Phosphorylation of Bcl10 negatively regulates T-cell receptor-mediated NF-kappaB activation

Abstract

Bcl10 (B-cell lymphoma 10) is an adaptor protein comprised of an N-terminal caspase recruitment domain and a C-terminal serine/threonine-rich domain. Bcl10 plays a critical role in antigen receptor-mediated NF-kappaB activation and lymphocyte development and functions. Our current study has discovered that T-cell activation induced monophosphorylation and biphosphorylation of Bcl10 and has identified S138 within Bcl10 as one of the T-cell receptor-induced phosphorylation sites. Alteration of S138 to an alanine residue impaired T-cell activation-induced ubiquitination and subsequent degradation of Bcl10, ultimately resulting in prolongation of TCR-mediated NF-kappaB activation and enhancement of interleukin-2 production. Taken together, our findings demonstrate that phosphorylation of Bcl10 at S138 down-regulates Bcl10 protein levels and thus negatively regulates T-cell receptor-mediated NF-kappaB activation.

FIG. 1.

Bcl10 is monophosphorylated and biphosphorylated following TCR stimulation. (A) Bcl10 is phosphorylated following TCR engagement in Jurkat cells. Jurkat cells were labeled with [³²P]orthophosphate followed by incubation in medium alone (−) or in the presence (+) of anti-CD3 (α-CD3) plus anti-CD28 (α-CD28) for 15 min. Cell lysates were immunoprecipitated with anti-Bcl10 (α-Bcl10) antibodies and then subjected to SDS-PAGE, followed by autoradiography or Western blot analysis with α-Bcl10 antibodies. (B) TCR engagement induces two phosphorylated isoforms of Bcl10. Jurkat cells were untreated (−) or stimulated (+) with α-CD3 plus α-CD28 for 15 min. Cell lysates were subjected to 2-D electrophoresis with IEF (pH 5 to 8) in one dimension and gradient SDS-PAGE in the second dimension, followed by Western blot analysis with α-Bcl10 antibodies. (C) The major Bcl10 isoform existing in T cells is a nonphosphorylated form. Jurkat cells were stimulated with α-CD3 plus α-CD28. Cell lysates were untreated or were treated with λ-PPase and subjected to 2-D electrophoresis followed by Western blot analysis with α-Bcl10 antibodies. Arrows point to Bcl10 isoforms, and phosphorylated Bcl10 isoforms are enclosed by rectangles.

FIG. 2.

S138 is one of the activation-dependent phosphorylation sites. (A) Bcl10 expressed in GP+E86 cells contains a phosphorylation site(s) between residues 130 and 157. Cell lysates (20 μg) from the retrovirus packaging cells GP+E86 that express wild-type Bcl10, truncation mutant Bcl10(1-157), or truncation mutant Bcl10(1-130) were treated with (+) λ-PPase or were left untreated (−), followed by SDS-PAGE and Western blot analysis with anti-Bcl10 (α-Bcl10) antibodies. (B) Bcl10 expressed in Jurkat cells contains a phosphorylation site(s) between residues 130 and 157. Jurkat cells expressing Bcl10(1-157) or Bcl10(1-130) were untreated or were stimulated with α-CD3 plus α-CD28 for 15 min. The cells were then collected, and cell lysates were resolved on SDS-PAGE followed by Western blot analysis with the indicated antibodies. (C) Flag-tagged wild-type Bcl10 has two TCR-induced phosphorylated isoforms, the same as endogenous Bcl10. Jurkat cells expressing wild-type Flag-tagged Bcl10 (Flag-Bcl10) were untreated (−) or were stimulated (+) with α-CD3 plus α-CD28 for 15 min. Cell lysates were subjected to 2-D electrophoresis, followed by Western blot analysis, first with α-Flag antibodies and later with α-Bcl10 antibodies. (D) Replacement of S141/S144 with alanine does not affect the two TCR-induced phosphorylated isoforms of Bcl10. Jurkat cells expressing Flag-Bcl10(S141A/S144A) were treated and analyzed as described for panel C. (E) Replacement of S134/S138 with alanine abolishes one of the two TCR-induced phosphorylated isoforms of Bcl10. Jurkat cells expressing Flag-Bcl10(S134A/S138A) were treated and analyzed as described for panel C. (F) Replacement of S138 with alanine abolishes one of the two TCR-induced phosphorylated isoforms of Bcl10. Jurkat cells expressing Flag-Bcl10(S138A) were treated and analyzed as described for panel C. (G) Replacement of S134 with alanine does not affect the two TCR-induced phosphorylated isoforms of Bcl10. Jurkat cells expressing Flag-Bcl10(S134A) were treated and analyzed as described for panel C. (H) S138 is an activation-dependent phosphorylation site in Bcl10, as demonstrated by a [³²P]orthophosphate labeling experiment. Jurkat cells expressing Flag-Bcl10 or Flag-Bcl10(S134A/S138A) were labeled with [³²P]orthophosphate, followed by incubation in medium alone (−) or in the presence (+) of α-CD3 plus α-CD28 for 15 min. Cell lysates were immunoprecipitated (IP) with α-Bcl10 antibodies and then subjected to SDS-PAGE, followed by autoradiography or Western blot analysis with α-Bcl10 antibodies. (C to G) Arrows point to Bcl10 isoforms, and phosphorylated Bcl10 isoforms are enclosed by rectangles.

FIG. 3.

Replacement of S134/S138 or S138 alone with alanine impairs activation-induced degradation of Bcl10 in lymphocytes. (A) The Bcl10 protein level is down-regulated after TCR stimulation. Jurkat cells expressing Flag-Bcl10 were stimulated with anti-CD3 (α-CD3) plus α-CD28 for the indicated times. Cell lysates were subjected to SDS-PAGE and Western blot analysis with α-Flag or α-actin antibodies. (B) Bcl10 degradation is insensitive to proteasome inhibitor treatment. Jurkat cells expressing Flag-Bcl10 were left untreated or were treated with MG132 for 40 min. The cells were then stimulated with α-CD3 plus α-CD28 for the indicated times, followed by Western blot analysis with the indicated antibodies. (C) Replacement of S134/S138 or S138 alone with alanine impairs TCR-induced Bcl10 degradation in Jurkat cells. The cells were then stimulated with α-CD3 plus α-CD28 for the indicated times, followed by Western blot analysis with the indicated antibodies. (D) Replacement of S134/S138 with alanine impairs activation-induced Bcl10 degradation in primary splenic B cells. LPS-activated Bcl10-deficient splenic B cells were reconstituted with wild-type (WT) Bcl10 or Bcl10(S134A/S138A) by retrovirus transduction. Subsequently, the retrovirus-transduced GFP⁺ cells were sorted and stimulated with PMA plus ionomycin for the indicated times. Cell lysates were subjected to SDS-PAGE and Western blot analysis with α-Bcl10 or α-actin antibodies. (E) Replacement of S134/S138 with alanine impairs activation-induced Bcl10 degradation in primary large pre-B cells. BM culture-derived Bcl10-deficient large pre-B cells were reconstituted with wild-type Bcl10 or Bcl10(S134A/S138A) by retrovirus transduction. Subsequently, the retrovirus-transduced GFP⁺ cells were sorted and stimulated with PMA plus ionomycin for the indicated times. Cell lysates were subjected to SDS-PAGE and Western blot analysis with α-Bcl10 or α-actin antibodies. E-Bcl10, endogenous Bcl10. DMSO, dimethyl sulfoxide.

FIG. 4.

Replacement of S134/S138 or S138 alone with alanine impairs activation-induced ubiquitination of Bcl10 in Jurkat cells. (A) Endogenous Bcl10 is ubiquitinated following activation in Jurkat cells. Jurkat cells were stimulated with PMA plus ionomycin (P + I) for the indicated times. Duplicate cell samples were collected, and cell lysates were immunoprecipitated (IP) with anti-Bcl10 (α-Bcl10) antibody (sc-9560). Subsequently, one set of samples was subjected to Western blot analysis with α-ubiquitin (α-Ubi) (sc-8017) antibody, whereas the other set was subjected to Western blot analysis with α-Bcl10 (sc-5273) antibody. (B). Anti-Flag antibody does not nonspecifically associate with ubiquitinated Bcl10. Parental Jurkat cells or Jurkat cells expressing Flag-Bcl10 were stimulated with α-CD3 plus α-CD28 for the indicated times. Duplicate cell samples were collected, and cell lysates were immunoprecipitated with α-Flag antibodies. Subsequently, one set of samples was subjected to Western blot analysis with α-ubiquitin antibody (sc-8017), whereas the other set was subjected to Western blot analysis with α-Bcl10 antibodies. (C) Replacement of S134/S138 or S138 with alanine directly reduces ubiquitination of Bcl10 but not Bcl10-associated protein. Jurkat cells expressing Flag-Bcl10, Flag-Bcl10(S134A/S138A), or Flag-Bcl10(S138A) were treated with 0.4 μM MG132 for 1 h and then were stimulated with α-CD3 plus α-CD28 for the indicated times. The samples were collected and lysed in the presence of 1% SDS and boiled for 5 min. The cell lysates were diluted 10-fold with the lysis buffer in the absence of SDS before being immunoprecipitated with α-Flag antibodies. Subsequently, the immunoprecipitates were subjected to Western blot analysis with α-ubiquitin antibodies (a gift from Arthur L. Hass). The data shown are representative of two independent experiments. *, immunoglobulin light chain; **, immunoglobulin heavy chain; Ubi_n, polyubiquitin; #, nonspecific band; WT, wild type.

FIG. 5.

Replacement of S134/S138 or S138 alone with alanine prolongs TCR-induced NF-κB activation. Jurkat cells expressing Flag-Bcl10, Flag-Bcl10(S134A/S138A) (A), or Flag-Bcl10(S138A) (B) were stimulated with anti-CD3 (α-CD3) plus α-CD28 for the indicated times. Subsequently, the cells were subjected to gel shift assays for NF-κB activity with the ³²P-labeled oligonucleotide containing the NF-κB binding site in the IL-2 gene promoter as a probe. Gel shift for Oct1 binding was used as a loading control. The data shown are representative of at least two independent experiments.

FIG. 6.

Replacement of S134/S138 or S138 alone with alanine enhances TCR-induced IL-2 production. (A) Replacement of S134/S138 with alanine enhances TCR-induced IL-2 production in Jurkat cells. Jurkat cells expressing Flag-Bcl10 or Flag-Bcl10(S134A/S138A) were stimulated in 96-well plates with anti-CD3 (α-CD3) or α-CD3 plus α-CD28, and then cell supernatants were harvested and subjected to assays for IL-2 biological activity. (B) Replacement of S138 with alanine enhances TCR-induced IL-2 production in Jurkat cells. Jurkat cells expressing Flag-Bcl10 or Flag-Bcl10(S138A) were stimulated, and IL-2 production was examined as described for panel A. (C) Flag-Bcl10, Flag-Bcl10(S134A/S138A), and Flag-Bcl10(S138A) are expressed at equivalent levels in Jurkat cells. (D) Replacement of S134/S138 with alanine enhances TCR-induced IL-2 production in splenic T cells. Bcl10-deficient splenic T cells were reconstituted with wild-type Bcl10 or Bcl10(S134A/S138A) by retrovirus transduction. Subsequently, the retrovirus-transduced GFP⁺ cells were sorted and rested overnight. Sorted cells (10⁵) were stimulated in 96-well plates with α-CD3 for 18 h, and the supernatants were harvested and subjected to assays for IL-2 biological activity. The bottom panel shows the FACS analysis of GFP fluorescence intensities in T cells reconstituted with wild-type Bcl10 or Bcl10(S134A/S138A). (E) Replacement of S134/S138 with alanine enhances TCR-induced IL-2 production by T cells in a BM transplantation system. GFP⁺ Thy1.2⁺ T cells were sorted from the splenocytes derived from the recipients of wild-type BM transduced with retrovirus expressing Bcl10 or Bcl10(S134A/S138A). Following stimulation of these T cells in 96-well plates with α-CD3, the supernatants were harvested and subjected to assays for IL-2 biological activity. The bottom panel shows the FACS analysis of GFP fluorescence intensities in T cells reconstituted with wild-type Bcl10 or Bcl10(S134A/S138A). Bars represent mean induction and standard deviation of triplicate samples within an experiment. Data presented in panels A to E are representative of at least two independent experiments. WT, wild type.

FIG. 7.

Schematic illustration of the role of Bcl10 phosphorylation in its degradation and NF-κB activation. Engagement of TCR and CD28 results in the recruitment of IKK to the CARMA1/Bcl10/MALT complex in the lipid rafts and subsequent activation of IKK. Activated IKK phosphorylates IκB and consequently leads to NF-κB activation. In the meantime, Bcl10 is phosphorylated at S138, likely by IKK, resulting in ubiquitination and degradation of Bcl10. Reduction of Bcl10 protein causes attenuation of NF-κB activation.