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.     

CD45 negatively regulates lyn activity by dephosphorylating both positive and negative regulatory tyrosine residues in immature B cells.

Using CD45-deficient clones from the immature B cell line, WEHI-231, we previously demonstrated that CD45 selectively dephosphorylates the Src-family protein tyrosine kinase Lyn and inhibits its kinase activity. To further define the mechanisms of CD45 action on Lyn, we metabolically labeled Lyn from CD45-positive and -negative WEHI-231 cells and analyzed cyanogen bromide fragments by SDS-PAGE analysis. Phosphoamino acid analysis confirmed that Lyn is tyrosine phosphorylated with little serine or threonine phosphorylation. In CD45-negative cells, two bands at 8.2 and 4.1 kDa were phosphorylated in the absence of B cell Ag receptor (BCR) ligation. The 8.2-kDa band corresponded to a fragment containing the positive regulatory site (Tyr397), as assessed by its size and its phosphorylation in an in vitro kinase assay. The 4.1-kDa band was phosphorylated by COOH-terminal Src kinase, suggesting that it contains the COOH-terminal negative regulatory site (Tyr508). CD45 was also shown to dephosphorylate autophosphorylated Lyn in vitro. Thus, CD45 dephosphorylates not only the negative but also the positive regulatory tyrosine residues of Lyn. Furthermore, coimmunoprecipitations using anti-Igalpha Ab demonstrated that Lyn associated with the resting BCR was constitutively phosphorylated and activated in CD45-negative cells. In the parental cells, both regulatory sites were phosphorylated on BCR ligation. Taken collectively, these results suggest that CD45 keeps both BCR-associated and total cytoplasmic pools of Lyn in an inactive state, and a mechanism by which Lyn is activated by relative reduction of CD45 effect may be operative on BCR ligation.     

A CD19-dependent signaling pathway regulates autoimmunity in Lyn-deficient mice

CD19 and the Src family protein tyrosine kinases (PTKs) are important regulators of intrinsic signaling thresholds in B cells. Regulation is achieved by cross-talk between Src family PTKs and CD19; Lyn is essential for CD19 phosphorylation, while CD19 establishes an Src family PTK activation loop that amplifies kinase activity. However, CD19-deficient (CD19(-/-)) B cells are hyporesponsive to transmembrane signals, while Lyn-deficient (Lyn(-/-)) B cells exhibit a hyper-responsive phenotype resulting in autoimmunity. To identify the outcome of interactions between CD19 and Src family PTKs in vivo, B cell function was examined in mice deficient for CD19 and Lyn (CD19/Lyn(-/-)). Remarkably, CD19 deficiency suppressed the hyper-responsive phenotype of Lyn(-/-) B cells and autoimmunity characterized by serum autoantibodies and immune complex-mediated glomerulonephritis in Lyn(-/-) mice. Consistent with Lyn and CD19 each regulating conventional B cell development, B1 cell development was markedly reduced by Lyn deficiency, with further reductions in the absence of CD19 expression. Tyrosine phosphorylation of Fyn and other cellular proteins induced following B cell Ag receptor ligation was dramatically reduced in CD19/Lyn(-/-) B cells relative to Lyn(-/-) B cells, while Syk phosphorylation was normal. In addition, the enhanced intracellular Ca(2+) responses following B cell Ag receptor ligation that typify Lyn deficiency were delayed by the loss of CD19 expression. BCR-induced proliferation and humoral immune responses were also markedly inhibited by CD19/Lyn deficiency. These findings demonstrate that while the CD19/Lyn amplification loop is a major regulator of signal transduction thresholds in B lymphocytes, CD19 regulation of other Src family PTKs also influences B cell function and the development of autoimmunity.     

CD22 regulates B cell receptor-mediated signals via two domains that independently recruit Grb2 and SHP-1

Recognition of antigen by the B cell antigen receptor (BCR) determines the subsequent fate of a B cell and is regulated in part by the involvement of other surface molecules, termed coreceptors. CD22 is a B cell-restricted coreceptor that gets rapidly tyrosyl-phosphorylated and recruits various signaling molecules to the membrane following BCR ligation. Although CD22 contains three immunoreceptor tyrosine-based inhibitory motifs (ITIMs), only the two carboxyl-terminal ITIM tyrosines are required for efficient recruitment of the SHP-1 phosphatase after BCR ligation. Furthermore, Grb2 is inducibly recruited to CD22 in human and murine B cells. Unlike SHP-1, Grb2 recruitment to CD22 is not inhibited by specific doses of the Src family kinase-specific inhibitor PP1. The tyrosine residue in CD22 required for Grb2 recruitment (Tyr-828) is distinct and independent from the two ITIM tyrosines required for efficient SHP-1 recruitment (Tyr-843 and Tyr-863). Individually both Lyn and Syk are required for maximal phosphorylation of CD22 following ligation of the BCR, and together Lyn and Syk are required for all of the constitutive and induced tyrosine phosphorylation of CD22. We propose that the cytoplasmic tail of CD22 contains two domains that regulate signal transduction pathways initiated by the BCR and B cell fate.     

CD19 amplification of B lymphocyte Ca2+ responses: a role for Lyn sequestration in extinguishing negative regulation

B lymphocyte antigen receptor (BCR) signals are regulated by CD19, with BCR-induced intracellular calcium ([Ca(2+)](i)) responses enhanced by CD19 co-ligation. In this study, CD19 engagement using a dimeric anti-CD19 antibody induced [Ca(2+)](i) mobilization and significantly enhanced BCR-induced [Ca(2+)](i) responses without a requirement for CD19/BCR co-ligation. Although simultaneous CD19 and BCR engagement significantly enhanced CD19/Lyn complex formation and [Ca(2+)](i) responses, downstream tyrosine phosphorylation of CD22 and multiple other cellular proteins was inhibited, as was SHP1 recruitment to phosphorylated CD22. CD19 overexpression also enhanced BCR-induced [Ca(2+)](i) responses, but down-regulated tyrosine phosphorylation of CD22 and multiple other cellular proteins following BCR ligation. Because CD19 and Lyn expression are genetically titrated in B cells, CD19 engagement may augment BCR-induced [Ca(2+)](i) responses by sequestering the available pool of functional Lyn away from downstream negative regulatory proteins such as CD22. Consistent with this, simultaneous CD19 engagement did not further enhance the BCR-induced [Ca(2+)](i) responses of Lyn- or CD22-deficient B cells. Thus, CD19 recruitment of Lyn may preferentially activate selective signaling pathways downstream of the CD19/Lyn complex to the exclusion of other downstream regulatory and effector pathways. Other receptors may also utilize a similar strategy to regulate kinase availability and downstream intermolecular signaling.     

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.     

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.     

CD72 negatively regulates signaling through the antigen receptor of B cells

The immunoreceptor tyrosine-based inhibition motif (ITIM) is found in various membrane molecules such as CD22 and the low-affinity Fc receptor for IgG in B cells and the killer cell-inhibitory receptor and Ly-49 in NK cells. Upon tyrosine phosphorylation at the ITIMs, these molecules recruit SH2 domain-containing phosphatases such as SH2-containing tyrosine phosphatase-1 and negatively regulate cell activity. The B cell surface molecule CD72 carries an ITIM and an ITIM-like sequence. We have previously shown that CD72 is phosphorylated and recruits SH2-containing tyrosine phosphatase-1 upon cross-linking of the Ag receptor of B cells (BCR). However, whether CD72 modulates BCR signaling has not yet been elucidated. In this paper we demonstrate that expression of CD72 down-modulates both extracellular signal-related kinase (ERK) activation and Ca2+ mobilization induced by BCR ligation in the mouse B lymphoma line K46micromlambda, whereas BCR-mediated ERK activation was not reduced by the ITIM-mutated form of CD72. Moreover, coligation with CD72 with BCR reduces BCR-mediated ERK activation in spleen B cells of normal mice. These results indicate that CD72 negatively regulates BCR signaling. CD72 may play a regulatory role in B cell activation, probably by setting a threshold for 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.     

Differential regulation of the B cell receptor-mediated signaling by the E3 ubiquitin ligase Cbl

The E3 ubiquitin ligase Cbl has been implicated in intracellular signaling pathways induced by the engagement of the B cell antigen receptor (BCR) as a negative regulator. Here we showed that Cbl deficiency results in a reduction of B cell proliferation. Cbl-/- B cells show impaired tyrosine phosphorylation, reduced Erk activation, and attenuated calcium mobilization in response to BCR engagement. The phosphorylation of Syk and Btk is also down-modulated. Interestingly, Cbl-/- B cells display enhanced BCR-induced phosphorylation of CD19 and its association with phosphatidylinositol 3-kinase. Importantly, Lyn kinase activity is up-regulated in Cbl-/- B cells, which correlates inversely with the Cbl-mediated ubiquitination of Lyn. Because Lyn has both negative and positive roles in B cells, our results suggested that Cbl differentially modulates the BCR-mediated signaling pathways through targeting Lyn ubiquitination, which affects B cell development and activation.     

Complementary roles for CD19 and Bruton's tyrosine kinase in B lymphocyte signal transduction

CD19 and Bruton's tyrosine kinase (Btk) may function along common signaling pathways in regulating intrinsic and B cell Ag receptor (BCR)-induced signals. To identify physical and functional interactions between CD19 and Btk, a CD19-negative variant of the A20 B cell line was isolated, and CD19-deficient (CD19(-/-)) and CD19-overexpressing mice with the X-linked immunodeficient (Xid; Btk) mutation were generated. In A20 cells, Btk physically associated with CD19 following BCR engagement. CD19 and Btk interactions were not required for initial Btk phosphorylation, but CD19 expression maintained Btk in an activated state following BCR engagement. In primary B cells, CD19 signaling also required downstream Btk function since CD19-induced intracellular Ca(2+) ([Ca(2+)](i)) responses were modest in Xid B cells. In addition, CD19 overexpression did not normalize the Xid phenotype and most phenotypic and functional hallmarks of CD19 overexpression were not evident in these mice. However, CD19 and Btk also regulate independent signaling pathways since their combined loss had additive inhibitory effects on BCR-induced [Ca(2+)](i) responses and CD19 deficiency induced a severe immunodeficiency in Xid mice. Thus, CD19 expression amplifies or prolongs Btk-mediated signaling, rather than serving as a required agent for Btk activation. Consistent with this, phosphatidylinositol 3-monophosphate kinase and Akt activation were normal in CD19(-/-) B cells following IgM engagement, although their kinetics of activation was altered. Thus, these biochemical and compound gene dosage studies indicate that Btk activation and [Ca(2+)](i) responses following BCR engagement are regulated through multiple pathways, including a CD19/Src family kinase-dependent pathway that promotes the longevity of Btk signaling.     

The B lymphocyte adaptor molecule of 32 kilodaltons (Bam32) regulates B cell antigen receptor internalization

The B lymphocyte adaptor molecule of 32 kDa (Bam32) is an adaptor that plays an indispensable role in BCR signaling. In this study, we found that upon BCR ligation, Bam32 is recruited to the plasma membrane where it associates with BCR complexes and redistributes and internalizes with BCRs. BCR ligation induced colocalization of Bam32 with lipid rafts, clathrin, and actin filaments. An inhibitor of Src family protein tyrosine kinases (PTKs) blocked both BCR-induced tyrosine phosphorylation of Bam32 and BCR internalization. Moreover, BCR internalization is impaired in Bam32-/- and Lyn-/- cells, and expression of Bam32 with a mutation of its tyrosine phosphorylation site (Y139F) inhibited BCR internalization. These data suggest that Bam32 functions downstream of Src family PTKs to regulate BCR internalization. Bam32 deficiency does not affect tyrosine phosphorylation of clathrin or the association of clathrin with lipid rafts upon BCR cross-linking. However, BCR-induced actin polymerization is impaired in Bam32-/- cells. Collectively, these findings indicate a novel role of Bam32 in connecting Src family PTKs to BCR internalization by an actin-dependent mechanism.     

Transient translocation of the B cell receptor and Src homology 2 domain-containing inositol phosphatase to lipid rafts: evidence toward a role in calcium regulation

Membrane microdomains (lipid rafts) are enriched in selected signaling molecules and may compartmentalize receptor-mediated signals. Here, we report that in primary human B lymphocytes and in Ramos B cells B cell receptor (BCR) stimulation induces rapid and transient redistribution of a subset of engaged BCRs to lipid rafts and phosphorylation of raft-associated tyrosine kinase substrates. Cholesterol sequestration disrupted the lipid rafts, preventing BCR redistribution, but did not inhibit tyrosine kinase activation or phosphorylation of mitogen-activated protein kinase/extracellular regulated kinase. However, raft disruption enhanced the release of calcium from intracellular stores, suggesting that rafts may sequester early signaling events that down-regulate calcium flux. Consistent with this, BCR stimulation induced rapid and transient translocation of the Src homology 2 domain-containing inositol phosphatase, SHIP, into lipid rafts.     

CD40-mediated activation of vascular smooth muscle cell chemokine production through a Src-initiated, MAPK-dependent pathway

The interaction between CD40 ligand (CD154) expressed on activated T cells and its receptor, CD40, has been shown to play a role in the onset and maintenance of autoimmune inflammation. Recent studies suggest that CD154+T cells also contribute to the regulation of atherogenesis due to their capacity to activate CD40+cells of the vasculature, including vascular smooth muscle cells (VSMC). The present study evaluated the signalling events initiated through CD40 ligation which culminate in VSMC chemokine production. CD40 ligation resulted in the phosphorylation/activation of mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinases 1 and 2 (ERK1/2), and p38, but not c-jun N-terminal kinase. Inhibition of both ERK1/2 and p38 activity abrogated CD40 stimulation of IL-8 and MCP-1 production. CD40-mediated induction of chemokines also showed dependence on the Src family kinase activity. The Src kinase inhibitor, PP2, was found to inhibit CD40-induced phosphorylation of ERK1/2 as well as activation of IkappaB kinase. An evaluation of Src kinases that may be important in CD40 signalling identified Lyn as a potential candidate. These data indicate that CD40 signalling in VSMC activates a Src family kinase-initiated pathway that results in the induction of MAPK activities required for successful induction of chemokine synthesis.     

The SH2 domain containing tyrosine phosphatase-1 down-regulates activation of Lyn and Lyn-induced tyrosine phosphorylation of the CD19 receptor in B cells

SHP-1 is a cytosolic tyrosine phosphatase implicated in down-regulation of B cell antigen receptor signaling. SHP-1 effects on the antigen receptor reflect its capacity to dephosphorylate this receptor as well as several inhibitory comodulators. In view of our observation that antigen receptor-induced CD19 tyrosine phosphorylation is constitutively increased in B cells from SHP-l-deficient motheaten mice, we investigated the possibility that CD19, a positive modulator of antigen receptor signaling, represents another substrate for SHP-1. However, analysis of CD19 coimmunoprecipitable tyrosine phosphatase activity in CD19 immunoprecipitates from SHP-1-deficient and wild-type B cells revealed that SHP-1 accounts for only a minor portion of CD19-associated tyrosine phosphatase activity. As CD19 tyrosine phosphorylation is modulated by the Lyn protein-tyrosine kinase, Lyn activity was evaluated in wild-type and motheaten B cells. The results revealed both Lyn as well as CD19-associated Lyn kinase activity to be constitutively and inducibly increased in SHP-1-deficient compared with wild-type B cells. The data also demonstrated SHP-1 to be associated with Lyn in stimulated but not in resting B cells and indicated this interaction to be mediated via Lyn binding to the SHP-1 N-terminal SH2 domain. These findings, together with cyanogen bromide cleavage data revealing that SHP-1 dephosphorylates the Lyn autophosphorylation site, identify Lyn deactivation/dephosphorylation as a likely mechanism whereby SHP-1 exerts its influence on CD19 tyrosine phosphorylation and, by extension, its inhibitory effect on B cell antigen receptor signaling.     

CTLA-4 suppresses proximal TCR signaling in resting human CD4(+) T cells by inhibiting ZAP-70 Tyr(319) phosphorylation: a potential role for tyrosine phosphatases

The balance between positive and negative signals plays a key role in determining T cell function. CTL-associated Ag-4 is a surface receptor that can inhibit T cell responses induced upon stimulation of the TCR and its CD28 coreceptor. Little is known regarding the signaling mechanisms elicited by CTLA-4. In this study we analyzed CTLA-4-mediated inhibition of TCR signaling in primary resting human CD4(+) T cells displaying low, but detectable, CTLA-4 cell surface expression. CTLA-4 coligation with the TCR resulted in reduced downstream protein tyrosine phosphorylation of signaling effectors and a striking inhibition of extracellular signal-regulated kinase 1/2 activation. Analysis of proximal TCR signaling revealed that TCR zeta-chain phosphorylation and subsequent zeta-associated protein of 70 kDa (ZAP-70) tyrosine kinase recruitment were not significantly affected by CTLA-4 engagement. However, the association of p56(lck) with ZAP-70 was inhibited following CTLA-4 ligation, correlating with reduced actions of p56(lck) in the ZAP-70 immunocomplex. Moreover, CTLA-4 ligation caused the selective inhibition of CD3-mediated phosphorylation of the positive regulatory ZAP-70 Y319 site. In addition, we demonstrate protein tyrosine phosphatase activity associated with the phosphorylated CTLA-4 cytoplasmic tail. The major phosphatase activity was attributed to Src homology protein 2 domain-containing tyrosine phosphatase 1, a protein tyrosine phosphatase that has been shown to be a negative regulator of multiple signaling pathways in hemopoietic cells. Collectively, our findings suggest that CTLA-4 can act early during the immune response to regulate the threshold of T cell activation.     

Regulation of signaling in B cells through the phosphorylation of Syk on linker region tyrosines. A mechanism for negative signaling by the Lyn tyrosine kinase

The B cell antigen receptor (BCR) is coupled to the mobilization of Ca(2+) by the protein-tyrosine kinase, Syk. Syk, recruited to the clustered BCR, becomes phosphorylated on three tyrosines (Tyr-317, Tyr-342, and Tyr-346) located within the linker region that separates the C-terminal catalytic domain from the N-terminal tandem Src homology 2 domains. Phosphorylation within the linker region can be either activating or inhibitory to Ca(2+) mobilization depending on the sites that are modified. Syk that is not phosphorylated on linker region tyrosines couples the BCR to Ca(2+) mobilization through a phosphoinositide 3-kinase-dependent pathway. The phosphorylation of Tyr-342 and -346 enhances the phosphorylation and activation of phospholipase C-gamma and the early phase of Ca(2+) mobilization via a phosphoinositide 3-kinase-independent pathway. The phosphorylation of Tyr-317 strongly dampens the Ca(2+) signal. In cells that lack the Src family kinase, Lyn, the phosphorylation of the inhibitory Tyr-317 is suppressed leading to elevated production of inositol 1,4,5-trisphosphate and an amplified Ca(2+) signal. This provides a novel mechanism by which Lyn functions as an inhibitor of BCR-stimulated signaling. Thus, Syk and Lyn combine to determine the pathway through which the BCR is coupled to Ca(2+) mobilization as well as the magnitude and duration of the Ca(2+) flux.     

Positive signaling through CD72 induces mitogen-activated protein kinase activation and synergizes with B cell receptor signals to induce X-linked immunodeficiency B cell proliferation

CD72 is a 45-kDa B cell transmembrane glycoprotein that has been shown to be important for B cell activation. However, whether CD72 ligation induces B cell activation by delivering positive signals or sequestering negative signals away from B cell receptor (BCR) signals remains unclear. Here, by comparing the late signaling events associated with the mitogen-activated protein kinase pathway, we identified many similarities and some differences between CD72 and BCR signaling. Thus, CD72 and BCR activated the extracellular signal-regulated kinase (ERK) and the c-Jun N-terminal kinase (JNK) but not p38 mitogen-activated protein kinase. Both CD72- and BCR-mediated ERK and JNK activation required protein kinase C activity, which was equally important for CD72- and BCR-induced B cell proliferation. However, CD72 induced stronger JNK activation compared with BCR. Surprisingly, the JNK activation induced by both BCR and CD72 is Btk independent. Although both CD72 and BCR induced Btk-dependent ERK activation, CD72-mediated proliferation is more resistant to blocking of ERK activity than that of BCR, as shown by the proliferation response of B cells treated with PD98059 and dibutyryl cAMP, agents that inhibit ERK activity. Most importantly, CD72 signaling compensated for defective BCR signaling in X-linked immunodeficiency B cells and partially restored the proliferation response of X-linked immunodeficiency B cells to anti-IgM ligation. These results suggest that CD72 signals B cells by inducing BCR-independent positive signaling pathways.     

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.