Beston F. Nore

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

Summary:  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‐κB (NF‐κB) and nuclear factor of activated T cells (NFAT). In B cells, NF‐κB was shown to bind to the Btk promoter and induce transcription, whereas the B‐cell receptor‐dependent NF‐κB 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.

Bruton's tyrosine kinase: cell biology, sequence conservation, mutation spectrum, siRNA modifications, and expression profiling

Summary:  Brutons tyrosine kinase (Btk) is encoded by the gene that when mutated causes the primary immunodeficiency disease X‐linked agammaglobulinemia (XLA) in humans and X‐linked immunodeficiency (Xid) in mice. Btk is a member of the Tec family of protein tyrosine kinases (PTKs) and plays a vital, but diverse, modulatory role in many cellular processes. Mutations affecting Btk block B‐lymphocyte development. Btk is conserved among species, and in this review, we present the sequence of the full‐length rat Btk and find it to be analogous to the mouse Btk sequence. We have also analyzed the wealth of information compiled in the mutation database for XLA (BTKbase), representing 554 unique molecular events in 823 families and demonstrate that only selected amino acids are sensitive to replacement (P < 0.001). Although genotype–phenotype correlations have not been established in XLA, based on these findings, we hypothesize that this relationship indeed exists. Using short interfering‐RNA technology, we have previously generated active constructs downregulating Btk expression. However, application of recently established guidelines to enhance or decrease the activity was not successful, demonstrating the importance of the primary sequence. We also review the outcome of expression profiling, comparing B lymphocytes from XLA‐, Xid‐, and Btk‐knockout (KO) donors to healthy controls. Finally, in spite of a few genes differing in expression between Xid‐ and Btk‐KO mice, in vivo competition between cells expressing either mutation shows that there is no selective survival advantage of cells carrying one genetic defect over the other. We conclusively demonstrate that for the R28C‐missense mutant (Xid), there is no biologically relevant residual activity or any dominant negative effect versus other proteins.

Redistribution of Bruton's tyrosine kinase by activation of phosphatidylinositol 3-kinase and Rho-family GTPases.

Brutons tyrosine kinase (Btk) is a member of the Tec family of protein tyrosine kinases (PTK) characterized by an N‐terminal pleckstrin homology domain (PH) thought to directly interact with phosphoinositides. We report here that wild‐type (wt) and also a gain‐of‐function mutant of Btk are redistributed following a wide range of receptor‐mediated stimuli through phosphatidylinositol 3‐kinase (PI 3‐K) activation. Employing chimeric Btk with green fluorescent protein in transient transfections resulted in Btk translocation to the cytoplasmic membrane of live cells through various forms of upstream PI 3‐K activation. The redistribution was blocked by pharmacological and biological inhibitors of PI 3‐K. A gain‐of‐function mutant of Btk was found to be a potent inducer of lamellipodia and / or membrane ruffle formation. In the presence of constitutively active forms of Rac1 and Cdc42, Btk is co‐localized with actin in these regions. Formation of the membrane structures was blocked by the dominant negative form of N17‐Rac1. Therefore, Btk forms a link between a vast number of cell surface receptors activating PI 3‐K and certain members of the Rho‐family of small GTPases. In the chicken B cell line, DT40, cells lacking Btk differed from wt cells in the actin pattern and showed decreased capacity to form aggregates, further suggesting that cytoskeletal regulation mediated by Btk may be of physiological relevance.

Silencing of Bruton’s tyrosine kinase (Btk) using short interfering RNA duplexes (siRNA)

Tec family tyrosine kinases, Brutons tyrosine kinase (Btk), Itk, Bmx, Tec, and Txk, are multi‐domain proteins involved in hematopoietic signaling. Here, we demonstrate that human Btk protein can transiently be depleted using double‐stranded short RNA interference (siRNA) oligonucleotides. Imaging and Western blotting analysis demonstrate that Btk expression is down regulated in heterologous systems as well as in hematopoietic lineages, following transfection or microinjection of Btk siRNA duplexes. The induction of histamine release, a pro‐inflammatory mediator, in RBL‐2H3 mast cells was reduced by 20–25% upon Btk down regulation. Similar, results were obtained when the Btk activity was inhibited using the kinase blocker LFM‐A13. These results demonstrate a direct role of Btk for the efficient secretion of histamine in allergic responses.

Inhibitors of BTK and ITK: State of the New Drugs for Cancer, Autoimmunity and Inflammatory Diseases

BTK and ITK are cytoplasmic tyrosine kinases of crucial importance for B and T cell development, with loss‐of‐function mutations causing X‐linked agammaglobulinemia and susceptibility to severe, frequently lethal, Epstein–Barr virus infection, respectively. Over the last few years, considerable efforts have been made in order to develop small‐molecule inhibitors for these kinases to treat lymphocyte malignancies, autoimmunity or allergy/hypersensitivity. The rationale is that even if complete lack of BTK or ITK during development causes severe immunodeficiency, inactivation after birth may result in a less severe phenotype. Moreover, therapy can be transient or only partially block the activity of BTK or ITK. Furthermore, a drug‐induced B cell deficiency is treatable by gamma globulin substitution therapy. The newly developed BTK inhibitor PCI‐32765, recently renamed Ibrutinib, has already entered several clinical trials for various forms of non‐Hodgkin lymphoma as well as for multiple myeloma. Experimental animal studies have demonstrated highly promising treatment effects also in autoimmunity. ITK inhibitors are still under the early developmental phase, but it can be expected that such drugs will also become very useful. In this study, we present BTK and ITK with their signalling pathways and review the development of the corresponding inhibitors.

Signalling of Bruton's tyrosine kinase, Btk.

Brutons tyrosine kinase, which is encoded by the BTK gene, is a cytoplasmic protein tyrosine kinase (PTK) crucial for B‐cell development and differentiation. It belongs to the Tec family of PTKs containing several domains that are characteristic of signalling molecules. In humans, mutations that disrupt the function of this gene lead to the classical XLA syndrome (X‐linked agammaglobulinaemia), a primary immunodeficiency mainly characterized by lack of mature B cells as well as low levels of immunoglobulins. In contrast, animal models of this disease such as the xid mice display profoundly milder XLA phenotype. BTK phosphorylation and activation in response to engagement of the B‐cell receptor (BCR) by antigen is a dynamic process whereby a variety of proteins interact with each other and recruit signalling molecules resulting in a physiological response such as B‐cell proliferation and antibody production. The main players, however, that participate in the intracellular downstream cascade have not yet been identified and are therefore under intense scrutiny in several laboratories. This review discusses certain aspects of BTK activation following receptor stimulation by agonists and how this event is translated into the biochemical signals within the cell that eventually lead to nuclear responses.

Identification of phosphorylation sites within the SH3 domains of Tec family tyrosine kinases

Tec family protein tyrosine kinases (TFKs) play a central role in hematopoietic cellular signaling. Initial activation takes place through specific tyrosine phosphorylation situated in the activation loop. Further activation occurs within the SH3 domain via a transphosphorylation mechanism, which for Brutons tyrosine kinase (Btk) affects tyrosine 223. We found that TFKs phosphorylate preferentially their own SH3 domains, but differentially phosphorylate other member family SH3 domains, whereas non-related SH3 domains are not phosphorylated. We demonstrate that SH3 domains are good and reliable substrates. We observe that transphosphorylation is selective not only for SH3 domains, but also for dual SH3SH2 domains. However, the dual domain is phosphorylated more effectively. The major phosphorylation sites were identified as conserved tyrosines, for Itk Y180 and for Bmx Y215, both sites being homologous to the Y223 site in Btk. There is, however, one exception because the Tec-SH3 domain is phosphorylated at a non-homologous site, nevertheless a conserved tyrosine, Y206. Consistent with these findings, the 3D structures for SH3 domains point out that these phosphorylated tyrosines are located on the ligand-binding surface. Because a number of Tec family kinases are coexpressed in cells, it is possible that they could regulate the activity of each other through transphosphorylation.

Missense mutations affecting a conserved cysteine pair in the TH domain of Btk

Tec family protein tyrosine kinases have in their N‐terminus two domains. The PH domain is followed by Tec homology (TH) domain, which consists of two motifs. The first pattern, Btk motif, is also present in some Ras GAP molecules. C‐terminal half of the TH domain, a proline‐rich region, has been shown to bind to SH3 domains. Mutations in Brutons tyrosine kinase (Btk) belonging to the Tec family cause X‐linked agammaglobulinemia (XLA) due to developmental arrest of B cells. Here we present the first missense mutations in the TH domain. The substitutions affect a conserved pair of cysteines, residues 154 and 155, involved in Zn2+ binding and thereby the mutations alter protein folding and stability.

Btk29A Promotes Wnt4 Signaling in the Niche to Terminate Germ Cell Proliferation in Drosophila

Wnt–β-Catenin in Germ Cells The Wnt–β-catenin pathway contributes to many signaling mechanisms during organismal development and carcinogenesis by regulating both transcription and cell adhesion. Hamada-Kawaguchi et al. (p. 294) demonstrate that this pathway must be activated in ovarian somatic cells to stop proliferation of germ cells in Drosophila. Phosphorylation of a tyrosine residue on β-catenin by the tyrosine kinase Btk turns on signaling in the niche cells by promoting transcriptional activity of β-catenin. Failure in this process resulted in ovarian tumors in the flies. Phosphorylation of β-catenin in somatic niche cells of the fly ovary stops germ cell division and prevents tumorigenesis. Btk29A is the Drosophila ortholog of the mammalian Bruton’s tyrosine kinase (Btk), mutations of which in humans cause a heritable immunodeficiency disease. Btk29A mutations stabilized the proliferating cystoblast fate, leading to an ovarian tumor. This phenotype was rescued by overexpression of wild-type Btk29A and phenocopied by the interference of Wnt4–β-catenin signaling or its putative downstream nuclear protein Piwi in somatic escort cells. Btk29A and mammalian Btk directly phosphorylated tyrosine residues of β-catenin, leading to the up-regulation of its transcriptional activity. Thus, we identify a transcriptional switch involving the kinase Btk29A/Btk and its phosphorylation target, β-catenin, which functions downstream of Wnt4 in escort cells to terminate Drosophila germ cell proliferation through up-regulation of piwi expression. This signaling mechanism likely represents a versatile developmental switch.

Glutathione transferase mu 2 protects glioblastoma cells against aminochrome toxicity by preventing autophagy and lysosome dysfunction

U373MG cells constitutively express glutathione S-transferase mu 2 (GSTM2) and exhibit 3H-dopamine uptake, which is inhibited by 2 µM of nomifensine and 15 µM of estradiol. We generated a stable cell line (U373MGsiGST6) expressing an siRNA against GSTM2 that resulted in low GSTM2 expression (26% of wild-type U373MG cells). A significant increase in cell death was observed when U373MGsiGST6 cells were incubated with 50 µM purified aminochrome (18-fold increase) compared with wild-type cells. The incubation of U373MGsiGST6 cells with 75 µM aminochrome resulted in the formation of autophagic vacuoles containing undigested cellular components, as determined using transmission electron microscopy. A significant increase in autophagosomes was determined by measuring endogenous LC3-II, a significant decrease in cell death was observed in the presence of bafilomycin A1, and a significant increase in cell death was observed in the presence of trehalose. A significant increase in LAMP2 immunostaining was observed, a significant decrease in bright red fluorescence of lysosomes with acridine orange was observed, and bafilomycin A1 pretreatment reduced the loss of lysosome acidity. A significant increase in cell death was observed in the presence of lysosomal protease inhibitors. Aggregation of TUBA/α-tubulin (tubulin, α) and SQSTM1 protein accumulation were also observed. Moreover, a significant increase in the number of lipids droplets was observed compared with U373MG cells with normal expression of GSTM2. These results support the notion that GSTM2 is a protective enzyme against aminochrome toxicity in astrocytes and that aminochrome cell death in U373MGsiGST6 cells involves autophagic-lysosomal dysfunction.