T cell receptor-dependent activation of mTOR signaling in T cells is mediated by Carma1 and MALT1, but not Bcl10

Abstract

Signaling to the mechanistic target of rapamycin (mTOR) regulates diverse cellular processes, including protein translation, cellular proliferation, metabolism, and autophagy. Most models place Akt upstream of the mTOR complex, mTORC1; however, in T cells, Akt may not be necessary for mTORC1 activation. We found that the adaptor protein Carma1 [caspase recruitment domain (CARD)-containing membrane-associated protein 1] and at least one of its associated proteins, the paracaspase MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1), were required for optimal activation of mTOR in T cells in response to stimulation of the T cell receptor (TCR) and the co-receptor CD28. However, Bcl10, which binds to Carma1 and MALT1 to form a complex that mediates signals from the TCR to the transcription factor NF-κB (nuclear factor κB), was not required. The catalytic activity of MALT1 was required for the proliferation of stimulated CD4+ T cells, but not for early TCR-dependent activation events. Consistent with an effect on mTOR, MALT1 activity was required for the increased metabolic flux in activated CD4+ T cells. Together, our data suggest that Carma1 and MALT1 play previously unappreciated roles in the activation of mTOR signaling in T cells after engagement of the TCR.

Fig. 1. Carma1 is required for TCR- and CD28-mediated phosphorylation of S6

(A) Jurkat cells, JPM 50.6 cells transfected with plasmid encoding Carma1 (JPM + Carma1), and JPM 50.6 cells were left unstimulated or were stimulated with antibodies against CD3 and CD28 (TCR/CD28) for the indicated times and then were analyzed by Western blotting with an antibody specific for phosphorylated substrates of Akt. β-actin was used as a loading control. (B) Top: JPM 50.6 cells and Carma1-reconstituted JPM 50.6 cells were left unstimulated or were stimulated with anti-CD3 and anti-CD28 antibodies for the indicated times, and then were analyzed by Western blotting with an antibody specific for S6 phosphorylated at S235/236. β-actin was used as a loading control. Bottom: Quantitation of the fold-increase in the abundance of pS6 protein. Densitometric analysis was used to determine the relative intensities of bands corresponding to pS6, which were normalized to those of β-actin. The relative fold-increases in pS6 abundance in the Carma1-reconstituted JPM 50.6 cells (filled bars) and JPM 50.6 cells (empty bars) at the indicated times were then calculated, based on three independent experiments. (C) Jurkat cells and JPM 50.6 cells were left unstimulated or were stimulated with anti-CD3 and anti-CD28 antibodies or with amino acids (10X AA). Cells were then analyzed by flow cytometry to detect pS6. (D) Jurkat cells expressing control shRNA or Carma1-specific shRNA (Carma1shRNA cells) were left unstimulated or were stimulated with anti-CD3 and anti-CD28 antibodies for 15 min before being analyzed by flow cytometry for pS6. The graph below shows mean MFI ± SEM of triplicate samples. (E) Primary CD4⁺ T cells isolated from wild-type (WT) or Carma1 KO mice were left unstimulated or were stimulated with PMA and ionomycin for 15 min before being analyzed by flow cytometry to detect pS6. Data in the bar graph represent mean fold-increases ± SEM in pS6 abundance in duplicate samples of stimulated WT or Carma1 KO T cells, relative to that in unstimulated cells. *P < 0.05, **P <0.01 by unpaired student's t test. Data in all panels are representative of three independent experiments.

Fig. 2. The phosphorylation of mTOR targets in response to stimulation of TCR and CD28 depends on Carma1

(A) JPM 50.6 cells and Carma1-reconstituted JPM 50.6 cells were left untreated or were treated with anti-CD3 and anti-CD28 antibodies (TCR/CD28) for the indicated times before being analyzed by Western blotting with antibodies against the indicated proteins. (B) Jurkat cells and JPM 50.6 cells were left untreated or were treated with anti-CD3 and anti-CD28 antibodies for 15 min before being subjected to immunoprecipitation (IP) of S6K with a specific antibody. IP'd samples were analyzed by in vitro kinase assays with GST-S6 as the substrate. Top panels: Reactions were analyzed by autoradiography to examine GST-S6 phosphorylation and S6K auto-phosphorylation. Bottom panels: Reactions were also analyzed by Western blotting to determine the relative amounts of IP'd S6K and GST-S6. Data in panels A and B are representative of three independent experiments. (C and D) Left: Untransfected Jurkat cells (C) or Jurkat cells transfected with a control shRNA (D) and Carma1shRNA cells (D) were left untreated or were stimulated with anti-CD3 and anti-CD28 antibodies for the indicated times and then were analyzed by Western blotting with antibodies against the indicated proteins. Right: Graphs show the fold-increase in the abundance of p4E-BP1 (normalized to that of total 4E-BP1) in the indicated cells at the indicated times. Data shown are the means ± SEM from three (panel C) or two (panel D) independent experiments.

Fig. 3. The TCR- and CD28-stimulated phosphorylation of S6 requires PKC

(A) D10 T cells were left unstimulated or were pretreated with vehicle, Akti1/2, or BIM before being stimulated with biotinylated anti-CD3, anti-CD28, and anti-CD4 antibodies and streptavidin for 30 min. Cells were then analyzed by flow cytometry to detect pS6. (B) Primary murine T cells were left untreated or were pretreated with BIM before being stimulated with anti-CD3 and anti-CD28 antibodies for 30 min. Cells were then analyzed by flow cytometry for pS6. Graph shows mean MFIs of pS6 ± SEM of triplicate measurements from a single experiment and are representative of three independent experiments. **P < 0.01, by unpaired Student's t-test. (C) Jurkat cells (top) and JPM 50.6 cells (bottom) were left untreated or were pretreated with the indicated concentrations of BIM before being stimulated with anti-CD3 and anti-CD28 antibodies for 15 min. Cells were then analyzed by flow cytometry for pS6. Right: Graphs show the fold-increase in mean fluorescence intensity (MFI) for each condition. Data are means ± SEM of three (Jurkat) and two (JPM 50.6) independent experiments.

Fig. 4. MALT1, but not Bcl10, is required for optimal S6 phosphorylation in response to TCR and CD28 stimulation

(A) Parental Jurkat cells, MALT1shRNA cells, and Bcl10shRNA cells were analyzed by Western blotting with antibodies against the indicated proteins. Data are representative of three independent experiments. (B and C) Parental Jurkat cells (B) and either Bcl10shRNA cells (B) or MALT1shRNA cells (C) were stimulated with anti-CD3 and anti-CD28 antibodies for 15 min and then were analyzed by flow cytometry for pS6. Bar graphs show the fold-increase in pS6 in the indicated cells. Data are means ± SEM from three independent experiments. ns, not significant. (D) MALT1shRNA and Bcl10shRNA cells were stimulated with anti-CD3 and anti-CD28 antibodies for 15 min, before being analyzed by flow cytometry for pS6. The bar graph shows the fold-increase in pS6 in the indicated cells. Data are means ± SEM from three independent experiments. (E) Primary CD4⁺ T cells from WT and Bcl10 KO mice were left untreated or were stimulated with PMA and ionomycin for 30 min before being analyzed by flow cytometry for pS6. The bar graph shows average MFIs for pS6 ± SEM of triplicate samples from one experiment, which is representative of three experiments. (F) Jurkat cells and MALT1shRNA cells were stimulated as indicated and then analyzed by Western blotting for p4E-BP1(T70). β-actin was used as a loading control. Blots are representative of three independent experiments. (G) Primary mouse CD4⁺ T cells were left untreated or were pretreated with the IKK-2 inhibitor before being stimulated with anti-CD3 and anti-CD28 antibodies for 30 min, and then analyzed by flow cytometry for pS6. Bar graph shows mean MFIs for pS6 ± SEM of triplicate samples from one experiment, which is representative of two experiments. (H) Jurkat cells were left untreated or were stimulated with anti-CD3 and anti-CD28 antibodies before being subjected to immunoprecipitation (IP) with anti-MATL1 antibody. IP's were then analyzed by Western blotting to detect MALT1-associated S6K (left) and mTOR (right). Blots are representative of three independent experiments. *P < 0.05, **P <0.01, by unpaired student's t test.

Fig. 5. The catalytic activity of MALT1 contributes to TCR- and CD28-stimulated phosphorylation of S6K and S6 in T cells

(A and B) Jurkat cells (A) and primary mouse CD4⁺ T cells (B) were left untreated, were (A) pretreated with the indicated concentrations of z-VRPR-fmk, or were (B) pretreated with either z-VRPR-fmk or rapamycin before being stimulated with anti-TCR and anti-CD28 antibodies (Jurkat cells) or anti-CD3 and anti-CD28 antibodies (mouse CD4⁺ T cells). (A and B) Cells were then analyzed by flow cytometry for pS6. Bar graph shows mean MFIs of pS6 ± SEM from (A) three independent experiments or (B) triplicate measurements from a single experiment, which is representative of three independent experiments. *P < 0.05, **P < 0.01, by unpaired student's t test. (C) Primary mouse CD4⁺ T cells were left untreated or were stimulated with anti-CD3 and anti-CD28 antibodies for indicated times. Cell lysates were then analyzed by Western blotting for p70S6K (top) or β-actin (bottom). Blots are representative of three independent experiments.

Fig. 6. MALT1 activity is required for T cell proliferation, but not for acute blasting or early activation

(A) Primary mouse CD4⁺ T cells were pretreated with vehicle or z-VRPR-fmk before being stimulated with anti-CD3 and anti-CD28 antibodies for the indicated times. Numbers of live cells were determined by trypan blue exclusion. Data are means ± SEM of three independent experiments. **P < 0.01, by unpaired student's t test. (B) Primary mouse CD4⁺ T cells pretreated with either vehicle or inhibitor in the presence of exogenous IL-2 were stimulated with anti-CD3 and anti-CD28 antibodies for the indicated times and then cell proliferation was assessed by MTT assay. Data are means ± SEM of three independent experiments. *P < 0.05; **P < 0.01, by unpaired student's t test. (C) Primary mouse CD4⁺ T cells were pretreated with vehicle or z-VRPR-fmk, stimulated for the indicated times, and then analyzed by flow cytometry for forward scatter (FSC), an indicator of cell size. Bar graph shows mean FSC values ± SEM of vehicle- and inhibitor-treated cells from three experiments. ns, not significant. (D) Mouse CD4⁺ T cells were pretreated with vehicle or z-VRPR-fmk before being stimulated with anti-CD3 and anti-CD28 antibodies for the indicated times, and then were analyzed for cell-surface abundance of CD69 by flow cytometry. Bar graph shows mean CD69 MFI values ± SEM of vehicle- and inhibitor-treated cells for the indicated times from three experiments.

Fig. 7. MALT1 activity is required for TCR-dependent changes in T cell metabolism

(A and B) Naïve CD4⁺ T cells were isolated from C57BL/6 mice, pretreated with vehicle or the indicated inhibitors, and stimulated with anti-CD3 and anti-CD28 antibodies. After 24 hours, cells were analyzed on a Seahorse XF24 analyzer to determine (A) oxygen consumption rate (OCR) and (B) extracellular acidification rate (ECAR), which are measures of oxidative phosphorylation and glycolysis, respectively. Spare respiratory capacity (SRC) refers to the amount of additional ATP (above baseline) that can be produced by mitochondria in the presence of the ionophore uncoupling agent FCCP. The maximal respiratory capacity (“Max Resp”) is determined by the difference between the response to FCCP and complete inhibition of the electron transport chain by rotenone. *P < 0.05, by unpaired student's t test. (C) After Seahorse analysis, the cells shown in (A) and (B) were analyzed by flow cytometry to determine the relative cell-surface abundance of CD69 on the indicated cells. Data are representative of three independent experiments.

Fig. 8. Model for the regulation of mTOR signaling by Carma1 and MALT1 in response to TCR signaling in T cells

Our data suggest that Carma1 and MALT1 participate in the TCR-dependent activation of mTOR downstream of PKC. The other CBM component, Bcl10, seems to be dispensable for this pathway, although it is possible that another protein substitutes for Bcl10 in mediating an association between Carma1 and MALT1 (indicated by the oval with a question mark). See the Discussion for more details.