Brutons's tyrosine kinase (Btk) is a non-receptor protein tyrosine kinase (nrPTK) essential for the development of B lymphocytes in humans and mice. Like Src and Abl PTKs, Btk contains a conserved cassette formed by SH3, SH2 and protein kinase domains, but differs from them by the presence of an N-terminal PH domain and the Tec homology region. The domain structure of Btk was analysed using X-ray synchrotron radiation scattering in solution. Low resolution shapes of the full-length protein and several deletion mutants determined ab initio from the scattering data indicate a linear arrangement of domains. This arrangement was further confirmed by rigid body modelling using known high resolution structures of individual domains. The final model of Btk displays an extended conformation with no, or little, inter-domain interactions. In agreement with these results, deletion of non-catalytic domains failed to enhance the activity of Btk. Taken together, these results indicate that, contrary to Src and Abl, Btk might not require an assembled conformation for the regulation of its activity (Marquez, 2003).
Interleukin-2 tyrosine kinase (Itk), is a T-cell specific tyrosine kinase of the Tec family. An intermolecular interaction between the SH3 and SH2 domains of Itk was examined. In addition to the interaction between the isolated domains, it was found that the dual SH3/SH2 domain-containing fragment of Itk self-associates in a specific manner in solution. Tec family members contain the SH3, SH2 and catalytic domains common to many kinase families but are distinguished by a unique amino-terminal sequence, which contains a proline-rich stretch. Previous work has identified an intramolecular regulatory association between the proline-rich region and the adjacent SH3 domain of Itk. The intermolecular interaction between the SH3 and SH2 domains of Itk that is described provides a possible mechanism for displacement of this intramolecular regulatory sequence, a step that may be required for full Tec kinase activation. Additionally, localization of the interacting surfaces on both the SH3 and SH2 domains by chemical shift mapping has provided information about the molecular details of this recognition event. The interaction involves the conserved aromatic binding pocket of the SH3 domain and a newly defined binding surface on the SH2 domain. The interacting residues on the SH2 domain do not conform to the consensus motif for an SH3 proline-rich ligand. Interestingly, a striking correlation is noted between the SH2 residues that mediate this interaction and those residues that, when mutated in the Tec family member Btk, cause the hereditary immune disorder, X-linked agammaglobulinemia (Brazin, 2000).
A protein fragment from the Tec family member Rlk (also known as Txk) containing a single proline-rich ligand adjacent to a Src homology 3 (SH3) domain has been investigated by nuclear magnetic resonance (NMR) spectroscopy. Analysis of the concentration dependence of the chemical shifts, NMR linewidths and self-diffusion coefficients reveals that the Rlk fragment dimerizes in solution. Mutation of two critical prolines in the proline-rich ligand abolishes dimerization. Furthermore, analysis of the extrapolated chemical shifts at infinite dilution reveals that intramolecular binding of the proline-rich ligand to the SH3 domain is disfavored. This is in contrast to the corresponding fragment of Itk, for which the proline-rich ligand/SH3 interaction occurs exclusively in an intramolecular fashion and no intermolecular binding is observed. Comparison of the Itk and Rlk sequences reveals that Rlk contains five fewer residues than Itk in the linker region between the proline-rich ligand and the SH3 domain. To assess whether linker length is a molecular determinant of intra- versus intermolecular self-association, the length of the linker was varied in both Rlk and Itk and the resulting variants were analyzed by NMR. Intramolecular binding in Itk is reduced by shortening the linker and conversely a longer linker between the proline-rich ligand and the SH3 domain in Rlk enhances intramolecular self-association. Association constants for the binding of peptides corresponding to the proline-rich ligand with their respective SH3 domains were also measured by NMR. The protein/peptide data combined with the association constants for binding of each proline-rich peptide to the corresponding SH3 domain provide an explanation for the opposing modes of self-association within the otherwise closely related Rlk and Itk proteins (Laederach, 2003).
Activation of phospholipase C-gamma2 (PLCgamma2) is the critical step in B cell antigen receptor (BCR)-coupled calcium signaling. Although genetic dissection experiments on B cells have demonstrated that Bruton's tyrosine kinase (Btk) and Syk are required for activating PLCgamma2, the exact activation mechanism of PLCgamma2 by these kinases has not been established. This study identifies the tyrosine residues 753, 759, 1197, and 1217 in rat PLCgamma2 as Btk-dependent phosphorylation sites by using an in vitro kinase assay. To evaluate the role of these tyrosine residues in phosphorylation-dependent activation of PLCgamma2, PLCgamma2-deficient DT40 cells were reconstituted with a series of mutant PLCgamma2s in which the phenylalanine was substituted for tyrosine. Substitution of all four tyrosine residues almost completely eliminates the BCR-induced PLCgamma2 phosphorylation, indicating that these residues include the major phosphorylation sites upon BCR engagement. Cells expressing PLCgamma2 with a single substitution exhibit some extent of reduction in calcium mobilization, whereas those expressing quadruple mutant PLCgamma2 show greatly reduced calcium response. These findings indicate that the phosphorylations of the tyrosine residues 753, 759, 1197, and 1217, that have been identified as Btk-dependent phosphorylation sites in vitro, coordinately contribute to BCR-induced activation of PLCgamma2 (Watanabe, 2001).
Tyrosine phosphorylation of phospholipase Cgamma2 (PLCgamma2) is a crucial activation switch that initiates and maintains intracellular calcium mobilization in response to B cell antigen receptor (BCR) engagement. Although members from three distinct families of non-receptor tyrosine kinases can phosphorylate PLCgamma in vitro, the specific kinase(s) controlling BCR-dependent PLCgamma activation in vivo remains unknown. Btk-deficient human B cells exhibit diminished inositol 1,4,5-trisphosphate production and calcium signaling despite a normal inducible level of total PLCgamma2 tyrosine phosphorylation. This suggested that Btk might modify a critical subset of residues essential for PLCgamma2 activity. To evaluate this hypothesis, site-specific phosphotyrosine antibodies were generated recognizing four putative regulatory residues within PLCgamma2. Whereas all four sites are rapidly modified in response to BCR engagement in normal B cells, Btk-deficient B cells exhibit a marked reduction in phosphorylation of the Src homology 2 (SH2)-SH3 linker region sites, Tyr(753) and Tyr(759). Phosphorylation of both sites is restored by expression of Tec, but not Syk, family kinases. In contrast, phosphorylation of the PLCgamma2 carboxyl-terminal sites, Tyr(1197) and Tyr(1217), is unaffected by the absence of functional Btk. Together, these data support a model whereby Btk/Tec kinases control sustained calcium signaling via site-specific phosphorylation of key residues within the PLCgamma2 SH2-SH3 linker (Humphries, 2004).
Loss of function of Btk causes X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency in mice (xid). By using MS analysis and phosphopeptide-specific antibodies, a tyrosine phosphorylation site (Y617) was identified near the carboxyl terminus of the Btk domain from Btk expressed in 293T as well as DT-40 cells. Y617 is conserved in all Tec family kinases except murine Tec. Replacement of Y617 with a negatively charged glutamic acid (E) suppresses Btk-mediated phospholipase Cgamma2 activation and calcium response in DT-40 cells, whereas Akt activation is not affected. The Btk Y617E mutant can partially restore conventional B cell development and proliferation in Btk(-)/Tec(-) mice but fails to rescue CD5(+) B-1 cell development and the TI-II immune response to 2,4,6,-trinitrophenyl-Ficoll. These data suggest that Y617 phosphorylation or a negative charge at this site may down-regulate the function of Btk by selectively suppressing the B cell calcium signaling pathway (Guo, 2004).
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. 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, it has been 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 regulates its signaling function (Saito, 2001).
Members of the Toll-like receptor (TLR) family (namely, TLRs 4, 6, 8, and 9) have been identified as proteins to which 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 is required for the interaction. Furthermore, co-immunoprecipitation experiments reveal 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 results in an increase in the level of tyrosine phosphorylation of Btk (indicative of activation). The autokinase activity of Btk is also stimulated after LPS stimulation. In addition, a dominant negative form of Btk inhibits TLR4-mediated activation of a NFkappaB-dependent reporter gene in HEK293 cells as well as LPS-induced activation of NFkappaB in an astrocytoma cell line and a monocytic cell line. Further investigation revealed that the Btk-specific inhibitor, LFM-A13, inhibits the activation of NFkappaB by LPS in THP-1 cells. These findings implicate Btk as a Toll/interleukin-1 receptor domain-binding protein that is important for NFkappaB activation by TLR4 (Jefferies, 2004).
Btk is essential for B cell development and function. BAP/TFII-I, a protein implicated in transcriptional regulation, is associated with Btk in B cells and is transiently phosphorylated on tyrosine following B cell receptor engagement. BAP/TFII-I is a substrate for Btk in vitro and is hyperphosphorylated on tyrosine upon coexpression with Btk in mammalian cells. In an effort to understand the physiologic consequences of BAP/TFII-I tyrosine phosphorylation following B cell receptor stimulation, site-directed mutagenesis and phosphopeptide mapping were used to locate the predominant sites of BAP/TFII-I phosphorylation by Btk in vitro. These residues, Tyr248, Tyr357, and Tyr462, were also found to be the major sites for Btk-dependent phosphorylation of BAP/TFII-I in vivo. Residues Tyr357 and Tyr462 are contained within the loop regions of adjacent helix-loop-helix-like repeats within BAP/TFII-I. Mutation of either Tyr248, Tyr357, or Tyr462 to phenylalanine reduces transcription from a c-fos promoter relative to wild-type BAP/TFII-I in transfected COS-7 cells, consistent with the interpretation that phosphorylation at these sites contributes to transcriptional activation. Phosphorylation of BAP/TFII-I by Btk may link engagement of receptors such as surface immunoglobulin to modulation of gene expression (Egloff, 2001).
TFII-I is a ubiquitously expressed multifunctional transcription factor with broad biological roles in transcription and signal transduction in a variety of cell types. TFII-I can interact physically and functionally with Bruton's tyrosine kinase (Btk), a hematopoietic non-receptor protein tyrosine kinase that is critical for B lymphocyte development. Although TFII-I-Btk interactions are impaired in B cells from X-linked immunodeficient mice, the precise molecular determinants governing TFII-I-Btk complex formation remain unknown. To this end, a structural analysis of TFII-I-Btk interactions was conducted by using a panel of TFII-I mutants. These studies have revealed that a region within the N-terminal 90 amino acids of TFII-I, which includes a putative leucine zipper motif, is primarily responsible for its interaction with Btk. Mutations in the leucine zipper region itself are not sufficient to abrogate binding of TFII-I to Btk, suggesting that regions/residues outside the leucine zipper are responsible for such interactions. Because the first 90 amino acids of TFII-I are required for its dimerization, it is proposed that Btk tethers TFII-I to the cytoplasm by preventing its dimerization and its subsequent nuclear localization. The requirement of tyrosine phosphorylation for TFII-I-Btk complex formation was further analzyed. The data show that Src-dependent tyrosine phosphorylation sites in TFII-I are not targeted by Btk, suggesting that multiple kinases can independently target TFII-I via distinct signaling pathways. These results provide a beginning step toward understanding the functional importance of the TFII-I-Btk pathway in B cell signaling and gene expression (Sacristan, 2004).
Although BTK plays multiple roles in the life of a B cell, its functional role in neuronal cells has not been elucidated. In the present study, BTK is shown to activate the transcription factor CREB and subsequent CRE-mediated gene transcription during basic fibroblast growth factor (bFGF)-induced neuronal differentiation in immortalized hippocampal progenitor cells (H19-7). The kinase activity of BTK is also induced by bFGF, and BTK directly phosphorylates CREB at Ser-133 residue, indicating that BTK has a dual protein kinase activity. In addition, blockading BTK activation significantly inhibits CREB phosphorylation as well as the neurite outgrowth induced by bFGF in H19-7 cells. These results suggest that the activation of BTK and the subsequent phosphorylation of CREB at Ser-133 are important in the neuronal differentiation of hippocampal progenitor cells (Yang, 2004).
Bruton's tyrosine kinase has been shown to participate in the induction of NFkappaB-dependent gene expression by the lipopolysaccharide (LPS) receptor Toll-like receptor-4 (TLR4). The mechanism whereby Btk participates in this response has been examined. Treatment of a murine monocytic cell line with LFM-A13, a specific Btk inhibitor, blocks LPS-induced NFkappaB-dependent reporter gene expression but not IkappaB alpha degradation. Transient transfection of HEK293 cells with Btk has no effect on NFkappaB-dependent reporter gene expression but strongly promotes transactivation of a reporter gene by a p65-Gal4 fusion protein. IkappaB alpha degradation activated by LPS is intact in macrophages from X-linked immunodeficiency (Xid) mice, which contain inactive Btk. Transfection of cells with a dominant negative form of Btk (BtkK430R) inhibits LPS-driven p65 mediated transactivation. Additionally LFM-A13 impairs phosphorylation of serine 536 on p65 induced by LPS in HEK293-TLR4 cells, and in Xid macrophages this response is impaired. This study therefore reveals a novel function for Btk. It is required for the signaling pathway activated by TLR4 that culminates in phosphorylation of p65 on serine 536 promoting transactivation by NFkappaB (Doyle, 2005).
Bright (B-cell regulator of immunoglobulin heavy chain transcription) binding to immunoglobulin heavy chain loci after B-cell activation is associated with increased heavy chain transcription. Bright coimmunoprecipitates with Bruton's tyrosine kinase (Btk), and these proteins associate in a DNA-binding complex in primary B cells. B cells from immunodeficient mice with a mutation in Btk fail to produce stable Bright DNA-binding complexes. In order to determine if Btk is important for Bright function, a transcription activation assay was established and analyzed using real-time PCR technology. Cells lacking both Bright and Btk were transfected with Bright and/or Btk along with an immunoglobulin heavy chain reporter construct. Immunoglobulin gene transcription is enhanced when Bright and Btk are coexpressed. In contrast, neither Bright nor Btk alone leads to activation of heavy chain transcription. Furthermore, Bright function requires both Btk kinase activity and sequences within the pleckstrin homology domain of Btk. Bright is not appreciably phosphorylated by Btk; however, a third tyrosine-phosphorylated protein coprecipitates with Bright. Thus, the ability of Bright to enhance immunoglobulin transcription critically requires functional Btk (Rajaiya, 2005).
Bruton's tyrosine kinase (Btk) has been shown to play a role in normal B-lymphocyte development. Defective expression of Btk leads to human and murine immunodeficiencies. However, the exact role of Btk in the cytoplasmic signal transduction in B cells is still unclear. This study represents a search for the substrate for Btk in vivo. One of the major phosphoproteins associated with Btk in the preB cell line NALM6 has been identified as the Wiskott-Aldrich syndrome protein (WASP), the gene product responsible for Wiskott-Aldrich syndrome, which is another hereditary immunodeficiency with distinct abnormalities in hematopoietic cells. WASP is transiently tyrosine-phosphorylated after B-cell antigen receptor cross-linking on B cells, suggesting that WASP is located downstream of cytoplasmic tyrosine kinases. An in vivo reconstitution system demonstrated that WASP is physically associated with Btk and can serve as the substrate for Btk. A protein binding assay suggested that the tyrosine-phosphorylation of WASP alters the association between WASP and a cellular protein. Furthermore, identification of the phosphorylation site of WASP in reconstituted cells allowed an evaluation of the catalytic specificity of Btk, the exact nature of which is still unknown (Baba, 1999b).
Actin polymerization at the immune synapse is required for T cell activation and effector function; however, the relevant regulatory pathways remain poorly understood. Binding to antigen presenting cells (APCs) induces localized activation of Cdc42 and Wiskott-Aldrich Syndrome protein (WASP) at the immune synapse. Several lines of evidence suggest that Tec kinases interact with WASP-dependent actin regulatory processes. Since T cells from Rlk-/-, Itk-/-, and Rlk-/- x Itk-/- mice have defects in signaling and development, it was asked whether Itk or Rlk function in actin polymerization at the immune synapse. Itk-/- and Rlk-/- x Itk-/- T cells are defective in actin polymerization and conjugate formation in response to antigen-pulsed APCs. Itk functions downstream of the TCR, since similar defects are observed upon TCR engagement alone. Using conformation-specific probes, it was shown that although the recruitment of WASP and Arp2/3 complex to the immune synapse proceeds normally, the localized activation of Cdc42 and WASP is defective. Finally, the defect in Cdc42 activation likely stems from a requirement for Itk in the recruitment of Vav to the immune synapse. These results identify Itk as a key element of the pathway leading to localized actin polymerization at the immune synapse (Labno, 2003).
Cell polarization and migration in response to chemokines is essential for proper development of the immune system and activation of immune responses. Recent studies of chemokine signaling have revealed a critical role for PI3-Kinase, which is required for polarized membrane association of pleckstrin homology (PH) domain-containing proteins and activation of Rho family GTPases that are essential for cell polarization and actin reorganization. Additional data argue that tyrosine kinases are also important for chemokine-induced Rac activation. However, how and which kinases participate in these pathways remain unclear. This study demonstrates that the Tec kinases Itk and Rlk play an important role in chemokine signaling in T lymphocytes. Chemokine stimulation induces transient membrane association of Itk and phosphorylation of both Itk and Rlk, and purified T cells from Rlk(-/-)Itk(-/-) mice exhibit defective migration to multiple chemokines in vitro and decreased homing to lymph nodes upon transfer to wt mice. Expression of a dominant-negative Itk impaires SDF-1alpha-induced migration, cell polarization, and activation of Rac and Cdc42. Thus, Tec kinases are critical components of signaling pathways required for actin polarization downstream from both antigen and chemokine receptors in T cells (Takesono, 2004).
The Tec protein tyrosine kinase is the founding member of a family that includes Btk, Itk, Bmx, and Txk. Btk is essential for B-cell receptor signaling; mutations in Btk are responsible for X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (xid) in mice, whereas Itk is involved in T-cell receptor signaling. Tec is expressed in both T and B cells, but its role in antigen receptor signaling is not clear. This study shows that Tec protein is expressed at substantially lower levels in primary T and B cells relative to Itk and Btk, respectively. However, Tec is up-regulated upon T-cell activation and in Th1 and Th2 cells. In functional experiments that mimic Tec up-regulation, Tec overexpression in lymphocyte cell lines was found to be sufficient to induce phospholipase Cgamma (PLC-gamma) phosphorylation and NFAT activation. In contrast, overexpression of Btk, Itk, or Bmx does not induce NFAT activation. Tec-induced NFAT activation requires PLC-gamma, but not the adapters LAT, SLP-76, and BLNK, that are required for Btk and Itk to couple to PLC-gamma. Finally, this study shows that the unique effector function for Tec correlates with a unique subcellular localization. It is hypothesized that Tec functions in activated and effector T lymphocytes to induce the expression of genes regulated by NFAT transcription factors (Tomlinson, 2004).
Tec family tyrosine kinases are key regulators of lymphocyte activation and effector function. Several Tec family kinases (Tec, Itk, Rlk/Txk) are expressed in T cells, but it is still not clear to what degree these are redundant or have unique functions. Tec alone, among the Tec kinase family members examined, can induce NFAT cell-dependent transcription. This unique functional characteristic correlates with a unique pattern of subcellular localization, since Tec (but not other family members) was found in small vesicles, the appearance of which requires signaling through the T cell receptor for antigen. This study reports on studies of these Tec-containing structures in live T cells, using total internal reflection fluorescence microscopy. With this technique, it has been shown that, in live T cells, the Tec vesicles are located at the plasma membrane, the vesicles are unique to Tec (and not the related kinase Itk), and their formation and maintenance require T cell receptor signaling through Src family kinases and PI 3-kinase. Finally, isolated T cell membranes have been imaged by confocal microscopy, confirming the membrane-proximal location of Tec vesicles, as well as demonstrating overlap of these vesicles with the tyrosine kinase Lck, the Tec substrate PLC-gamma1, and the early endosomal antigen 1 marker EEA1 (Kane, 2005).
Expression of the pre-B cell receptor (pre-BCR) leads to activation of the adaptor molecule SLP-65 and the cytoplasmic kinase Btk. Mice deficient for one of these signaling proteins have an incomplete block in B cell development at the stage of large cycling pre-BCR+CD43+ pre-B cells. The recent findings of defective SLP-65 expression in approximately 50% of childhood pre-B acute lymphoblastic leukemias and spontaneous pre-B cell lymphoma development in SLP-65-/- mice demonstrate that SLP-65 acts as a tumor suppressor. To investigate cooperation between Btk and SLP-65, the pre-B cell compartment in single and double mutant mice was characterized, and the two proteins were found to have a synergistic role in the developmental progression of large cycling into small resting pre-B cells. Btk/SLP-65 double mutant mice have a dramatically increased pre-B cell tumor incidence (approximately 75% at 16 wk of age), as compared with SLP-65 single deficient mice (<10%). These findings demonstrate that Btk cooperates with SLP-65 as a tumor suppressor in pre-B cells. Furthermore, transgenic low-level expression of a constitutive active form of Btk, the E41K-Y223F mutant, prevents tumor formation in Btk/SLP-65 double mutant mice, indicating that constitutive active Btk can substitute for SLP-65 as a tumor suppressor (Kerssevoom, 2003).
During B-cell development in the mouse, Bruton tyrosine kinase (Btk) and the adaptor protein SLP-65 (Src homology 2 [SH2] domain-containing leukocyte protein of 65 kDa) limit the expansion and promote the differentiation of pre-B cells. Btk is thought to mainly function by phosphorylating phospholipase Cgamma2, which is brought into close proximity of Btk by SLP-65. However, this model was recently challenged by the identification of a role for Btk as a tumor suppressor in the absence of SLP-65 and by the finding that Btk function is partially independent of its kinase activity. To investigate if enzymatic activity is critical for the tumor suppressor function of Btk, transgenic mice expressing the kinase-inactive K430R-Btk mutant were crossed onto a Btk/SLP-65 double-deficient background. It was found that K430R-Btk expression rescues the severe developmental arrest at the pre-B-cell stage in Btk/SLP-65 double-deficient mice. Moreover, K430R-Btk can functionally replace wild-type Btk as a tumor suppressor in SLP-65- mice: at 6 months of age, the observed pre-B-cell lymphoma frequencies were approximately 15% for SLP-65- mice, 44% for Btk/SLP-65-deficient mice, and 14% for K430R-Btk transgenic mice on the Btk/SLP-65-deficient background. Therefore, it is concluded that Btk exerts its tumor suppressor function in pre-B cells as an adaptor protein, independent of its catalytic activity (Middendorp, 2005).
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