Bruton's tyrosine kinase mediates NF-kappa B activation and B cell survival by B cell-activating factor receptor of the TNF-R family

Loss of Bruton's tyrosine kinase (Btk) function results in mouse Xid disease characterized by a reduction in mature B cells and impaired humoral immune responses. These defects have been mainly attributed to impaired BCR signaling including reduced activation of the classical NF-kappaB pathway. In this study we show that Btk also couples the receptor for B cell-activating factor (BAFF) of the TNF family (BAFF-R) to the NF-kappaB pathway. Loss of Btk results in defective BAFF-mediated activation of both classical and alternative NF-kappaB pathways. Btk appears to regulate directly the classical pathway in response to BAFF such that Btk-deficient B cells exhibit reduced kinase activity of IkappaB kinase gamma-containing complexes and defective IkappaBalpha degradation. In addition, Btk-deficient B cells produce reduced levels of NF-kappaB2 (p100) basally and in response to stimulation via the BCR or BAFF-R, resulting in impaired activation of the alternative NF-kappaB pathway by BAFF. These results suggest that Btk regulates B cell survival by directly regulating the classical NF-kappaB pathway under both BCR and BAFF-R, as well as by inducing the expression of the components of alternative pathway for sustained NF-kappaB activation in response BAFF. Thus, impaired BCR- and BAFF-induced signaling to NF-kappaB may contribute to the observed defects in B cell survival and humoral immune responses in Btk-deficient mice.     

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.     

Syk and Bruton's tyrosine kinase are required for B cell antigen receptor-mediated activation of the kinase Akt.

Activation of Akt by multiple stimuli including B cell antigen receptor (BCR) engagement requires phosphatidylinositol 3-kinase and regulates processes including cell survival, proliferation, and metabolism. BCR cross-linking activates three families of non-receptor protein tyrosine kinases (PTKs) and these are transducers of signaling events including phospholipase C and mitogen-activated protein kinase activation; however, the relative roles of PTKs in BCR-mediated Akt activation are unknown. We examined Akt activation in Lyn-, Syk- and Btk-deficient DT40 cells and B cells from Lyn(-/-) mice. BCR-mediated Akt activation required Syk and was partially dependent upon Btk. Increased BCR-induced Akt phosphorylation was observed in Lyn-deficient DT40 cells and Lyn(-/-) mice compared with wild-type cells suggesting that Lyn may negatively regulate Akt function. BCR-induced tyrosine phosphorylation of the phosphatidylinositol 3-kinase catalytic subunit was abolished in Syk-deficient cells consistent with a receptor-proximal role for Syk in BCR-mediated phosphatidylinositol 3-kinase activation; in contrast, it was maintained in Btk-deficient cells, suggesting Btk functions downstream of phosphatidylinositol 3-kinase. Calcium depletion did not influence BCR-induced Akt phosphorylation/activation, showing that neither Syk nor Btk mediates its effects via changes in calcium levels. Thus, BCR-mediated Akt stimulation is regulated by multiple non-receptor PTK families which regulate Akt both proximal and distal to phosphatidylinositol 3-kinase activation.     

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.     

Cutting edge: CD40 engagement eliminates the need for Bruton's tyrosine kinase in B cell receptor signaling for NF-kappa B

The Tec kinase Bruton's tyrosine kinase (Btk) represents a key intermediary for B cell receptor (BCR) signaling. Btk mutation produces B cell deficiency in mice with X-linked immunodeficiency (xid), and surface Ig-mediated responses of mature B cells are seriously deranged. The central role that Btk plays in directing downstream events produced by BCR engagement is demonstrated by the complete failure of NF-kappa B induction and cellular proliferation following anti-Ig treatment of B cells obtained from xid mice. In this study, we report that the block in BCR signaling produced by Btk mutation is reversed by CD40 engagement. Prior treatment with CD40 ligand normalized subsequent responses of xid B cells to BCR cross-linking, so that typical outcomes of BCR signaling such as NF-kappa B activation and cell cycle progression occurred in a Btk-independent fashion. These results demonstrate that a specific genetic lesion interrupting BCR-mediated intracellular signaling is circumvented through stimulation of CD40.     

B cell activation triggered by the formation of the small receptor cluster: a computational study

We proposed a spatially extended model of early events of B cell receptors (BCR) activation, which is based on mutual kinase-receptor interactions that are characteristic for the immune receptors and the Src family kinases. These interactions lead to the positive feedback which, together with two nonlinearities resulting from the double phosphorylation of receptors and Michaelis-Menten dephosphorylation kinetics, are responsible for the system bistability. We demonstrated that B cell can be activated by a formation of a tiny cluster of receptors or displacement of the nucleus. The receptors and Src kinases are activated, first locally, in the locus of the receptor cluster or the region where the cytoplasm is the thinnest. Then the traveling wave of activation propagates until activity spreads over the whole cell membrane. In the models in which we assume that the kinases are free to diffuse in the cytoplasm, we found that the fraction of aggregated receptors, capable to initiate B cell activation decreases with the decreasing thickness of cytoplasm and decreasing kinase diffusion. When kinases are restricted to the cell membrane - which is the case for most of the Src family kinases - even a cluster consisting of a tiny fraction of total receptors becomes activatory. Interestingly, the system remains insensitive to the modest changes of total receptor level. The model provides a plausible mechanism of B cells activation due to the formation of small receptors clusters collocalized by binding of polyvalent antigens or arising during the immune synapse formation.     

Cellular maturation defects in Bruton's tyrosine kinase-deficient immature B cells are amplified by premature B cell receptor expression and reduced by receptor editing

In the mouse, Bruton's tyrosine kinase (Btk) is essential for efficient developmental progression of CD43(+)CD2(-) large cycling into CD43(-)CD2(+) small resting pre-B cells in the bone marrow and of IgM(high) transitional type 2 B cells into IgM(low) mature B cells in the spleen. In this study, we show that the impaired induction of cell surface changes in Btk-deficient pre-B cells was still noticeable in kappa(+) immature B cells, but was largely corrected in lambda(+) immature B cells. As lambda gene rearrangements are programmed to follow kappa rearrangements and lambda expression is associated with receptor editing, we hypothesized that the transit time through the pre-B cell compartment or receptor editing may affect the extent of the cellular maturation defects in Btk-deficient B cells. To address this issue, we used 3-83 mu delta transgenic mice, which prematurely express a complete B cell receptor and therefore manifest accelerated B cell development. In Btk-deficient 3-83 mu delta mice, the IgM(+) B cells in the bone marrow exhibited a very immature phenotype (pre-BCR(+)CD43(+)CD2(-)) and were arrested at the transitional type 1 B cell stage upon arrival in the spleen. However, these cellular maturation defects were largely restored when Btk-deficient 3-83 mu delta B cells were on a centrally deleting background and therefore targeted for receptor editing. Providing an extended time window for developing B cells by enforced expression of the antiapoptotic gene Bcl-2 did not alter the Btk dependence of their cellular maturation. We conclude that premature B cell receptor expression amplifies the cellular maturation defects in Btk-deficient B cells, while extensive receptor editing reduces these defects.     

Function of Bruton's tyrosine kinase during B cell development is partially independent of its catalytic activity

The Tec family member Bruton's tyrosine kinase (Btk) is a cytoplasmic protein tyrosine kinase that transduces signals from the pre-B and B cell receptor (BCR). Btk is involved in pre-B cell maturation by regulating IL-7 responsiveness, cell surface phenotype changes, and the activation of lambda L chain gene rearrangements. In mature B cells, Btk is essential for BCR-mediated proliferation and survival. Upon BCR stimulation, Btk is transphosphorylated at position Y551, which promotes its catalytic activity and subsequently results in autophosphorylation at position Y223 in the Src homology 3 domain. To address the significance of Y223 autophosphorylation and the requirement of enzymatic activity for Btk function in vivo, we generated transgenic mice that express the autophosphorylation site mutant Y223F and the kinase-inactive mutant K430R, respectively. We found that Y223 autophosphorylation was not required for the regulation of IL-7 responsiveness and cell surface phenotype changes in differentiating pre-B cells, or for peripheral B cell differentiation. However, expression of the Y223F-Btk transgene could not fully rescue the reduction of lambda L chain usage in Btk-deficient mice. In contrast, transgenic expression of kinase-inactive K430R-Btk completely reconstituted lambda usage in Btk-deficient mice, but the defective modulation of pre-B cell surface markers, peripheral B cell survival, and BCR-mediated NF-kappaB induction were partially corrected. From these findings, we conclude that: 1) autophosphorylation at position Y223 is not essential for Btk function in vivo, except for regulation of lambda L chain usage, and 2) during B cell development, Btk partially acts as an adapter molecule, independent of its catalytic activity.     

Inducible T cell tyrosine kinase regulates actin-dependent cytoskeletal events induced by the T cell antigen receptor

The tec family kinase, inducible T cell tyrosine kinase (Itk), is critical for both development and activation of T lymphocytes. We have found that Itk regulates TCR/CD3-induced actin-dependent cytoskeletal events. Expression of Src homology (SH) 2 domain mutant Itk transgenes into Jurkat T cells inhibits these events. Furthermore, Itk(-/-) murine T cells display significant defects in TCR/CD3-induced actin polymerization. In addition, Jurkat cells deficient in linker for activation of T cells expression, an adaptor critical for Itk activation, display impaired cytoskeletal events and expression of SH3 mutant Itk transgenes reconstitutes this impairment. Interestingly, expression of an Itk kinase-dead mutant transgene into Jurkat cells has no effect on cytoskeletal events. Collectively, these data suggest that Itk regulates TCR/CD3-induced actin-dependent cytoskeletal events, possibly in a kinase-independent fashion.     

Bruton's tyrosine kinase is dispensable for the Toll-like receptor-mediated activation of mast cells

Bruton's tyrosine kinase (Btk) represents an important signaling element downstream of ITAM-containing receptors, e.g. FcepsilonR1 and BCR. Btk is part of the calcium signalosome and thus, critically involved in intracellular calcium mobilization. Loss of Btk or expression of mutant forms results in severe disease phenotypes, X-linked agammaglobulinemia (XLA) and Xid in humans and mice, respectively. Previously, roles for Btk in TLR-mediated signal transduction have been found in monocytes/macrophages. In the present study we show that Btk deficiency moderately enhances or has no influence on the LPS- or lipopeptide-induced secretion of IL-6 and TNF-alpha from murine bone marrow-derived mast cells (BMMCs). Furthermore, activation of p38 kinase, which is required for cytokine production, is comparable in WT and Btk-/- BMMCs. Moreover, stability of the adaptor protein Mal as well as LPS-induced H(2)O(2) production does not vary between WT and Btk-/- cells. Interestingly, PKC-beta deficiency, which results in a Xid-like phenotype as well, has also no negative effect on LPS-induced cytokine secretion, suggesting that proteins of the calcium signalosome are not involved in TLR-mediated BMMC activation. In conclusion, the study reveals that Btk is dispensable for TLR signaling and function in murine BMMCs.     

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.     

Insulin receptor serine kinase activation by casein kinase 2 and a membrane tyrosine kinase

The insulin receptor (IR) tyrosine kinase can apparently directly phosphorylate and activate one or more serine kinases. The identities of such serine kinases and their modes of activation are still unclear. We have described a serine kinase (here designated insulin receptor serine (IRS) kinase) from rat liver membranes that co-purifies with IR on wheat germ agglutinin-agarose. The kinase was activated after phosphorylation of the membrane glycoproteins by casein kinase-1, casein kinase-2, or casein kinase-3 (Biochem Biophys Res Commun 171:75–83, 1990). In this study, IRS kinase was further characterized. The presence of vanadate or phosphotyrosine in reaction mixtures was required for activation to be observed. Phosphoserine and phosphothreonine are only about 25% as effective as phosphotyrosine, whereas sodium fluoride and molybdate were ineffective in supporting activation. Vanadate and phosphotyrosine support IRS kinase activation by apparently inhibiting phosphotyrosine protein phosphatases present among the membrane glycoproteins. IR -subunit, myelin basic protein, and microtubule-associated protein-2 are good substrates for IRS kinase. The kinase prefers Mn²⁺ (K_a=1.3 mM) as a metal cofactor. Mg²⁺ (K_a=3.3 mM) is only 30% as effective as Mn²⁺. The kinase activity is stimulated by basic polypeptides, with greater than 30-fold activation achieved with polylysine and protamine. Our results suggest that both serine/threonine and tyrosine phosphorylation are required for activation of IRS kinase. Serine phosphorylation is catalyzed by one of the casein kinases, whereas tyrosine phosphorylation is catalyzed by a membrane tyrosine kinase, possibly IR tyrosine kinase. (Mol Cell Biochem121: 167–174, 1993)     

Structural basis for activation of the receptor tyrosine kinase KIT by stem cell factor

Stem Cell Factor (SCF) initiates its multiple cellular responses by binding to the ectodomain of KIT, resulting in tyrosine kinase activation. We describe the crystal structure of the entire ectodomain of KIT before and after SCF stimulation. The structures show that KIT dimerization is driven by SCF binding whose sole role is to bring two KIT molecules together. Receptor dimerization is followed by conformational changes that enable lateral interactions between membrane proximal Ig-like domains D4 and D5 of two KIT molecules. Experiments with cultured cells show that KIT activation is compromised by point mutations in amino acids critical for D4-D4 interaction. Moreover, a variety of oncogenic mutations are mapped to the D5-D5 interface. Since key hallmarks of KIT structures, ligand-induced receptor dimerization, and the critical residues in the D4-D4 interface, are conserved in other receptors, the mechanism of KIT stimulation unveiled in this report may apply for other receptor activation.     

Reduced dosage of Bruton's tyrosine kinase uncouples B cell hyperresponsiveness from autoimmunity in lyn-/- mice

The development of autoimmunity is correlated with heightened sensitivity of B cells to B cell Ag receptor (BCR) cross-linking. BCR signals are down-regulated by Lyn, which phosphorylates inhibitory receptors. lyn(-/-) mice have reduced BCR signaling thresholds and develop autoantibodies, glomerulonephritis, splenomegaly due to myeloid hyperplasia, and increased B-1 cell numbers. Bruton's tyrosine kinase (Btk), a critical component of BCR signaling pathways, is required for autoantibody production in lyn(-/-) mice. It is unclear whether Btk mediates autoimmunity at the level of BCR signal transduction or B cell development, given that lyn(-/-)Btk(-/-) mice have a severe reduction in conventional B and B-1 cell numbers. To address this issue, we crossed a transgene expressing a low dosage of Btk (Btk(low)) in B cells to lyn(-/-)Btk(-/-) mice. Conventional B cell populations were restored to levels similar to those in lyn(-/-) mice. These cells were as hypersensitive to BCR cross-linking as lyn(-/-) B cells as measured by proliferation, Ca(2+) flux, and activation of extracellular signal-regulated kinase and Akt. However, lyn(-/-)Btk(low) mice did not produce anti-ssDNA, anti-dsDNA, anti-histone, or anti-histone/DNA IgM or IgG. They also lacked B-1 cells and did not exhibit splenomegaly. Thus, B cell hyperresponsiveness is insufficient for autoimmunity in lyn(-/-) mice. These studies implicate B-1 and/or myeloid cells as key contributors to the lyn(-/-) autoimmune phenotype.     

6-Phosphogluconate dehydrogenase regulates tumor cell migration in vitro by regulating receptor tyrosine kinase c-Met

6-Phosphogluconate dehydrogenase (6PGD) is the third enzyme in the oxidative pentose phosphate pathway (PPP). Recently, we reported that knockdown of 6PGD inhibited lung tumor growth in vitro and in a xenograft model in mice. In this study, we continued to examine the functional role of 6PGD in cancer. We show that 6PGD expression positively correlates with advancing stage of lung carcinoma. In search of functional signals related to 6PGD, we discovered that knockdown of 6PGD significantly inhibited phosphorylation of c-Met at tyrosine residues known to be critical for activity. This downregulation of c-Met phosphorylation correlated with inhibition of cell migration in vitro. Overexpression of a constitutively active c-Met specifically rescued the migration but not proliferation phenotype of 6PGD knockdown. Therefore, 6PGD appears to be required for efficient c-Met signaling and migration of tumor cells in vitro.     

Signaling by Toll-like receptors 8 and 9 requires Bruton's tyrosine kinase

Toll-like receptors (TLRs) are a primary surveillance system for the detection of pathogens and are crucial to the activation of host defense. TLR7 and TLR8 sense single-stranded RNA from viruses or host ribonucleoproteins and synthetic imidazoquinolines such as R848, whereas TLR9 senses unmethylated CpG motifs in viral and bacterial DNA and in host DNA. Here we report the endogenous interaction between Brutons's tyrosine kinase (Btk) and human TLR8 and TLR9 in the monocytic cell line THP1. We also show that R848, single-stranded RNA, and CpGB-DNA activate Btk in THP1 cells as shown by phosphorylation of the tyrosine 223 residue of Btk and also by increased autokinase activity. We demonstrate that Btk is required for NFkappaB activation, participating in the pathway to increased phosphorylation of p65 on serine 536 activated by TLR8 and TLR9. Finally we demonstrate that peripheral blood mononuclear cells from patients with X-linked agammaglobulinaemia (XLA) that have dysfunctional Btk are impaired in the induction of interleukin-6 by CpGB-DNA. This study therefore establishes Btk as a key signaling molecule that interacts with and acts downstream of TLR8 and TLR9. Lack of functioning Btk in XLA patients downstream of TLR8 and TLR9 might explain the susceptibility of XLA patients to viral infections.     

Protein kinase B activation by reactive oxygen species is independent of tyrosine kinase receptor phosphorylation and requires SRC activity

Reactive oxygen species (ROS) participate as second messengers in the mitogenic signal transduction. Most of the experimental data supporting the role of ROS as signaling molecules have been obtained by using H2O2. Exposure of cells to H2O2 rapidly increases tyrosine phosphorylation of tyrosine kinase receptors (TKRs) in the absence of growth factor binding, thus inducing the activation of downstream signaling cascades, like that of protein kinase B (AKT). Another molecule able to induce an increase of intracellular ROS levels is diethylmaleate (DEM), which acts by depleting the ROS scavenger reduced glutathione (GSH). A comparison of the effects exerted by H2O2 and DEM shows that the latter induces redox modifications milder than those generated by H2O2. We also demonstrated that DEM-induced redox modifications are not accompanied by platelet-derived growth factor-receptor (PDGF-R) and epidermal growth factor-receptor Tyr phosphorylation, although they are able to activate ERKs and AKT, with kinetics different from those observed following H2O2 treatment. The activation of these two pathways is not blocked by AG1296, a selective inhibitor of PDGF-R Tyr kinase, thus confirming that the effects of DEM are not mediated by the TKR phosphorylation. On the contrary, PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazole[3,4-d]pyrimidine), an inhibitor of Src kinase, completely prevents DEM- and H2O2-induced AKT activation but has no effect on the pathway of ERKs. Finally, nitration of Tyr residues in PDGF-R is observed in DEM-treated cells, thus suggesting that ROS-induced modifications different from Tyr phosphorylation can occur at the growth factor-receptor level and can be involved in the regulation of signaling pathways.