title: Dual phosphorylation of Btk by Akt/protein kinase b provides docking for 14-3-3ζ, regulates shuttling, and attenuates both tonic and induced signaling in B cells.
abstract: Bruton's tyrosine kinase (Btk) is crucial for B-lymphocyte activation and development. Mutations in the Btk gene cause X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (Xid) in mice. Using tandem mass spectrometry, 14-3-3ζ was identified as a new binding partner and negative regulator of Btk in both B-cell lines and primary B lymphocytes. The activated serine/threonine kinase Akt/protein kinase B (PKB) phosphorylated Btk on two sites prior to 14-3-3ζ binding. The interaction sites were mapped to phosphoserine pS51 in the pleckstrin homology domain and phosphothreonine pT495 in the kinase domain. The double-alanine, S51A/T495A, replacement mutant failed to bind 14-3-3ζ, while phosphomimetic aspartate substitutions, S51D/T495D, caused enhanced interaction. The phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY294002 abrogated S51/T495 phosphorylation and binding. A newly characterized 14-3-3 inhibitor, BV02, reduced binding, as did the Btk inhibitor PCI-32765 (ibrutinib). Interestingly, in the presence of BV02, phosphorylation of Btk, phospholipase Cγ2, and NF-κB increased strongly, suggesting that 14-3-3 also regulates B-cell receptor (BCR)-mediated tonic signaling. Furthermore, downregulation of 14-3-3ζ elevated nuclear translocation of Btk. The loss-of-function mutant S51A/T495A showed reduced tyrosine phosphorylation and ubiquitination. Conversely, the gain-of-function mutant S51D/T495D exhibited intense tyrosine phosphorylation, associated with Btk ubiquitination and degradation, likely contributing to the termination of BCR signaling. Collectively, this suggests that Btk could become an important new candidate for the general study of 14-3-3-mediated regulation.
authors.affiliation: Department of Laboratory Medicine, Clinical Research Center, Karolinska Institutet, Karolinska Hospital Huddinge, Huddinge, Stockholm, Sweden.
authors.forename: Dara K
authors.initials: DK
authors.lastname: Mohammad
authors.forename: Beston F
authors.initials: BF
authors.lastname: Nore
authors.forename: Alamdar
authors.initials: A
authors.lastname: Hussain
authors.forename: Manuela O
authors.initials: MO
authors.lastname: Gustafsson
authors.forename: Abdalla J
authors.initials: AJ
authors.lastname: Mohamed
authors.forename: C I Edvard
authors.initials: CI
authors.lastname: Smith
mesh_terms: D048948:14-3-3 Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D000225:Adenine / Q000031:analogs & derivatives; D000077329:Agammaglobulinaemia Tyrosine Kinase; D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism*; D019556:COS Cells; D002460:Cell Line; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D006801:Humans; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism; D051966:Phospholipase C gamma / Q000378:metabolism; D010766:Phosphorylation; D010880:Piperidines; D060066:Protein Interaction Maps / Q000187:drug effects; D021381:Protein Transport; D011505:Protein-Tyrosine Kinases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D011720:Pyrazoles / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D034622:RNA Interference; D015398:Signal Transduction; D054875:Ubiquitination
chemical_list: D048948:14-3-3 Proteins; D016328:NF-kappa B; D010880:Piperidines; D011720:Pyrazoles; D011743:Pyrimidines; C551803:ibrutinib; D011505:Protein-Tyrosine Kinases; D000077329:Agammaglobulinaemia Tyrosine Kinase; C000625949:BTK protein, human; C000625951:Btk protein, mouse; D051057:Proto-Oncogene Proteins c-akt; D051966:Phospholipase C gamma; D000225:Adenine