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.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.00247-13.

ACKNOWLEDGMENTS

We are indebted to Vivian Nguyen, Pavel Metalnikov, Karen Colwill, and Tony Pawson, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada, and Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada, for assistance in the proteomics analysis. We thank Martin J. Humphries for the mRFP–14-3-3ζ plasmid and Brian Hemmings for the Akt (PKB) plasmids.

Dara Khorshed Mohammad holds a Ph.D. fellowship from the Ministry of Higher Education and Scientific Research in the Kurdistan Regional Government (ERBIL-Iraq). Alamdar Hussein has received a Ph.D. fellowship from COMSATS, Institute of Information Technology, Islamabad, Pakistan, and Manuela O. Gustafsson receives a Ph.D. scholarship from Södertörn University College. This work was further supported by the Swedish Cancer Society, the Swedish Research Council, the Stockholm County Council (research grant ALF), and the European Council FP7 grant EURO-PADnet.

We declare no competing financial interests.

Dara K. Mohammad performed and conceived the majority of the experiments, analyzed data, and wrote the paper; Beston F. Nore conceived the research, designed the experiments and revised the manuscript; Alamdar Hussain and Manuela O. Gustafsson conducted some laboratory work; Abdalla J. Mohamed analyzed the data and revised the manuscript; C. I. Edvard Smith conceived the project, provided supervision throughout, interpreted data, assisted with manuscript editing, and obtained research funding.