). Cell lysates were immunoblotted. (G) Expression of CARD14^E138A was induced with tetracycline (Tet; 3 h) in CARD14^E138A 3xFLAG HaCaT-TR cells ± GDC-0941 (GDC) treatment (at indicated concentrations) to inhibit PI 3-kinase. Control cells were treated with DMSO vehicle (Con). Cell lysates were immunoblotted. (H) Expression of CARD14^WT-3xFLAG (WT) and CARD14^E138A-3xFLAG (E138A) was induced with tetracycline (Tet; 3 h) in HaCaT-TR cells. Confocal imaging determined localization of anti-mTOR and anti-LAMP1 staining. Nuclei were stained with DAPI. Pixel data from three independent experiments were used to determine the degree of signal correlation via Pearson's correlation with Costes method in relevant regions of interest. Means (±SD) are presented and tested by unpaired two tailed t-test to respective control mean for each cell line. (I) CARD14^E138A 3xFLAG HaCaT-TR cells were pre-treated with AP2M1 or control (Con) siRNAs. Cell lysates ± tetracycline induction (Tet; 3 h) were immunoblotted for the indicated antigens. Data for S6 kinase phosphorylation from three independent experiments normalized to tubulin loading control are plotted (means ± SD) and analyzed by one-way ANOVA, with correction for multiple testing.

Figure 6.. CARD11^L232LI activates mTORC1 independently of AP2M1 and CARD14^E138A induces cell metabolism via mTOR in HaCaT-TR cells.

(A) Expression of CARD11^L232LI-3xFLAG and CARD14^E138A-3xFLAG were induced with tetracycline (Tet; 3 h) in HaCaT-TR cells. Cell lysates were immunoblotted for the indicated antigens. (B) Parental HaCaT-TR cells (HaCaT-TR), CARD14^E138A-3xFLAG HaCaT-TR cells (CARD14^E138A) and CARD11^L232LI-3xFLAG HaCaT-TR cells (CARD11^L232LI) were induced with tetracycline (Tet; 3 h). Cell lysates were immunoprecipitated with anti-FLAG. Immunoprecipitates (IP) and input lysates were immunoblotted for the indicated antigens. (C) Confocal imaging of anti-FLAG localization in CARD14^E138A-3xFLAG HaCaT-TR cells cultured for the indicated times with tetracycline (Tet). Nuclei were stained with DAPI. (D) CARD14^E138A-3xFLAG HaCaT-TR cells (CARD14^E138A) and CARD11^L232LI-3xFLAG HaCaT-TR cells (CARD11^L232LI) were pre-treated with AP2M1 or control (Con) siRNAs. Cell lysates were prepared after tetracycline induction (Tet; 3 h) and immunoblotted for the indicated antigens. S6 kinase phosphorylation was quantified and normalized to tubulin loading control for three independent experiments. Means (±SD) are presented and tested statistically by unpaired two-tailed t-test relative to control means for each cell line. (E) Triplicate cultures of HOIP-deficient (HOIP Def.) and control non-targeted (Con) CARD14^E138A 3xFLAG HaCaT-TR (Cas9) cells were induced for with tetracycline (Tet) for the indicated times. Metabolic activity was monitored by MTT assay (mean ± SD). (F) Triplicate cultures of CARD14^E138A 3xFLAG HaCaT-TR cells were induced with tetracycline (Tet; 8 h) plus AZD8055 (AZD) or DMSO vehicle control. Metabolic activity was monitored by MTT assay (mean ± SD). Statistical significance was tested by two tailed, unpaired t-test. (G) Triplicate cultures of HOIP-deficient (HOIP Def) and control non-targeted (Con) CARD14^E138A 3xFLAG HaCaT-TR (Cas9) cells were induced with tetracycline (Tet; 6 h). Nascent protein synthesis was monitored by OPP assay (mean ± SD). (H) Triplicate cultures of CARD14^E138A 3xFLAG HaCaT-TR cells were induced with tetracycline (Tet; 6 h) plus AZD8055 (AZD) or DMSO vehicle control. Nascent protein synthesis was monitored by OPP assay (mean ± SD). Statistical significance was tested by two tailed, unpaired t-test.

Figure 7.. Rapamycin reduces CARD14^E138A-induced epidermal acanthosis in mice.

(A) Schematic representation of the experimental procedure for CARD14^E138A induction and rapamycin treatment. CARD14^E138A expression was induced in keratinocytes of Tg CARD14^E138A by daily tamoxifen injections up to 3 days. Rapamycin or control vehicle was injected daily intraperitoneally on d1–d4. Mice were culled on d5 for analysis. Tg control mice were injected with tamoxifen ± rapamycin for comparison. (B) Ear sections were produced from mice 5 days after tamoxifen induction ± vehicle (Control) or rapamycin co-injection. Representative sections are shown, stained with hematoxylin and eosin (H&E) and anti-phospho-S6. Scale bar represents 100 µM. (C) Skin extracts from ears were produced from mice 5d after tamoxifen induction ± vehicle control (Con) or rapamycin (Rapa) injection and immunoblotted for the indicated antigens. (D) Ear sections were produced from mice 5d after tamoxifen induction plus vehicle (Control) or rapamycin treatment. Representative images are shown of ear sections stained with H&E. Scale bar represents 100 µM. (E) Changes in ear thickness in mice treated with vehicle (Con) or rapamycin (Rapa) following tamoxifen induction of Tg CARD14^E138A and Tg Control mice. Combined results of two independent experiments are shown. Ear thickness data were analyzed as repeated measurements using the residual maximum likelihood (REML). (Tg Control/vehicle, n = 6; Tg Control/rapamycin n = 7, Tg CARD14^E138A/vehicle, n = 10, Tg CARD14^E138A/rapamycin n = 10). (F) Changes in epidermal thickness in Tg CARD14^E138A and Tg Control mice treated with vehicle (Con) or rapamycin (Rapa) following tamoxifen induction. Each symbol represents the mean of at least 10 epidermal thickness measurements for each ear, the line represents the mean value. (E and F) Statistical differences were determined using a modified F-test. P values shown. (G) Ear sections were produced from Tg CARD14^E138A and Tg Control mice 5d after tamoxifen induction plus vehicle (Control) or rapamycin treatment. Representative images are shown of ear sections stained with H&E and anti-Ki67. Scale bar represents 100 µM. (H) Quantification of Ki67 positive cells from control (Tg Control) and Tg CARD14^E138A mice treated with vehicle (Con) or rapamycin (Rapa). Each symbol represents one mouse, the line represents the mean value. Statistical difference between two groups was determined using one-way ANOVA test. *P < 0.01. (I) Analysis of infiltrating immune cells in single cell suspension of the ears from Tg Control and Tg CARD14^E138A mice treated or non-treated with rapamycin were analysed by flow cytometry. Cell counts of neutrophils (CD45⁺CD3⁻CD19⁻CD11b⁺Ly6G⁺), DCs (CD45⁺CD3/CD19⁻CD11b⁺CD64⁻MHCII⁺CD11c⁺), eosinophils (CD45⁺CD3/CD19⁻CD11b⁺CD64⁻SiglecF⁺), T-cells (CD45⁺CD3/CD19⁺,MHCII⁻). The data was log-transformed and analyzed using a modified F-test. Significant P-values shown. NS, not significant. (Tg Control/vehicle, n = 4; Tg Control/rapamycin, n = 4; Tg CARD14^E138A/vehicle, n = 6; Tg CARD14^E138A/rapamycin, n = 6).