The CARMA1-Bcl10 signaling complex selectively regulates JNK2 kinase in the T cell receptor-signaling pathway

Abstract

Members of the c-Jun NH(2)-terminal kinase (JNK) family play crucial roles in cell activation, differentiation, and apoptosis. Although many studies have indicated that JNK1 and JNK2 have functional differences and redundancy, the upstream signaling pathway that selectively activates JNK1 or JNK2 remains unknown. In this study, we have revealed a selective mechanism of JNK activation, in which JNK2, but not JNK1, was regulated by CARMA1, a scaffold molecule, after stimulation of the T cell receptor (TCR). This CARMA1-dependent regulation of JNK2 worked through the scaffold molecule Bcl10, which was inducibly associated with JNK2 and served as a JNK-interacting protein (JIP)-like scaffold to assemble the kinases JNK2, MKK7, and TAK1. Finally, we showed that CARMA1- and Bcl10-mediated JNK2 activation had a critical role in regulating the amount of c-Jun protein. Together, our studies provide genetic evidence that JNK1 and JNK2 are differentially regulated in the TCR-signaling pathway and play different functions.

Figure 1

CARMA1-deficient (JPM50.6) cells are defective in TCR-induced JNK2 phosphorylation. (A-C) Jurkat or JPM50.6 cells (6×10⁶/sample) were stimulated with or without anti-CD3 plus anti-CD28 antibodies (6 and 3 μg/ml, respectively) for various time points (A, B) or with PMA (40 ng/ml) plus ionomycin (100 ng/ml) or with sorbitol (0.2 mM) (C). Whole cell lysates were subjected to SDS-PAGE and analyzed by Western blot using anti-phospho-JNK, anti-JNK, anti-CARMA1, anti-phospho-ERK or anti-ERK antibodies. (D) The cells (1×10⁷/sample) were treated with different stimulators for 20 min and cell lysates were analyzed using phospho-JNK2 ELISA kit (R&D System). (E) CARMA1-deficient cells (JPM50.6) or JPM50.6 cells reconstituted with CARMA1 (50.6 WT) were stimulated with or without anti-CD3 (6 μg/ml) and anti-CD28 antibodies (3 μg/ml) or PMA (40 ng) plus ionomycin (100 ng) or sorbitol (0.2 mM) for 20 min. The cell lysates were subjected to SDS-PAGE and examined by Western blots using anti-phospho-JNK, anti-JNK or anti-CARMA1 antibodies.

Figure 2

JNK2 kinase activity is defective in the absence of CARMA1. (A) Schematic diagram of JNK isoforms. (B) In vitro kinase assay. The cells (1×10⁷/sample) were stimulated with or without anti-CD3 plus anti-CD28 antibodies and cell lysates were precipitated with anti-JNK1 or JNK2 antibodies for 4 hours. The immunocomplexes were incubated with GST-c-Jun(1-79) substrate (1 μg/sample) and [γ-³²P]ATP in kinase buffer at 30°C for 30 min. The phosphorylated proteins were resolved by SDS-PAGE, transferred to Nylon membranes, and analyzed by autoradiography and immunoblotting. (C-E) Jurkat or JPM50.6 cells were transfected with Flag-JNK1p46, HA-JNK2p54 or Myc-JNK1p54 by electroporation. 24 hours later cells were stimulated and overexpressed proteins were immunopracipitated with anti-Flag, anti-HA or anti-Myc antibodies. Kinase assay was performed as described in 2B.

Figure 3

Primary cells from CARMA1 or Bcl10 null mice are defective in JNK2 phosphorylation. Lymph nodes T cells from wild type, CARMA1^−/− (A) or Bcl10^−/− (B) mice were stimulated with or without PMA (20 ng/ml) plus ionomycin (200 ng/ml). (C) Splenic B cells and bone marrow-derived mast cells were isolated from wild type, CARMA1^−/−, and Bcl10^−/− mice and stimulated with or without PMA (20 ng/ml) plus ionomycin (200 ng/ml). Whole cell lysates were subjected to SDS-PAGE and analyzed by Western blot using indicated antibodies.

Figure 4

Bcl10 inducibly associates with JNK2, but not with JNK1. Jurkat or JPM50.6 cells (3×10⁷/sample) were stimulated with or without PMA (40 ng/ml) plus ionomycin (100 ng/ml). The cells were lysed and precipitated with anti-JNK2, anti-Bcl10, or anti-JNK1 antibodies (A, B). Immunocomplexes (top and middle panels) or whole cell lysates (bottom panel) were subjected to SDS-PAGE and analyzed by Western blot using indicated antibodies. (C) Plasmids used in overexpression experiments. (D, E) HEK293 cells (7×10⁵) were transiently transfected with 0.5 μg of expression plasmids encoding several constructs: HA-JNK2p54, HA-JNK2p46, Myc-Bcl10 (D) or Myc-Bcl10(L47A) (E). Cells were lysed and precipitated with anti-HA antibodies. The immunocomplexes were subjected to SDS-PAGE and analyzed by Western blot using indicated antibodies. The levels of expression of the transfected products were assessed with anti-Myc and anti-HA immunoblots of total cell lysates.

Figure 5

Bcl10 inducibly associates with TAK1 and MKK7 upon PMA/ionomycin stimulation. Jurkat or JPM50.6 cells (3×10⁷/sample) were stimulated with or without PMA (40 ng/ml) plus ionomycin (100 ng/ml). The cells were lysed and precipitated with anti-TAK1 (A), or anti-MKK7 antibodies (B). Immunocomplexes or whole cell lysates were subjected to SDS-PAGE and analyzed by Western blot using indicated antibodies. (C) Timed immunoprecipitation. Jurkat cells (12×10⁷/time point) were stimulated with or without PMA plus ionomycin for 10 or 20min and lyzed in 1 ml of lysis buffer. Each lysate was divided into four portions and immunoprecipitated with anti-JNK2, anti-MKK7, anti-TAK1, and anti-MALT1 antibodies. The immunocomplexes were subjected to SDS-PAGE and analyzed by Western blot using indicated antibodies.

Figure 6

TCR-induced c-Jun accumulation is supressed in CARMA1-deficient cells. (A) Jurkat or JPM50.6 cells were stimulated with or without anti-CD3 and anti-CD28 antibodies (6 and 3 μg/ml, respectively) for various time points, or (B) NEMO-deficient Jurkat cells or JPM50.6 cells were stimulated with or without PMA (40 ng/ml) plus anti-CD28 antibodies (3 μg/ml) for various time points. Cell lysates were subjected to SDS PAGE and Western blot analysis using indicated antibodies. (C and D) Jurkat T cells were pre-incubated with cycloheximide (CHX) (5 μg/ml), MG132 (5 μg/ml) (C), or actinomycin D (ActD) (5 μg/ml) (D) for 30 min, and then stimulated with or without anti-CD3 plus anti-CD28 antibodies. Cell lysates were subjected to SDS PAGE and Western blot analysis using indicated antibodies. (E) Cells were stimulated with or without anti-CD3 plus anti-CD28 antibodies (30 or 60 min) and total RNA was isolated using RNA isolation kit (Qiagen) followed by reverse transcription polymerase chain reaction (RT-PCR). An aliquot (2 μl) of first-strand cDNA was used as a template in PCR amplifications of c-Jun and GAPDH transcripts. Amplification products were resolved on a 2% agarose gel.

Figure 7

AP-1 activation is not affected in activated CARMA1-deficient cells. (A) Jurkat or JPM50.6 cells (1×10⁷/sample) were pre-incubated with DMSO or MG132 for 30 min and than stimulated with or without anti-CD3 plus anti-CD28 antibodies (6 and 3 μg/ml, respectively) for 30 min. Nuclear extracts were prepared and AP-1 binding activities were analyzed by electrophoretic mobility shift assay (EMSA) using ³²P-labeled probes containing AP-1 or Oct-1 binding sites. (B) Cells were transfected by electroporation with reporter plasmid encoding 4xAP-1-luc (10 μg) and pEF-Renilla-luc (200 ng). Twenty hours later, the transfected cells were either untreated or stimulated with anti-CD3 plus anti-CD28 antibodies or PMA plus ionomycin for 6 hr. Cell lysates were prepared and luciferase activities were measured with Dual-Luciferase assay kits (Promega, Madison, WI). AP-1 activities were determined by normalization of AP-1-dependent luciferase to Renilla luciferase activity. (C) The model of TCR-induced JNK phosphorylation. CD3-CD28 costimulation activates various proximal signaling components that lead to activation of PKC and CARMA1, which, in turn, recruits Bcl10 and induces its oligomerization. The activated (oligomerized) Bcl10 serves as a template for TAK1, MKK7 and JNK2. Phosphorylated JNK2 and independently activated JNK1 lead to accumulation of c-Jun and activation of AP-1.