Epstein-Barr virus latent membrane protein 2A (LMP2A) employs the SLP-65 signaling module.

In latently infected B lymphocytes, the Epstein-Barr virus (EBV) suppresses signal transduction from the antigen receptor through expression of the integral latent membrane protein 2A (LMP2A). At the same time, LMP2A triggers B cell survival by a yet uncharacterized maintenance signal that is normally provided by the antigen receptor. The molecular mechanisms are unknown as LMP2A-regulated signaling cascades have not been described so far. Using a novel mouse model we have identified the intracellular adaptor protein Src homology 2 (SH2) domain-containing leukocyte protein (SLP)-65 as a critical downstream effector of LMP2A in vivo. Biochemical analysis of the underlying signaling pathways revealed that EBV infection causes constitutive tyrosine phosphorylation of one of the two SLP-65 isoforms and complex formation between SLP-65 and the protooncoprotein CrkL (CT10 regulator of kinase like). This leads to antigen receptor-independent phosphorylation of Cbl (Casitas B lineage lymphoma) and C3G. In contrast, phospholipase C-gamma2 (PLC-gamma2) activation is completely blocked. Our data show that in order to establish a latent EBV infection, LMP2A selectively activates or represses SLP-65-regulated signaling pathways.

SLP-65: a new signaling component in B lymphocytes which requires expression of the antigen receptor for phosphorylation.

The B cell antigen receptor (BCR) consists of the membrane-bound immunoglobulin (Ig) molecule as antigen-binding subunit and the Ig-alpha/Ig-beta heterodimer as signaling subunit. BCR signal transduction involves activation of protein tyrosine kinases (PTKs) and phosphorylation of several proteins, only some of which have been identified. The phosphorylation of these proteins can be induced by exposure of B cells either to antigen or to the tyrosine phosphatase inhibitor pervanadate/H2O2. One of the earliest substrates in B cells is a 65-kD protein, which we identify here as a B cell adaptor protein. This protein, named SLP-65, is part of a signaling complex involving Grb-2 and Vav and shows homology to SLP-76, a signaling element of the T cell receptor. In pervanadate/H2O2-stimulated cells, SLP-65 becomes phosphorylated only upon expression of the BCR. These data suggest that SLP-65 is part of a BCR transducer complex.

Regulation of in vitro and in vivo immune functions by the cytosolic adaptor protein SKAP-HOM.

SKAP-HOM is a cytosolic adaptor protein representing a specific substrate for the Src family protein tyrosine kinase Fyn. Previously, several groups have provided experimental evidence that SKAP-HOM (most likely in cooperation with the cytosolic adaptor protein ADAP) is involved in regulating leukocyte adhesion. To further assess the physiological role of SKAP-HOM, we investigated the immune system of SKAP-HOM-deficient mice. Our data show that T-cell responses towards a variety of stimuli are unaffected in the absence of SKAP-HOM. Similarly, B-cell receptor (BCR)-mediated total tyrosine phosphorylation and phosphorylation of Erk, p38, and JNK, as well as immunoreceptor-mediated Ca(2+) responses, are normal in SKAP-HOM(-/-) animals. However, despite apparently normal membrane-proximal signaling events, BCR-mediated proliferation is strongly attenuated in the absence of SKAP-HOM(-/-). In addition, adhesion of activated B cells to fibronectin (a ligand for beta1 integrins) as well as to ICAM-1 (a ligand for beta2 integrins) is strongly reduced. In vivo, the loss of SKAP-HOM results in a less severe clinical course of experimental autoimmune encephalomyelitis following immunization of mice with the encephalitogenic peptide of MOG (myelin oligodendrocyte glycoprotein). This is accompanied by strongly reduced serum levels of MOG-specific antibodies and lower MOG-specific T-cell responses. In summary, our data suggest that SKAP-HOM is required for proper activation of the immune system, likely by regulating the cross-talk between immunoreceptors and integrins.

SH3P7 is a cytoskeleton adapter protein and is coupled to signal transduction from lymphocyte antigen receptors.

Lymphocytes respond to antigen receptor engagement with tyrosine phosphorylation of many cellular proteins, some of which have been identified and functionally characterized. Here we describe SH3P7, a novel substrate protein for Src and Syk family kinases. SH3P7 migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a 55-kDa protein that is preferentially expressed in brain, thymus, and spleen. It contains multiple amino acid sequence motifs, including two consensus tyrosine phosphorylation sites of the YXXP type and one SH3 domain. A region of sequence similarity, which we named SCAD, was found in SH3P7 and three actin-binding proteins. The SCAD region may represent a new type of protein-protein interaction domain that mediates binding to actin. Consistent with this possibility, SH3P7 colocalizes with actin filaments of the cytoskeleton. Altogether, our data implicate SH3P7 as an adapter protein which links antigen receptor signaling to components of the cytoskeleton.

Characterization of the B cell-specific adaptor SLP-65 and other protein tyrosine kinase substrates by two-dimensional gel electrophoresis.

The identification of substrates for protein tyrosine kinases in B cells is a critical step to a better understanding of the molecular mechanism(s) of lymphocyte activation through the antigen receptor. The substrate proteins were immunopurified from stimulated B cells and separated by two-dimensional gel electrophoresis techniques using either the isoelectric focussing (IEF)/SDS-PAGE or the non-equilibrium PH gradient electrophoresis (NEPHGE)/SDS-PAGE method. The biochemical characteristics of the proteins (isoelectric point and relative molecular mass) obtained and the subsequent use of antibodies that are specific for different cellular proteins confirmed the participation of HS1, Vav, Ig-alpha, Lyn and Btk in antigen receptor-mediated signal transduction. The heat shock cognate protein HSC70 was identified as a novel substrate protein in activated B cells. An important signaling function has previously been suggested for a 65-kDa protein (p65), whose phosphorylation can be detected before that of other substrate proteins. The analysis identified p65 as a so far unknown protein. Based on p65 peptide sequences, the full length cDNA was isolated and found to encode a B cell-specific adaptor protein, called SLP-65.

B cell signaling. Introduction.

The B cell antigen receptor (BCR) is composed of the membrane form of the immunoglobulin (Ig) and the Ig-alpha/Ig-beta heterodimer, which function as the antigen recognition component and the signaling component, respectively. A signal transmitted by BCR modulates gene expression, adhesion or survival, thereby determining the fate of antigen-encountered B cells. BCR proximal signaling occurs within cholesterol- and sphingolipid-rich plasma membrane microdomains termed lipid rafts, and involves tyrosine kinases such as Lyn, Syk and Btk and the adapter molecule SLP65/BLNK. Although the distal signaling cascades via BCR are not yet fully elucidated, various components are already identified, such as lipid kinases and small G-proteins. BCR signaling is regulated by various membrane molecules termed co-receptors such as CD19 and CD22. The BCR co-receptors appear to be required for normal immune functions. Viral proteins such as LMP2 also regulate BCR signaling to maintain viral latency. Various aspects of BCR signaling and its regulatory mechanisms are discussed in this issue.

Multitasking of Ig-alpha and Ig-beta to regulate B cell antigen receptor function.

Since their discovery as signaling subunits of the B cell antigen receptor (BCR), Ig-alpha and Ig-beta are discussed to serve either a redundant or distinct function for B cell development, maintenance, and activation. Dependent upon the experimental system that has been used to address this issue, evidence could be provided to support both possibilities. Only recently has it become clear that Ig-alpha and Ig-beta possess a unique signaling identity but that both together are required to orchestrate proper B cell function in vivo. Here we discuss some of the underlying mechanisms that may involve direct coupling to discrete subsets of BCR effector proteins, such as protein tyrosine kinases or the intracellular adaptor SLP-65/BLNK.

Signal transduction elements of the B cell antigen receptor and their role in immunodeficiencies.

The primary function of B lymphocytes is to contribute to the elimination of foreign antigens by producing large amounts of soluble antibodies. The activation of B cells through their antigen receptor triggers a dynamic network of intracellular signaling proteins. The recent identification of the cytoplasmic adaptor protein SLP-65 (also called BLNK or BASH) provided insight in how the antigen receptor-regulated protein tyrosine kinases couple to downstream signaling cascades, including the mobilization of Ca2+ ions, activation of mitogen-activated kinases and reorganization of the cytoskeleton architecture. While these events have been mostly studied in mature B cells, it is now clear that the components of the antigen receptor and its downstream effector elements play also a central role during early and late B cell development, and in the apoptotic elimination of B cells with reactivity to self-antigens. Thus, genetic defects affecting the expression of antigen receptor subunits or its intracellular signaling proteins can interfere with B cell development and activation, and can cause severe antibody deficiencies in mouse and man. In this article I summarize our current picture of the B cell antigen receptor, how the extracellular signal is transported into the cell interior, and how dysregulation of these processes contribute to immune defects.

PDK1 controls upstream PI3K expression and PIP3 generation.

The PI3K/PDK1/Akt signaling axis is centrally involved in cellular homeostasis and controls cell growth and proliferation. Due to its key function as regulator of cell survival and metabolism, the dysregulation of this pathway is manifested in several human pathologies including cancers and immunological diseases. Thus, current therapeutic strategies target the components of this signaling cascade. In recent years, numerous feedback loops have been identified that attenuate PI3K/PDK1/Akt-dependent signaling. Here, we report the identification of an additional level of feedback regulation that depends on the negative transcriptional control of phosphatidylinositol 3-kinase (PI3K) class IA subunits. Genetic deletion of 3-phosphoinositide-dependent protein kinase 1 (PDK1) or the pharmacological inhibition of its downstream effectors, that is, Akt and mammalian target of rapamycin (mTOR), relieves this suppression and leads to the upregulation of PI3K subunits, resulting in enhanced generation of phosphatidylinositol-3,4,5-trisphosphate (PIP3). Apparently, this transcriptional induction is mediated by the concerted action of different transcription factor families, including the transcription factors cAMP-responsive element-binding protein and forkhead box O. Collectively, we propose that PDK1 functions as a cellular sensor that balances basal PIP3 generation at levels sufficient for survival but below a threshold being harmful to the cell. Our study suggests that the efficiency of therapies targeting the aberrantly activated PI3K/PDK1/Akt pathway might be increased by the parallel blockade of feedback circuits.

The B cell antigen receptor of class IgD can be expressed on the cell surface in two different forms.

Membrane-bound immunoglobulins of the IgM and IgD class are expressed on the B cell surface in association with a disulfide-linked heterodimer consisting of alpha and beta subunits. While the alpha component of the IgM antigen receptor (IgM-alpha, 34 kDa) is encoded by the B cell-specific gene mb-1, the gene coding for IgD-alpha (35 kDa) has not yet been identified. We show here that the alpha component of the IgD antigen receptor is also encoded by the mb-1 gene. The difference in molecular weight between IgM-alpha and IgD-alpha thus seems to be due to post-translational modifications of the mb-1 gene product. We also demonstrate that the previously described myeloma variant J558L delta m2.6 expresses an alternative form of the IgD antigen receptor, which does not contain an alpha/beta heterodimer.

Glycosyl-phosphatidylinositol linkage as a mechanism for cell-surface expression of immunoglobulin D.

The B-cell antigen receptor of the IgM and IgD class is a multimeric complex consisting of the membrane-bound form of the immunoglobulin molecule and two other proteins, Ig-alpha and Ig-beta. The Ig-alpha and Ig-beta proteins form a disulphide-linked alpha/beta heterodimer and are encoded by the mb-1 (ref 9, 10) and B29 genes, respectively. Surface expression of the membrane-bound IgM molecule requires assembly with the alpha/beta heterodimer. The IgD molecule, however, can be expressed on the cell surface in an alpha/beta-dependent and -independent form. We show here that in the alpha/beta-independent form the IgD molecule is anchored in the plasma membrane through a glycosyl-phosphatidylinositol linker. In the presence of the alpha/beta heterodimer, most of the otherwise glycosyl-phosphatidylinositol-linked IgD molecule is expressed on the cell surface as transmembrane proteins.

Initiation and processing of signals from the B cell antigen receptor.

Current models of signal transduction from the antigen receptors on B and T cells still resemble equations with several unknown elements. Data from recent knockout experiments in cell lines and mice contradict the assumption that Src-family kinase and tyrosine kinases of the Syk/Zap-70 family are the transducer elements that set signaling from these receptors in motion. Using a functional definition of signaling elements, we discuss the current knowledge of signaling events from the BCR and suggest the existence of an as-yet-unknown BCR transducer complex.

Association of SLP-65/BLNK with the B cell antigen receptor through a non-ITAM tyrosine of Ig-alpha.

The cytoplasmic adaptor protein SLP-65 (BLNK or BASH) is a critical downstream effector of the B cell antigen receptor (BCR). Tyrosine-phosphorylated SLP-65 assembles intracellular signaling complexes such as the Ca(2 +) initiation complex encompassing phospholipase C-gamma2 and Bruton's tyrosine kinase. It is, however, unclear how the SLP-65 signaling module can be recruited to the plasma membrane. Here we show that following B cell stimulation, SLP-65 associates directly with the BCR signaling subunit, the Ig-alpha / Ig-beta heterodimer. The interaction is mediated by the Src homology 2 domain of SLP-65 and the phosphorylated Ig-alpha tyrosine 204, which is located outside of the immunoreceptor tyrosine-based activation motif. Our data identify an unexpected BCR phosphorylation pattern and indicate that Ig-alpha has the capability to serve as transmembrane adaptor in BCR signaling.

The serine and threonine residues in the Ig-alpha cytoplasmic tail negatively regulate immunoreceptor tyrosine-based activation motif-mediated signal transduction.

The B cell antigen receptor (BCR) is a multiprotein complex consisting of the membrane-bound Ig molecule and the Ig-alpha/Ig-beta heterodimer. On BCR engagement, Ig-alpha and Ig-beta become phosphorylated not only on tyrosine residues of the immunoreceptor tyrosine-based activation motif but also on serine and threonine residues. We have mutated all serine and threonine residues in the Ig-alpha tail to alanine and valine, respectively. The mutated Ig-alpha sequence was expressed either as a single-chain Fv/Ig-alpha molecule or in the context of the complete BCR. In both cases, the mutated Ig-alpha showed a stronger tyrosine phosphorylation than the wild-type Ig-alpha and initiated increased signaling on stimulation. These findings suggest that serine/threonine kinases can negatively regulate signal transduction from the BCR.

Tyrosine-phosphorylated forms of Ig beta, CD22, TCR zeta and HOSS are major ligands for tandem SH2 domains of Syk.

The protein tyrosine kinase Syk plays an important role in signal transduction from the B cell antigen receptor and possibly also from the TCR. We have examined the binding specificity of Syk-derived SH2 domains in vitro and found that the tandem SH2 domains have two major ligands in activated Ramos B cells as well as in activated Jurkat T cells. The SH2-binding proteins in Ramos B cells were identified as the tyrosine-phosphorylated forms of the Ig alpha beta heterodimer and of CD22. Binding to the Ig alpha beta heterodimer seems to occur predominantly via Ig beta, indicating that the two receptor components might couple to distinct signaling pathways. In Jurkat T cells one of the SH2-binding proteins represents the tyrosine-phosphorylated TCR zeta chain. The identity of the second SH2 ligand, called HOSS, is not known. HOSS is discussed as a putative member of the receptor family characterized by the immunoreceptor tyrosine-based activation motif.

Evidence for a preformed transducer complex organized by the B cell antigen receptor.

The B cell antigen receptor (BCR) consists of the membrane-bound immunoglobulin (mIg) molecule and the Ig-alpha/Ig-beta heterodimer, which functions as signaling subunit of the receptor. Stimulation of the BCR activates protein tyrosine kinases (PTKs) that phosphorylate a number of substrate proteins, including the Ig-alpha/Ig-beta heterodimer of the BCR itself. How the PTKs become activated after BCR engagement is not known at present. Here, we show that BCR-negative J558L cells treated with the protein tyrosine phosphatase inhibitor pervanadate/H2O2 display only a weak substrate phosphorylation. However, in BCR-positive transfectants of J558L, treatment with pervanadate/H2O2 induces a strong phosphorylation of several substrate proteins. Treatment with pervanadate/H2O2 does not result in receptor crosslinking, yet the pattern of protein phosphorylation is similar to that observed after BCR stimulation by antigen. The response requires cellular integrity because tyrosine phosphorylation of most substrates is not visible in cell lysates. Cells that express a BCR containing an Ig-alpha subunit with a mutated immunoreceptor tyrosine-based activation motif display a delayed response. The data suggest that, once expressed on the surface, the BCR organizes protein tyrosine phosphatases, PTKs, and their substrates into a transducer complex that can be activated by pervanadate/H202 in the absence of BCR crosslinking. Assembly of this preformed complex seems to be a prerequisite for BCR-mediated signal transduction.

An unsolved problem of the clonal selection theory and the model of an oligomeric B-cell antigen receptor.

The B cell antigen receptor (BCR) plays a central role in the development, survival and activation of B lymphocytes. As the pre-BCR, it controls allelic exclusion of heavy chains and the expansion of pre-B cells. As the BCR, it controls the positive and negative selection of immature B cells as well as the survival and activation of mature B cells. Recent studies of receptors have shown that it is the ligand that brings them into the conformation necessary for signaling. How the multiple and structurally diverse antigens could fulfill this task for the BCR is unknown, and we regard this as an unsolved problem of Burnet's clonal selection theory This question and our recent biochemical studies lead us to propose a new model for the BCR, according to which the BCR exists as a precise oligomeric complex on the B cell surface. In this form, it can signal positive selection and survival of B cells. Binding to self- or foreign antigen results in a distortion of the oligomeric complex that gives the signal for negative selection of immature and activation of mature B cells.