B cell receptor signaling involves physical and functional association of FAK with Lyn and IgM

B cell receptor (BCR) stimulation induces phosphorylation of a number of proteins, leading to functional activation of B lymphocytes. Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase, involved in a variety of signaling pathways. In this study, we show that FAK is tyrosine-phosphorylated and activated following BCR stimulation. We also demonstrate constitutive association of FAK with the Src-family kinase Lyn and with components of the BCR. Association of Lyn with FAK which was not correlated with BCR-induced activation of both kinases, appeared to be mediated via the binding of Lyn to the COOH-terminal part of the FAK molecule. Our results indicate that FAK is a component of the BCR complex and that it participates in BCR signaling.     

A computational model for early events in B cell antigen receptor signaling: analysis of the roles of Lyn and Fyn

BCR signaling regulates the activities and fates of B cells. BCR signaling encompasses two feedback loops emanating from Lyn and Fyn, which are Src family protein tyrosine kinases (SFKs). Positive feedback arises from SFK-mediated trans phosphorylation of BCR and receptor-bound Lyn and Fyn, which increases the kinase activities of Lyn and Fyn. Negative feedback arises from SFK-mediated cis phosphorylation of the transmembrane adapter protein PAG1, which recruits the cytosolic protein tyrosine kinase Csk to the plasma membrane, where it acts to decrease the kinase activities of Lyn and Fyn. To study the effects of the positive and negative feedback loops on the dynamical stability of BCR signaling and the relative contributions of Lyn and Fyn to BCR signaling, we consider in this study a rule-based model for early events in BCR signaling that encompasses membrane-proximal interactions of six proteins, as follows: BCR, Lyn, Fyn, Csk, PAG1, and Syk, a cytosolic protein tyrosine kinase that is activated as a result of SFK-mediated phosphorylation of BCR. The model is consistent with known effects of Lyn and Fyn deletions. We find that BCR signaling can generate a single pulse or oscillations of Syk activation depending on the strength of Ag signal and the relative levels of Lyn and Fyn. We also show that bistability can arise in Lyn- or Csk-deficient cells.     

CD19 amplifies B lymphocyte signal transduction by regulating Src-family protein tyrosine kinase activation.

Ligation of the B cell Ag receptor (BCR) induces cellular activation by stimulating Src-family protein tyrosine kinases (PTKs) to phosphorylate members of the BCR complex. Subsequently, Src-family PTKs, particularly Lyn, are proposed to phosphorylate and bind CD19, a cell-surface costimulatory molecule that regulates mature B cell activation. Herein, we show that B cells from CD19-deficient mice have diminished Lyn kinase activity and BCR phosphorylation following BCR ligation. Tyrosine phosphorylation of other Src-family PTKs was also decreased in CD19-deficient B cells. In wild-type B cells, CD19 was constitutively complexed with Vav, Lyn, and other Src-family PTKs, with CD19 phosphorylation and its associations with Lyn and Vav increased after BCR ligation. Constitutive CD19/Lyn/Vav complex signaling may therefore be responsible for the establishment of baseline signaling thresholds in B cells before Ag receptor ligation, in addition to accelerating signaling following BCR engagement or other transmembrane signals. In vitro kinase assays using purified CD19 and purified Lyn revealed that the kinase activity of Lyn was significantly increased when coincubated with CD19. Thus, constitutive and induced CD19/Lyn complexes are likely to regulate basal signaling thresholds and BCR signaling by amplifying the kinase activity of Lyn and other Src-family PTKs. These in vivo and in vitro findings demonstrate a novel mechanism by which CD19 regulates signal transduction in B lymphocytes. The absence of this CD19/Src-family kinase amplification loop may account for the hyporesponsive phenotype of CD19-deficient B cells.     

Regulation of microtubule formation in activated mast cells by complexes of gamma-tubulin with Fyn and Syk kinases

Aggregation of the high-affinity IgE receptors (FcepsilonRIs) on the surface of granulated mast cells initiates a chain of signaling events culminating in the release of allergy mediators. Although microtubules are involved in mast cell degranulation, the molecular mechanism that controls microtubule rearrangement after FcepsilonRI triggering is poorly understood. In this study, we show that the activation of bone marrow-derived mast cells (BMMCs) induced by FcepsilonRI aggregation or treatment with pervanadate leads to a rapid polymerization of microtubules. This polymerization was not dependent on the presence of Lyn kinase as determined by experiments with BMMCs isolated from Lyn-negative mice. One of the key regulators of microtubule polymerization is gamma-tubulin. Immunoprecipitation experiments revealed that gamma-tubulin from activated cells formed complexes with Fyn and Syk protein tyrosine kinases and several tyrosine phosphorylated proteins from both wild-type and Lyn(-/-) BMMCs. Pretreatment of the cells with Src-family or Syk-family selective tyrosine kinase inhibitors, PP2 or piceatannol, respectively, inhibited the formation of microtubules and reduced the amount of tyrosine phosphorylated proteins in gamma-tubulin complexes, suggesting that Src and Syk family kinases are involved in the initial stages of microtubule formation. This notion was corroborated by pull-down experiments in which gamma-tubulin complex bounds to the recombinant Src homology 2 and Src homology 3 domains of Fyn kinase. We propose that Fyn and Syk kinases are involved in the regulation of binding properties of gamma-tubulin and/or its associated proteins, and thus modulate the microtubule nucleation in activated mast cells.     

Functional analysis of Csk in signal transduction through the B-cell antigen receptor.

In B cells, two classes of protein tyrosine kinases (PTKs), the Src family of PTKs (Lyn, Fyn, Lck, and Blk) and non-Src family of PTKs (Syk), are known to be involved in signal transduction induced by the stimulation of the B-cell antigen receptor (BCR). Previous studies using Lyn-negative chicken B-cell clones revealed that Lyn is necessary for transduction of signals through the BCR. The kinase activity of the Src family of PTKs is negatively regulated by phosphorylation at the C-terminal tyrosine residue, and the PTK Csk has been demonstrated to phosphorylate this C-terminal residue of the Src family of PTKs. To investigate the role of Csk in BCR signaling, Csk-negative chicken B-cell clones were generated. In these Csk-negative cells, Lyn became constitutively active and highly phosphorylated at the autophosphorylation site, indicating that Csk is necessary to sustain Lyn in an inactive state. Since the C-terminal tyrosine phosphorylation of Lyn is barely detectable in the unstimulated, wild-type B cells, our data suggest that the activities of Csk and a certain protein tyrosine phosphatase(s) are balanced to maintain Lyn at a hypophosphorylated and inactive state. Moreover, we show that the kinase activity of Syk was also constitutively activated in Csk-negative cells. The degree of activation of both the Lyn and Syk kinases in Csk-negative cells was comparable to that observed in wild-type cells after BCR stimulation. However, BCR stimulation was still necessary in Csk-negative cells to elicit tyrosine phosphorylation of cellular proteins, as well as calcium mobilization and inositol 1,4,5-trisphosphate generation. These results suggest that not only activation of the Lyn and Syk kinases but also additional signals induced by the cross-linking of the BCR are required for full transduction of BCR signaling.     

Dual role of the tyrosine activation motif of the Ig-alpha protein during signal transduction via the B cell antigen receptor.

The B cell antigen receptor (BCR) is a multimeric protein complex consisting of the ligand binding immunoglobulin molecule and the Ig-alpha/beta heterodimer that mediates intracellular signalling by coupling the receptor to protein tyrosine kinases (PTKs). Transfection of the Ig-alpha deficient myeloma cell line J558L microns with expression vectors coding for mutated Ig-alpha allowed us to test the function of the tyrosines in the cytoplasmic region of Ig-alpha in the context of the BCR. Furthermore we expressed Ig-alpha mutations as chimeric CD8-Ig-alpha molecules on K46 B lymphoma cells and tested their signalling capacity in terms of PTK activation and release of calcium. We show here that the conserved tyrosine residues in the cytoplasmic portion of Ig-alpha have a dual role. First, they are required for efficient activation of PTKs during signal induction and second, one of them is subject to phosphorylation by activated src-related PTKs. Phosphorylation on tyrosine in the cytoplasmic portion of Ig-alpha is discussed as a possible mechanism to couple the BCR to SH2 domain-carrying molecules.     

B-cell antigen receptor motifs have redundant signalling capabilities and bind the tyrosine kinases PTK72, Lyn and Fyn

BACKGROUND: The 13 cell antigen receptor (BCR) is a multimeric protein complex consisting of an antigen recognition structure (membrane immunoglobulin) and two associated proteins, lg-alpha and Ig-beta It has been proposed that signalling through the BCR involves Ig-alpha and Ig-beta. Both of these proteins contain within their cytoplasmic domains an amino-acid motif that is present in a number of immune recognition receptors, including the BCR, T-cell antigen receptor and Fc receptor complexes. This motif, termed the antigen-receptor homology motif (ARH1), appears to have signal transduction ability. RESULTS: We now show that the presence of cytoplasmic regions containing the ARM motif from either Ig-alpha or Ig-beta is sufficient to confer signalling capability on an otherwise non-functional fusion protein. Both Ig-alpha- and Ig-beta-containing chimeras induced, in an apparently redundant fashion, signalling events seen upon membrane immunoglobulin crosslinking, including tyrosine phosphorylation of particular proteins, phosphoinositicle breakdown and calcium mobilization. Furthermore, crosslinking of the chimeras resulted in tyrosine phosphorylation of the Ig-alpha and Tg-beta tails and their association with the tyrosine kinases PTK72, p53/56(lyn) and p59(fyn). CONCLUSIONS: These observations indicate that Ig-alpha and Ig-beta are responsible for coupling membrane immunoglobulin to intracellular signalling components. Moreover, they demonstrate that a number of tyrosine kinases associate directly with the cytoplasmic domains of both Ig-alpha and Ig-beta. Stimulation of the chimeras, which results in tyrosine phosphorylation of the ig-alpha and Ig-beta tails, is a prerequisite for some of these associations. The implications of these findings for the mechanism by which the BCR initiates the signalling reactions are discussed.     

Amplification of B cell antigen receptor signaling by a Syk/ITAM positive feedback loop

We have established a protocol allowing transient and inducible coexpression of many foreign genes in Drosophila S2 Schneider cells. With this powerful approach of reverse genetics, we studied the interaction of the protein tyrosine kinases Syk and Lyn with the B cell antigen receptor (BCR). We find that Lyn phosphorylates only the first tyrosine whereas Syk phosphorylates both tyrosines of the BCR immunoreceptor tyrosine-based activation motif (ITAM). Furthermore, we show that Syk is a positive allosteric enzyme, which is strongly activated by the binding to the phosphorylated ITAM tyrosines, thus initiating a positive feedback loop at the receptor. The BCR-dependent Syk activation and signal amplification is efficiently counterbalanced by protein tyrosine phosphatases, the activity of which is regulated by H(2)O(2) and the redox equilibrium inside the cell.     

Substrate recognition by the Lyn protein-tyrosine kinase. NMR structure of the immunoreceptor tyrosine-based activation motif signaling region of the B cell antigen receptor

The immunoreceptor tyrosine-based activation motif (ITAM) plays a central role in transmembrane signal transduction in hematopoietic cells by mediating responses leading to proliferation and differentiation. An initial signaling event following activation of the B cell antigen receptor is phosphorylation of the CD79a (Ig-alpha) ITAM by Lyn, a Src family protein-tyrosine kinase. To elucidate the structural basis for recognition between the ITAM substrate and activated Lyn kinase, the structure of an ITAM-derived peptide bound to Lyn was determined using exchange-transferred nuclear Overhauser NMR spectroscopy. The bound substrate structure has an irregular helix-like character. Docking based on the NMR data into the active site of the closely related Lck kinase strongly favors ITAM binding in an orientation similar to binding of cyclic AMP-dependent protein kinase rather than that of insulin receptor tyrosine kinase. The model of the complex provides a rationale for conserved ITAM residues, substrate specificity, and suggests that substrate binds only the active conformation of the Src family tyrosine kinase, unlike the ATP cofactor, which can bind the inactive form.     

Tyrosine kinases Lyn and Syk regulate B cell receptor-coupled Ca2+ mobilization through distinct pathways.

Stimulation of B lymphocytes through their antigen receptor (BCR) results in rapid increases in tyrosine phosphorylation on a number of proteins and induces both an increase of phosphatidylinositol and mobilization of cytoplasmic free calcium. The BCR associates with two classes of tyrosine kinase: Src-family kinase (Lyn, Fyn, Blk or Lck) and Syk kinase. To dissect the functional roles of these two types of kinase in BCR signaling, lyn-negative and syk-negative B cell lines were established. Syk-deficient B cells abolished the tyrosine phosphorylation of phospholipase C-gamma 2, resulting in the loss of both inositol 1,4,5-trisphosphate (IP3) generation and calcium mobilization upon receptor stimulation. Crosslinking of BCR on Lyn-deficient cells evoked a delayed and slow Ca2+ mobilization, despite the normal kinetics of IP3 turnover. These results demonstrate that Syk mediates IP3 generation, whereas Lyn regulates Ca2+ mobilization through a process independent of IP3 generation.     

In vivo and in vitro specificity of protein tyrosine kinases for immunoglobulin G receptor (FcgammaRII) phosphorylation.

Human B cells express four immunoglobulin G receptors, FcgammaRIIa, FcgammaRIIb1, FcgammaRIIb2, and FcgammaRIIc. Coligation of either FcgammaRII isoform with the B-cell antigen receptor (BCR) results in the abrogation of B-cell activation, but only the FcgammaRIIa/c and FcgammaIIb1 isoforms become phosphorylated. To identify the FcgammaRII-phosphorylating protein tyrosine kinase (PTK), we used the combination of an in vitro and an in vivo approach. In an in vitro assay using recombinant cytoplasmic tails of the different FcgammaRII isoforms as well as tyrosine exchange mutants, we show that each of the BCR-associated PTKs (Lyn, Blk, Fyn, and Syk) shows different phosphorylation patterns with regard to the different FcgammaR isoforms and point mutants. While each PTK phosphorylated FcgammaRIIa/c, FcgammaRIIb1 was phosphorylated by Lyn and Blk whereas FcgammaRIIb2 became phosphorylated only by Blk. Mutants lacking both tyrosine residues of the immune receptor tyrosine-based activation motif (ITAM) of FcgammaRIIa/c were not phosphorylated by Blk and Fyn, while Lyn-mediated phosphorylation was dependent on the presence of the C-terminal tyrosine of the ITAM. Results obtained in assays using an FcgammaR- B-cell line transfected with wild-type or mutated FcgammaRIIa demonstrated that exchange of the C-terminal tyrosine of the ITAM of FcgammaRIIa/c was sufficient to abolish FcgammaRIIa/c phosphorylation in B cells. Additionally, we could show that Lyn and Fyn bind to FcgammaRIIa/c, with the ITAM being necessary for association. Comparison of the phosphorylation pattern of each PTK observed in vitro with the phosphorylation pattern observed in vivo suggests that Lyn is the most likely candidate for FcgammaRIIa/c and FcgammaRIIb1 phosphorylation in vivo.     

Floating the raft hypothesis for immune receptors: access to rafts controls receptor signaling and trafficking

The B cell antigen receptor (BCR) is a member of an important family of multichain immune recognition receptors, which are complexes composed of ligand-binding domains associated with signal-transduction complexes. The signaling components of these receptors have no inherent kinase activity but become tyrosine phosphorylated in their cytoplasmic domains by Src-family kinases upon oligomerization, thus initiating signaling cascades. The BCR is unique in this family in that, in addition to its signaling function, it also serves to deliver antigen to intracellular compartments where the antigen is processed and presented bound to major histocompatibility complex (MHC) class II molecules. Recent evidence indicates that both the signaling and antigen-trafficking functions of the BCR are regulated by cholesterol- and sphingolipid-rich plasma membrane microdomains termed rafts. Indeed, upon oligomerization, the BCR translocates into rafts that concentrate the Src-family kinase Lyn and is subsequently internalized directly from the rafts. Thus, translocation into rafts allows the association of the oligomerized BCR with Lyn and the initiation of both signaling and trafficking. Significantly, the access of the BCR to rafts appears to be controlled by a variety of B lymphocyte co-receptors, as well as factors including the developmental state of the B cell and viral infection. Thus, the translocation of the immune receptors into signaling-competent microdomains may represent a novel mechanism to initiate and regulate immune-cell activation.     

Cbl-mediated degradation of Lyn and Fyn induced by constitutive fibroblast growth factor receptor-2 activation supports osteoblast differentiation

Fibroblast growth factors (FGFs) play an important regulatory role in skeletal development and bone formation. However, the FGF signaling mechanisms controlling osteoblast function are poorly understood. Here, we identified a role for the Src family members Lyn and Fyn in osteoblast differentiation promoted by constitutive activation of FGF receptor-2 (FGFR2). We show that the overactive FGFR2 S252W mutation induced decreased Src family kinase tyrosine phosphorylation and activity associated with decreased Lyn and Fyn protein expression in human osteoblasts. Pharmacological stimulation of Src family kinases or transfection with Lyn or Fyn vectors repressed alkaline phosphatase (ALP) up-regulation induced by overactive FGFR2. Inhibition of proteasome activity restored normal Lyn and Fyn expression and ALP activity in FGFR2 mutant osteoblasts. Immunoprecipitation studies showed that Lyn, Fyn, and FGFR2 interacted with the ubiquitin ligase c-Cbl and ubiquitin. Transfection with c-Cbl in which the RING finger was disrupted or with c-Cbl with a point mutation that abolishes the binding ability of the Cbl phosphotyrosine-binding domain restored Src kinase activity and Lyn, Fyn, and FGFR2 levels and reduced ALP up-regulation in mutant osteoblasts. Thus, constitutive FGFR2 activation induces c-Cbl-dependent Lyn and Fyn proteasome degradation, resulting in reduced Lyn and Fyn kinase activity, increased ALP expression, and FGFR2 down-regulation. This reveals a common Cbl-mediated negative feedback mechanism controlling Lyn, Fyn, and FGFR2 degradation in response to overactive FGFR2 and indicates a role for Cbl-dependent down-regulation of Lyn and Fyn in osteoblast differentiation induced by constitutive FGFR2 activation.     

Phosphorylation of CD19 Y484 and Y515, and linked activation of phosphatidylinositol 3-kinase, are required for B cell antigen receptor-mediated activation of Bruton's tyrosine kinase

Bruton's tyrosine kinase (Btk) plays a critical role in B cell Ag receptor (BCR) signaling, as indicated by the X-linked immunodeficiency and X-linked agammaglobulinemia phenotypes of mice and men that express mutant forms of the kinase. Although Btk activity can be regulated by Src-family and Syk tyrosine kinases, and perhaps by phosphatidylinositol 3,4,5-trisphosphate, BCR-coupled signaling pathways leading to Btk activation are poorly understood. In view of previous findings that CD19 is involved in BCR-mediated phosphatidylinositol 3-kinase (PI3-K) activation, we assessed its role in Btk activation. Using a CD19 reconstituted myeloma model and CD19 gene-ablated animals we found that BCR-mediated Btk activation and phosphorylation are dependent on the expression of CD19, while BCR-mediated activation of Lyn and Syk is not. Wortmannin preincubation inhibited the BCR-mediated activation and phosphorylation of Btk. Btk activation was not rescued in the myeloma by expression of a CD19 mutant in which tyrosine residues previously shown to mediate CD19 interaction with PI3-K, Y484 and Y515, were changed to phenylalanine. Taken together, the data presented indicate that BCR aggregation-driven CD19 phosphorylation functions to promote Btk activation via recruitment and activation of PI3-K. Resultant phosphatidylinositol 3,4,5-trisphosphate probably functions to localize Btk for subsequent phosphorylation and activation by Src and Syk family kinases.     

Evidence for glycosylphosphatidylinositol (GPI)-anchored eosinophil-derived neurotoxin (EDN) on human granulocytes

MIST (mast cell immunoreceptor signal transducer; also termed Clnk) is an adaptor protein structurally related to SLP-76-family hematopoietic cell-specific adaptor proteins. We demonstrate here that two major MIST-associated phosphoproteins expressed in mast cell lines are SLAP-130 and SKAP55, adaptors known to interact with the Src-homology (SH) 2 domain of Src-family protein tyrosine kinases (PTKs). MIST directly associated with SLAP-130 via its SH2 domain, and collaboration of SLAP-130 with SKAP55 was required for the recruitment of MIST to Lyn. Furthermore, MIST was preferentially recruited to Fyn rather than Lyn, which is regulated by higher affinity binding of SLAP-130 and SKAP55 with the Fyn-SH2 domain than the Lyn-SH2 domain. Our results suggest that the MIST–SLAP-130–SKAP55 adaptor complex functions downstream of high-affinity IgE receptor-associated Src-PTKs in mast cells.     

Affinity of Src family kinase SH3 domains for HIV Nef in vitro does not predict kinase activation by Nef in vivo

Nef is an HIV accessory protein required for high-titer viral replication and AIDS progression. Previous studies have shown that the SH3 domains of Hck and Lyn bind to Nef via proline-rich sequences in vitro, identifying these Src-related kinases as potential targets for Nef in vivo. Association of Nef with Hck causes displacement of the intramolecular interaction between the SH3 domain and the SH2-kinase linker, leading to kinase activation both in vitro and in vivo. In this study, we investigated whether interaction with Nef induces activation of other Src family kinases (Lyn, Fyn, Src, and Lck) following coexpression with Nef in Rat-2 fibroblasts. Coexpression with Nef induced Hck kinase activation and fibroblast transformation, consistent with previous results. In contrast, coexpression of Nef with Lyn was without effect, despite equivalent binding of Nef to full-length Lyn and Hck. Furthermore, Nef was found to suppress the kinase and transforming activities of Fyn, the SH3 domain of which exhibits low affinity for Nef. Coexpression with Nef did not alter c-Src or Lck tyrosine kinase or transforming activity in this system. Differential modulation of Src family members by Nef may produce unique downstream signals depending on the profile of Src kinases expressed in a given cell type.     

Association of Fyn and Lyn with the proline-rich domain of glycoprotein VI regulates intracellular signaling

The glycoprotein VI (GPVI)-Fc receptor (FcR) gamma-chain complex, a key activatory receptor for collagen on platelet surface membranes, is constitutively associated with the Src family kinases Fyn and Lyn. Molecular cloning of GPVI has revealed the presence of a proline-rich domain in the sequence of GPVI cytoplasmic tail which has the consensus for interaction with the Src homology 3 (SH3) domains of Fyn and Lyn. A series of in vitro experiments demonstrated the ability of the SH3 domains of both Src kinases to bind the proline-rich domain of GPVI. Furthermore, depletion of the proline-rich domain in GPVI (Pro(-)-GPVI) prevented binding of Fyn and Lyn and markedly reduced phosphorylation of FcR gamma-chain in transiently transfected COS-7 cells, but did not affect the association of the gamma-chain with GPVI. Jurkat cells stably transfected with wild type GPVI show robust increases in tyrosine phosphorylation and intracellular Ca2+ in response to the snake venom convulxin that targets GPVI. Importantly, convulxin is not able to activate cells transfected with Pro(-)-GPVI, even though the association with the immunoreceptor tyrosine-based activation motif-containing chains is maintained. These findings demonstrate that the proline-rich domain of GPVI mediates the association with Fyn/Lyn via their SH3 domain and that this interaction initiates activation signals through GPVI.