The Btk inhibitor LFM-A13 is a potent inhibitor of Jak2 kinase activity

LFM-A13, or alpha-cyano-beta-hydroxy-beta-methyl-N-(2,5-dibromophenyl)propenamide, was shown to inhibit Bruton's tyrosine kinase (Btk). Here we show that LFM-A13 efficiently inhibits erythropoietin (Epo)-induced phosphorylation of the erythropoietin receptor, Janus kinase 2 (Jak2) and downstream signalling molecules. However, the tyrosine kinase activity of immunoprecipitated or in vitro translated Btk and Jak2 was equally inhibited by LFM-A13 in in vitro kinase assays. Finally, Epo-induced signal transduction was also inhibited in cells lacking Btk. Taken together, we conclude that LFM-A13 is a potent inhibitor of Jak2 and cannot be used as a specific tyrosine kinase inhibitor to study the role of Btk in Jak2-dependent cytokine signalling.     

Dual functions of Bruton's tyrosine kinase and Tec kinase during Fcgamma receptor-induced signaling and phagocytosis

Tec family nonreceptor tyrosine kinases are expressed by hematopoietic cells, activate phospholipase C (PLC)gamma, and regulate cytoskeletal rearrangement, yet their role in FcgammaR-induced signaling and phagocytosis remains unknown. We demonstrate in this study that Bruton's tyrosine kinase (Btk) and Tec, the only Tec kinases expressed by RAW 264.7 cells, are activated throughout phagocytosis. Activated Btk and Tec kinase accumulate at an early stage at the base of phagocytic cups and inhibition of their activity by the specific inhibitor LFM-A13 or expression by small interfering RNA significantly inhibited FcgammaR-induced phagocytosis. Similarly, a significant role for these kinases in phagocytosis was found in primary macrophages. FcgammaR-induced activation of Mac-1, which is required for optimal phagocytosis, was markedly inhibited and our findings suggest that the roles of kinases Btk and Tec in Mac-1 activation account for their functions in the early stages of phagocytosis. Initial activation of PLCgamma2, the predominant PLC isoform in RAW 264.7 cells, is dependent on Syk. In contrast, a late and prolonged activation of PLCgamma2 was dependent on Btk and Tec. We found accumulation of diacylglycerol (DAG), a PLCgamma product, in phagosome membranes, and activated Btk, but not Tec, colocalized with phagosomal DAG. Inhibition of Tec family kinase activity increased the level of DAG in phagosomes, suggesting a negative regulatory role for Btk. Tec, in contrast, clustered at sites near phagosome formation. In summary, we elucidated that Tec family kinases participate in at least two stages of FcgammaR-mediated phagocytosis: activation of Mac-1 during ingestion, and after phagosome formation, during which Btk and Tec potentially have distinct roles.     

Tyrosine kinase Btk is required for NK cell activation

Bruton tyrosine kinase (Btk) is not only critical for B cell development and differentiation but is also involved in the regulation of Toll-like receptor-triggered innate response of macrophages. However, whether Btk is involved in the regulation of natural killer (NK) cell innate function remains unknown. Here, we show that Btk expression is up-regulated during maturation and activation of mouse NK cells. Murine Btk(-/-) NK cells have decreased innate immune responses to the TLR3 ligand, with reduced expressions of IFN-γ, perforin, and granzyme-B and decreased cytotoxic activity. Furthermore, Btk is found to promote TLR3-triggered NK cell activation mainly by activating the NF-κB pathway. Poly(I:C)-induced NK cell-mediated acute hepatitis was observed to be attenuated in Btk(-/-) mice or the mice with in vivo administration of the Btk inhibitor. Correspondingly, liver damage was aggravated in Btk(-/-) mice after the adoptive transfer of Btk(+/+) NK cells, further indicating that Btk-mediated NK cell activation contributes to TLR3-triggered acute liver injury. Importantly, reduced TLR3-triggered activation of human NK cells was observed in Btk-deficient patients with X-linked agammaglobulinemia, as evidenced by the reduced IFN-γ, CD69, and CD107a expression and cytotoxic activity. These results indicate that Btk is required for activation of NK cells, thus providing insight into the physiological significance of Btk in the regulation of immune cell functions and innate inflammatory response.     

Bruton's tyrosine kinase regulates B cell antigen receptor-mediated JNK1 response through Rac1 and phospholipase C-gamma2 activation

Bruton's tyrosine kinase (Btk) is essential for B cell development and B cell antigen receptor (BCR) function. Recent studies have shown that Btk plays an important role in BCR-mediated c-Jun NH(2)-terminal kinase (JNK) 1 activation; however, the mechanism by which Btk participates in the JNK1 response remains elusive. Here we show that the BCR-mediated Rac1 activation is significantly inhibited by loss of Btk, while this Rac1 activation is not affected by loss of phospholipase C-gamma2 (PLC-gamma2). Since PLC-gamma2 is also required for BCR-mediated JNK1 response, our results suggest that Btk regulates Rac1 pathway as well as PLC-gamma2 pathway, both of which contribute to the BCR-mediated JNK1 response.     

Bruton's tyrosine kinase associates with the actin-based cytoskeleton in activated platelets

Bruton's tyrosine kinase (Btk) plays a crucial role in the maturation and differentiation of B-lymphocytes and immunoglobulin synthesis. Recently Btk has been described to be present in significant amount in human platelets. To investigate the regulation of this kinase in the platelets we studied its subcellular redistribution in the resting and activated cells. In the resting platelets Btk was almost absent from the actin-based cytoskeleton. Upon challenge of the platelet thrombin receptor upto 30% of total Btk appeared in the cytoskeleton and the protein underwent phosphorylation on tyrosine. Translocation of Btk to the cytoskeleton but not aggregation was prevented by cytochalasin B, which inhibits actin polymerization. Wortmannin and genistein (inhibitors of phosphoinositide 3-kinase and protein tyrosine kinase, respectively) decreased while phenylarsine oxide (a tyrosine phosphatase inhibitor) increased the cytoskeletal content of Btk. The association of Btk with the cytoskeleton was regulated by integrin alpha(IIb)beta(3) and partly reversible. Taken together, these data suggest that Btk might be a component of a signaling complex containing specific cytoskeletal proteins in the activated platelets.     

Interaction between the Btk PH domain and phosphatidylinositol-3,4,5-trisphosphate directly regulates Btk

Bruton's tyrosine kinase (Btk) binds to phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P(3)) through the Btk pleckstrin homology (PH) domain, an interaction thought to be required for Btk membrane translocation during B cell receptor signaling. Here, we report that interaction of PtdIns-3,4,5-P(3) with the PH domain of Btk directly induces Btk enzymatic activation in an in vitro kinase assay. A point mutation that reduces interaction of PtdIns-3,4,5-P(3) with the Btk PH domain blocks in vitro PtdIns-3,4,5-P(3)-dependent Btk activation, whereas the PH domain deletion enhances Btk basal activity but eliminates the PtdIns-3,4,5-P(3)-dependent stimulation. Btk kinase activity and the Btk activation loop phosphorylation site are both required for the PtdIns-3,4,5-P(3)-mediated stimulation of Btk kinase activity. Together, these results suggest that the Btk PH domain is positioned such that it normally suppresses both Btk kinase activity and access to substrates; when interacting with PtdIns-3,4,5-P(3), this suppression is relieved, producing apparent Btk activation. In addition, using Src family kinase inhibitors and Btk catalytically inactive mutants, we demonstrate that in vivo, the activation of Btk is due to both Lyn phosphorylation and PtdIns-3,4,5-P(3)-mediated direct activation. Thus, the Btk-PtdIns-3,4,5-P(3) interaction serves to translocate Btk to the membrane and directly regulate its signaling function.     

PKCbeta modulates antigen receptor signaling via regulation of Btk membrane localization

Mutations in Bruton's tyrosine kinase (Btk) result in X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (xid) in mice. While targeted disruption of the protein kinase C-beta (PKCbeta) gene in mice results in an immunodeficiency similar to xid, the overall tyrosine phosphorylation of Btk is significantly enhanced in PKCbeta-deficient B cells. We provide direct evidence that PKCbeta acts as a feedback loop inhibitor of Btk activation. Inhibition of PKCbeta results in a dramatic increase in B-cell receptor (BCR)-mediated Ca2+ signaling. We identified a highly conserved PKCbeta serine phosphorylation site in a short linker within the Tec homology domain of Btk. Mutation of this phosphorylation site led to enhanced tyrosine phosphorylation and membrane association of Btk, and augmented BCR and FcepsilonRI-mediated signaling in B and mast cells, respectively. These findings provide a novel mechanism whereby reversible translocation of Btk/Tec kinases regulates the threshold for immunoreceptor signaling and thereby modulates lymphocyte activation.     

Bruton's tyrosine kinase is a Toll/interleukin-1 receptor domain-binding protein that participates in nuclear factor kappaB activation by Toll-like receptor 4

In this study we have identified members of the Toll-like receptor (TLR) family (namely, TLRs 4, 6, 8, and 9) as proteins to which the intracellular protein tyrosine kinase, Bruton's tyrosine kinase (Btk), binds. Detailed analysis of the interaction between Btk and TLR8 demonstrates that the presence of both Box 2 and 3 motifs in the Toll/interleukin-1 receptor domain was required for the interaction. Furthermore, co-immunoprecipitation experiments revealed that Btk can also interact with key proteins involved in TLR4 signal transduction, namely, MyD88, Mal (MyD88 adapter-like protein), and interleukin-1 receptor-associated kinase-1, but not TRAF-6. The ability of Btk to interact with TLR4 and Mal suggests a role for Btk in lipopolysaccharide (LPS) signal transduction. Stimulation of the human monocytic cell line THP-1 with LPS resulted in an increase in the level of tyrosine phosphorylation of Btk (indicative of activation). The autokinase activity of Btk was also stimulated after LPS stimulation. In addition, a dominant negative form of Btk inhibited TLR4-mediated activation of a nuclear factor kappaB (NFkappaB)-dependent reporter gene in HEK293 cells as well as LPS-induced activation of NFkappaB in the astrocytoma cell line U373 and the monocytic cell line RAW264.7. Further investigation revealed that the Btk-specific inhibitor, LFM-A13, inhibited the activation of NFkappaB by LPS in THP-1 cells. Our findings implicate Btk as a Toll/interleukin-1 receptor domain-binding protein that is important for NFkappaB activation by TLR4.     

Molecular and structural characterization of five novel mutations in the Bruton's tyrosine kinase gene from patients with X-linked agammaglobulinemia.

BACKGROUND: The Btk (Bruton's tyrosine kinase) gene has been shown to be mutated in the human immunodeficiency disease, XLA (X-linked agammaglobulinemia). Btk is a member of the Tec family of cytosolic protein tyrosine kinases with distinct functional domains PH, TH, SH3, SH2, and kinase. Mutations have been observed in each of the Btk subdomains in XLA. We have analyzed the Btk gene in six XLA patients from five unrelated families. MATERIALS AND METHODS: DNA was prepared from the patients peripheral blood. The Btk exons including the junctional sequences were analyzed by single-strand conformation polymorphism (SSCP) followed by direct nucleotide sequencing after PCR-amplification. For structural analysis, the missense mutations were introduced into three-dimensional models of the PH and kinase domains of Btk and the outcome was predicted based on the knowledge of the protein function. RESULTS: Five novel mutations and two novel polymorphisms, all of which resulted from single-base alterations, were identified. Three of the five mutations were in the PH domain and two were in the kinase domain of Btk. Three of these mutations were of the missense type, two of which altered the same codon in the PH domain; the third one was located in the kinase domain. The fourth mutation was a point deletion in the PH domain causing a frameshift followed by premature termination. The fifth mutation was a splice donor-site mutation within the kinase domain which could result in an exon skipping. In four of the five instances, mothers of the patients were shown to be obligate carriers. In one instance, a sibling sister was identified as a heterozygote establishing her as a carrier. CONCLUSIONS: Functional consequences of the mutations causing frameshifts and altered splicing can be inferred directly. Functional consequences of the missense mutations were interpreted by 3-dimensional structural modeling of Btk domains. It is proposed that the two PH domain mutations will interfere with membrane localization while the kinase domain mutation will interfere with the enzymatic function of Btk. This study provides further insight into the role of Btk in XLA.     

Rational design and purification of human Bruton's tyrosine kinase SH3-SH2 protein for structure-function studies

Bruton's tyrosine kinase (Btk) is a cytoplasmic protein tyrosine kinase consisting of N-terminal pleckstrin homology (PH) domain followed by Tec homology (TH) domain, Src homology 3 and 2 (SH3 and SH2) domains, and a C-terminal kinase domain. Mutations in the human BTK gene cause the severe immunodeficiency disease X-linked agammaglobulinemia (XLA). The structural and functional basis of several XLA-causing mutations remains unknown, since only the structures of the PH and SH3 domains of human Btk are currently available. In this study, we overexpressed and purified a protein consisting of the SH3 and SH2 domains of human Btk for biochemical and structural analysis. The purified protein was only partially soluble and had a tendency to dimerize, which made it unsuitable for further studies. To overcome the problems of low solubility and dimerization, subdomain interactions were engineered without altering the function of the protein.     

Role of the PHTH module in protein substrate recognition by Bruton's agammaglobulinemia tyrosine kinase

Defects in Bruton's tyrosine kinase (Btk) are responsible for X chromosome-linked agammaglobulinemia in patients. Mutations in each of the structural domains of Btk have been detected in patients, yet a mechanistic explanation for most of these mutant phenotypes is lacking. To understand the possible role of the unique pleckstrin homology and Tec homology (PHTH) module of Btk, we have compared the enzymatic properties of full-length Btk and a Btk mutant lacking the PHTH module (BtkDeltaPHTH). Here we show that Btk and BtkDeltaPHTH have similar basal catalytic activity but very different abilities to recognize protein substrates. Furthermore, the catalytic domain of Btk is inactive, in contrast to the catalytic domain of the prototypical Src tyrosine kinase that retains full catalytic ability. These data suggest that the PHTH module plays an important role in protein substrate recognition, that Btk and Src likely have different interdomain organizations and regulations, and that alterations in substrate recognition might play a role in X chromosome-linked agammaglobulinemia.     

Phosphorylation of Bruton's tyrosine kinase by c-Abl

Bruton's tyrosine kinase (Btk) is necessary for B-lymphocyte development. Mutation in the gene coding for Btk causes X-linked agammaglobulinemia (XLA) in humans. Similar to Btk, c-Abl is a tyrosine kinase shuttling between the cytoplasm and the nucleus where it is involved in different functions depending on the localization. In this report we describe for the first time that c-Abl and Btk physically interact and that c-Abl can phosphorylate tyrosine 223 in the SH3 domain of Btk. Interestingly, the Btk sequence matched a v-Abl substrate [correction] identified from a randomized peptide library and was also highly related to a number of previously found c-Abl substrates.     

Src family protein tyrosine kinases induce autoactivation of Bruton's tyrosine kinase.

Bruton's tyrosine kinase (Btk) is tyrosine phosphorylated and enzymatically activated following ligation of the B-cell antigen receptor. These events are temporally regulated, and Btk activation follows that of various members of the Src family of protein tyrosine kinases, thus raising the possibility that Src kinases participate in the Btk activation process. We have evaluated the mechanism underlying Btk enzyme activation and have explored the potential regulatory relationship between Btk and Src protein kinases. We demonstrate in COS transient-expression assays that Btk can be activated through intramolecular autophosphorylation at tyrosine 551 and that Btk autophosphorylation is required for Btk catalytic functions. Coexpression of Btk with members of the Src family of protein tyrosine kinases, but not Syk, led to Btk tyrosine phosphorylation and activation. Using a series of point mutations in Blk (a representative Src protein kinase) and Btk, we show that Src kinases activate Btk through an indirect mechanism that requires membrane association of the Src enzymes as well as functional Btk SH3 and SH2 domains. Our results are compatible with the idea that Src protein tyrosine kinases contribute to Btk activation by indirectly stimulating Btk intramolecular autophosphorylation.     

The Bruton tyrosine kinase inhibitor PCI-32765 ameliorates autoimmune arthritis by inhibition of multiple effector cells

Introduction  

The aim was to determine the effect of the Bruton tyrosine kinase (Btk)-selective inhibitor PCI-32765, currently in Phase I/II studies in lymphoma trials, in arthritis and immune-complex (IC) based animal models and describe the underlying cellular mechanisms.     

Ca2+-independent activation of Bruton's tyrosine kinase is required for store-mediated Ca2+ entry in human platelets

Store-mediated Ca(2+) entry (SMCE), which is rapidly activated by depletion of the intracellular Ca(2+) stores, is a major mechanism for Ca(2+) influx. Several studies have involved tyrosine kinases in the activation of SMCE, such as pp60(src), although at present those involved in the early activation steps are unknown. Here we report the involvement of Bruton's tyrosine kinase (Btk) in the early stages of SMCE in human platelets. Cell treatment with thrombin or thapsigargin (TG) plus ionomycin (Iono) results in rapid activation of Btk, which was independent of rise in intracellular Ca(2+) concentration ([Ca(2+)](i)) but dependent on H(2)O(2) generation. Platelet treatment with Btk inhibitors, LFM-A13 or terreic acid, significantly reduced TG+Iono- and thrombin-evoked SMCE. Btk was rapidly activated by addition of low concentrations of H(2)O(2), whose effect on Ca(2+) entry was prevented by Btk inhibitors. Our results indicate that pp60(src) and Btk co-immunoprecipitate after platelet stimulation with TG+Iono, thrombin or H(2)O(2). In addition, we have found that LFM-A13 impaired actin filament reorganization after store depletion and agonist-induced activation of pp60(src), while the inhibitor of pp60(src), a protein that requires actin reorganization for its activation, did not modify Btk activation, suggesting that Btk is upstream of pp60(src). We propose a role for Btk in the early steps of activation of SMCE in human platelets.     

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

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 {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}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.     

Structure of the PH domain and Btk motif from Bruton's tyrosine kinase: molecular explanations for X-linked agammaglobulinaemia.

Bruton''s tyrosine kinase (Btk) is an enzyme which is involved in maturation of B cells. It is a target for mutations causing X-linked agammaglobulinaemia (XLA) in man. We have determined the structure of the N-terminal part of Btk by X-ray crystallography at 1.6 A resolution. This part of the kinase contains a pleckstrin homology (PH) domain and a Btk motif. The structure of the PH domain is similar to those published previously: a seven-stranded bent beta-sheet with a C-terminal alpha-helix. Individual point mutations within the Btk PH domain which cause XLA can be classified as either structural or functional in the light of the three-dimensional structure and biochemical data. All functional mutations cluster into the positively charged end of the molecule around the predicted binding site for phosphatidylinositol lipids. It is likely that these mutations inactivate the Btk pathway in cell signalling by reducing its affinity for inositol phosphates, which causes a failure in translocation of the kinase to the cell membrane. A small number of signalling proteins contain a Btk motif that always follows a PH domain in the sequence. This small module has a novel fold which is held together by a zinc ion bound by three conserved cysteines and a histidine. The Btk motif packs against the second half of the beta-sheet of the PH domain, forming a close contact with it. Our structure opens up new ways to study the role of the PH domain and Btk motif in the cellular function of Btk and the molecular basis of its dysfunction in XLA patients.