FLICE is activated by association with the CD95 death-inducing signaling complex (DISC).

Upon activation, the apoptosis-inducing cell membrane receptor CD95 (APO-1/Fas) recruits a set of intracellular signaling proteins (CAP1-4) into a death-inducing signaling complex (DISC). In the DISC, CAP1 and CAP2 represent FADD/MORT1. CAP4 was identified recently as an ICE-like protease, FLICE, with two death effector domains (DED). Here we show that FLICE binds to FADD through its N-terminal DED. This is an obligatory step in CD95 signaling detected in the DISC of all CD95-sensitive cells tested. Upon prolonged triggering of CD95 with agonistic antibodies all cytosolic FLICE gets proteolytically activated. Physiological FLICE cleavage requires association with the DISC and occurs by a two-step mechanism. Initial cleavage generates a p43 and a p12 fragment further processed to a p10 fragment. Subsequent cleavage of the receptor-bound p43 results in formation of the prodomain p26 and the release of the active site-containing fragment p18. Activation of FLICE is blocked by the peptide inhibitors zVAD-fmk, zDEVD-fmk and zIETD-fmk, but not by crmA or Ac-YVAD-CHO. Taken together, our data indicate that FLICE is the first in a cascade of ICE-like proteases activated by CD95 and that this activation requires a functional CD95 DISC.     

The role of CAP3 in CD95 signaling: new insights into the mechanism of procaspase-8 activation

Formation of the CD95 (APO-1/Fas) death inducing signaling complex (DISC) plays a central role in CD95 signaling. Previously, CD95 DISC composition was analyzed by two-dimensional gel electrophoresis and four major cytotoxicity-associated proteins (CAP1-4) were found. CAP1 and CAP2 were defined to be unmodified and phosphorylated FADD, respectively. CAP4 was identified as procaspase-8a. CAP3, however, has remained elusive. In this study, we demonstrate that CAP3 is an intermediate of procaspase-8 processing. CAP3 is generated within seconds of DISC formation and subsequently processed to the prodomain of procaspase-8a that is known as p26 (CAP5). These findings lead to new insights into the mechanism of procaspase-8 processing and apoptosis initiation.     

CD95 death receptor and epidermal growth factor receptor (EGFR) in liver cell apoptosis and regeneration

Recent evidence suggests that signaling pathways towards cell proliferation and cell death are much more interconnected than previously thought. Whereas not only death receptors such as CD95 (Fas, APO-1) can couple to both, cell death and proliferation, also growth factor receptors such as the epidermal growth factor receptor (EGFR) are involved in these opposing kinds of cell fate. EGFR is briefly discussed as a growth factor receptor involved in liver cell proliferation during liver regeneration. Then the role of EGFR in activating CD95 death receptor in liver parenchymal cells (PC) and hepatic stellate cells (HSC), which represent a liver stem/progenitor cell compartment, is described summarizing different ways of CD95- and EGFR-dependent signaling in the liver. Here, depending on the hepatic cell type (PC vs. HSC) and the respective signaling context (sustained vs. transient JNK activation) CD95-/EGFR-mediated signaling ends up in either liver cell apoptosis or cell proliferation.     

Association between GRB2/Sos and insulin receptor substrate 1 is not sufficient for activation of extracellular signal-regulated kinases by interleukin-4: implications for Ras activation by insulin.

Insulin receptor substrate 1 (IRS-1) mediates the activation of a variety of signaling pathways by the insulin and insulin-like growth factor 1 receptors by serving as a docking protein for signaling molecules with SH2 domains. We and others have shown that in response to insulin stimulation IRS-1 binds GRB2/Sos and have proposed that this interaction is important in mediating Ras activation by the insulin receptor. Recently, it has been shown that the interleukin (IL)-4 receptor also phosphorylates IRS-1 and an IRS-1-related molecule, 4PS. Unlike insulin, however, IL-4 fails to activate Ras, extracellular signal-regulated kinases (ERKs), or mitogen-activated protein kinases. We have reconstituted the IL-4 receptor into an insulin-responsive L6 myoblast cell line and have shown that IRS-1 is tyrosine phosphorylated to similar degrees in response to insulin and IL-4 stimulation in this cell line. In agreement with previous findings, IL-4 failed to activate the ERKs in this cell line or to stimulate DNA synthesis, whereas the same responses were activated by insulin. Surprisingly, IL-4's failure to activate ERKs was not due to a failure to stimulate the association of tyrosine-phosphorylated IRS-1 with GRB2/Sos; the amounts of GRB2/Sos associated with IRS-1 were similar in insulin- and IL-4-stimulated cells. Moreover, the amounts of phosphatidylinositol 3-kinase activity associated with IRS-1 were similar in insulin- and IL-4-stimulated cells. In contrast to insulin, however, IL-4 failed to induce tyrosine phosphorylation of Shc or association of Shc with GRB2. Thus, ERK activation correlates with Shc tyrosine phosphorylation and formation of an Shc/GRB2 complex. Thus, ERK activation correlates with Shc tyrosine phosphorylation and formation of an Shc/GRB2 complex. Previous studies have indicated that activation of ERks in this cell line is dependent upon Ras since a dominant-negative Ras (Asn-17) blocks ERK activation by insulin. Our findings, taken in the context of previous work, suggest that binding of GRB2/Sos to Shc may be the predominant mechanism whereby insulin as well as cytokine receptors activate Ras.     

Isolation and analysis of components of CD95 (APO-1/Fas) death-inducing signaling complex

CD95 (APO-1/Fas) is an apoptosis-inducing receptor belonging to the tumor necrosis factor receptor superfamily. Multimerization of CD95 leads to instant recruitment of the signaling molecules FADD and caspase-8 to the activated receptor forming the death-inducing signaling complex (DISC). DISC formation is the first essential step of CD95 signaling and results in activation of caspase-8 starting a signaling cascade that leads to apoptosis. Here we describe a method for analyzing the CD95 DISC. The method is based on coimmunoprecipitation of the signaling molecules with the activated CD95 receptor followed by Western blot detection of associated molecules. Therefore, this method can analyze the very first signaling events during CD95-mediated apoptosis.     

T cell regulation of p62(dok) (Dok1) association with Crk-L.

In addition to engagement of the T cell receptor-CD3 complex, T lymphocytes can be activated by a variety of cell surface molecules including the approximately 50-kDa surface receptor CD2. While the majority of biochemical signaling elements are triggered by either CD2 or TcR-CD3 receptors, a small number of proteins are engaged by only one receptor. Recently, p62(dok) (Dok1), a member of the Dok family of adapter molecules, has been reported to be activated by CD2 and not by CD3 engagement. Here we have examined the role of p62(dok) in CD2-dependent signaling in Jurkat T cells. As previously reported, we find that ligation of the CD2 molecule by mitogenic pairs of anti-CD2 mAbs led to phosphorylation of p62(dok). While CD2-induced p62(dok) tyrosine phosphorylation was independent of both the p36/38 membrane adapter protein linker of activated T cells (LAT) and the ZAP70/Syk family of kinases, it was dependent upon the Src family of kinases including Lck and Fyn. We find further that CD2 engagement induced the association of tyrosine-phosphorylated p62(dok) to Crk-L. The CD2-dependent association of p62(dok) to Crk-L was independent of expression of the ZAP70/Syk family of kinases. Of note, while T cell receptor-CD3 engagement did not induce either p62(dok) phosphorylation or Crk-L association in Jurkat T cells, it did inhibit CD2-dependent p62(dok)-Crk-L complexes; this TcR-CD3-mediated regulation was dependent upon ZAP70 kinase activity. Our data suggest that phosphorylation of p62(dok) and its interaction with other signaling proteins may depend upon integrated signals emanating from the CD2 receptor, utilizing a ZAP70/LAT-independent pathway, and the TcR-CD3 receptor, which is ZAP70/Syk-dependent.     

RIP mediates tumor necrosis factor receptor 1 activation of NF-kappaB but not Fas/APO-1-initiated apoptosis.

The CD95 (Fas/APO-1) and tumor necrosis factor (TNF) receptor pathways share many similarities, including a common reliance on proteins containing 'death domains' for elements of the membrane-proximal signal relay. We have created mutant cell lines that are unable to activate NF-kappaB in response to TNF. One of the mutant lines lacks RIP, a 74 kDa Ser/Thr kinase originally identified by its ability to associate with Fas/APO-1 and induce cell death. Reconstitution of the line with RIP restores responsiveness to TNF. The RIP-deficient cell line is susceptible to apoptosis initiated by anti-CD95 antibodies. An analysis of cells reconstituted with mutant forms of RIP reveals similarities between the action of RIP and FADD/MORT-1, a Fas-associated death domain protein.     

Cell surface sialylation plays a role in modulating sensitivity towards APO-1-mediated apoptotic cell death

APO-1/Fas(CD95), a member of the tumour necrosis factor (TNF)/nerve growth factor (NGF) receptor superfamily transduces apoptotic signals into apoptosis sensitive cells. In metabolic labelling experiments using the highly APO-1 positive cell lines HUT78 (adultT cell leukemia) and SKW6.4 (Blymphoblastoid cell line) APO-1 was characterised as a long living protein with a complex glycosylation pattern involving terminal sialic acid groups which account for 8-kDa of its apparent molecular weight on SDS-PAGE. APO-1 expression and the degree of sialylation were determined in additionalT and B cell lines. On the group I Burkitt's lymphoma cell line BL60 transfected with human APO-1 (K50) low sialylated species were detected only on the cell surface, suggesting that sialylation might be functionally important. Removal of terminal sialic acid groups by treatment of B and T cell lines with Vibrio cholerae neuraminidase (VCN) augmented sensitivity towards anti-APO-1 and human APO-1 ligand induced apoptosis. Similarly, VCN-treated U937 cells were rendered more sensitive to TNFalpha-induced cell death. Thus, sialylation may be one mechanism to regulate sensitivity towards ligand-mediated cell death in this receptor family.     

Toll-like receptor (TLR) signaling in response to Aspergillus fumigatus

Aspergillus fumigatus causes life-threatening infections in patients with qualitative and quantitative defects in phagocytic function. Here, we examined the contribution of Toll-like receptor (TLR)-2, TLR4, the adapter protein MyD88, and CD14 to signaling in response to the three forms of A. fumigatus encountered during human disease: resting conidia (RC), swollen conidia (SC), and hyphae (H). Compared with elicited peritoneal macrophages obtained from wild-type and heterozygous mice, TLR2(-/-) and MyD88(-/-) macrophages produced significantly less tumor necrosis factor-alpha (TNFalpha) following A. fumigatus stimulation. In contrast, following stimulation with RC, SC, and H, TLR4(-/-) and CD14(-/-) macrophages exhibited no defects in tumor necrosis factor-alpha release. TLR2(-/-), TLR4(-/-), MyD88(-/-), and CD14(-/-) macrophages bound similar numbers of RC and SC compared with wild-type macrophages. RC, SC, and H stimulated greater activation of a nuclear factor kappa B (NFkappaB)-dependent reporter gene and greater release of tumor necrosis factor-alpha from the human monocytic THP-1 cell line stably transfected with CD14 compared with control cells stably transfected with empty vector. A. fumigatus stimulated NFkappaB-dependent reporter gene activity in the human embryonic kidney cell line, HEK293, only if the cells were transfected with TLR2. Moreover, activity increased when TLR2 and CD14 were co-transfected. Taken together, these data suggest that optimal signaling responses to A. fumigatus require TLR2 in both mouse and human cells. In contrast, a role for CD14 was found only in the human cells. MyD88 acts as a central adapter protein mediating signaling responses following stimulation with RC, SC, and H.     

Two CD95 (APO-1/Fas) signaling pathways.

We have identified two cell types, each using almost exclusively one of two different CD95 (APO-1/Fas) signaling pathways. In type I cells, caspase-8 was activated within seconds and caspase-3 within 30 min of receptor engagement, whereas in type II cells cleavage of both caspases was delayed for approximately 60 min. However, both type I and type II cells showed similar kinetics of CD95-mediated apoptosis and loss of mitochondrial transmembrane potential (DeltaPsim). Upon CD95 triggering, all mitochondrial apoptogenic activities were blocked by Bcl-2 or Bcl-xL overexpression in both cell types. However, in type II but not type I cells, overexpression of Bcl-2 or Bcl-xL blocked caspase-8 and caspase-3 activation as well as apoptosis. In type I cells, induction of apoptosis was accompanied by activation of large amounts of caspase-8 by the death-inducing signaling complex (DISC), whereas in type II cells DISC formation was strongly reduced and activation of caspase-8 and caspase-3 occurred following the loss of DeltaPsim. Overexpression of caspase-3 in the caspase-3-negative cell line MCF7-Fas, normally resistant to CD95-mediated apoptosis by overexpression of Bcl-xL, converted these cells into true type I cells in which apoptosis was no longer inhibited by Bcl-xL. In summary, in the presence of caspase-3 the amount of active caspase-8 generated at the DISC determines whether a mitochondria-independent apoptosis pathway is used (type I cells) or not (type II cells).     

Transient receptor potential vanilloid subtype 1 mediates microglial cell death in vivo and in vitro via Ca2+-mediated mitochondrial damage and cytochrome c release

The present study examined the expression of transient receptor potential vanilloid subtype 1 (TRPV1) in microglia, and its association with microglial cell death. In vitro cell cultures, RT-PCR, Western blot analysis, and immunocytochemical staining experiments revealed that rat microglia and a human microglia cell line (HMO6) showed TRPV1 expression. Furthermore, exposure of these cells to TRPV1 agonists, capsaicin (CAP) and resiniferatoxin (RTX), triggered cell death. This effect was ameliorated by the TRPV1 antagonists, capsazepine and iodo-resiniferatoxin (I-RTX), suggesting that TRPV1 is directly involved. Further examinations revealed that TRPV1-induced toxicity was accompanied by increases in intracellular Ca(2+), and mitochondrial damage; these effects were inhibited by capsazepine, I-RTX, and the intracellular Ca(2+) chelator BAPTA-AM. Treatment of cells with CAP or RTX led to increased mitochondrial cytochrome c release and enhanced immunoreactivity to cleaved caspase-3. In contrast, the caspase-3 inhibitor z-DEVD-fmk protected microglia from CAP- or RTX-induced toxicity. In vivo, we also found that intranigral injection of CAP or 12-hydroperoxyeicosatetraenoic acid, an endogenous agonist of TRPV1, into the rat brain produced microglial damage via TRPV1 in the substantia nigra, as visualized by immunocytochemistry. To our knowledge, this study is the first to demonstrate that microglia express TRPV1, and that activation of this receptor may contribute to microglial damage via Ca(2+) signaling and mitochondrial disruption.     

Cytokine-independent Jak3 activation upon T cell receptor (TCR) stimulation through direct association of Jak3 and the TCR complex

Jak3 is responsible for growth signals by various cytokines such as interleukin (IL)-2, IL-4, and IL-7 through association with the common gamma chain (gammac) in lymphocytes. We found that T cells from Jak3-deficient mice exhibit impairment of not only cytokine signaling but also early activation signals and that Jak3 is phosphorylated upon T cell receptor (TCR) stimulation. TCR-mediated phosphorylation of Jak3 is independent of IL-2 receptor/gammac but is dependent on Lck and ZAP-70. Jak3 was found to be assembled with the TCR complex, particularly through direct association with CD3zeta via its JH4 region, which is a different region from that for gammac association. These results suggest that Jak3 plays a role not only in cell growth but also in T cell activation and represents cross-talk of a signaling molecule between TCR and growth signals.     

Phosphatidylinositol 3-Kinase (PI3K) Activity Bound to Insulin-like Growth Factor-I (IGF-I) Receptor, which Is Continuously Sustained by IGF-I Stimulation, Is Required for IGF-I-induced Cell Proliferation

Continuous stimulation of cells with insulin-like growth factors (IGFs) in G(1) phase is a well established requirement for IGF-induced cell proliferation; however, the molecular components of this prolonged signaling pathway that is essential for cell cycle progression from G(1) to S phase are unclear. IGF-I activates IGF-I receptor (IGF-IR) tyrosine kinase, followed by phosphorylation of substrates such as insulin receptor substrates (IRS) leading to binding of signaling molecules containing SH2 domains, including phosphatidylinositol 3-kinase (PI3K) to IRS and activation of the downstream signaling pathways. In this study, we found prolonged (>9 h) association of PI3K with IGF-IR induced by IGF-I stimulation. PI3K activity was present in this complex in thyrocytes and fibroblasts, although tyrosine phosphorylation of IRS was not yet evident after 9 h of IGF-I stimulation. IGF-I withdrawal in mid-G(1) phase impaired the association of PI3K with IGF-IR and suppressed DNA synthesis the same as when PI3K inhibitor was added. Furthermore, we demonstrated that Tyr(1316)-X-X-Met of IGF-IR functioned as a PI3K binding sequence when this tyrosine is phosphorylated. We then analyzed IGF signaling and proliferation of IGF-IR(-/-) fibroblasts expressing exogenous mutant IGF-IR in which Tyr(1316) was substituted with Phe (Y1316F). In these cells, IGF-I stimulation induced tyrosine phosphorylation of IGF-IR and IRS-1/2, but mutated IGF-IR failed to bind PI3K and to induce maximal phosphorylation of GSK3β and cell proliferation in response to IGF-I. Based on these results, we concluded that PI3K activity bound to IGF-IR, which is continuously sustained by IGF-I stimulation, is required for IGF-I-induced cell proliferation.     

The congenic mutant B6.C-H-2bm-1 (H-2bm-1) serological response to the T-cell receptor on EL4

Antisera reactive with the C57Bl lymphoma EL4 were induced by hyperimmunization of B6.C-H-2bm-1 (H-2bm-1) mice, a congenic mutant strain of C57Bl/6 (B6). Molecular weight determinations of EL4 surface structures detected with the congenic mutant antibodies were accomplished by electrophoretic analyses (one and two dimensions) on sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE). Analysis of immunoprecipitates in the first dimension under reducing conditions revealed protein bands corresponding to gp 70 and its 33-kDa cleavage fragment, and two-three bands (40-45 kDa) that overlapped with those precipitated from EL4 with AS 8177, an antiserum which detects framework determinants on the T-cell heterodimeric receptor. Further analysis by diagonal electrophoresis confirmed identity of the 40- to 45-kDa proteins precipitated from EL4 with either AS 8177 or H-2bm-1 anti-EL4 sera. Additional diagonal electrophoretic studies showed that the congenic mutant antibodies do not precipitate T-cell receptor molecules from C6VL, an in vitro line derived from the C6XL T-cell lymphoma of C57Bl/ka origin, which was previously shown by some of us to express the heterodimeric T-cell receptor. Together these results demonstrate detection of the heterodimeric T-cell receptor on EL4 with congenic mutant antibodies directed against nonconstant region determinant(s), and indicate a possible linkage between MHC haplotype and the immune response to intraspecies variable regions of the T-cell receptor.     

Molecular networks in FGF signaling: flotillin-1 and cbl-associated protein compete for the binding to fibroblast growth factor receptor substrate 2

Fibroblast growth factor receptor substrate 2 (FRS2α) is a signaling adaptor protein that regulates downstream signaling of many receptor tyrosine kinases. During signal transduction, FRS2 can be both tyrosine and threonine phosphorylated and forms signaling complexes with other adaptor proteins and tyrosine phosphatases. We have here identified flotillin-1 and the cbl-associated protein/ponsin (CAP) as novel interaction partners of FRS2. Flotillin-1 binds to the phosphotyrosine binding domain (PTB) of FRS2 and competes for the binding with the fibroblast growth factor receptor. Flotillin-1 knockdown results in increased Tyr phosphorylation of FRS2, in line with the inhibition of ERK activity in the absence of flotillin-1. CAP directly interacts with FRS2 by means of its sorbin homology (SoHo) domain, which has previously been shown to interact with flotillin-1. In addition, the third SH3 domain in CAP binds to FRS2. Due to the overlapping binding domains, CAP and flotillin-1 appear to compete for the binding to FRS2. Thus, our results reveal a novel signaling network containing FRS2, CAP and flotillin-1, whose successive interactions are most likely required to regulate receptor tyrosine kinase signaling, especially the mitogen activated protein kinase pathway.     

c-Jun N-terminal kinase 2 (JNK2) enhances cell migration through epidermal growth factor substrate 8 (EPS8)

Membrane-bound receptors induce biochemical signals to remodel the actin cytoskeleton and mediate cell motility. In association with receptor tyrosine kinases, several downstream mitogen-induced kinases facilitate cell migration. Here, we show a role for c-Jun N-terminal kinase 2 (JNK2) in promoting mammary cancer cell migration through inhibition of epidermal growth factor substrate 8 (EPS8) expression, a key regulator of EGF receptor (R) signaling and trafficking. Using jnk2(-/-) mice, we found that EPS8 expression is higher in polyoma middle T antigen (PyVMT)jnk2(-/-) mammary tumors and jnk2(-/-) mammary glands compared with the respective jnk2(+/+) controls. The inverse relationship between the jnk2 and eps8 expression was also associated with cancer progression in that patients with basal-type breast tumors expressing high jnk2 and low eps8 experienced poor disease-free survival. In mammary tumor cell lines, the absence of jnk2 greatly reduces cell migration that is rescued by EPS8 knockdown. Subsequent studies show that JNK2 enhances formation of the EPS8-Abi-1-Sos-1 complex to augment EGFR activation of Akt and ERK, whereas the absence of JNK2 promotes ESP8/RN-Tre association to inhibit endocytotic trafficking of the EGFR. Together, these studies unveil a critical role for JNK2 and EPS8 in receptor tyrosine kinase signaling and trafficking to convey distinctly different effects on cell migration.     

Multiple pathways originate at the Fas/APO-1 (CD95) receptor: sequential involvement of phosphatidylcholine-specific phospholipase C and acidic sphingomyelinase in the propagation of the apoptotic signal.

The early signals generated following cross-linking of Fas/APO-1, a transmembrane receptor whose engagement by ligand results in apoptosis induction, were investigated in human HuT78 lymphoma cells. Fas/APO-1 cross-linking by mAbs resulted in membrane sphingomyelin hydrolysis and ceramide generation by the action of both neutral and acidic sphingomyelinases. Activation of a phosphatidylcholine-specific phospholipase C (PC-PLC) was also detected which appeared to be a requirement for subsequent acidic sphingomyelinase (aSMase) activation, since PC-PLC inhibitor D609 blocked Fas/APO-1-induced aSMase activation, but not Fas/APO-1-induced neutral sphingomyelinase (nSMase) activation. Fas/APO-1 cross-linking resulted also in ERK-2 activation and in phospholipase A2 (PLA2) induction, independently of the PC-PLC/aSMase pathway. Evidence for the existence of a pathway directly involved in apoptosis was obtained by selecting HuT78 mutant clones spontaneously expressing a newly identified death domain-defective Fas/APO-1 splice isoform which blocks Fas/APO-1 apoptotic signalling in a dominant negative fashion. Fas/APO-1 cross-linking in these clones fails to activate PC-PLC and aSMase, while nSMase, ERK-2 and PLA2 activates are induced. These results strongly suggest that a PC-PLC/aSMase pathway contributes directly to the propagation of Fas/APO-1-generated apoptotic signal in lymphoid cells.