Satoshi Tsukada

Paired immunoglobulin-like receptor B (PIR-B) inhibits BCR-induced activation of Syk and Btk by SHP-1.

Coligation of paired immunoglobulin-like receptor B (PIR-B) with B cell antigen receptor (BCR) blocks antigen-induced B cell activation. This inhibition is mediated in part by recruitment of SHP-1 and SHP-2 to the phosphorylated ITIMs in the cytoplasmic domain of PIR-B; however the molecular target(s) of these phosphatases remain elusive. Here we show that PIR-B ligation inhibits the BCR-induced tyrosine phosphorylation of Igα/Igβ, Syk, Btk and phospholipase C (PLC)-γ2. Overexpression of a catalytically inactive form of SHP-1 prevents the PIR-B-mediated inhibition of tyrosine phosphorylation of Syk, Btk, and PLC-γ2. Dephosphorylation of Syk and Btk mediated by SHP-1 leads to a decrease of their kinase activity, which in turn inhibits tyrosine phosphorylation of PLC-γ2. Furthermore, we define a requirement for Lyn in mediating tyrosine phosphorylation of PIR-B. Based on these results, we propose a model of PIR-B-mediated inhibitory signaling in which coligation of PIR-B and BCR results in phosphorylation of ITIMs by Lyn, subsequent recruitment of SHP-1, and a resulting inhibition of the BCR-induced inositol 1,4,5-trisphosphate generation by dephosphorylation of Syk and Btk.

BLNK mediates Syk-dependent Btk activation.

Btk is a critical molecule in B cell antigen receptor (BCR)-coupled signaling, and its activity is regulated by Lyn and Syk. Although the molecular mechanism of Lyn-dependent Btk activation has been investigated, that of Syk-dependent Btk activation has remained unidentified. We have demonstrated that BLNK mediates Syk-dependent Btk activation. In a reconstitution cell system, coexpression of BLNK allows Syk to phosphorylate Btk on its tyrosine 551, leading to the enhancement of Btk activity. This phosphorylation depends on the interaction of Btk and BLNK by means of the Btk-Src homology 2 domain. The existence of such an activation mechanism is supported by the observation that the BCR-induced Btk phosphorylation and activation are significantly reduced in BLNK-deficient B cells as well as in Syk-deficient B cells. Although previous observations have identified the function of BLNK as the linker that integrates the action of Btk and Syk into downstream effectors such as phospholipase Cγ2, our present study indicates another function of BLNK that connects the activity of Syk to that of Btk.

Four Tyrosine Residues in Phospholipase C-γ2, Identified as Btk-dependent Phosphorylation Sites, Are Required for B Cell Antigen Receptor-coupled Calcium Signaling

Activation of phospholipase C-γ2 (PLCγ2) is the critical step in B cell antigen receptor (BCR)-coupled calcium signaling. Although genetic dissection experiments on B cells have demonstrated that Brutons tyrosine kinase (Btk) and Syk are required for activating PLCγ2, the exact activation mechanism of PLCγ2 by these kinases has not been established. We identify the tyrosine residues 753, 759, 1197, and 1217 in rat PLCγ2 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 PLCγ2, PLCγ2-deficient DT40 cells were reconstituted with a series of mutant PLCγ2s in which the phenylalanine was substituted for tyrosine. Substitution of all four tyrosine residues almost completely eliminated the BCR-induced PLCγ2 phosphorylation, indicating that these residues include the major phosphorylation sites upon BCR engagement. Cells expressing PLCγ2 with a single substitution exhibited some extent of reduction in calcium mobilization, whereas those expressing quadruple mutant PLCγ2 showed greatly reduced calcium response. These findings indicate that the phosphorylations of the tyrosine residues 753, 759, 1197, and 1217, which have been identified as Btk-dependent phosphorylation sites in vitro, coordinately contribute to BCR-induced activation of PLCγ2.

Detection of Bruton's tyrosine kinase mutations in hypogammaglobulinaemic males registered as common variable immunodeficiency (CVID) in the Japanese Immunodeficiency Registry

CVID is frequently diagnosed in male and female individuals with hypogammaglobulinaemia of unknown aetiology. To examine the possibility that sporadic male cases with X‐linked agammaglobulinaemia (XLA), which is caused by mutations in the Brutons tyrosine kinase (Btk) gene, might be misregistered as having CVID, we employed a flow cytometric test to identify XLA in hypogammaglobulinaemic males registered as CVID in the Japanese Immunodeficiency Registry. From 30 male cases registered as having CVID between 1992 and 1998, we selected 21 males with low or unreported peripheral B cell counts. Blood samples could be obtained from 11 patients and their mothers. Using flow cytometric analysis, the Btk‐deficient status in monocytes was demonstrated in seven out of nine cases with decreased numbers of peripheral B cells. The diagnosis of XLA was confirmed in each of the seven patients by demonstration of Btk gene mutations in the patients or cellular mosaicism in the mother. This study demonstrates misregistration of XLA as CVID.

A new c-Jun N-terminal kinase (JNK)-interacting protein, Sab (SH3BP5), associates with mitochondria.

We have identified a novel c-Jun N-terminal kinase (JNK)-interacting protein, Sab, by yeast two-hybrid screening. Sab binds to and serves as a substrate for JNK in vitro, and was previously found to interact with the Src homology 3 (SH3) domain of Brutons tyrosine kinase (Btk). Inspection of the sequence of Sab reveals the presence of two putative mitogen-activated protein kinase interaction motifs (KIMs) similar to that found in the JNK docking domain of the c-Jun transcription factor, and four potential serine-proline JNK phosphorylation sites in the C-terminal half of the molecule. Using deletion and site-directed mutagenesis, we demonstrate that the most N-terminal KIM in Sab is essential for JNK binding, and that, as with c-Jun, physical interaction with JNK is necessary for Sab phosphorylation. Interestingly, confocal immunocytochemistry and cell fractionation studies indicate that Sab is associated with mitochondria, where it co-localizes with a fraction of active JNK. These and previously reported properties of Sab suggest a possible role in targeting JNK to this subcellular compartment and/or mediating cross-talk between the Btk and JNK signal transduction pathways.

Bruton's tyrosine kinase is present in normal platelets and its absence identifies patients with X-linked agammaglobulinaemia and carrier females

X‐linked agammaglobulinaemia (XLA) is a primary immunodeficiency caused by mutations in the gene coding for Brutons tyrosine kinase (Btk) and is characterized by an arrest of B‐cell development. We analysed Btk protein expression in platelets using flow cytometry and found that normal platelets express large amounts of Btk. Assessment of affected males from 45 unrelated XLA families revealed that platelets of the majority of the patients (37 out of 45 families) had decreased or absent Btk expression, and that platelets from carrier females of these families had both normal and mutated Btk expression, indicating that megakaryocytes in XLA carriers undergo random X‐chromosome inactivation. These observations demonstrate that Btk is not crucial for maturation of megakaryocytes and the production of platelets. No correlation between Btk expression in platelets and clinical phenotype was observed in this study. Flow cytometric evaluation using platelets is a simple and rapid method to test Btk expression. It may be used as a screening test for XLA and for carrier detection, followed, if necessary, by more expensive mutation analyses.

Functional and possible physical association of scavenger receptor with cytoplasmic tyrosine kinase Lyn in monocytic THP-1-derived macrophages

Acetyl LDL (modified low‐density lipoprotein), which is thought to be taken up through scavenger receptor A (SR‐A), rapidly induced the appearance of phosphotyrosine proteins in monocytic THP‐1‐derived macrophages in vitro. The two alternative forms of Lyn (p53 and p56) were found to be tyrosine‐phosphorylated within 30 s after the stimulation with acetyl LDL. The catalytic activity of Lyn measured by an in vitro kinase assay had also increased in acetyl LDL‐stimulated THP‐1‐derived macrophages. Furthermore, Lyn could be co‐immunoprecipitated with SR‐A from the cell lysate. These observations suggest a functional and possible physical association of SR‐A with Lyn in THP‐1‐derived macrophages, and also imply a possible involvement of Lyn in SR‐A signal transduction.

Btk and BLNK in B cell development.

Publisher Summary This chapter discusses the major advances in the study of Brutons tyrosine kinase (Btk), especially the identification of the link between Btk and BLNK in the context of B cell development, with an attempt to incorporate these studies into a complete picture of the molecular framework of the Btk signaling pathway. The development of B-lineage cells is tightly regulated by the sequential expressions of immunoreceptors whereupon growth factors such as cytokines or other receptor ligands promote developmental progression. In particular, studies have demonstrated that the expression of the B cell antigen receptor (BCR) and of the pre–B cell receptor (pre-BCR) at appropriate differentiation stages is mandatory for B cell development. Not only the signals resulting from ligand binding to the receptors, but also continuous signals from the BCR and pre-BCR, are thought to be important for the proliferation and survival of cells. One of the most significant advances in the study of Btk signaling is the identification of the functional connection of Btk and BLNK. BLNK integrates two distinct protein tyrosine kinases (PTKs)—Btk and Syk—into the downstream effectors such as PLCγ 2 that allows for the intracellular calcium store to be released upon BCR cross-linking.

Recurrent pneumonia with mild hypogammaglobulinemia diagnosed as X-linked agammaglobulinemia in adults

BackgroundX-linked agammaglobulinemia (XLA) is a humoral immunodeficiency caused by disruption of the Brutons tyrosine kinase (BTK) gene. Typical XLA patients suffer recurrent and severe bacterial infections in childhood.MethodsFlow cytometric analysis of the peripheral monocytes using the anti-BTK antibody was used to characterize a 27 year old male patient with mild hypogammaglobulinemia (IgG, 635 mg/dl; IgM, 11 mg/dl; IgA, <5 mg/dl). He had suffered from frequent pneumonia since age 25 but had no history of frequent infections in his childhood or in adolescence. Sequencing of the BTK cDNA obtained from an Epstein–Barr virus-transformed B lymphoblastoid cell line derived from the bone marrow of the patient was performed to confirm a genetic defect.ResultsFlow cytometric analysis of cytoplasmic BTK protein in peripheral monocytes indicated that the patient presents a rare case of adult-onset XLA and that his mother is an XLA carrier. Sequencing of the BTK gene revealed a deletion of AG in the codon for Glu605 (AGT), resulting in an aberrant stop codon that truncates the BTK protein in its kinase domain.ConclusionsThis case suggests that some XLA cases may remain undiagnosed because they only show mild hypogammaglobulinemia and they lack repeated infections in childhood. Flow cytometric analysis is a powerful method to screen these patients.

Bruton's tyrosine kinase regulates B cell antigen receptor‐mediated JNK1 response through Rac1 and phospholipase C‐γ2 activation

Brutons 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 NH2‐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‐γ2 (PLC‐γ2). Since PLC‐γ2 is also required for BCR‐mediated JNK1 response, our results suggest that Btk regulates Rac1 pathway as well as PLC‐γ2 pathway, both of which contribute to the BCR‐mediated JNK1 response.