The adaptor protein BLNK is required for b cell antigen receptor-induced activation of nuclear factor-kappa B and cell cycle entry and survival of B lymphocytes

B lymphocytes lacking the adaptor protein B cell linker (BLNK) do not proliferate in response to B cell antigen receptor (BCR) engagement. We demonstrate here that BCR-activated BLNK(-)/- B cells fail to enter the cell cycle, and this is due to their inability to induce the expression of the cell cycle regulatory proteins such as cyclin D2 and cyclin-dependent kinase 4. BCR-stimulated BLNK(-)/- B cells also do not up-regulate the cell survival protein Bcl-x(L), which may be necessary for the cells to complete the cell cycle. In addition, BLNK(-)/- B cells exhibit a high rate of spontaneous apoptosis in culture. Examination of the various BCR-activated signaling pathways in mouse BLNK(-)/- B cells reveals the intact activation of Akt and mitogen-activated protein kinases but the impaired activation of nuclear factor (NF)-kappaB that is known to regulate genes involved in cell proliferation and survival. The inability to activate NF-kappaB in BCR-stimulated BLNK(-)/- B cells is due to a failure to induce the degradation of the inhibitory kappaB protein. In all these aspects, BLNK(-)/- B cells resemble xid B cells that have a mutation in Bruton's tyrosine kinase (Btk). Recently, phospholipase C (PLC)-gamma2 has also been demonstrated to be essential for NF-kappaB activation. Since BLNK has been shown separately to interact with both Btk and PLC-gamma2, our finding of normal Btk but impaired PLC-gamma2 activation in BCR-stimulated BLNK(-)/- B cells strongly suggests that BLNK orchestrates the formation of a Btk-PLC-gamma2 signaling axis that regulates NF-kappaB activation. Taken together, the NF-kappaB activation defect may be sufficient to explain the similar defects in BCR-induced B cell proliferation and T cell-independent immune responses in BLNK(-)/-, Btk(-)/-, and PLC-gamma2(-)/- mice.     

A mutation in Epstein-Barr virus LMP2A reveals a role for phospholipase D in B-Cell antigen receptor trafficking

Epstein-Barr virus (EBV) latent infection of B cells blocks the interrelated signaling and antigen-trafficking functions of the BCR through the activity of its latent membrane protein 2A (LMP2A). At present, the molecular mechanisms by which LMP2A exerts its control of BCR functions are only poorly understood. Earlier studies showed that in B cells expressing LMP2A containing a tyrosine mutation at position 112 in its cytoplasmic domain (Y112-LMP2A), the BCR could initiate signaling but could not properly traffic antigen for processing. Here, we show that BCR signaling in Y112-LMP2A-expressing cells is attenuated with a reduction in both the degree and duration of phosphorylation of key components of the BCR signaling cascade including Syk, BLNK, PI3K, and Btk. Notably, Y112-LMP2A expression completely blocked the BCR-induced activation of phospholipase D (PLD), a lipase implicated in the intracellular trafficking of a variety of surface receptors. We show that blocking PLD activity, by expressing Y112-LMP2A, treating cells with the PLD inhibitor 1-butanol or reducing PLD expression by siRNA, blocked BCR trafficking to class II-containing compartments. Moreover, Y112-LMP2A expression blocked the recruitment of phosphorylated forms of the downstream BCR signaling components, Erk and JNK, through both PLD-dependent and PLD-independent mechanisms. Thus, the investigation of the mechanism by which Y112-LMP2A blocks BCR function revealed an essential role for PLD in BCR trafficking for antigen processing.     

Synergistic activation of PKD by the B cell antigen receptor and CD19 requires PI3K, Vav1 and PLCgamma

Antigens coated with complement fragments coligate the B cell receptor (BCR) with the CD21/CD19 complex which results in synergistic activation of B cells. Previous studies identified PI3K, Vav proteins and PLCgamma as important components of this synergy. We now show that protein kinase D (also known as PKCmu) is also a point of convergence of these signalling pathways. We found that PKD activation upon BCR engagement or coligation of the BCR with CD19 is entirely dependent on PI3K and PLCgamma but differ in the requirement for Vav proteins. Whereas PKD activation is Vav1 and Vav2 dependent in response to BCR cross-linking, PKD activation is sensitive to the lack of Vav1 under synergistic stimulation of BCR and CD19. These findings show that Vav proteins and PI3K regulation of PLCgamma contributes to the activation of PKD in response to BCR and or CD19 cross-linking.     

Phospholipase Cgamma2 provides survival signals via Bcl2 and A1 in different subpopulations of B cells

PLCgamma2 plays a critical role in B cell receptor (BCR) signaling and its targeted deletion results in defective B cell development and function. Here, we show that PLCgamma2 deficiency specifically blocks B cell maturation at the transitional type 2 (T2) to follicular (FO) B cell transition and the PLCgamma2 pathway regulates survival of B cells. BCR-induced apoptosis is dramatically enhanced in all subsets of splenic PLCgamma2-deficient B cells, especially in T2 and FO B cell subpopulations. We also find that all splenic PLCgamma2-deficient B cell subpopulations express abnormally low levels of Bcl-2 protein. In addition, PLCgamma2 deficiency disrupts BCR-mediated induction of A1 expression. Enforced expression of Bcl-2 prevents BCR-induced apoptosis in all splenic PLCgamma2-deficient B cell subpopulations and partially restores the numbers of PLCgamma2-deficient FO B cells. In contrast to Bcl-2, enforced expression of A1 preferentially prevents BCR-induced apoptosis in PLCgamma2-deficient FO B cells and partially restores the numbers of these B cells. Therefore, the PLCgamma2 pathway provides a survival signal via regulation of Bcl-2 in all splenic B cell subpopulations and via additional induction of A1 in mature FO B cells.     

Syk and Bruton's tyrosine kinase are required for B cell antigen receptor-mediated activation of the kinase Akt.

Activation of Akt by multiple stimuli including B cell antigen receptor (BCR) engagement requires phosphatidylinositol 3-kinase and regulates processes including cell survival, proliferation, and metabolism. BCR cross-linking activates three families of non-receptor protein tyrosine kinases (PTKs) and these are transducers of signaling events including phospholipase C and mitogen-activated protein kinase activation; however, the relative roles of PTKs in BCR-mediated Akt activation are unknown. We examined Akt activation in Lyn-, Syk- and Btk-deficient DT40 cells and B cells from Lyn(-/-) mice. BCR-mediated Akt activation required Syk and was partially dependent upon Btk. Increased BCR-induced Akt phosphorylation was observed in Lyn-deficient DT40 cells and Lyn(-/-) mice compared with wild-type cells suggesting that Lyn may negatively regulate Akt function. BCR-induced tyrosine phosphorylation of the phosphatidylinositol 3-kinase catalytic subunit was abolished in Syk-deficient cells consistent with a receptor-proximal role for Syk in BCR-mediated phosphatidylinositol 3-kinase activation; in contrast, it was maintained in Btk-deficient cells, suggesting Btk functions downstream of phosphatidylinositol 3-kinase. Calcium depletion did not influence BCR-induced Akt phosphorylation/activation, showing that neither Syk nor Btk mediates its effects via changes in calcium levels. Thus, BCR-mediated Akt stimulation is regulated by multiple non-receptor PTK families which regulate Akt both proximal and distal to phosphatidylinositol 3-kinase activation.     

Functional Independence and Interdependence of the Src Homology Domains of Phospholipase C-γ1 in B-Cell Receptor Signal Transduction

B-cell receptor (BCR)-induced activation of phospholipase C-gamma1 (PLCgamma1) and PLCgamma2 is crucial for B-cell function. While several signaling molecules have been implicated in PLCgamma activation, the mechanism coupling PLCgamma to the BCR remains undefined. The role of PLCgamma1 SH2 and SH3 domains at different steps of BCR-induced PLCgamma1 activation was examined by reconstitution in a PLCgamma-negative B-cell line. PLCgamma1 membrane translocation required a functional SH2 N-terminal [SH2(N)] domain, was decreased by mutation of the SH3 domain, but was unaffected by mutation of the SH2(C) domain. Tyrosine phosphorylation did not require the SH2(C) or SH3 domains but depended exclusively on a functional SH2(N) domain, which mediated the association of PLCgamma1 with the adapter protein, BLNK. Forcing PLCgamma1 to the membrane via a myristoylation signal did not bypass the SH2(N) domain requirement for phosphorylation, indicating that the phosphorylation mediated by this domain is not due to membrane anchoring alone. Mutation of the SH2(N) or the SH2(C) domain abrogated BCR-stimulated phosphoinositide hydrolysis and signaling events, while mutation of the SH3 domain partially decreased signaling. PLCgamma1 SH domains, therefore, have interrelated but distinct roles in BCR-induced PLCgamma1 activation.     

Four tyrosine residues in phospholipase C-gamma 2, identified as Btk-dependent phosphorylation sites, are required for B cell antigen receptor-coupled calcium signaling

Activation of phospholipase C-gamma2 (PLCgamma2) is the critical step in B cell antigen receptor (BCR)-coupled calcium signaling. Although genetic dissection experiments on B cells have demonstrated that Bruton's tyrosine kinase (Btk) and Syk are required for activating PLCgamma2, the exact activation mechanism of PLCgamma2 by these kinases has not been established. We identify the tyrosine residues 753, 759, 1197, and 1217 in rat PLCgamma2 as Btk-dependent phosphorylation sites by using an in vitro kinase assay. To evaluate the role of these tyrosine residues in phosphorylation-dependent activation of PLCgamma2, PLCgamma2-deficient DT40 cells were reconstituted with a series of mutant PLCgamma2s in which the phenylalanine was substituted for tyrosine. Substitution of all four tyrosine residues almost completely eliminated the BCR-induced PLCgamma2 phosphorylation, indicating that these residues include the major phosphorylation sites upon BCR engagement. Cells expressing PLCgamma2 with a single substitution exhibited some extent of reduction in calcium mobilization, whereas those expressing quadruple mutant PLCgamma2 showed greatly reduced calcium response. These findings indicate that the phosphorylations of the tyrosine residues 753, 759, 1197, and 1217, which have been identified as Btk-dependent phosphorylation sites in vitro, coordinately contribute to BCR-induced activation of PLCgamma2.     

Down-regulation of B cell receptor signaling by hematopoietic progenitor kinase 1 (HPK1)-mediated Phosphorylation and Ubiquitination of Activated B cell linker protein (BLNK)

Hematopoietic progenitor kinase 1 (HPK1) is a Ste20-like serine/threonine kinase that suppresses immune responses and autoimmunity. B cell receptor (BCR) signaling activates HPK1 by inducing BLNK/HPK1 interaction. Whether HPK1 can reciprocally regulate BLNK during BCR signaling is unknown. Here, we show that HPK1-deficient B cells display hyper-proliferation and hyper-activation of IκB kinase and MAPKs (ERK, p38, and JNK) upon the ligation of BCR. HPK1 attenuates BCR-induced cell activation via inducing BLNK threonine 152 phosphorylation, which mediates BLNK/14-3-3 binding. Furthermore, threonine 152-phosphorylated BLNK is ubiquitinated at lysine residues 37, 38, and 42, leading to attenuation of MAPK and IκB kinase activation in B cells during BCR signaling. These results reveal a novel negative feedback regulation of BCR signaling by HPK1-mediated phosphorylation, ubiquitination, and subsequent degradation of the activated BLNK.     

B cell receptor-induced cAMP-response element-binding protein activation in B lymphocytes requires novel protein kinase Cdelta

The cAMP-response element-binding protein (CREB) is activated by phosphorylation on Ser-133 and plays a key role in the proliferative and survival responses of mature B cells to B cell receptor (BCR) signaling. The signal link between the BCR and CREB activation depends on a phorbol ester (phorbol 12-myristate 13-acetate)-sensitive protein kinase C (PKC) activity and not protein kinase A or calmodulin kinase; however, the identity and role of the PKC(s) activity has not been elucidated. We found the novel PKCdelta (nPKCdelta) activator bistratene A is sufficient to induce CREB phosphorylation in murine splenic B cells. The pharmacological inhibitor G?6976, which targets conventional PKCs and PKCmu, has no effect on CREB phosphorylation, whereas the nPKCdelta inhibitor rottlerin blocks CREB phosphorylation following BCR cross-linking. Bryostatin 1 selectively prevents nPKCdelta depletion by phorbol 12-myristate 13-acetate when coapplied, coincident with protection of BCR-induced CREB phosphorylation. Ectopic expression of a kinase-inactive nPKCdelta blocks BCR-induced CREB phosphorylation in A20 B cells. In addition, BCR-induced CREB phosphorylation is significantly diminished in nPKCdelta-deficient splenic B cells in comparison with wild type mice. Consistent with the essential role for Bruton's tyrosine kinase and phospholipase Cgamma2 in mediating PKC activation, Bruton's tyrosine kinase- and phospholipase Cgamma2-deficient B cells display defective CREB phosphorylation by the BCR. We also found that p90 RSK directly phosphorylates CREB on Ser-133 following BCR cross-linking and is positioned downstream of nPKCdelta. Taken together, these results suggest a model in which BCR engagement leads to the phosphorylation of CREB via a signaling pathway that requires nPKCdelta and p90 RSK in mature B cells.     

The Gab1 docking protein links the b cell antigen receptor to the phosphatidylinositol 3-kinase/Akt signaling pathway and to the SHP2 tyrosine phosphatase

B cell antigen receptor (BCR) signaling causes tyrosine phosphorylation of the Gab1 docking protein. This allows phosphatidylinositol 3-kinase (PI3K) and the SHP2 tyrosine phosphatase to bind to Gab1. In this report, we tested the hypothesis that Gab1 acts as an amplifier of PI3K- and SHP2-dependent signaling in B lymphocytes. By overexpressing Gab1 in the WEHI-231 B cell line, we found that Gab1 can potentiate BCR-induced phosphorylation of Akt, a PI3K-dependent response. Gab1 expression also increased BCR-induced tyrosine phosphorylation of SHP2 as well as the binding of Grb2 to SHP2. We show that the pleckstrin homology (PH) domain of Gab1 is required for BCR-induced phosphorylation of Gab1 and for Gab1 participation in BCR signaling. Moreover, using confocal microscopy, we show that BCR ligation can induce the translocation of Gab1 from the cytosol to the plasma membrane and that this requires the Gab1 PH domain as well as PI3K activity. These findings are consistent with a model in which the binding of the Gab1 PH domain to PI3K-derived lipids brings Gab1 to the plasma membrane, where it can be tyrosine-phosphorylated and then act as an amplifier of BCR signaling.     

Src homology region 2 domain-containing phosphatase 1 positively regulates B cell receptor-induced apoptosis by modulating association of B cell linker protein with Nck and activation of c-Jun NH2-terminal kinase

Src homology region 2 domain-containing phosphatase 1 (SHP-1) is a key mediator in lymphocyte differentiation, proliferation, and activation. We previously showed that B cell linker protein (BLNK) is a physiological substrate of SHP-1 and that B cell receptor (BCR)-induced activation of c-Jun NH(2)-terminal kinase (JNK) is significantly enhanced in cells expressing a form of SHP-1 lacking phosphatase activity (SHP-1-C/S). In this study, we confirmed that SHP-1 also exerts negative regulatory effects on JNK activation in splenic B cells. To further clarify the role of SHP-1 in B cells, we examined how dephosphorylation of BLNK by SHP-1 affects downstream signaling events. When a BLNK mutant (BLNK Delta N) lacking the NH(2)-terminal region, which contains four tyrosine residues, was introduced in SHP-1-C/S-expressing WEHI-231 cells, the enhanced JNK activation was inhibited. Among candidate proteins likely to regulate JNK activation through BLNK, Nck adaptor protein was found to associate with tyrosine-phosphorylated BLNK and this association was more pronounced in SHP-1-C/S-expressing cells. Furthermore, expression of dominant-negative forms of Nck inhibited BCR-induced JNK activation. Finally, BCR-induced apoptosis was suppressed in SHP-1-C/S-expressing cells and coexpression of Nck SH2 mutants or a dominant-negative form of SEK1 reversed this phenotype. Collectively, these results suggest that SHP-1 acts on BLNK, modulating its association with Nck, which in turn negatively regulates JNK activation but exerts a positive effect on apoptosis.     

Bruton's tyrosine kinase is essential for hydrogen peroxide-induced calcium signaling.

Using Btk-deficient DT40 cells and the transfectants expressing wild-type Btk or Btk mutants in either kinase (Arg(525) to Gln), Src homology 2 (SH2, Arg(307) to Ala), or pleckstrin homology (PH, Arg(28) to Cys) domains, we investigated the roles and structure-function relationships of Btk in hydrogen peroxide-induced calcium mobilization. Our genetic evidence showed that Btk deficiency resulted in a significant reduction in hydrogen peroxide-induced calcium response. This impaired calcium signaling is correlated with the complete elimination of IP3 production and the significantly reduced tyrosine phosphorylation of PLCgamma2 in Btk-deficient DT40 cells. All of these defects were fully restored by the expression of wild-type Btk in Btk-deficient DT40 cells. The data from the point mutation study revealed that a defect at any one of the three functional domains would prevent a full recovery of Btk-mediated hydrogen peroxide-induced intracellular calcium mobilization. However, mutation at either the SH2 or PH domain did not affect the hydrogen peroxide-induced activation of Btk. Mutation at the SH2 domain abrogates both IP3 generation and calcium release, while the mutant with the nonfunctional PH domain can partially activate PLCgamma2 and catalyze IP3 production but fails to produce significant calcium mobilization. Thus, these observations suggest that Btk-dependent tyrosine phosphorylation of PLCgamma2 is required but not sufficient for hydrogen peroxide-induced calcium mobilization. Furthermore, hydrogen peroxide stimulates a Syk-, but not Btk-, dependent tyrosine phosphorylation of B cell linker protein BLNK. The overall results, together with those reported earlier [Qin et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 7118], are consistent with the notion that functional SH2 and PH domains are required for Btk to form a complex with PLCgamma2 through BLNK in order to position the Btk, PLCgamma2, and phosphatidylinositol 4,5-bisphosphate in close proximity for efficient activation of PLCgamma2 and to maximize its catalytic efficiency for IP3 production.     

Distinct roles of phosphoinositide-3 kinase and phospholipase Cgamma2 in B-cell receptor-mediated signal transduction

During B-cell receptor (BCR) signaling, phosphoinositide-3 kinase (PI3K) is thought to function upstream of phospholipase Cgamma2 (PLCgamma2). PLCgamma2 deficiency specifically impedes transitional type 2 (T2) to follicular (FO) mature B-cell transition. Here, we demonstrate that PI3K deficiency specifically impaired T2-to-FO mature B-cell transition and marginal zone B-cell development. Furthermore, we investigated the functional relationship between PI3K and PLCgamma2 using PI3K-/-, PLCgamma2-/-, and PI3K-/- PLCgamma2-/- B cells. Interestingly, PLCgamma2 deficiency had no effect on BCR-mediated PI3K activation, whereas PI3K deficiency only partially blocked activation of PLCgamma2. Moreover, whereas PI3K-/- PLCgamma2-/- double deficiency did not affect hematopoiesis, it resulted in embryonic lethality. PI3K-/- PLCgamma2-/- fetal liver cells transplanted into B-cell null JAK3-/- mice failed to restore development of peripheral B cells and failed to progress through early B-cell development at the pro-B- to pre-B-cell transition, a more severe phenotype than was observed with either PI3K or PLCgamma2 single-deficiency B cells. Consistent with this finding, BCR signaling was more severely impaired in the absence of both PI3K and PLCgamma2 genes than in the absence of either one alone. Taken together, these results demonstrate that whereas PI3K functions upstream of PLCgamma2, activation of PLCgamma2 can occur independently of PI3K and that PI3K and PLCgamma2 also have distinct functions in BCR signal transduction.     

Btk/Tec kinases regulate sustained increases in intracellular Ca2+ following B-cell receptor activation.

Bruton's tyrosine kinase (Btk) is essential for B-lineage development and represents an emerging family of non-receptor tyrosine kinases implicated in signal transduction events initiated by a range of cell surface receptors. Increased dosage of Btk in normal B cells resulted in a striking enhancement of extracellular calcium influx following B-cell antigen receptor (BCR) cross-linking. Ectopic expression of Btk, or related Btk/Tec family kinases, restored deficient extracellular Ca2+ influx in a series of novel Btk-deficient human B-cell lines. Btk and phospholipase Cgamma (PLCgamma) co-expression resulted in tyrosine phosphorylation of PLCgamma and required the same Btk domains as those for Btk-dependent calcium influx. Receptor-dependent Btk activation led to enhanced peak inositol trisphosphate (IP3) generation and depletion of thapsigargin (Tg)-sensitive intracellular calcium stores. These results suggest that Btk maintains increased intracellular calcium levels by controlling a Tg-sensitive, IP3-gated calcium store(s) that regulates store-operated calcium entry. Overexpression of dominant-negative Syk dramatically reduced the initial phase calcium response, demonstrating that Btk/Tec and Syk family kinases may exert distinct effects on calcium signaling. Finally, co-cross-linking of the BCR and the inhibitory receptor, FcgammaRIIb1, completely abrogated Btk-dependent IP3 production and calcium store depletion. Together, these data demonstrate that Btk functions at a critical crossroads in the events controlling calcium signaling by regulating peak IP3 levels and calcium store depletion.     

Basal B cell receptor-directed phosphatidylinositol 3-kinase signaling turns off RAGs and promotes B cell-positive selection

PI3K plays key roles in cell growth, differentiation, and survival by generating the second messenger phosphatidylinositol-(3,4,5)-trisphosphate (PIP3). PIP3 activates numerous enzymes, in part by recruiting them from the cytosol to the plasma membrane. We find that in immature B lymphocytes carrying a nonautoreactive Ag receptor, PI3K signaling suppresses RAG expression and promotes developmental progression. Inhibitors of PI3K signaling abrogate this positive selection. Furthermore, immature primary B cells from mice lacking the p85alpha regulatory subunit of PI3K suppress poorly RAG expression, undergo an exaggerated receptor editing response, and, as in BCR-ligated cells, fail to progress into the G1 phase of cell cycle. Moreover, immature B cells carrying an innocuous receptor have sustained elevation of PIP3 levels and activation of the downstream effectors phospholipase C (PLC)gamma2, Akt, and Bruton's tyrosine kinase. Of these, PLCgamma2 appears to play the most significant role in down-regulating RAG expression. It therefore appears that when the BCR of an immature B cell is ligated, PIP3 levels are reduced, PLCgamma2 activation is diminished, and receptor editing is promoted by sustained RAG expression. Taken together, our results provide evidence that PI3K signaling is an important cue required for fostering development of B cells carrying a useful BCR.