The kinase Syk as an adaptor controlling sustained calcium signalling and B-cell development

Upon B-cell antigen receptor (BCR) activation, the protein tyrosine kinase Syk phosphorylates the adaptor protein SH2 domain-containing leukocyte protein of 65 kDa (SLP-65), thus coupling the BCR to diverse signalling pathways. Here, we report that SLP-65 is not only a downstream target and substrate of Syk but also a direct binding-partner and activator of this kinase. This positive feedback is mediated by the binding of the SH2 domain of SLP-65 to an autophosphorylated tyrosine of Syk. The mutant B cells that cannot form the Syk/SLP-65 complex are defective in BCR-induced extracellular signal-regulated kinase, nuclear factor kappa B and nuclear factor of activated T cells, but not Akt activation, and are blocked in B-cell development. Furthermore, we show that formation of the Syk/SLP-65 complex is required for sustained Ca(2+) responses in activated B cells. We suggest that after activation and internalization of the BCR, Syk remains active as part of a membrane-bound Syk/SLP-65 complex controlling sustained signalling and calcium influx.     

B cell receptor-mediated Syk-independent activation of phosphatidylinositol 3-kinase,Ras, and mitogen-activated protein kinase pathways

The Syk tyrosine kinase is a key molecule in the development of the B cell lineage and the activation of B lymphocytes after Ag recognition by the B cell Ag receptor (BCR). Several genetic studies with chicken B cells have reported that the recruitment of Syk by BCR is essential for activation of a cascade of signaling molecules including phosphatidylinositol 3-kinase, mitogen-activated protein kinases, Ras signaling pathways, phospholipase C-gamma2 activation, and calcium mobilization. The identification of a Syk-deficient mouse IIA1.6/A20 B cell line provided us the opportunity to investigate Syk-mediated signaling in mouse. Surprisingly, phosphatidylinositol 3-kinase, Ras, and mitogen-activated protein kinases were activated upon BCR cross-linking in these Syk-deficient mouse B cells, whereas, as expected from results obtained in chicken B cells, phospholipase C-gamma2 activation and calcium mobilization were impaired as well as the NF-kappaB pathway. These results indicate that BCR signaling is not strictly dependent on Syk expression in mouse IIA1.6/A20 B cells. Thus, B lymphocyte activation may be initiated by Syk-dependent and Syk-independent signaling cascades.     

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.     

The dynamics of protein kinase B regulation during B cell antigen receptor engagement.

This study has used biochemistry and real time confocal imaging of green fluorescent protein (GFP)-tagged molecules in live cells to explore the dynamics of protein kinase B (PKB) regulation during B lymphocyte activation. The data show that triggering of the B cell antigen receptor (BCR) induces a transient membrane localization of PKB but a sustained activation of the enzyme; active PKB is found in the cytosol and nuclei of activated B cells. Hence, PKB has three potential sites of action in B lymphocytes; transiently after BCR triggering PKB can phosphorylate plasma membrane localized targets, whereas during the sustained B cell response to antigen, PKB acts in the nucleus and the cytosol. Membrane translocation of PKB and subsequent PKB activation are dependent on BCR activation of phosphatidylinositol 3-kinase (PI3K). Moreover, PI3K signals are both necessary and sufficient for sustained activation of PKB in B lymphocytes. However, under conditions of continuous PI3K activation or BCR triggering there is only transient recruitment of PKB to the plasma membrane, indicating that there must be a molecular mechanism to dissociate PKB from sites of PI3K activity in B cells. The inhibitory Fc receptor, the FcgammaRIIB, mediates vital homeostatic control of B cell function by recruiting an inositol 5 phosphatase SHIP into the BCR complex. Herein we show that coligation of the BCR with the inhibitory FcgammaRIIB prevents membrane targeting of PKB. The FcgammaRIIB can thus antagonize BCR signals for PKB localization and prevent BCR stimulation of PKB activity which demonstrates the mechanism for the inhibitory action of the FcgammaRIIB on the BCR/PKB response.     

Syk-mediated tyrosine phosphorylation is required for the association of hematopoietic lineage cell-specific protein 1 with lipid rafts and B cell antigen receptor signalosome complex

Hematopoietic lineage cell-specific protein 1 (HS1) is an F-actin- and actin-related proteins 2 and 3 (Arp2/3)-binding protein that undergoes a rapid tyrosine phosphorylation upon B cell antigen receptor (BCR) activation. Density gradient centrifugation of Triton X-100 lysates from B lymphocytes demonstrated that HS1 was translocated in response to BCR cross-linking into lipid raft microdomain along with Arp2/3 complex and Wiskott-Aldrich syndrome protein. HS1-green fluorescent protein was localized in membrane patches enriched with GM1 gangliosides and BCR in the cells treated with anti-IgM antibody. Colocalization of HS1-green fluorescent protein with BCR was also correlated with tyrosine phosphorylation of HS1. Interestingly a murine HS1 mutant at the tyrosine residues Tyr388 and Tyr405 targeted by Syk failed to respond to BCR cross-linking for either translocation into lipid rafts or colocalization with BCR within cells. Furthermore HS1 was unable to translocate into lipid rafts in a chicken B cell line deficient in Syk. Reintroducing a Syk construct into the Syk knock-out cells recovered effectively both tyrosine phosphorylation and translocation of HS1 into lipid rafts. In contrast, translocation of HS1 into rafts was normal in a Lyn knock-out B cell line, and an HS1 mutant at the tyrosine residue Tyr222 targeted by Lyn maintained the ability to partition into rafts upon BCR cross-linking. These data indicate that Syk plays an important role in the translocation of HS1 into lipid rafts and may be responsible for actin assembly recruitment to rafts and subsequent antigen presentations.     

Syk is required for the activation of Akt survival pathway in B cells exposed to oxidative stress

Syk has been demonstrated to play a crucial role in oxidative stress signaling in B cells. Here we report that Syk is required for the activation of the phosphatidylinositol (PI) 3-kinase-Akt survival pathway in B cells exposed to oxidative stress. Phosphorylation and activation of the serine-threonine kinase Akt were markedly increased in B cells treated with H(2)O(2). In Syk-deficient DT40 cells treated with low doses of H(2)O(2) (10-100 microm), Akt activation was considerably reduced. Pretreatment with wortmannin, a PI 3-kinase-specific inhibitor, completely blocked the Syk-dependent Akt activation. Following stimulation by low doses of H(2)O(2), a significant increase in PI 3-kinase activity was found in wild-type but not in Syk-deficient cells. These findings suggest that PI 3-kinase mediates Syk-dependent Akt activation pathway. Furthermore, viability of Syk-deficient cells, after exposure to H(2)O(2), was dramatically decreased and caspase-9 activity was greatly increased compared with that of the wild-type cells. These results suggest that Syk is essential for the Akt survival pathway in B cells and enhances cellular resistance to oxidative stress-induced apoptosis.     

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.     

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.     

CD19 and BAFF‐R can signal to promote B‐cell survival in the absence of Syk

The development and function of B lymphocytes is regulated by numerous signaling pathways, some emanating from the B‐cell antigen receptor (BCR). The spleen tyrosine kinase (Syk) plays a central role in the activation of the BCR, but less is known about its contribution to the survival and maintenance of mature B cells. We generated mice with an inducible and B‐cell‐specific deletion of the Syk gene and found that a considerable fraction of mature Syk‐negative B cells can survive in the periphery for an extended time. Syk‐negative B cells are defective in BCR, RP105 and CD38 signaling but still respond to an IL‐4, anti‐CD40, CpG or LPS stimulus. Our in vivo experiments show that Syk‐deficient B cells require BAFF receptor and CD19/PI3K signaling for their long‐term survival. These studies also shed a new light on the signals regulating the maintenance of the normal mature murine B‐cell pool.     

The adaptor protein APS modulates BCR signalling in mature B cells

Activation process of mature B cell is predominantly driven by specific BCR-mediated pathways, switched on and off all through late B cell differentiation stages. Mice deficient for APS, a member of the Lnk/SH2B family of adaptor proteins, showed that this adaptor plays a BCR-mediated regulatory role in mature B cells. However, the intermediates involved in this adaptor modulating functions in B cells are still unknown. In the present study, we investigated the role of APS in regulating BCR signalling notably through cytoskeleton remodeling in mature B cells. Herein, we showed that APS function is stage specific, as it exclusively intervenes in mature B cells. Upon activation, APS colocalizes with the BCR and associates with important regulators of BCR signalling, such as Syk and Cbl kinase. Importantly, APS interferes, as a scaffold protein, with the stability of Syk kinase by recruiting Cbl. This function is mainly mediated by APS SH2 domain, which regulates BCR-evoked cell dynamics. Our findings thus reveal that APS plays a regulatory role in BCR-induced responses by specifically modulating its interacting partners, which positions APS as a relevant modulator of BCR signalling in mature B cells.     

A-kinase-anchoring protein 95 functions as a potential carrier for the nuclear translocation of active caspase 3 through an enzyme-substrate-like association

Caspase-mediated proteolysis is a critical and central element of the apoptotic process, and caspase 3, one of the effector caspases, is proposed to play essential roles in the nuclear morphological changes of apoptotic cells. Although many substrates for caspase 3 localize in the nucleus and caspase 3 translocates from the cytoplasm to the nuclei after activation in apoptotic cells, the molecular mechanisms of nuclear translocation of active caspase 3 have been unclear. Recently, we suggested that a substrate-like protein(s) served as a carrier to transport caspase 3 from the cytoplasm into the nucleus. In the present study, we identified A-kinase-anchoring protein 95 (AKAP95) as a caspase 3-binding protein. Small interfering RNA-mediated depletion of AKAP95 reduced apoptotic nuclear morphological changes, suggesting that AKAP95 is involved in the process of apoptotic nuclear morphological changes. The association of AKAP95 with active caspase 3 was analogous to an enzyme-substrate interaction. Furthermore, overexpression of AKAP95 with nuclear localization sequence mutations inhibited nuclear morphological changes in apoptotic cells. These results indicate that AKAP95 is a potential carrier protein for active caspase 3 from the cytoplasm into the nuclei in apoptotic cells.     

Mitochondrial Telomerase Protects Cancer Cells from Nuclear DNA Damage and Apoptosis

Most cancer cells express high levels of telomerase and proliferate indefinitely. In addition to its telomere maintenance function, telomerase also has a pro-survival function resulting in an increased resistance against DNA damage and decreased apoptosis induction. However, the molecular mechanisms for this protective function remain elusive and it is unclear whether it is connected to telomere maintenance or is rather a non-telomeric function of the telomerase protein, TERT. It was shown recently that the protein subunit of telomerase can shuttle from the nucleus to the mitochondria upon oxidative stress where it protects mitochondrial function and decreases intracellular oxidative stress. Here we show that endogenous telomerase (TERT protein) shuttles from the nucleus into mitochondria upon oxidative stress in cancer cells and analyzed the nuclear exclusion patterns of endogenous telomerase after treatment with hydrogen peroxide in different cell lines. Cell populations excluded TERT from the nucleus upon oxidative stress in a heterogeneous fashion. We found a significant correlation between nuclear localization of telomerase and high DNA damage, while cells which excluded telomerase from the nucleus displayed no or very low DNA damage. We modeled nuclear and mitochondrial telomerase using organelle specific localization vectors and confirmed that mitochondrial localization of telomerase protects the nucleus from inflicted DNA damage and apoptosis while, in contrast, nuclear localization of telomerase correlated with higher amounts of DNA damage and apoptosis. It is known that nuclear DNA damage can be caused by mitochondrially generated reactive oxygen species (ROS). We demonstrate here that mitochondrial localization of telomerase specifically prevents nuclear DNA damage by decreasing levels of mitochondrial ROS. We suggest that this decrease of oxidative stress might be a possible cause for high stress resistance of cancer cells and could be especially important for cancer stem cells.     

Activation of Syk protein tyrosine kinase in response to osmotic stress requires interaction with p21-activated protein kinase Pak2/gamma-PAK

The p21-activated serine/threonine protein kinase Pak2/gamma-PAK and the nonreceptor type of protein tyrosine kinase Syk are known to be activated when the cells are exposed to osmotic stress. The purpose of the present study was to examine whether Pak2 and Syk functionally cooperate in cellular signaling. Cotransfection studies revealed that Pak2 associates with Syk in COS cells. The constitutively active form of Cdc42 increases the association of Pak2 with Syk. Pak2 coexpressed with an inactive form of Cdc42 or kinase-inactive Pak2 interacts to a lesser extent with Syk, suggesting that Pak2-Syk association is enhanced by Pak2 activation. Interaction with Pak2 enhances the intrinsic kinase activity of Syk. This is supported by in vitro studies showing that Pak2 phosphorylates and activates Syk. Treatment of cells with sorbitol to induce hyperosmolarity results in the translocation of Pak2 and Syk to the region surrounding the nucleus and in dramatic enhancement of their association. Furthermore, cotransfection of Pak2 and Syk leads to the activation of c-Jun N-terminal kinase (JNK) under hyperosmotic conditions. Pak2 short interfering RNA suppresses sorbitol-mediated activation of endogenous Syk and JNK, thus identifying a novel pathway for JNK activation by Cdc42. These results demonstrate that Pak2 and Syk positively cooperate to regulate cellular responses to stress.     

B cell antigen receptor-induced activation of Akt promotes B cell survival and is dependent on Syk kinase

Signaling through the B cell Ag receptor (BCR) is a key determinant in the regulation of B cell physiology. Depending on additional factors, such as microenvironment and developmental stage, ligation of the BCR can trigger B lymphocyte activation, proliferation, or apoptosis. The regulatory mechanisms determining B cell apoptosis and survival are not known. Using the chicken B lymphoma cell line DT40 as a model system, we investigated the role of the serine/threonine kinase Akt in B cell activation. While parental DT40 cells undergo apoptosis in response to BCR cross-linking, cells overexpressing Akt show a greatly diminished apoptotic response. By contrast, limiting the activation of Akt, either by inhibiting phosphatidylinositol 3-kinase or by ectopic expression of the phospholipid phosphatase MMAC1, results in a significant increase in the percentage of apoptotic cells after BCR cross-linking. Using various DT40 knockout cell lines, we further demonstrate that the tyrosine kinase Syk is required for Akt activation and that Lyn tyrosine kinase inhibits Akt activation. Taken together, the data demonstrate that Akt plays an important role in B cell survival and that Akt is activated in a Syk-dependent pathway.     

Oxidative stress induces protein kinase C-mediated activation loop phosphorylation and nuclear redistribution of protein kinase D

Oxidative stress induced by cell treatments with H(2)O(2) activates protein kinase D (PKD) via a protein kinase C (PKC)-dependent signal transduction pathway (Waldron, R. T., and Rozengurt, E. (2000) J. Biol. Chem. 275, 17114-17121). Here we show that oxidative stress induces PKC-dependent activation loop Ser(744) and Ser(748) phosphorylation to mediate dose- and time-dependent activation of PKD, both endogenously expressed in Swiss 3T3 cells and stably overexpressed in Swiss 3T3-GFP.PKD cells. Although oxidative stress induced PKD activation loop phosphorylation and activation with identical kinetics, both were dose-dependently blocked by preincubation of cells with selective inhibitors of PKC (GF109203X and G?6983) or c-Src (PP2). Inhibition of Src tyrosine kinase activity eliminated oxidative stress-induced direct PKD tyrosine phosphorylation, but only partially attenuated activation loop phosphorylation and activation. Mutation of a putative tyrosine phosphorylation site on PKD, Tyr(469) to phenylalanine, had no effect on its activation by oxidative stress in transfected COS-7 cells. Similarly, a mutant with Tyr(469) replaced by aspartic acid had increased basal activity but was also further activated by oxidative stress. Thus, PKD tyrosine phosphorylation at this site neither produced full activation by itself nor was required for oxidative stress-induced activation mediated by activation loop phosphorylation. In addition to PKD activation, activation loop phosphorylation in response to oxidative stress also redistributed activated PKD to cell nuclei, as revealed by PKD indirect immunofluorescence, imaging of a PKD-green fluorescent protein fusion construct (GFP-PKD), and analysis of nuclear pellets. Cell preincubation with G?6983 strongly diminished H(2)O(2)-induced nuclear relocalization of GFP-PKD. Taken together, these results indicate that PKC-mediated PKD Ser(744) and Ser(748) phosphorylation induced by oxidative stress integrates PKD activation with redistribution to the nucleus.