Activation of NF-κB in B cell receptor signaling through Bruton’s tyrosine kinase-dependent phosphorylation of IκB-α
- 244 Downloads
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.
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.
KeywordsB-cell receptor NF-κB BTK IκB-α tyrosine phosphorylation
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.
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
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.
- 5.Colomer C, Marruecos L, Vert A, Bigas A, Espinosa L (2017) NF-kappaB members left home: NF-kappaB-independent roles in cancer. Biomedicines 5(2):E26Google Scholar
- 8.Takada Y, Mukhopadhyay A, Kundu GC, Mahabeleshwar GH, Singh S, Aggarwal BB (2003) Hydrogen peroxide activates NF-kappa B through tyrosine phosphorylation of I kappa B alpha and serine phosphorylation of p65: evidence for the involvement of I kappa B alpha kinase and Syk protein-tyrosine kinase. J Biol Chem 278:24233–24241CrossRefGoogle Scholar
- 19.Janda E, Palmieri C, Pisano A, Pontoriero M, Iaccino E, Falcone C, Fiume G, Gaspari M, Nevolo M, di Salle E, Rossi A, de Laurentiis A, Greco A, di Napoli D, Verheij E, Britti D, Lavecchia L, Quinto I, Scala G (2011) Btk regulation in human and mouse B cells via protein kinase C phosphorylation of IBtkgamma. Blood 117:6520–6531CrossRefGoogle Scholar
- 25.Giordano V, De Falco G, Chiari R, Quinto I, Pelicci PG et al (1997) Shc mediates IL-6 signaling by interacting with gp130 and Jak2 kinase. J Immunol 158:4097–4103Google Scholar
- 26.Mimmi S, Vecchio E, Iaccino E, Rossi M, Lupia A, Albano F, Chiurazzi F, Fiume G, Pisano A, Ceglia S, Pontoriero M, Golino G, Tassone P, Quinto I, Scala G, Palmieri C (2016) Evidence of shared epitopic reactivity among independent B-cell clones in chronic lymphocytic leukemia patients. Leukemia 30:2419–2422CrossRefGoogle Scholar
- 27.Albano F, Chiurazzi F, Mimmi S, Vecchio E, Pastore A, Cimmino C, Frieri C, Iaccino E, Pisano A, Golino G, Fiume G, Mallardo M, Scala G, Quinto I (2018) The expression of inhibitor of bruton’s tyrosine kinase gene is progressively up regulated in the clinical course of chronic lymphocytic leukaemia conferring resistance to apoptosis. Cell Death Dis 9:13CrossRefGoogle Scholar
- 28.Fiume G, Scialdone A, Rizzo F, De Filippo MR, Laudanna C, et al. (2016) IBTK differently modulates gene expression and RNA splicing in HeLa and K562 cells. Int J Mol Sci 17(11):E1848Google Scholar
- 29.Pisano A, Ceglia S, Palmieri C, Vecchio E, Fiume G, de Laurentiis A, Mimmi S, Falcone C, Iaccino E, Scialdone A, Pontoriero M, Masci FF, Valea R, Krishnan S, Gaspari M, Cuda G, Scala G, Quinto I (2015) CRL3IBTK regulates the tumor suppressor Pdcd4 through ubiquitylation coupled to proteasomal degradation. J Biol Chem 290:13958–13971CrossRefGoogle Scholar
- 31.Capasso A, Cerchia C, Di Giovanni C, Granato G, Albano F et al (2015) Ligand-based chemoinformatic discovery of a novel small molecule inhibitor targeting CDC25 dual specificity phosphatases and displaying in vitro efficacy against melanoma cells. Oncotarget 6:40202–40222Google Scholar
- 33.Schiavone M, Fiume G, Caivano A, de Laurentiis A, Falcone C, Masci FF, Iaccino E, Mimmi S, Palmieri C, Pisano A, Pontoriero M, Rossi A, Scialdone A, Vecchio E, Andreozzi C, Trovato M, Rafay J, Ferko B, Montefiori D, Lombardi A, Morsica G, Poli G, Quinto I, Pavone V, de Berardinis P, Scala G (2012) Design and characterization of a peptide mimotope of the HIV-1 gp120 bridging sheet. Int J Mol Sci 13:5674–5699CrossRefGoogle Scholar
- 37.de Laurentiis A, Gaspari M, Palmieri C, Falcone C, Iaccino E, Fiume G, Massa O, Masullo M, Tuccillo FM, Roveda L, Prati U, Fierro O, Cozzolino I, Troncone G, Tassone P, Scala G, Quinto I (2011) Mass spectrometry-based identification of the tumor antigen UN1 as the transmembrane CD43 sialoglycoprotein. Mol Cell Proteomics 10:M111 007898CrossRefGoogle Scholar
- 43.Frenzel LP, Claus R, Plume N, Schwamb J, Konermann C, Pallasch CP, Claasen J, Brinker R, Wollnik B, Plass C, Wendtner CM (2011) Sustained NF-kappaB activity in chronic lymphocytic leukemia is independent of genetic and epigenetic alterations in the TNFAIP3 (A20) locus. Int J Cancer 128:2495–2500CrossRefGoogle Scholar