FADD is a cytoplasmic adapter molecule that links the family of death receptors to the activation of caspases during apoptosis. We have produced transgenic mice expressing a dominantly interfering mutant of FADD, lacking the caspase-dimerizing death effector domain, as well as mice overexpressing the poxvirus serpin, CrmA, an inhibitor of caspases downstream of FADD. While thymocytes from either line of mice were completely protected from CD95-dependent cytotoxicity, neither transgene afforded protection from apoptosis induced during thymocyte selection and neither led to the lymphoproliferative disorders associated with deficiencies in CD95. However, in FADD dominant negative (FADDdd) mice, early thymocyte development was retarded and peripheral lymphocyte pools were devoid of normal populations of T cells. We show that thymocytes and peripheral T cells from FADDdd display signaling anomalies, implying that FADD plays a previously uncharacterized role in T cell development and activation.

T-cell receptors (TCRs) are created by a stochastic gene rearrangement process during thymocyte development, generating thymocytes bearing useful, as well as unwanted, specificities. Within the latter group, autoreactive thymocytes arise which are subsequently eliminated via a thymocyte-specific apoptotic mechanism, termed negative selection. The molecular basis of this deletion is unknown. Here, we show that TCR triggering by peptide/MHC ligands activates a caspase in double-positive (DP) CD4+ CD8+ thymocytes, resulting in their death. Inhibition of this enzymatic activity prevents antigen-induced death of DP thymocytes in fetal thymic organ culture (FTOC) from TCR transgenic mice as well as apoptosis induced by anti-CD3epsilon monoclonal antibody and corticosteroids in FTOC of normal C57BL/6 mice. Hence, a common caspase mediates immature thymocyte susceptibility to cell death.

The ability of thrombospondin (TSP), an extracellular matrix glycoprotein, and two proteolytic fragments to support adhesion and neurite outgrowth from embryonic dorsal root ganglia, spinal cord neurons, and PC12 cells was examined. Anti-TSP antibodies or a synthetic peptide (GRGDS) containing an RGD cell-binding region was also added to cells plated on TSP. TSP and its 140 kd fragment were more efficient than laminin controls in supporting adhesion. Neurites formed on laminin, on varying concentrations of TSP, and particularly the 140 kd fragment. The amino-terminal heparin-binding domain supported little adhesion and outgrowth. Both adhesion and process outgrowth on TSP were inhibited by addition of anti-TSP antibodies, but not GRGDS.

During an immune response naive T helper (Th) cells differentiate into two functionally distinct subsets, Th1 and Th2, based on their cytokine secretion profile and immunomodulatory function. c-Jun amino terminal kinase (JNK) regulates Th cell differentiation by activating a transcriptional program required for cytokine production. We have recently identified a TNFR superfamily death domain-containing molecule, death receptor (DR)6, which potently activates JNK. T cells from DR6-deficient mice are substantially impaired in JNK activation. When DR6(-/-) mice were challenged with protein antigen, their T cells hyperproliferate and display a profound polarization toward a Th2 response whereas Th1 differentiation is not equivalently affected. In addition, DR6(-/)- T cells showed preference toward Th2 differentiation in vitro. The phenotype seen in the DR6(-/)- mice is not due to the apoptotic pathway. Therefore, DR6, working through JNK, rather than apoptosis, functions to attenuate the Th2 response. This is the first demonstration of a role in the activation and differentiation of Th cells by DR6 in particular and DRs in general.

Although the mechanism of mammalian apoptosis has not been elucidated, a protease of the CED-3/ICE family is anticipated to be a component of the death machinery. Several lines of evidence predict that this protease cleaves the death substrate poly(ADP-ribose) polymerase (PARP) to a specific 85 kDa form observed during apoptosis, is inhibitable by the CrmA protein, and is distinct from ICE. We cloned a ced-3/ICE-related gene, designated Yama, that encodes a protein identical to CPP32 beta. Purified Yama was a zymogen that, when activated, cleaved PARP to generate the 85 kDa apoptotic fragment. Cleavage of PARP by Yama was inhibited by CrmA but not by an inactive point mutant of CrmA. Furthermore, CrmA blocked cleavage of PARP in cells undergoing apoptosis. We propose that Yama may represent an effector component of the mammalian cell death pathway and suggest that CrmA blocks apoptosis by inhibiting Yama.

In this report, we describe the cloning and characterization of Boo, a novel anti-apoptotic member of the Bcl-2 family. The expression of Boo was highly restricted to the ovary and epididymis implicating it in the control of ovarian atresia and sperm maturation. Boo contains the conserved BH1 and BH2 domains, but lacks the BH3 motif. Like Bcl-2, Boo possesses a hydrophobic C-terminus and localizes to intracellular membranes. Boo also has an N-terminal region with strong homology to the BH4 domain found to be important for the function of some anti-apoptotic Bcl-2 homologues. Chromosomal localization analysis assigned Boo to murine chromosome 9 at band d9. Boo inhibits apoptosis, homodimerizes or heterodimerizes with some death-promoting and -suppressing Bcl-2 family members. More importantly, Boo interacts with Apaf-1 and forms a multimeric protein complex with Apaf-1 and caspase-9. Bak and Bik, two pro-apoptotic homologues disrupt the association of Boo and Apaf-1. Furthermore, Boo binds to three distinct regions of Apaf-1. These results demonstrate the evolutionarily conserved nature of the mechanisms of apoptosis. Like Ced-9, the mammalian homologues Boo and Bcl-xL interact with the human counterpart of Ced-4, Apaf-1, and thereby regulate apoptosis.

Initiation of T cell antigen receptor (TCR) signaling is dependent on Lck, a Src family kinase. The Src-like adaptor protein (SLAP) contains Src homology (SH)3 and SH2 domains, which are highly homologous to those of Lck and other Src family members. Because of the structural similarity between Lck and SLAP, we studied its potential role in TCR signaling. Here, we show that SLAP is expressed in T cells, and that when expressed in Jurkat T cells it can specifically inhibit TCR signaling leading to nuclear factor of activated T cells (NFAT)-, activator protein 1 (AP-1)-, and interleukin 2-dependent transcription. The SH3 and SH2 domains of SLAP are required for maximal attenuation of TCR signaling. This inhibitory activity can be bypassed by the combination of phorbol myristate acetate (PMA) and ionomycin, suggesting that SLAP acts proximally in the TCR signaling pathway. SLAP colocalizes with endosomes in Jurkat and in HeLa cells, and is insoluble in mild detergents. In stimulated Jurkat cells, SLAP associates with a molecular signaling complex containing CD3zeta, ZAP-70, SH2 domain-containing leukocyte protein of 76 kD (SLP-76), Vav, and possibly linker for activation of T cells (LAT). These results suggest that SLAP is a negative regulator of TCR signaling.

CD95 (Fas/APO-1) and tumor necrosis factor receptor-1 (TNFR-1) are related molecules that signal apoptosis. Recently, a number of novel binding proteins have been proposed to mediate the signaling of these death receptors. Here we report that an N-terminal truncation of one of these candidate signal transducers, FADD/MORT1, abrogates CD95-induced apoptosis, ceramide generation, and activation of the cell death protease Yama/CPP32. In addition, this dominant-negative derivative of FADD (FADD-DN) blocked TNF-induced apoptosis while not affecting NF- kappaB activation. FADD-DN bound both receptors, and in the case of CD95, it disrupted the assembly of a signaling complex. Taken together, our results functionally establish FADD as the apoptotic trigger of CD95 and TNFR-1.

Inhibitors of apoptosis (IAPs) are a family of cell death inhibitors found in viruses and metazoans. All IAPs have at least one baculovirus IAP repeat (BIR) motif that is essential for their anti-apoptotic activity. IAPs physically interact with a variety of pro-apoptotic proteins and inhibit apoptosis induced by diverse stimuli. This allows them to function as sensors and inhibitors of death signals that emanate from a variety of pathways.