EphB receptors trigger Akt activation and suppress Fas receptor-induced apoptosis in malignant T lymphocytes

Treatment of hematopoietic malignancies often requires allogeneic bone marrow transplantation, and the subsequent graft-versus-leukemia response is crucial for the elimination of malignant cells. Cytotoxic T lymphocytes and NK cells responsible for the immunoelimination express Fas ligand and strongly rely on the induction of Fas receptor-mediated apoptosis for their action. Although cancer cells are removed successfully by graft-versus-leukemia reactions in myeloid malignancies, their efficiency is low in T cell leukemias. This may be partially because of the ability of malignant T cells to escape apoptosis. Our work shows that Eph family receptor EphB3 is consistently expressed by malignant T lymphocytes, most frequently in combination with EphB6, and that stimulation with their common ligands, ephrin-B1 and ephrin-B2, strongly suppresses Fas-induced apoptosis in these cells. This effect is associated with Akt activation and with the inhibition of the Fas receptor-initiated caspase proteolytic cascade. Akt proved to be crucial for the prosurvival response, because inhibition of Akt, but not of other molecules central to T cell biology, including Src kinases, MEK1 and MEK2, blocked the antiapoptotic effect. Overall, this demonstrates a new role for EphB receptors in the protection of malignant T cells from Fas-induced apoptosis through Akt engagement and prevention of caspase activation. Because Fas-triggered apoptosis is actively involved in the graft-versus-leukemia response and cytotoxic T cells express ephrin-Bs, our observations suggest that EphB receptors are likely to support immunoevasivenes of T cell malignancies and may represent promising targets for therapies, aiming to enhance immunoelimination of cancerous T cells.     

Inhibition of Daxx-mediated apoptosis by heat shock protein 27

Heat shock protein 27 (HSP27) confers cellular protection against a variety of cytotoxic stresses and also against physiological stresses associated with growth arrest or receptor-mediated apoptosis. Phosphorylation modulates the activity of HSP27 by causing a major change in the supramolecular organization of the protein, which shifts from oligomers to dimers. Here we show that phosphorylated dimers of HSP27 interact with Daxx, a mediator of Fas-induced apoptosis, preventing the interaction of Daxx with both Ask1 and Fas and blocking Daxx-mediated apoptosis. No such inhibition was observed with an HSP27 phosphorylation mutant that is only expressed as oligomers or when apoptosis was induced by transfection of a Daxx mutant lacking its HSP27 binding domain. HSP27 expression had no effect on Fas-induced FADD- and caspase-dependent apoptosis. However, HSP27 blocked Fas-induced translocation of Daxx from the nucleus to the cytoplasm and Fas-induced Daxx- and Ask1-dependent apoptosis. The observations revealed a new level of regulation of the Fas pathway and suggest a mechanism for the phosphorylation-dependent protective function of HSP27 during stress and differentiation.     

Selective inhibition of protein kinase C isozymes by Fas ligation.

Activation of protein kinase C (PKC) can protect cells from apoptosis induced by various agents, including Fas ligation. To elucidate a possible interaction between Fas-mediated apoptotic signals and activation-related protective signals, we investigated the impact of Fas ligation on PKC activity. We demonstrate that engagement of Fas on human lymphoid Jurkat cells triggered apoptosis, and Fas ligation resulted in partial blockade of cellular PKC activity. The phorbol 12-myristate 13-acetate-mediated translocation of PKCtheta from the cytoplasm to the membrane was inhibited by treatment with anti-Fas antibody, whereas the translocation of PKCalpha or epsilon was not affected. In vitro kinase assay of PKCalpha or epsilon phosphotransferase activity demonstrated that Fas ligation inhibited the ability of PKCalpha to phosphorylate histone H1 as substrate but did not inhibit epsilon isozyme activity. This inhibition of PKCalpha activity mediated by Fas ligation was reversed by okadaic acid, a phosphatase inhibitor, suggesting the involvement of a member of the protein phosphatase 2A subfamily in this component of Fas signaling. Identical patterns of PKC isozyme inhibition were obtained using mouse thymoma cells overexpressing the fas gene (LF(+)). These results suggest that the selective inhibition of a potentially protective, PKC-mediated pathway by Fas activation may, to some extent, contribute to Fas-induced apoptotic signaling.     

Gadd45b Mediates Fas-induced Apoptosis by Enhancing the Interaction between p38 and Retinoblastoma Tumor Suppressor

Gadd45b has been known as a positive mediator of apoptosis induced by certain cytokines and oncogenes. Here, we identified Gadd45b as an effector of Fas-induced apoptosis and found that p38-mediated Rb hyperphosphorylation is one of the mechanisms of Fas-induced apoptosis in murine hepatocyte AML12 cells. Gadd45b has been shown to activate p38 through its physical interaction with MTK1 and induce apoptosis. However, in this study, we have showed that the function of Gadd45b during Fas-induced apoptosis in AML12 cells is different from that reported in previous studies. Depletion of Gadd45b expression did not inhibit the phosphorylation of p38, but it suppressed p38-mediated Rb phosphorylation and apoptosis in response to Fas stimulation by reducing the interaction between p38 and Rb. Ectopic expression of Gadd45b was sufficient to enhance this interaction. These findings suggest that Gadd45b mediates p38-induced Rb phosphorylation by enhancing the interaction between p38 and Rb during Fas-induced apoptosis in murine hepatocytes.     

NFkappaB activation by Fas is mediated through FADD, caspase-8, and RIP and is inhibited by FLIP

Fas (APO-1/CD95) is the prototypic death receptor, and the molecular mechanisms of Fas-induced apoptosis are comparably well understood. Here, we show that Fas activates NFkappaB via a pathway involving RIP, FADD, and caspase-8. Remarkably, the enzymatic activity of the latter was dispensable for Fas-induced NFkappaB signaling pointing to a scaffolding-related function of caspase-8 in nonapoptotic Fas signaling. NFkappaB was activated by overexpressed FLIPL and FLIPS in a cell type-specific manner. However, in the context of Fas signaling both isoforms blocked FasL-induced NFkappaB activation. Moreover, down-regulation of both endogenous FLIP isoforms or of endogenous FLIPL alone was sufficient to enhance FasL-induced expression of the NFkappaB target gene IL8. As NFkappaB signaling is inhibited during apoptosis, FasL-induced NFkappaB activation was most prominent in cells that were protected by Bcl2 expression or caspase inhibitors and expressed no or minute amounts of FLIP. Thus, protection against Fas-induced apoptosis in a FLIP-independent manner converted a proapoptotic Fas signal into an inflammatory NFkappaB-related response.     

Sequence-specific 1H, 13C and 15N backbone resonance assignments of the 34 kDa catalytic domain of human PTPN7

PTPN7 is a protein tyrosine phosphatase responsible for inactivation of MAPK in leukocytes. Here we report the backbone resonance assignments of the 34 kDa phosphatase domain of human PTPN7, which is amplified in myeloid malignancies and deleted in lymphoproliferative disorders.     

Calmodulin binding to cellular FLICE-like inhibitory protein modulates Fas-induced signalling

We and others have demonstrated that Fas-mediated apoptosis is a potential therapeutic target for cholangiocarcinoma. Previously, we reported that CaM (calmodulin) antagonists induced apoptosis in cholangiocarcinoma cells through Fas-related mechanisms. Further, we identified a direct interaction between CaM and Fas with recruitment of CaM into the Fas-mediated DISC (death-inducing signalling complex), suggesting a novel role for CaM in Fas signalling. Therefore we characterized the interaction of CaM with proteins recruited into the Fas-mediated DISC, including FADD (Fas-associated death domain)-containing protein, caspase 8 and c-FLIP {cellular FLICE [FADD (Fas-associated death domain)-like interleukin 1beta-converting enzyme]-like inhibitory protein}. A Ca(2+)-dependent direct interaction between CaM and FLIP(L), but not FADD or caspase 8, was demonstrated. Furthermore, a 37.3+/-5.7% increase (n=6, P=0.001) in CaM-FLIP binding was observed at 30 min after Fas stimulation, which returned to the baseline after 60 min and correlated with a Fas-induced increase in intracellular Ca(2+) that reached a peak at 30 min and decreased gradually over 60 min in cholangiocarcinoma cells. A CaM antagonist, TFP (trifluoperazine), inhibited the Fas-induced increase in CaM-FLIP binding concurrent with inhibition of ERK (extracellular-signal-regulated kinase) phosphorylation, a downstream signal of FLIP. Direct binding between CaM and FLIP(L) was demonstrated using recombinant proteins, and a CaM-binding region was identified in amino acids 197-213 of FLIP(L). Compared with overexpression of wild-type FLIP(L) that resulted in decreased spontaneous as well as Fas-induced apoptosis, mutant FLIP(L) with deletion of the CaM-binding region resulted in increased spontaneous and Fas-induced apoptosis in cholangiocarcinoma cells. Understanding the biology of CaM-FLIP binding may provide new therapeutic targets for cholangiocarcinoma and possibly other cancers.     

ErbB2, EphrinB1, Src kinase and PTPN13 signaling complex regulates MAP kinase signaling in human cancers

In non-cancerous cells, phosphorylated proteins exist transiently, becoming de-phosphorylated by specific phosphatases that terminate propagation of signaling pathways. In cancers, compromised phosphatase activity and/or expression occur and contribute to tumor phenotype. The non-receptor phosphatase, PTPN13, has recently been dubbed a putative tumor suppressor. It decreased expression in breast cancer correlates with decreased overall survival. Here we show that PTPN13 regulates a new signaling complex in breast cancer consisting of ErbB2, Src, and EphrinB1. To our knowledge, this signaling complex has not been previously described. Co-immunoprecipitation and localization studies demonstrate that EphrinB1, a PTPN13 substrate, interacts with ErbB2. In addition, the oncogenic V660E ErbB2 mutation enhances this interaction, while Src kinase mediates EphrinB1 phosphorylation and subsequent MAP Kinase signaling. Decreased PTPN13 function further enhances signaling. The association of oncogene kinases (ErbB2, Src), a signaling transmembrane ligand (EphrinB1) and a phosphatase tumor suppressor (PTPN13) suggest that EphrinB1 may be a relevant therapeutic target in breast cancers harboring ErbB2-activating mutations and decreased PTPN13 expression.     

Potential role of protein tyrosine phosphatase nonreceptor type 13 in the control of oocyte meiotic maturation

Protein tyrosine phosphatase nonreceptor type 13 (PTPN13) is a tyrosine phosphatase with multiple interacting domains that has been implicated previously in the regulation of apoptosis. We provide evidence that PTPN13 plays an important role in the control of the meiotic cell cycle. A cDNA coding for PTPN13 was isolated during the screening for the substrate of protein kinase A expressed in mammalian oocytes. PTPN13 is expressed in both mouse and Xenopus oocytes and is a substrate for protein kinase A in vitro and in vivo. Expression of a truncated constitutively-active PTPN13 in Xenopus oocytes synergizes with progesterone in the induction of germinal vesicle breakdown, the translation of Mos, the phosphorylation of Erk and the dephosphorylation of Cdc2. The phosphatase activity of PTPN13 is required for this synergism. Oocyte injection with specific small interference RNA downregulates the expression of mRNA for PTPN13 and blocks oocyte maturation induced by progesterone, a blockade that can be overcome by Cdc25 overexpression. These findings indicate that PTPN13 is involved in the regulation of the meiotic cell cycle.     

Fas-induced in vivo apoptosis in bone marrow: anti-Fas mAb-induced elimination and successive proliferation of Fas-expressing cells especially those of myeloid lineage.

A single administration of agonistic anti-Fas mAb RK8 into mice decreased the number of bone marrow cells especially Mac1+ and Gr1+ cells of myeloid lineage. These cells, which were shown to be Fas-positive in normal bone marrow, were directly eliminated in vivo by Fas-mediated apoptosis. After the elimination of Fas-positive bone marrow cells, bone marrow was reconstituted by successive increase of numbers of Gr1(low) and Mac1(low) myeloid precursor cells expressing high levels of Fas, which are minor constituents in normal bone marrow. The increased cells consisted at least two components, Gr1(dull) Mac1+ cKit+ cells and Gr1(intermediate) Mac1+ cKit- cells, both of which were shown to be sensitive to Fas-induced apoptosis in vivo. Thus, Fas is functional in normal bone marrow and Fas-induced apoptosis in bone marrow enhances marked proliferation of Fas-expressing myeloid precursor cells in vivo.