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Journal of Molecular Medicine

, Volume 97, Issue 5, pp 675–690 | Cite as

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

  • Marilena Pontoriero
  • Giuseppe FiumeEmail author
  • Eleonora Vecchio
  • Annamaria de Laurentiis
  • Francesco Albano
  • Enrico Iaccino
  • Selena Mimmi
  • Antonio Pisano
  • Valter Agosti
  • Emilia Giovannone
  • Annalisa Altobelli
  • Carmen Caiazza
  • Massimo Mallardo
  • Giuseppe Scala
  • Ileana QuintoEmail author
Original Article

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.

Keywords

B-cell receptor NF-κB BTK IκB-α tyrosine phosphorylation 

Notes

Author contribution

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.

Funding information

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.

Compliance with ethical standards

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.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

109_2019_1777_MOESM1_ESM.docx (353 kb)
ESM 1 (DOCX 352 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Marilena Pontoriero
    • 1
  • Giuseppe Fiume
    • 1
    Email author
  • Eleonora Vecchio
    • 1
  • Annamaria de Laurentiis
    • 1
  • Francesco Albano
    • 1
  • Enrico Iaccino
    • 1
  • Selena Mimmi
    • 1
  • Antonio Pisano
    • 1
  • Valter Agosti
    • 1
  • Emilia Giovannone
    • 1
  • Annalisa Altobelli
    • 2
  • Carmen Caiazza
    • 2
  • Massimo Mallardo
    • 2
  • Giuseppe Scala
    • 1
  • Ileana Quinto
    • 1
    Email author
  1. 1.Department of Clinical and Experimental MedicineUniversity of Catanzaro “Magna Græcia”CatanzaroItaly
  2. 2.Department of Molecular Medicine and Medical BiotechnologyUniversity of Naples “Federico II”NaplesItaly

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