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Comparative Study
. 2008 Feb 26;105(8):3023-8.
doi: 10.1073/pnas.0712313105. Epub 2008 Feb 19.

NEMO recognition of ubiquitinated Bcl10 is required for T cell receptor-mediated NF-kappaB activation

Chuan-Jin Wu  1 , Jonathan D Ashwell
Affiliations
Comparative Study

NEMO recognition of ubiquitinated Bcl10 is required for T cell receptor-mediated NF-kappaB activation

Chuan-Jin Wu et al. Proc Natl Acad Sci U S A. .

Abstract

The mechanism by which the Carma1-Bcl10-MALT1 (CBM) complex couples T cell antigen receptor (TCR) signaling to IkappaB kinase (IKK) and NF-kappaB activation is not known. Here, we show that Bcl10 undergoes K63-linked polyubiquitination in response to T cell activation and subsequently binds NEMO, the regulatory subunit of IKK. This interaction requires the ubiquitin-binding activity of NEMO. The sites of Bcl10 ubiquitination were mapped to K31 and K63. Mutation of these residues did not affect TCR signaling-induced CBM complex assembly but prevented Bcl10 ubiquitination, NEMO binding, and NF-kappaB activation. Therefore, the regulated ubiquitination of Bcl10 and its recognition by NEMO are a critical link between the CBM complex, IKK recruitment, and NF-kappaB activation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
NEMO binding to K63-linked polyubiquitin chains is required for TCR-mediated IKK/NF-κB activation. (A) NEMO-deficient Jurkat cells and NEMO-deficient Jurkat cell clones stably transfected with HA-NEMO or HA-NEMO(L329P) were treated with PMA/iono for the indicated times, and IκB levels were determined by immunoblotting. (B) NEMO-deficient Jurkat cells were transiently transfected with NF-κB-driven luciferase reporter and β-gal plasmids in combination with pMSCVpuro or pMSCVpuro encoding HA-NEMO or HA-NEMO(L329P). After 36 h, the cells were treated with or without PMA/iono for 5 h. Cell lysate luciferase activities were determined and normalized to β-gal activity.
Fig. 2.
Fig. 2.
Bcl10 in activated T cells undergoes K63-linked polyubiquitination and is bound by recombinant NEMO. (A) Jurkat cells or Carma1-deficient Jurkat cells were stimulated with or without PMA/iono for 20 min. Bcl10 was immunoprecipitated (IP) from denatured cell lysates and immunoblotted (IB) with anti-ubiquitin. WT, wild type. (B) Jurkat cells stably expressing MALT1 shRNA were immunoblotted with anti-MALT1 (Upper). Jurkat cells or the MALT1 shRNA-expressing Jurkat cells were treated with or without PMA/iono for 15 min and analyzed for Bcl10 ubiquitination as in A (Lower). (C) Jurkat cells transfected with HA-tagged K63-only or K48-only ubiquitin were stimulated or not with PMA/iono for the indicated times, harvested, and lysed. The lysates were treated as in A to disrupt protein–protein interactions, and Bcl10 was immunoprecipitated. The amount of K63-linked or K48-linked polyubiquitinated Bcl10 was determined by immunoblotting with anti-HA (Left). Expression of both HA-Ub mutants was similar, as judged by immunoblotting unstimulated cell lysates with anti-HA (data not shown). (D) Jurkat cells were stimulated with or without PMA/iono or anti-TCR C305/anti-CD28 for 20 min, harvested, and lysed. Cell lysates containing equal amounts of protein were incubated with GST-NEMO-coated beads. The bound material (Upper) and cell lysates (Lower) were immunoblotted with anti-Bcl10. (E) Cell lysates from Jurkat cells treated with or without PMA/iono for 20 min were incubated with GS-beads bound to GST-NEMO or GST-NEMO(L329P). The bound material was immunoblotted with anti-Bcl10. Ponceau S staining showed equal loading of the GST fusion proteins (data not shown).
Fig. 3.
Fig. 3.
NEMO binds to activation-induced polyubiquitinated Bcl10 in vivo. (A) Lysates of Jurkat cells that were stimulated with PMA/iono for the indicated times and lysates immunoprecipitated (IP) with anti-NEMO, resolved on SDS/PAGE, and immunoblotted (IB) with anti-Bcl10 or anti-NEMO. (B) Jurkat cells were treated with or without C305/anti-CD28 for 15 min, and lysates were immunoprecipitated and analyzed for Bcl10 and NEMO as in A. (C) NEMO-deficient Jurkat cells stably reconstituted with HA-NEMO (C1, see Fig. 1A) or HA-NEMO(L329P) (C1, see Fig. 1A) were stimulated with PMA/iono for 15 min, immunoprecipitated, and analyzed for Bcl10 and HA as in A.
Fig. 4.
Fig. 4.
Bcl10 K31 and K63 are required for activation-induced Bcl10 ubiquitination and binding to NEMO. Jurkat cells transiently expressing (A) FLAG-Bcl10 or FLAG-Bcl10(K31R), or (B) FLAG-Bcl10, FLAG-Bcl10(K63R/K67R), or FLAG-Bcl10(K98R), or (C) FLAG-Bcl10, FLAG-Bcl10(K31R/K63R), FLAG-Bcl10(K31R/K67R), FLAG-Bcl10(K44R/K45R), or FLAG-Bcl10(K105R/K110R/K115R) for 36 h were treated with PMA/iono for 15 min. Cell lysates were incubated with bead-bound GST-NEMO, and the proteins pulled down were immunoblotted (IB) with anti-FLAG. Cell lysates used for GST pulldown were also immunoblotted with anti-FLAG to determine the expression of the transfected FLAG-Bcl10 constructs. Equal amounts of GST-NEMO were used in each pulldown as confirmed by Ponceau S staining or immunoblotting with anti-GST (data not shown).
Fig. 5.
Fig. 5.
Bcl10 K31 and K63 are required for activation-induced Bcl10 ubiquitination and Bcl10 degradation. (A) Jurkat cells were transfected with FLAG-Bcl10 or FLAG-Bcl10(K31R/K63R) for 36 h and stimulated with PMA/iono for 15 min. Cell lysates were immunoprecipitated (IP) with anti-FLAG. The immunoprecipitates were eluted in lysis buffer containing SDS by heating, diluted with lysis buffer, reimmunoprecipitated with anti-Bcl10, and immunoblotted with anti-Ub or anti-Bcl10. (B) Jurkat cells were transfected with FLAG-Bcl10 and FLAG-Bcl10(K31R/K63R) for 36 h, then stimulated with PMA/iono (P+I) for the indicated times. Cell lysates were immunoblotted with anti-Bcl10.
Fig. 6.
Fig. 6.
Bcl10 K31 and K63 are required for activation-induced IKK/NF-κB activation but not CBM assembly. (A) Jurkat cells were infected with virus containing pRSMX-Bcl10 shRNA. A stable clone with very low levels of Bcl10 (Bcl10KD) as detected by immunoblotting was selected for further analysis. (B) Bcl10 shRNA-expressing Bcl10KD cells were transfected with a plasmid encoding EGFP plus pMSCVpuro or pMSCVpuro encoding Bcl10 shRNA-resistant FLAG-Bcl10 or FLAG-Bcl10(K31R/K63R) for 24 h, and EGFP-positive cells were isolated by cell sorting. Twenty-four h later, the EGFP-positive cells were treated with PMA/iono for the indicated times, and cell lysates were immunoblotted with anti-phospho-IκB or anti-FLAG. (C and D) Bcl10 shRNA-expressing Bcl10KD cells were transiently transfected with an NF-κB luciferase reporter and a β-gal plasmid, in combination with vector, Bcl10 shRNA-resistant FLAG-Bcl10, or the indicated FLAG-Bcl10 mutants for 36 h. The transfected cells were treated with PMA/iono or C305/anti-CD28 (C), or C305/anti-CD28 or TNF-α (D), for 6 h, and NF-κB luciferase activity was measured and normalized to β-gal activity. The experiment was performed in triplicate, and error bars represent the SEM. FLAG-Bcl10 expression from one set of transfected cells is shown. (E) Jurkat cells were transfected with the indicated plasmids and stimulated with PMA/iono for 15 min. Cell lysates were subjected to immunoprecipitation with anti-FLAG and immunoblotting with anti-Carma1, anti-MALT1, and anti-Bcl10.
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