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.

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.

The CD2v protein of African swine fever virus interacts with the actin-binding adaptor protein SH3P7.

The predicted extracellular domain of the CD2v protein of African swine fever virus (ASFV) shares significant similarity to that of the CD2 protein in T cells but has a unique cytoplasmic domain of unknown function. Here we have shown that CD2v is expressed as a glycoprotein of approximately 105 kDa in ASFV-infected cells. In the absence of an extracellular ligand, the majority of CD2v appears to localize to perinuclear membrane compartments. Furthermore, we have shown using the yeast two-hybrid system and by direct binding studies that the cytoplasmic tail of CD2v binds to the cytoplasmic adaptor protein SH3P7 (mAbp1, HIP55), which has been reported to be involved in diverse cellular functions such as vesicle transport and signal transduction. A cDNA clone encoding a variant form of SH3P7 could also be identified and was found to be expressed in a wide range of porcine tissues. Deletion mutagenesis identified proline-rich repeats of sequence PPPKPC in the ASFV CD2v protein to be necessary and sufficient for binding to the SH3 domain of SH3P7. In ASFV-infected cells, CD2v and SH3P7 co-localized in areas surrounding the perinuclear virus factories. These areas also stained with an antibody that recognizes a Golgi network protein, indicating that they contained membranes derived from the Golgi network. Our data provide a first molecular basis for the understanding of the immunomodulatory functions of CD2v in ASFV-infected animals.

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.

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.

SHP-1 requires inhibitory co-receptors to down-modulate B cell antigen receptor-mediated phosphorylation of cellular substrates.

Signaling through the B cell antigen receptor (BCR) is negatively regulated by the SH2 domain-containing protein-tyrosine phosphatase SHP-1, which requires association with tyrosine-phosphorylated proteins for activation. Upon BCR ligation, SHP-1 has been shown to associate with the BCR, the cytoplasmic protein-tyrosine kinases Lyn and Syk, and the inhibitory co-receptors CD22 and CD72. How SHP-1 is activated by BCR ligation and regulates BCR signaling is, however, not fully understood. Here we demonstrate that, in the BCR-expressing myeloma line J558L mu 3, CD72 expression reduces the BCR ligation-induced phosphorylation of the BCR component Ig alpha/Ig beta and its cytoplasmic effectors Syk and SLP-65. Substrate phosphorylation was restored by expression of dominant negative mutants of SHP-1, whereas the SHP-1 mutants failed to enhance phosphorylation of the cellular substrates in the absence of CD72. This indicates that SHP-1 is efficiently activated by CD72 but not by other pathways in J558L mu m3 cells and that inhibition of SHP-1 specifically activated by CD72 reverses CD72-induced dephosphorylation of cellular substrates in these cells. Taken together, BCR-induced SHP-1 activation is likely to require inhibitory co-receptors such as CD72, and SHP-1 appears to mediate the negative regulatory effect of CD72 on BCR signaling by dephosphorylating Ig alpha/Ig beta and its downstream signaling molecules Syk and 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.

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.

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.

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.

Abnormal development and function of B lymphocytes in mice deficient for the signaling adaptor protein SLP-65.

During signal transduction through the B cell antigen receptor (BCR), several signaling elements are brought together by the adaptor protein SLP-65. We have investigated the role of SLP-65 in B cell maturation and function in mice deficient for SLP-65. While the mice are viable, B cell development is affected at several stages. SLP-65-deficient mice show increased proportions of pre-B cells in the bone marrow and immature B cells in peripheral lymphoid organs. B1 B cells are lacking. The mice show lower IgM and IgG3 serum titers and poor IgM but normal IgG immune responses. Mutant B cells show reduced Ca2+ mobilization and reduced proliferative responses to B cell mitogens. We conclude that while playing an important role, SLP-65 is not always required for signaling from the BCR.

Interaction of SLP adaptors with the SH2 domain of Tec family kinases.

Activation of lymphocytes through their antigen receptors leads to mobilization of intracellular Ca(2+) ions. This process requires expression of SLP adaptors and involves phosphorylation of phospholipase C-gamma isoforms by the Tec-related protein tyrosine kinase Btk in B cells and Itk in T cells. The SH2 domain of Btk and Itk is essential for phospholipase C-gamma phosphorylation and mutations in this domain lead to the X-linked agammaglobulinemia immuno deficiency in humans. Here we show that, in contrast to SH2 domains from other signaling proteins, the Btk and Itk SH2 domains exhibit a restricted binding specificity. They bind selectively to tyrosine-phosphorylated SLP-65 and SLP-76 in activated B and T cells, respectively. Our findings suggest that Btk/Itk and phospholipase C-gamma both bind via their SH2 domain to phosphorylated SLP adaptors, and that this association is required for the activation of phospholipase C-gamma.

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.

Analysis of tyrosine phosphorylation-dependent interactions between stimulatory effector proteins and the B cell co-receptor CD22.

The B cell-restricted transmembrane glycoprotein CD22 is rapidly phosphorylated on tyrosine in response to cross-linking of the B cell antigen receptor, thereby generating phosphotyrosine motifs in the cytoplasmic domain which recruit intracellular effector proteins that contain Src homology 2 domains. By virtue of its interaction with these effector proteins CD22 modulates signal transduction through the B cell antigen receptor. To define further the molecular mechanism by which CD22 mediates its co-receptor function, phosphopeptide mapping experiments were conducted to determine which of the six tyrosine residues in the cytoplasmic domain are involved in recruitment of the stimulatory effector proteins phospholipase Cgamma (PLCgamma), phosphoinositide 3-kinase (PI3K), Grb2, and Syk. The results obtained indicate that the protein tyrosine kinase Syk interacts with multiple CD22-derived phosphopeptides in both immunoprecipitation and reverse Far Western assays. In contrast, the Grb2.Sos complex was observed to bind exclusively to the fourth phosphotyrosine motif (Y828ENV) from CD22 and does so via a direct interaction based on Far Western and reverse Far Western blotting. Although both PLCgamma and PI3K were observed to bind to multiple phosphopeptides in precipitation experiments, subsequent studies using reverse Far Western blot analysis demonstrated that only the carboxyl-terminal phosphopeptide of CD22 (Y863VTL) binds directly to either one. This finding suggests that PLCgamma and PI3K may be recruited to CD22 either through a direct interaction with Tyr863 or indirectly through an association with one or more intermediate proteins.

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.

Induction of the antigen receptor expression on B lymphocytes results in rapid competence for signaling of SLP-65 and Syk.

The binding of antigen to the B cell antigen receptor (BCR) results in the activation of protein tyrosine kinases (PTKs) such as Lyn and Syk, and the phosphorylation of several substrate proteins including HS1 and SLP-65. How these signaling elements are connected to the BCR is not well understood. Using an expression vector for a tamoxifen-regulated Cre recombinase, we have developed a method that allows the inducible expression of the BCR. Disruption of the VH leader reading frame of the immunoglobulin heavy chain by two loxP sites is overcome by Cre-mediated DNA recombination and results in the cell surface expression of the BCR starting 4 h after exposure of transfected B cells to tamoxifen. This method can, in principle, be employed for the inducible expression of any secreted or type I transmembrane protein. By monitoring the activation of signaling elements in pervanadate-stimulated B cells expressing different levels of the BCR, we show here that phosphorylation of SLP-65 and Syk, but not of Lyn, is strictly dependent on the expression of the BCR on the cell surface. These data suggest that the BCR reorganizes its signaling molecules as soon as it appears on the cell surface.