BLNK required for coupling Syk to PLC gamma 2 and Rac1-JNK in B cells.
(1/2715)
Signaling through the B cell receptor (BCR) is essential for B cell function and development. Despite the key role of Syk in BCR signaling, little is known about the mechanism by which Syk transmits downstream effectors. BLNK (B cell LiNKer protein), a substrate for Syk, is now shown to be essential in activating phospholipase C (PLC)gamma 2 and JNK. The BCR-induced PLC gamma 2 activation, but not the JNK activation, was restored by introduction of PLC gamma 2 membrane-associated form into BLNK-deficient B cells. As JNK activation requires both Rac1 and PLC gamma 2, our results suggest that BLNK regulates the Rac1-JNK pathway, in addition to modulating PLC gamma 2 localization. (+info)
Rel-dependent induction of A1 transcription is required to protect B cells from antigen receptor ligation-induced apoptosis.
(2/2715)
In response to different extracellular signals, Rel/NF-kappaB transcription factors are critical regulators of apoptosis in a variety of cell types. Here we show that in normal B and T cells, expression of the Bcl-2 prosurvival homolog, A1, is rapidly induced in a Rel-dependent manner by mitogens. In B-cell lines derived from c-rel-/- mice, which like primary cells lacking Rel undergo apoptosis in response to antigen receptor ligation, constitutive expression of an A1 transgene inhibits this pathway to cell death. These findings are the first to show that Rel/NF-kappaB regulates physiologically the expression of a Bcl-2-like protein that is critical for the control of cell survival during lymphocyte activation. (+info)
Interaction of B cells with activated T cells reduces the threshold for CD40-mediated B cell activation.
(3/2715)
CD154-CD40 interactions are of central importance for the induction of antibody responses to T-dependent antigens. Since most anti-CD40 mAb are only weak B cell mitogens, it is believed that under physiological conditions, signals through CD40 synergize with those from other receptors on B cells to induce B cell activation. We show here that the interaction of either normal B cells, or those from CBA/N (xid) mice, with CD3-activated primary T cells in whole spleen cell cultures markedly reduces the threshold for B cell activation via CD40. Hence, these pre-activated cells undergo vigorous proliferation when stimulated with either optimal or suboptimal concentrations of weakly mitogenic anti-CD40 mAb, or with soluble CD40 ligand. Blocking experiments indicate that the establishment of this priming effect requires stimulation via CD40 itself, plus T cell-derived IL-2. In support of this concept, only CD3/CD28-pre-activated, but not CD3-pre-activated T cells induce this effect, unless the co-cultures of B cells with the latter T cells are supplemented with IL-2. Although B cells activated in this fashion do express higher levels of CD40 than naive cells, we believe that this is insufficient to explain the observed dramatic effects on their proliferative capacity. Rather we propose that T cell-dependent B cell activation induces fundamental changes in the signalling machinery invoked by ligation of CD40. It is likely that this amplification loop could play an important role during the initiation of antibody responses to T-dependent antigens, when activated CD4 T cells only express low levels of CD154. (+info)
Induction of human immunoglobulin synthesis and secretion in somatic cell hybrids of mouse myeloma and human B lymphocytes from patients with agammaglobulinemia.
(4/2715)
Somatic cell hybrid clones were isolated from the fusion of RPC 5,4 mouse myeloma cells and B lymphocytes from three patients with agammaglobulinemia. One patient had X-linked agammaglobulinemia; the remaining two patients had common varied agammaglobulinemia. All three patients had B lymphocytes which fail to secrete immunoglobulin. The hybrid nature of the clones was established by examination of metaphase chromosome spreads. Most of the clones from all three patients expressed surface immunoglobulin of mouse and human parental origin. Clones from two of the patients had fewer cells with surface Ig than hybrids from normal persons, while clones from the third patient had large numbers of surface Ig fluorescent cells. Most of the clones from all three patients synthesized and secreted human and mouse immunoglobulin. As determined by sodium dodecyl sulfate acrylamide gel electrophoresis of radioactively labeled proteins, clones from each of the patients produced human gamma, alpha, and mu-heavy chains. These studies demonstrate the presence of functional structural genes coding for human immunoglobulin heavy chains in B lymphocytes of patients with agammaglobulinemia. Further, they represent induction in the somatic cell hybrids of a gene product not expressed in the parental B lymphocytes. (+info)
Encoding of Ca2+ signals by differential expression of IP3 receptor subtypes.
(5/2715)
Inositol 1,4,5-trisphosphate (IP3) plays a key role in Ca2+ signalling, which exhibits a variety of spatio-temporal patterns that control important cell functions. Multiple subtypes of IP3 receptors (IP3R-1, -2 and -3) are expressed in a tissue- and development-specific manner and form heterotetrameric channels through which stored Ca2+ is released, but the physiological significance of the differential expression of IP3R subtypes is not known. We have studied the Ca2+-signalling mechanism in genetically engineered B cells that express either a single or a combination of IP3R subtypes, and show that Ca2+-signalling patterns depend on the IP3R subtypes, which differ significantly in their response to agonists, i.e. IP3, Ca2+ and ATP. IP3R-2 is the most sensitive to IP3 and is required for the long lasting, regular Ca2+ oscillations that occur upon activation of B-cell receptors. IP3R-1 is highly sensitive to ATP and mediates less regular Ca2+ oscillations. IP3R-3 is the least sensitive to IP3 and Ca2+, and tends to generate monophasic Ca2+ transients. Furthermore, we show for the first time functional interactions between coexpressed subtypes. Our results demonstrate that differential expression of IP3R subtypes helps to encode IP3-mediated Ca2+ signalling. (+info)
The SH2 domain-containing inositol 5'-phosphatase (SHIP) recruits the p85 subunit of phosphoinositide 3-kinase during FcgammaRIIb1-mediated inhibition of B cell receptor signaling.
(6/2715)
Coligation of FcgammaRIIb1 with the B cell receptor (BCR) or FcepsilonRI on mast cells inhibits B cell or mast cell activation. Activity of the inositol phosphatase SHIP is required for this negative signal. In vitro, SHIP catalyzes the conversion of the phosphoinositide 3-kinase (PI3K) product phosphatidylinositol 3,4, 5-trisphosphate (PIP3) into phosphatidylinositol 3,4-bisphosphate. Recent data demonstrate that coligation of FcgammaRIIb1 with BCR inhibits PIP3-dependent Btk (Bruton's tyrosine kinase) activation and the Btk-dependent generation of inositol trisphosphate that regulates sustained calcium influx. In this study, we provide evidence that coligation of FcgammaRIIb1 with BCR induces binding of PI3K to SHIP. This interaction is mediated by the binding of the SH2 domains of the p85 subunit of PI3K to a tyrosine-based motif in the C-terminal region of SHIP. Furthermore, the generation of phosphatidylinositol 3,4-bisphosphate was only partially reduced during coligation of BCR with FcgammaRIIb1 despite a drastic reduction in PIP3. In contrast to the complete inhibition of Tec kinase-dependent calcium signaling, activation of the serine/threonine kinase Akt was partially preserved during BCR and FcgammaRIIb1 coligation. The association of PI3K with SHIP may serve to activate PI3K and to regulate downstream events such as B cell activation-induced apoptosis. (+info)
Gab-family adapter proteins act downstream of cytokine and growth factor receptors and T- and B-cell antigen receptors.
(7/2715)
We previously found that the adapter protein Gab1 (110 kD) is tyrosine-phosphorylated and forms a complex with SHP-2 and PI-3 kinase upon stimulation through either the interleukin-3 receptor (IL-3R) or gp130, the common receptor subunit of IL-6-family cytokines. In this report, we identified another adapter molecule (100 kD) interacting with SHP-2 and PI-3 kinase in response to various stimuli. The molecule displays striking homology to Gab1 at the amino acid level; thus, we named it Gab2. It contains a PH domain, proline-rich sequences, and tyrosine residues that bind to SH2 domains when they are phosphorylated. Gab1 is phosphorylated on tyrosine upon stimulation through the thrombopoietin receptor (TPOR), stem cell factor receptor (SCFR), and T-cell and B-cell antigen receptors (TCR and BCR, respectively), in addition to IL-3R and gp130. Tyrosine phosphorylation of Gab2 was induced by stimulation through gp130, IL-2R, IL-3R, TPOR, SCFR, and TCR. Gab1 and Gab2 were shown to be substrates for SHP-2 in vitro. Overexpression of Gab2 enhanced the gp130 or Src-related kinases-mediated ERK2 activation as that of Gab1 did. These data indicate that Gab-family molecules act as adapters for transmitting various signals. (+info)
Involvement of wiskott-aldrich syndrome protein in B-cell cytoplasmic tyrosine kinase pathway.
(8/2715)
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. We identified one of the major phosphoproteins associated with Btk in the preB cell line NALM6 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. We demonstrated that WASP was 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 us to evaluate the catalytic specificity of Btk, the exact nature of which is still unknown. (+info)