Btk regulation in human and mouse B cells via protein kinase C phosphorylation of IBtkγ

Abstract

The inhibitor of Bruton tyrosine kinase ␥ (IBtk␥) is a negative regulator of the Bruton tyrosine kinase (Btk), which plays a major role in B-cell differentiation; however, the mechanisms of IBtk␥-mediated regulation of Btk are unknown. Here we report that B-cell receptor (BCR) triggering caused serine-phosphorylation of IBtk␥ at protein kinase C consensus sites and dissociation from Btk. By liquid chromatography and mass-mass spectrometry and functional analysis, we identified IBtk␥-S87 and -S90 as the critical amino acid residues that regulate the IBtk␥ binding affinity to Btk. Consistently, the mutants IBtk␥ carrying S87A and S90A mutations bound constitutively to Btk and down-regulated Ca 2؉ IntroductionBruton tyrosine kinase (Btk) is a member of the Tec family of nonreceptor protein tyrosine kinases and is expressed in B cells, macrophages, and neutrophils. 1 Btk sustains the developmental program of pre-B cells by limiting the pre-B cell expansion and by promoting B-cell differentiation. 1,2 Consistently, mutations of BTK cause the human X-linked agammaglobulinemia and the murine X-linked immunodeficiency syndromes, which are characterized by increased susceptibility to recurrent bacterial infections as a consequence of the impaired generation of mature B cells and low production of immunoglobulin. 3,4 Btk is a crucial component of the immunoglobulin B-cell receptor (BCR) signaling pathway. Evidence from 5 indicates that Btk is required for a proper tyrosine phosphorylation of phospholipase C-␥ (PLC-␥), which in turn leads to inositol-3,4,5-triphosphate, a major mediator of [Ca 2ϩ ]i mobilization, and to diacylglycerol, an activator of protein kinase C (PKC). 6 These pathways activate specific transcription factors, including NF-B, 7,8 which regulate the gene transcription program required for B-cell survival and cell-cycle progression. Accordingly, DT40 Btk Ϫ/Ϫ chicken B cells show a drastic decrease in Ca 2ϩ signaling and NF-B activation on antigen stimulation, 9 and Btk Ϫ/Ϫ mice have a significant reduction of B cells. 10,11 Btk harbors the Pleckstrin homology domain (PH) and the Src homology domain 2 (SH2) and SH3, suggesting that its regulation occurs through protein-protein interaction. 12 The PH domain mediates the binding of Btk to phosphatidylinositol-(3,4,5)-trisphosphate (PIP 3 ) resulting in the recruitment of Btk to the plasma membrane. 13 The kinase activity of Btk is up-regulated by the Src-mediated phosphorylation of Y551 at the activation loop of the Btk kinase domain and by autophosphorylation of Y223 in the Src homology domain 3, 14,15 whereas it is inactivated by the PKC␤-mediated phosphorylation of S180. 16 Genetic evidence indicates that Syk, BLNK, and Btk are all required for the full activation of PLC␥ and Ca 2ϩ signaling on BCR triggering, [17][18][19][20] which suggests the occurrence of a multimolecular complex for PLC␥ activation. 21,22 In this scenario, it was proposed that the BCR triggering causes the Syk-mediated tyrosine phosphorylation of the signaling adaptor BLNK, w...

Selective mRNA translation during eIF2 phosphorylation induces expression of IBTKα

“…IBTKγ, a 26-kDa protein that is highly expressed in hematopoietic tissues, was previously reported to bind to and repress Btk protein kinase, hence the acronym IBTK for “inhibitor of BTK.” IBTKγ suppression of Btk lowers BTK-mediated calcium mobilization and reduces activation of nuclear factor-κB–targeted transcription in B cells (Liu et al. , 2001; Janda et al. , 2011).…”

IBTK Differently Modulates Gene Expression and RNA Splicing in HeLa and K562 Cells

“…The human inhibitor of Bruton’s tyrosine kinase ( IBTK ) gene (ENSG00000005700) includes 29 exons with two promoters and transcriptional start sites, and expresses three major transcripts, named IBTK α (ENST00000306270), IBTK β (ENST00000369751), and IBTK γ (ENST00000471036) [1,2]. The intron 26 of the IBTK gene encodes a pre-miR- IBTK 3, which is cleaved by Dicer to generate 22 nucleotide-long products [3].…”

Proteomics analysis of siRNA-mediated silencing of Wilms’ tumor 1 in the MDA-MB-468 breast cancer cell line

“…The relationship between WT1 and these proteins in the signaling pathway in MDA-MB-468 has not previously been elucidated. WT1 may behave as a negative-regulator of IBtK that binds to SH2 domain of BtK tyrosine kinase receptor resulting in IBtK inactivate leading to B-cell differentiation (27). …”