Bruton's Agammaglobulinemia Tyrosine Kinase Research Articles

Bruton’s tyrosine kinase (Btk): function, regulation, and transformation with special emphasis on the PH domain

Bruton's agammaglobulinemia tyrosine kinase (Btk) is a cytoplasmic tyrosine kinase important in B-lymphocyte development, differentiation, and signaling. Btk is a member of the Tec family of kinases. Mutations in the Btk gene lead to X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (Xid) in mice. Activation of Btk triggers a cascade of signaling events that culminates in the generation of calcium mobilization and fluxes, cytoskeletal rearrangements, and transcriptional regulation involving nuclear factor-kappaB (NF-kappaB) and nuclear factor of activated T cells (NFAT). In B cells, NF-kappaB was shown to bind to the Btk promoter and induce transcription, whereas the B-cell receptor-dependent NF-kappaB signaling pathway requires functional Btk. Moreover, Btk activation is tightly regulated by a plethora of other signaling proteins including protein kinase C (PKC), Sab/SH3BP5, and caveolin-1. For example, the prolyl isomerase Pin1 negatively regulates Btk by decreasing tyrosine phosphorylation and steady state levels of Btk. It is intriguing that PKC and Pin1, both of which are negative regulators, bind to the pleckstrin homology domain of Btk. To this end, we describe here novel mutations in the pleckstrin homology domain investigated for their transforming capacity. In particular, we show that the mutant D43R behaves similar to E41K, already known to possess such activity.

Hematopoietic engraftment of XLA bone marrow CD34+ cells in NOG/SCID mice

X-linked agammaglobulinemia (XLA) is a rare primary immunodeficiency associated with mutations of the BTK (Bruton agammaglobulinemia tyrosine kinase) gene. Non-functional BTK leads to a severe decline in peripheral B cells and profound pan-hypogammaglobulinemia. Substitutive immunoglobulin replacement therapy improves long-term survival but remains a symptomatic rather than curative treatment that does not provide an optimal quality of life. Hematopoietic stem cell gene therapy represents a potentially curative treatment. Thorough pre-clinical testing of innovative therapies requires that adequate disease models are available. Invalidation of the murine btk gene produces a phenotype that is less severe than the human disease; alternatively, xenotransplantation of human hematopoietic progenitors obtained from XLA patients may provide a model for testing new treatment procedures. The standard of care for XLA patients rarely offers an opportunity to collect peripheral blood or bone marrow (BM) hematopoietic progenitors; however, we had access to two BM samples obtained from such individuals. We analyzed hematopoietic engraftment of immunoselected CD34(+) cells from these samples in NOD/SCID/ gammac(null) (NOG) mice. In both cases, human hematopoietic cells were readily detected in BM and thymus, and at low levels in spleen and peripheral blood. Unexpectedly, the early defect in B-lymphoid differentiation associated with XLA was not accurately reproduced in NOG mice, as large amounts of pre-B cells were found in BM. These results support the existence of differences in environmental regulation of B-cell ontogeny between mice and humans. This questions the relevance of the NOG xenograft model for pre-clinical study of XLA gene therapy.

A progressive, fatal dystonia‐Parkinsonism syndrome in a patient with primary immunodeficiency receiving chronic IVIG therapy

X-linked agammaglobulinemia (XLA) is a primary immunodeficiency disorder caused by a mutation in the Bruton agammaglobulinemia tyrosine kinase gene that results in severe B-cell deficiency. So far, neurological complications of XLA have been primarily related to acute and/or chronic central nervous system enteroviral infections. In the last few years a progressive neurodegenerative syndrome of unknown etiology has been described in XLA patients. We describe and present a video of an XLA patient who developed a fatal dementing, dystonia-Parkinsonism syndrome 14 years into his immune disorder. Physician awareness of this rare syndrome may lead to its better characterization and management.

Primary Immunodeficiency: Complex Genetic Disorders?

Classical primary immunodeficiencies (PIDs) are usually monogenic (Mendelian) disorders affecting host defenses. More than 200 clinical phenotypes of PID have been described, and about 100 of them now have a well-defined molecular genetic basis (1). The classical example is X-linked agammaglobulinemia, in which disease-causing variants in the gene ( BTK , Bruton agammaglobulinemia tyrosine kinase) coding for Bruton’s tyrosine kinase lead to arrest of B-cell development at the pre-B-cell stage (2). Identification and characterization of such monogenic diseases are not only helpful for diagnosis and genetic counseling but will be valuable in development of mechanism-based therapies or gene therapy. Gene therapy has been used to insert a functional gene in hematopoietic stem cells in children with severe combined immunodeficiency, and it is hoped that this procedure will supplement or even replace allogeneic bone marrow transplantations as a treatment option in many diseases in the …

BTK: 22 novel and 25 recurrent mutations in European patients with X-linked agammaglobulinemia.

X linked agammaglobulinemia (XLA) is an immunodeficiency disease caused by mutations in the gene coding for Bruton's agammaglobulinemia tyrosine kinase (BTK), that is involved in signal transduction pathways regulating survival, activation, proliferation, and differentiation of B lineage lymphoid cells. XLA is a primary immunodeficiency disorder characterized by lack of mature, circulating B lymphocytes, and recurrent infections. Using Single Strand Conformation Polymorphism (SSCP) followed by direct sequencing we investigated 57 patients with XLA phenotype, with or without a positive family history, from 52 unrelated families enrolled in the Italian XLA Multicenter Clinical Study. We have identified 25 recurrent mutations, 22 novel mutations including one large deletion comprising the coding sequence from exon 11 to 18. Among the mutations identified, three were detected in different unrelated families, whereas all the others were private mutations.

G Protein βγ Subunits Act on the Catalytic Domain to Stimulate Bruton's Agammaglobulinemia Tyrosine Kinase

G proteins are critical cellular signal transducers for a variety of cell surface receptors. Both alpha and betagamma subunits of G proteins are able to transduce receptor signals. Several direct effect molecules for Gbetagamma subunits have been reported; yet the biochemical mechanism by which Gbetagamma executes its modulatory role is not well understood. We have shown that Gbetagamma could directly increase the kinase activity of Bruton's tyrosine kinase (Btk) whose defects are responsible for X chromosome-linked agammaglobulinemia in patients. The well characterized interaction of Gbetagamma with the PH (pleckstrin homology)/TH (Tec-homology) module of Btk was proposed to be the underlying activation mechanism. Here we show that Gbetagamma also interacts with the catalytic domain of Btk leading to increased kinase activity. Furthermore, we showed that the PH/TH module is required for Gbetagamma-induced membrane translocation of Btk. The membrane anchorage is also dependent on the interaction of Btk with phosphatidylinositol 3,4,5-trisphosphate, the product of phosphoinositide 3-kinase. These data support a dual role for Gbetagamma in the activation of Btk signaling function, namely membrane translocation and direct regulation of Btk catalytic activity.

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.

The cellular phenotype conditions Btk for cell survival or apoptosis signaling

Bruton's agammaglobulinemia tyrosine kinase (Btk) was identified as the gene mutated in X-linked agammaglobulinemia. Btk is involved in B-cell receptor signaling and B cell ontogeny as the disease is characterized by a block in B-cell development. Cell proliferation and apoptosis are integral parts of B-cell development. We have demonstrated that overexpression of Btk in HeLa cells led to apoptosis, whereas in B cells, en dogenous levels of Btk protected the cells from apoptosis. We suggest a dual role for Btk in apoptosis and cell survival. We further propose that the phenotype of the cell may direct Btk for either cell survival or apoptosis. Our model is in line with the general feature of mammalian cells that have the inherent property to die unless the survival signals are triggered. The interplay between survival and apoptotic signaling is regulated by cell sur face receptors, cytoplasmic and nuclear regulatory molecules. These regu latory molecules may be simple unidirectional regulators or bidirectional regulators such as Btk. The different molecules involved in these pathways bring about an orchestrated signal depending on the phenotype of the cell, and a cell type-specific biological response is achieved that decides the fate of the cell.

Bruton tyrosine kinase (BTK) in X-linked agammaglobulinemia (XLA).

X-linked agammaglobulinemia (XLA) is a heritable immunodeficiency disorder that is caused by a differentiation block leading to almost complete absence of B lymphocytes and plasma cells. The affected protein is a cytoplasmic protein tyrosine kinase, Bruton's agammaglobulinemia tyrosine kinase (Btk). Btk along with Tec, Itk, Bmx and Txk belong to a distinct family of protein kinases. These proteins contain five regions; PH, TH, SH3, SH2 and kinase domains. Mutations causing XLA may affect any of these domains. About 380 unique mutations have been identified and are collected in a mutation database, BTKbase. Here, we describe the structure, function, and interactions of the affected signaling molecules in atomic detail.

Synergistic Activation of the Human Btk Promoter by Transcription Factors Sp1/3 and PU.1

Analysis of the human Bruton's agammaglobulinemia tyrosine kinase (Btk) gene promoter revealed that 280 bp upstream of the transcriptional start site is sufficient for a cell restricted expression pattern. Here, the interplay of the transcription factors Sp1, Sp3, and PU.1 binding to this promoter area was analysed. All three proteins are able to independently activate the promoter in Drosophila Schneider (SL2) cells lacking endogenous Sp- or PU.1-like activities. Furthermore, PU.1 is able to act synergistically with Sp1 as well as Sp3 to transactivate the promoter. This transactivation is mediated through adjacent binding sites rather than through the more distant Sp binding site, suggesting a possible direct interaction between PU.1 and Sp1/3. Expression of Btk was found in ES cells and levels of expression were the same as in ES cells with a targeted deletion of the Sp1 gene, suggesting that Sp3 acts as a positive regulator of Btk in vivo, in the absence of Sp1.

Mutations of the human BTK gene coding for bruton tyrosine kinase in X-linked agammaglobulinemia.

X-linked agammaglobulinemia (XLA) is an immunodeficiency caused by mutations in the gene coding for Bruton agammaglobulinemia tyrosine kinase (BTK). A database (BTKbase) of BTK mutations lists 544 mutation entries from 471 unrelated families showing 341 unique molecular events. In addition to mutations, a number of variants or polymorphisms have been found. Mutations in all the five domains of BTK cause the disease, the single most common event being missense mutations. Most mutations lead to truncation of the enzyme. The mutations appear almost uniformly throughout the molecule. About one-third of point mutations affect CpG sites, which usually code for arginine residues. The putative structural implications of all the missense mutations are provided in the database. BTKbase is available at http://www.uta.fi/imt/bioinfo.

BTKbase, mutation database for X-linked agammaglobulinemia (XLA)

X-linked agammaglobulinemia (XLA) is an immunodeficiency caused by mutations in the gene coding for Bruton's agammaglobulinemia tyrosine kinase (BTK). A database (BTKbase) of BTK mutations has been compiled and the recent update lists 463 mutation entries from 406 unrelated families showing 303 unique molecular events. In addition to mutations, the database also lists variants or polymorphisms. Each patient is given a unique patient identity number (PIN). Information is included regarding the phenotype including symptoms. Mutations in all the five domains of BTK have been noticed to cause the disease, the most common event being missense mutations. The mutations appear almost uniformly throughout the molecule and frequently affect CpG sites that code for arginine residues. The putative structural implications of all the missense mutations are given in the database. The improved version of the registry having a number of new features is available at http://www. helsinki.fi/science/signal/btkbase.html

BTK, the tyrosine kinase affected in X-linked agammaglobulinemia.

X-linked agammaglobulinemia (XLA) is a heritable immunodeficiency disorder that is caused by a differentiation block leading to almost complete absence of B lymphocytes and plasma cells. The affected protein is a cytoplasmic protein tyrosine kinase, Bruton's agammaglobulinemia tyrosine kinase (Btk). Btk along with Tec, Itk and Bmx belong to a distinct family of protein kinases. These proteins contain five regions; PH, TH, SH3, SH2 and kinase domains. Mutations causing XLA may affect any of these domains. About 200 unique mutations have been identified and are collected in a mutation database, BTKbase. Here, we describle, the structure, function, and interactions of the affected signaling molecules in atomic detail.

BTKbase, mutation database for X-linked agammaglobulinemia (XLA)

X-linked agammaglobulinemia (XLA) is an immunodeficiency caused by mutations in the gene coding for Bruton's agammaglobulinemia tyrosine kinase (BTK). A database (BTKbase) of BTK mutations has been compiled and the recent update lists 368 entries from 318 unrelated families showing 228 unique molecular events. In addition to mutations the database lists also some polymorphisms and site-directed mutations. Each patient is given a unique patient identity number (PIN). Information is provided regarding the phenotype including symptoms. Mutations in all the five domains of BTK have been noticed to cause the disease, the most common event being missense mutations. The mutations appear almost uniformly throughout the molecule and frequently affect CpG sites forming arginine residues. These hot spots have generally pyrimidines 5'and purines 3'to the mutated cytosine. A decreased frequency of missense mutations was found in the TH, SH3 and the upper lobe of the kinase domain. The putative structural implications of all the missense mutations are given in the database showing 228 unique molecular events, including a novel missense mutation causing an R28C substitution as previously seen in the Xid mouse.

BTKbase: XLA-mutation registry

X-linked agammaglobulinemia (XLA) is an immunodeficiency caused by mutations in the gene coding for Bruton's agammaglobulinemia tyrosine kinase (Btk). A new edition of the database (BTKbase) of Btk mutations has been compiled containing 282 patient entries representing 175 unique molecular events.

BTKbase: XLA-mutation registry.

X-linked agammaglobulinemia (XLA) is an immunodeficiency caused by mutations in the gene coding for Bruton's agammaglobulinemia tyrosine kinase (Btk). A new edition of the database (BTKbase) of Btk mutations has been compiled containing 282 patient entries representing 175 unique molecular events.

X-linked agammaglobulinemia (XLA): a genetic tyrosine kinase (Btk) disease.

X-linked agammaglobulinemia is a heritable immunodeficiency disease caused by a differentiation abnormality, resulting in the virtual absence of B lymphocytes and plasma cells. The affected gene encodes a cytoplasmic protein tyrosine kinase, Bruton's agammaglobulinemia tyrosine kinase, designated Btk. Btk and the other family members, Tec, ltk and Bmx, contain five regions, four of which are common structural and functional modules that are found in other signaling proteins. Mutations affect all domains of the gene, but amino acid substitutions seem to be confined to certain regions. More than 150 unique mutations have been identified and are collected in a mutation database, BTKbase. Here we discuss the three-dimensional structural implications of such mutations and their putative functional role. Of special interest are mutations affecting the pleckstrin homology domain, as Btk is the only disease-associated protein so far reported to carry mutations in this particular module.

BTKbase, mutation database for X-linked agammaglobulinemia (XLA).

X-linked agammaglobulinemia (XLA) is an immunodeficiency caused by mutations in the gene coding for Bruton's agammaglobulinemia tyrosine kinase (BTK). A database (BTKbase) of BTK mutations has been compiled and the recent update lists 225 entries from 189 unrelated families showing 148 unique molecular events. Each patient is given a unique patient identity number (PIN). Information is included regarding the phenotype including symptoms. Mutations in all the five domains of BTK have been noticed to cause the disease, the most common event being missense mutations. The mutations appear almost uniformly throughout the molecule and frequently affect CpG sites forming arginine residues. A decreased frequency of missense mutations was found in the TH, SH3 and upper lobe of the kinase domain. The putative structural implications of all the missense mutations are given in the database.

Structural basis for X-linked agammaglobulinemia (XLA): mutations at interacting Btk residues R562, W563, and A582

It has been suggested that tryptophan 563 is sandwiched between residues R562 and A582 in Bruton's agammaglobulinemia tyrosine kinase (Btk). Mutations of the surrounding residues have been shown to cause X-linked agammaglobulinemia. Substitutions R562P and A582V were noticed to have impaired kinase activity. However, based on Western blot analysis, the mutant proteins were expressed at normal levels. Molecular modeling of the kinase domain has previously indicated that these residues presumably govern the position of the W563 side chain, which is thought to interact with the catalytic loop. W563 is inside the molecule and too far away from the catalytic center to interact directly with the substrate or cofactors. To prove these model-based conclusions, a conservative substitution with phenylalanine for W563 was made, and the resultant mutant lacked kinase activity. These results confirm our previous assumption that the side chain of W563, invariant in protein tyrosine kinases, is crucial for Btk kinase activity. Mutations in the surrounding residues seem to inactivate Btk by affecting the location of W563.

Molecular and Cellular Aspects of X-Linked Agammaglobulinemia

Publisher Summary The chapter discusses the current knowledge in the field of X-Linked Agammaglobulinemia (XLA) in the context of the recent developments. XLA, by being the first human disease caused by a defect in the function of a cytoplasmic tyrosine kinase (PTK)– the XLA product designated Btk (Bruton's agammaglobulinemia tyrosine kinase)– has attracted various investigators with interest in cellular immunology, clinical immunology, genetics, gene therapy, structural biochemistry, and signal transduction. The clinical observations in patients with XLA have been of great importance for the understanding of the role of antibodies in the immune defense. One way of studying these phenomena is to compare various forms of immune deficiencies. Although most manifestations are similar in patients with different immunoglobulin deficiencies there are several examples of those being more as well as less frequent in XLA. It is apparent that the virtual absence of both immunoglobulin and B lymphocytes in the majority of patients distinguishes XLA from most other deficiencies. This defect, as demonstrated by experimental immunizations, results in a typical feature of XLA– namely, the lack of an antibody response and the failure to clear microbial antigens, such as bacteriophage φX174..