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Activation of NF-κB in B cell receptor signaling through Bruton’s tyrosine kinase-dependent phosphorylation of IκB-α

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Abstract

The antigen-mediated triggering of B cell receptor (BCR) activates the transcription factor NF-κB that regulates the expression of genes involved in B cell differentiation, proliferation, and survival. The tyrosine kinase Btk is essentially required for the activation of NF-κB in BCR signaling through the canonical pathway of IKK-dependent phosphorylation and proteasomal degradation of IκB-α, the main repressor of NF-κB. Here, we provide the evidence of an additional mechanism of NF-κB activation in BCR signaling that is Btk-dependent and IKK-independent. In DeFew B lymphoma cells, the anti-IgM stimulation of BCR activated Btk and NF-κB p50/p65 within 0.5 min in absence of IKK activation and IκB-α degradation. IKK silencing did not affect the rapid activation of NF-κB. Within this short time, Btk associated and phosphorylated IκB-α at Y289 and Y305, and, concomitantly, p65 translocated from cytosol to nucleus. The mutant IκB-α Y289/305A inhibited the NF-κB activation after BCR triggering, suggesting that the phosphorylation of IκB-α at tyrosines 289 and 305 was required for NF-κB activation. In primary chronic lymphocytic leukemia cells, Btk was constitutively active and associated with IκB-α, which correlated with Y305-phosphorylation of IκB-α and increased NF-κB activity compared with healthy B cells. Altogether, these results describe a novel mechanism of NF-κB activation in BCR signaling that could be relevant for Btk-targeted therapy in B-lymphoproliferative disorders.

Key messages

  • Anti-IgM stimulation of BCR activates NF-κB p50/p65 within 30 s by a Btk-dependent and IKK-independent mechanism.

  • Btk associates and phosphorylates IκB-α at Y289 and Y305, promoting NF-κB activation.

  • In primary CLLs, the binding of Btk to IκB-α correlates with tyrosine phosphorylation of IκB-α and increased NF-κB activity.

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Funding

This work was supported by the following grants: Ministero della Salute RF-2010-2306943 (to G. S.), MIUR-POR CALABRIA FSE 2007/2013 (to G. S.), MIUR-PRIN 2012CK5RPF (to G. S.), MIUR-PRIN 2006052835_004 and MIUR-PRIN 2012CK5RPF_002 (to I. Q.), and MIUR-Finanziamento individuale attività base di ricerca (to G. F.). S. M. was supported by a fellowship from the Fondazione Italiana per la Ricerca sul Cancro.

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Authors and Affiliations

  1. Department of Clinical and Experimental Medicine, University of Catanzaro “Magna Græcia”, Viale Europa, Germaneto, 88100, Catanzaro, Italy

    Marilena Pontoriero, Giuseppe Fiume, Eleonora Vecchio, Annamaria de Laurentiis, Francesco Albano, Enrico Iaccino, Selena Mimmi, Antonio Pisano, Valter Agosti, Emilia Giovannone, Giuseppe Scala & Ileana Quinto

  2. Department of Molecular Medicine and Medical Biotechnology, University of Naples “Federico II”, Via S. Pansini, 80131, Naples, Italy

    Annalisa Altobelli, Carmen Caiazza & Massimo Mallardo

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  1. Marilena Pontoriero
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  2. Giuseppe Fiume
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  3. Eleonora Vecchio
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  4. Annamaria de Laurentiis
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  5. Francesco Albano
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Contributions

M.P. and G.F. analyzed the physical and functional interactions of Btk and IκB-α in the DeFew and CLL cells; M.P. generated the appropriate mutants; E.V. performed gene expression microarray and ChIP; F.A., A.P., E.I., and S.M. produced expression vectors; V.A. and E.G. gave technical advice for flow cytometry; C.C., A.A., and M.M. performed some Western blottings; I.Q., G.S., and G.F. conceived the experimental plan and wrote the manuscript.

Corresponding authors

Correspondence to Giuseppe Fiume or Ileana Quinto.

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Ethics statements

Experiments involving human subjects were approved by the Italian Regional “Calabria” Ethics Committee (Protocol N. 75, 23/03/17), in accordance with the ethical and safety rules and guidelines provided by the relevant Italian laws (art. 4–5 of D.lgs 116/92, DD.MM. of 29/09/1995 and 26/04/2000), and in accordance with the ethical guidelines of the European Community Council (directive n. 86/609/ECC). Blood samples from healthy donors or CLL patients were obtained upon written and oral informed consent from the participants to the study.

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The authors declare that they have no conflict of interest.

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Marilena Pontoriero and Giuseppe Fiume are co-first authors

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Pontoriero, M., Fiume, G., Vecchio, E. et al. Activation of NF-κB in B cell receptor signaling through Bruton’s tyrosine kinase-dependent phosphorylation of IκB-α. J Mol Med 97, 675–690 (2019). https://doi.org/10.1007/s00109-019-01777-x

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