PH Domain Of Btk Research Articles

Monitoring the Dynamic Change of Inositol Lipid Pools upon EGFR and M3R Activation in Live Cells

Phosphoinositides (PI) in the plasma membrane (PM) can dynamically change upon hormonal stimulation which can influence several cellular processes thus measuring the level of PIs can help us to better understand their distinct functions. We developed a highly sensitive method which enables us to follow the dynamic change of these lipids in living cells. We performed BRET measurements between various luciferase‐labeled PI‐binding domains and a PM‐targeted Venus in HEK 293T cells. To monitor the inositol lipid pools the followings were used: the 2xP4M domain of SidM for PI(4)P, the PH domain of PLCδ1 for PI(4,5)P2, the PH domain of BTK for PI(3,4,5)P3 and an intramolecular IP3 BRET sensor based on the ligand binding domain of the type‐1 IP3 receptor. Stimulation of cells with 100 ng/ml EGF resulted in a sustained increase of PI(3,4,5)P3, a slowly developing IP3 signal a transiently decreased PI(4,5)P2 level and an unexpected increase of PI(4)P. We also tested the lipid changes in case of M3R activation and we found that 100 µM carbachol causes more robust increase in the IP3, and decrease in the PI(4,5)P2 and PI4P levels however decreasing the carbachol concentration to 100 nM resulted in a very similar lipid changes as were seen in case of the EGF stimulation. Using different PI4K inhibitors we found that the increase in the PI(4)P level was caused by the activation of PI4KIIIα, and could be prevented by PKC inhibitors. With the help of our highly sensitive approach we were able to follow the dynamic inositol lipid changes upon stimulation of the cells with various compounds, and we could identify a possible signaling route how the cells can resynthesize PIs.

Elucidation of different inhibition mechanism of small chemicals on PtdInsP-binding domains using in silico docking experiments

Phosphatidylinositides, most negatively charged lipids in cellular membranes, regulate diverse effector proteins through the interaction with their lipid binding domains. We have previously reported inhibitory effect of small chemicals on the interaction between PtdIns(3,4,5)P3 and Btk PH domain. Here, we report that the inhibitory effects of same sets of chemicals on Grp1 PH domain and epsin1 ENTH domain to elucidate diversity of inhibitory mechanisms upon different lipid binding domains. Among the chemicals, chemical 8 showed best inhibition in vitro assay for Grp1 PH domain and epsin1 ENTH domain, and then the interaction between small chemicals and lipid binding domains was further investigated by in silico docking experiments. As a result, it was concluded that the diverse inhibitory effects on different lipid binding domains were dependent on not only the number of interactions between small chemical and domain, but also additional interaction with positively charged surfaces as the secondary binding sites. This finding will help to develop lipid binding inhibitors as antagonists for lipid–protein interactions, and these inhibitors would be novel therapeutic drug candidates via regulating effector proteins involved in severe human diseases.

Small chemicals with inhibitory effects on PtdIns(3,4,5)P3 binding of Btk PH domain

Phosphatidylinositol-3,4-5-triphosphates (PtdIns(3,4,5)P3) formed by phosphoinositide-3-kinase (PI3K) had been known as a signaling molecule that plays important roles in diverse cellular processes such as cell signaling, metabolism, cell differentiation, and apoptosis. PtdIns(3,4,5)P3 regulates diverse cellular processes by recruiting effector proteins to the specific cellular locations for correct functions. In this study, we reported the inhibitory effect of small chemicals on the interaction between PtdIns(3,4,5)P3–Btk PH domain. Small chemicals were synthesized based on structural similarity of PtdInsP head-groups, and tested the inhibitory effects in vitro via surface plasmon resonance (SPR). As a result, the chemical 8 showed highest inhibitory effect with 17μM of IC50 value. To elucidate diverse inhibitory effects of different small chemicals we employed in silico docking experiment using molecular modeling and simulation. The result of docking experiments showed chemical 8 has more hydrogen bonding with the residues in PtdIns(3,4,5)P3 binding site of Btk PH domain than others. Overall, our studies demonstrate the efficient approach to develop lipid binding inhibitors, and further we can use these chemicals to regulate effector proteins. In addition, our study would provide new insight that lipid binding domain may be the attractive therapeutic targets to treat severe human diseases.

Biomedical Informatics and Computational Biology for High-Throughput Data Analysis

The last few years have witnessed the emergence of several high-throughput (HTP) platforms that are based on the evolution of omic science through transcriptomics, proteomics, and metabolomics. With the huge data accumulation, the informatics and computational methods have become essential to understand the complexity of biomedical data. We lunched the special issue to address the demand for statistical models, biological omic data analyses, and meta-analysis of biomarker for complex diseases. Statistical models are always important for the understanding of complex data; in this issue, Y. Liang et al. proposed a new adaptive L1/2 shooting regularization method for variable selection based on the Cox's proportional hazards mode. Simulation and the real gene expression dataset analysis showed that the method is more accurate for variable selection than Lasso and adaptive Lasso methods. L. Tian et al. employed a nonlinear model to analyze time course gene expression data. They firstly developed a method for estimating the parameters in the nonlinear model and then utilized the model to perform the significance analysis of individually differentially expressed genes and clustering analysis of a set of gene expression profiles. The simulation and real-life biological data analyses showed that their methods outperform some existing methods. For the application of bioinformatics tools, Z. E et al. analyzed the gene expression in rice leaf blades at different temperatures. They analyzed the next-generation sequencing (NGS) datasets and characterized the transcription profiles of the samples in rice seedling leaf blades at 25°C and 30°C. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were applied to study the effect of the temperature. Finally, they observed that temperature markedly regulates several superfamilies of transcription factors, including bZIP, MYB, and WRKY. Chronobiology is a field of biology that studies biological rhythms. R. Lopes et al. reviewed the bioinformatics or computational systems biology methods, tools, and databases which are very helpful to the understanding of the patterns and biological rhythms found in living organisms and the review will benefit the community for the application of bioinformatics in their future researches. For the understanding of protein structure and structure-function relationship, two papers are selected in this issue. M. Shambhu et al. studied the surface accessibility of hydrophobic residues by considering their conservation score and knowledge of flanking regions. The accuracy of prediction is therefore improved. In the other work, D. Lu et al. applied molecular dynamics to investigate the effects of X-linked agammaglobulinaemia associated with amino acid mutations in pleckstrin homology (PH) domain on the binding of the domain with inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4). The mutations were then classified as “functional mutations”, and “folding mutations.” The result provided new insights into the biological function of the Btk-PH domain and related mutation-causing diseases. Finally, two works in this special issue are medical studies. The first is the work by B. Lv et al. They analyzed the 347 studies for the diagnostic value of tumor necrosis factor-α (TNF-α) and found that the northern hemisphere group in the TNF-α test has higher sensitivity and specificity than those of southern hemisphere group. The second medical study is an association study. Z. Liu et al. reported the significantly association of NCK2 with opiates addiction in African-origin men by a comprehensive analysis of a dataset from the Study of Addiction: Genetics and Environment (SAGE). They employed both SNP and gene based methods of analysis and identified a strong and significant association between a SNP in the NCK2 gene on chromosome 2 with opiates addiction in African-origin men. Bairong Shen Jian Ma Jiajun Wang Junbai Wang

Molecular Dynamic Simulation to Explore the Molecular Basis of Btk‐PH Domain Interaction with Ins(1,3,4,5)P4

Bruton's tyrosine kinase contains a pleckstrin homology domain, and it specifically binds inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4), which is involved in the maturation of B cells. In this paper, we studied 12 systems including the wild type and 11 mutants, K12R, S14F, K19E, R28C/H, E41K, L11P, F25S, Y40N, and K12R-R28C/H, to investigate any change in the ligand binding site of each mutant. Molecular dynamics simulations combined with the method of molecular mechanics/Poisson-Boltzmann solvent-accessible surface area have been applied to the twelve systems, and reasonable mutant structures and their binding free energies have been obtained as criteria in the final classification. As a result, five structures, K12R, K19E, R28C/H, and E41K mutants, were classified as “functional mutations,” whereas L11P, S14F, F25S, and Y40N were grouped into “folding mutations.” This rigorous study of the binding affinity of each of the mutants and their classification provides some new insights into the biological function of the Btk-PH domain and related mutation-causing diseases.

Physical and functional characterization of the genetic locus of IBtk, an inhibitor of Bruton's tyrosine kinase: evidence for three protein isoforms of IBtk

Bruton's tyrosine kinase (Btk) is required for B-cell development. Btk deficiency causes X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (Xid) in mice. Btk lacks a negative regulatory domain and may rely on cytoplasmic proteins to regulate its activity. Consistently, we identified an inhibitor of Btk, IBtk, which binds to the PH domain of Btk and down-regulates the Btk kinase activity. IBtk is an evolutionary conserved protein encoded by a single genomic sequence at 6q14.1 cytogenetic location, a region of recurrent chromosomal aberrations in lymphoproliferative disorders; however, the physical and functional organization of IBTK is unknown. Here, we report that the human IBTK locus includes three distinct mRNAs arising from complete intron splicing, an additional polyadenylation signal and a second transcription start site that utilizes a specific ATG for protein translation. By northern blot, 5′RACE and 3′RACE we identified three IBTKα, IBTKβ and IBTKγ mRNAs, whose transcription is driven by two distinct promoter regions; the corresponding IBtk proteins were detected in human cells and mouse tissues by specific antibodies. These results provide the first characterization of the human IBTK locus and may assist in understanding the in vivo function of IBtk.

Analysis of X-linked agammaglobulinemia patients in Mexico (B222)

Abstract Humoral immunodeficiences are very common in the National Registry of Primary Immunodeficiences in Mexico. Among the humoral defects, X-linked agammaglobulinemia (XLA) is one of the most frequently found. In this work we report Btk mutations for 13 unrelated XLA patients some of wich has not been reported previously, most of the mutations affect Btk kinase domain, and PH and SH2 domain affecting mutations were also detected. Btk protein expression was found to be present in the majority of patients, in some cases we detect low expression levels and it was absent in other patients.

Regulation of phosphoinositide 3-kinase signaling by oxidants: Hydrogen peroxide selectively enhances immunoreceptor-induced recruitment of phosphatidylinositol (3,4) bisphosphate-binding PH domain proteins

Phosphoinositide 3-kinases (PI3Ks) generate several distinct lipid second messengers including phosphatidylinositol (3,4,5) trisphosphate (PIP3) and phosphatidylinositol (3,4) bisphosphate PI(3,4)P2. PI(3,4)P2 is produced with distinct kinetics and binds to distinct PH domain effector proteins; however, the regulation of this signaling pathway is poorly understood. Superoxides such as hydrogen peroxide are transiently produced after activation through various cell surface receptors and play important roles in immune and inflammatory responses. Here we use quantitative microscopy to examine the effect of peroxide on PI(3,4)P2-mediated mobilization of signaling proteins in B lymphocytes. Peroxide was found to induce dose-dependant membrane recruitment of the PI(3,4)P2-binding PH domain proteins Bam32, TAPP2 and Akt/PKB but not the PIP3-binding PH domain of Btk. Peroxide-induced membrane recruitment was found to be dependant on PI3K activity, with the p110δ isoform contributing much of the activity in the BJAB human B lymphoma model. Strikingly, peroxide co-stimulation enhanced antigen receptor-induced membrane recruitment of Bam32 and TAPP2, with combined stimulation exceeding the maximum achievable with either stimulus alone. Expression of the lipid phosphatase PTEN led to reduction of antigen receptor-induced membrane recruitment of TAPP2; however, peroxide costimulation could overcome the inhibitory effect of PTEN. Inhibition of the NADPH oxidase led to reduction of antigen receptor-induced membrane recruitment of TAPP2. Our results indicate that exogenous and endogenous superoxides can modulate the quality of the PI3K signal in lymphocytes by selectively increasing PI(3,4)P2-dependant signaling.

Bruton's Tyrosine Kinase Is Involved in p65-mediated Transactivation and Phosphorylation of p65 on Serine 536 during NFκB Activation by Lipopolysaccharide

Bruton's tyrosine kinase (Btk) has recently been shown to participate in the induction of nuclear factor kappaB (NFkappaB)-dependent gene expression by the lipopolysaccharide (LPS) receptor Toll-like receptor-4 (TLR4). In this study we have examined the mechanism whereby Btk participates in this response. Treatment of the murine monocytic cell line Raw264.7 with LFM-A13, a specific Btk inhibitor, blocked LPS-induced NFkappaB-dependent reporter gene expression but not IkappaB alpha degradation. Transient transfection of HEK293 cells with Btk had no effect on NFkappaB-dependent reporter gene expression but strongly promoted transactivation of a reporter gene by a p65-Gal4 fusion protein. IkappaB alpha degradation activated by LPS was intact in macrophages from X-linked immunodeficiency (Xid) mice, which contain inactive Btk. Transfection of cells with a dominant negative form of Btk (BtkK430R) inhibited LPS-driven p65 mediated transactivation. Additionally LFM-A13 impaired phosphorylation of serine 536 on p65 induced by LPS in HEK293-TLR4 cells, and in Xid macrophages this response was impaired. This study therefore reveals a novel function for Btk. It is required for the signaling pathway activated by TLR4, which culminates in phosphorylation of p65 on serine 536 promoting transactivation by NFkappaB.

A Dual Function Anti-leukemic Agent with Anti-thrombotic Activity

Here we report that treatment with the anti-leukemic compound, LFM-A13 resulted in SYK kinase activation and caused distinct shape changes in platelets. Also provided is electron microscopic evidence that similar shape changes are observed in platelets from XID mice. We propose that LFM-A13 induces a conformational change in the PH domain of BTK and causes BTK to associate with PIP2 which effects actin bundling and shape change.

1I1445 分子動力学法を用いたBtkタンパク質のPHドメインの基質認識に関する研究(1.蛋白質(C)物性,一般演題,日本生物物理学会第40回年会)

1I1445 分子動力学法を用いたBtkタンパク質のPHドメインの基質認識に関する研究(1.蛋白質(C)物性,一般演題,日本生物物理学会第40回年会)

Direct inhibition of Bruton's tyrosine kinase by IBtk, a Btk-binding protein.

Bruton's tyrosine kinase (Btk) is required for human and mouse B cell development. Btk deficiency causes X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency in mice. Unlike Src proteins, Btk lacks a negative regulatory domain at the COOH terminus and may rely on cytoplasmic Btk-binding proteins to regulates its kinase activity by trans-inhibitor mechanisms. Consistent with this possibility, IBtk, which we identified as an inhibitor of Btk, bound to the PH domain of Btk. IBtk downregulated Btk kinase activity, Btk-mediated calcium mobilization and nuclear factor-kappaB-driven transcription. These results define a potential mechanism for the regulation of Btk function in B cells.

Phosphatidylinositol 3,4,5-trisphosphate regulates Ca2+ entry via Btk in platelets and megakaryocytes without increasing phospholipase C activity

The role of phosphatidylinositol 3,4,5-trisphosphate (PI3,4,5P(3)) and Btk in signalling by the collagen receptor glycoprotein VI was investigated. PI3,4,5P(3) was increased in platelets from mice deficient in the SH2 domain-containing inositol 5-phosphatase (SHIP), in response to collagen related peptide (CRP). Tyrosine phosphorylation and activation of phospholipase Cgamma2 (PLCgamma2) were unaltered in SHIP(-/-) platelets, whereas Btk was heavily tyrosine phosphorylated under basal conditions and maximally phosphorylated by low concentrations of CRP. There was an increase in basal Ca(2+), maximal expression of P-selectin, and potentiation of Ca(2+) and aminophospholipid exposure to CRP in SHIP(-/-) platelets in the presence of Ca(2+) (1 mM). Microinjection of PI3,4, 5P(3) into megakaryocytes caused a 3-fold increase in Ca(2+) in response to CRP, which was absent in X-linked immunodeficiency (Xid) mice, which have a mutation in the PH domain of Btk. There was a corresponding partial reduction in the sustained level of intracellular Ca(2+) in response to CRP in Xid mice but no change in PLC activity. These results demonstrate a novel pathway of Ca(2+) entry that involves PI3,4,5P(3) and Btk, and which is independent of increased PLC activity.

Regulation of nuclear localization and transcriptional activity of TFII-I by Bruton's tyrosine kinase.

Bruton's tyrosine kinase (Btk) is required for normal B-cell development, as defects in Btk lead to X-linked immunodeficiency (xid) in mice and X-linked agammaglobulinemia (XLA) in humans. Here we demonstrate a functional interaction between the multifunctional transcription factor TFII-I and Btk. Ectopic expression of wild-type Btk enhances TFII-I-mediated transcriptional activation and its tyrosine phosphorylation in transient-transfection assays. Mutation of Btk in either the PH domain (R28C, as in the murine xid mutation) or the kinase domain (K430E) compromises its ability to enhance both the tyrosine phosphorylation and the transcriptional activity of TFII-I. TFII-I associates constitutively in vivo with wild-type Btk and kinase-inactive Btk but not xid Btk. However, membrane immunoglobulin M cross-linking in B cells leads to dissociation of TFII-I from Btk. We further show that while TFII-I is found in both the nucleus and cytoplasm of wild-type and xid primary resting B cells, nuclear TFII-I is greater in xid B cells. Most strikingly, receptor cross-linking of wild-type (but not xid) B cells results in increased nuclear import of TFII-I. Taken together, these data suggest that although the PH domain of Btk is primarily responsible for its physical interaction with TFII-I, an intact kinase domain of Btk is required to enhance transcriptional activity of TFII-I in the nucleus. Thus, mutations impairing the physical and/or functional association between TFII-I and Btk may result in diminished TFII-I-dependent transcription and contribute to defective B-cell development and/or function.

A site of interaction between pleckstrin's PH domains and G βγ

Pleckstrin is a 40 kDa substrate for protein kinase C found in platelets and neutrophils. Based upon its sequence, pleckstrin contains two of the recently-described PH domains that are thought to be binding motifs for phosphatidyl 4,5-bisphosphate (PIP 2) and/or G protein βγ heterodimers (G βγ). In the present studies we have examined the interaction between pleckstrin and G βγ by incubating pleckstrin fusion proteins with lysates from human platelets. In this analysis, both the N-terminal and C-terminal PH domains from pleckstrin bound G βγ in vitro, as did peptides containing as little as the first 30 residues of the C-terminal pleckstrin PH domain. Introduction of a point mutation into this region, analogous to the mutation in the Btk PH domain that causes X-linked immunodeficiency disease (XID) in mice, dramatically disrupted this interaction. We propose that pleckstrin may interact with G βγ, and that one potential site for this interaction involves the first 30 residues of pleckstrin's C-terminal PH domain.

Structural Basis for Pleckstrin Homology Domain Mutations in X-Linked Agammaglobulinemia

Deficiencies in a tyrosine kinase, designated Btk, cause X-linked agammaglobulinemia (XLA) in man, a hereditary defect of B-cell differentiation. Mutations in the newly found PH domain located at the N-terminus of Btk have been shown to be the direct cause of XLA, and here two new mutations, T33P and V64F, are presented. Btk is thus far the only protein in which mutations of the PH domain have been found to cause a disease. The three-dimensional structure of the Btk PH domain was modeled on the basis of the dynamin PH structure. Despite a relatively low sequence similarity the Btk PH domain seems to have the same two beta-sheet structure observed in the known structures. The model was used to interpret the structural basis for disease in five independent point mutations and in an insertion in patients with XLA. The mutated residues F25, V64, and V113, and possibly residue(s) around Q103, could form a binding site, since these amino acids are located close to each other on the surface of the molecule.