The effector arm of the cell-death pathway is composed of cysteine proteases belonging to the ICE/CED-3 family. In metazoan cells these exist as inactive polypeptide precursors (zymogens), each composed of a prodomain, which is cleaved to activate the protease, and a large and small catalytic subunit. The coupling of these 'death' proteases to signalling pathways is probably mediated by adaptor molecules that contain protein-protein interaction motifs such as the death domain. Here we describe such an adaptor molecule, RAIDD, which has an unusual bipartite architecture comprising a carboxy-terminal death domain that binds to the homologous domain in RIP, a serine/threonine kinase component of the death pathway. The amino-terminal domain is surprisingly homologous with the sequence of the prodomain of two ICE/CED-3 family members, human ICH-1 (ref. 5) and Caenorhabditis elegans CED-3 (ref. 6). This similar region mediates the binding of RAIDD to ICH-1 and CED-3, serving as a direct link to the death proteases, indicating that the prodomain may, through homophilic interactions, determine the specificity of binding of ICE/CED-3 zymogens to regulatory adaptor molecules. Finally, alternations in the sequence of the N-terminal domain that are equivalent to inactivating mutations in the C. elegans ced-3 gene prevent homophilic binding, highlighting the potentially primordial nature of this interaction.
Members of the ICE/ced-3 gene family have been implicated as components of the cell death pathway. Based on similarities with the structural prototype interleukin-1-converting enzyme (ICE), family members are synthesized as proenzymes that are proteolytically processed to form active heterodimeric enzymes. In this report, we describe a novel member of this growing gene family, ICE-LAP3, which is closely related to the death effector Yama/CPP32/Apopain. Pro-ICE-LAP3 is a 35-kDa protein localized to the cytoplasm and expressed in a variety of tissues and cell lines. Overexpression of a truncated version of ICE-LAP3 (missing the pro-domain) induces apoptosis in MCF7 breast carcinoma cells. Importantly, upon receipt of a death stimulus, endogenous ICE-LAP3 is processed to its subunit forms, suggesting a physiological role in cell death. This is the first report to demonstrate processing of a native ICE/ ced-3 family member during execution of the death program and the first description of the subcellular localization of an ICE/ced-3 family member.
Members of the ICE/Ced-3 gene family are likely effector components of the cell death machinery. Here, we characterize a novel member of this family designated ICE-LAP6. By phylogenetic analysis, ICE-LAP6 is classified into the Ced-3 subfamily which includes Ced-3, Yama/ CPP32/apopain, Mch2, and ICE-LAP3/Mch3/CMH-1. Interestingly, ICE-LAP6 contains an active site QACGG pentapeptide, rather than the QACRG pentapeptide shared by other family members. Overexpression of ICE-LAP6 induces apoptosis in MCF7 breast carcinoma cells. More importantly, ICE-LAP6 is proteolytically processed into an active cysteine protease by granzyme B, an important component of cytotoxic T cell-mediated apoptosis. Once activated, ICE-LAP6 is able to cleave the death substrate poly(ADP-ribose) polymerase into signature apoptotic fragments.Apoptosis, or programmed cell death, is a physiologic process important in the normal development and homeostasis of metazoans (1). It is becoming apparent that a class of cysteine proteases homologous to Caenorhabditis elegans Ced-3 play the role of "executioner" in the apoptotic mechanism (2-4). In the nematode, two proteins, encoded by ced-3 and ced-4, are required for all somatic cell deaths that occur during development (5). Mutations of ced-3 and ced-4 abolish the apoptotic capability of cells that normally die during C. elegans embryogenesis (6). While no mammalian homologs of ced-4 have been identified, ced-3 shares sequence similarity with interleukin-1 converting enzyme (ICE) 1 (7), a cysteine protease involved in the processing and activation of pro-interleukin-1 to an active cytokine (8, 9). Recently, numerous homologs of ICE/ Ced-3 have been characterized, comprising a new gene family of cysteine proteases.To date, seven members of the ICE/Ced-3 family have been identified and include ICE (8), TX/ICH2/ICE rel-II (10 -12), ICE rel-III (10), ICH1/Nedd-2 (13, 14), Yama/CPP32/Apopain (15-17), Mch2 (16), and ICE-LAP3/Mch3/CMH-1 (18 -20). All family members share sequence homology with ICE/Ced-3 and contain an active site QACRG pentapeptide in which the cysteine residue is catalytic. Ectopic expression of these proteases in a variety of cells causes apoptosis. Phylogenetic analysis of the ICE/Ced-3 3gene family revealed three subfamilies (3, 18). Yama, ICE-LAP3, and Mch2 are closely related to C. elegans Ced-3 and comprise the Ced-3 subfamily. ICE and the ICErelated genes, ICE rel-II, and ICE rel-III form the ICE subfamily, while ICH1 and its mouse homologue, NEDD-2 form the NEDD-2 subfamily.Based on similarities with the structural prototype interleukin-1 converting enzyme, ICE/Ced-3 family members are synthesized as zymogens that are capable of being processed to form active heterodimeric enzymes (9). It will be important to determine which family members are in fact activated in response to apoptotic stimuli. Previous studies have demonstrated that pro-Yama and pro-ICE-LAP3 are processed into active subunits in response to various death stimuli including engagement of Fas/APO-1 or treatment w...
SAG (sensitive to apoptosis gene) was cloned as an inducible gene by 1,10-phenanthroline (OP), a redoxsensitive compound and an apoptosis inducer. SAG encodes a novel zinc RING finger protein that consists of 113 amino acids with a calculated molecular mass of 12.6 kDa. SAG is highly conserved during evolution, with identities of 70% between human and Caenorhabditis elegans sequences and 55% between human and yeast sequences. In human tissues, SAG is ubiquitously expressed at high levels in skeletal muscles, heart, and testis. SAG is localized in both the cytoplasm and the nucleus of cells, and its gene was mapped to chromosome 3q22-24. Bacterially expressed and purified human SAG binds to zinc and copper metal ions and prevents lipid peroxidation induced by copper or a free radical generator. When overexpressed in several human cell lines, SAG protects cells from apoptosis induced by redox agents (the metal chelator OP and zinc or copper metal ions). Mechanistically, SAG appears to inhibit and/or delay metal ion-induced cytochrome c release and caspase activation. Thus, SAG is a cellular protective molecule that appears to act as an antioxidant to inhibit apoptosis induced by metal ions and reactive oxygen species.
The Eph family of receptor protein tyrosine kinases (RPTKs) is the largest family of RPTKs. The signal transduction pathways initiated by this family have only recently begun to be explored. Using a yeast two-hybrid screen to identify molecules that interact with the cytoplasmic domain of Eck, it was previously shown that activated Eck RPTK bound to and stimulated phosphatidylinositol 3-kinase (Pandey, A., Lazar, D.F., Saltiel, A. R., and Dixit, V.M. (1994) J. Biol. Chem. 269, 30154-30157). Also isolated from this same screen was a novel protein containing SH3 and SH2 adapter modules that had striking homology to those found in the Src family of non-receptor tyrosine kinases. However, unlike other Src family members, it lacked a catalytic tyrosine kinase domain. Hence, this protein was designated SLAP for Src-like adapter protein. Using glutathione S-transferase fusion Proteins, it was demonstrated that SLAP bound to activated Eck receptor tyrosine kinase. Therefore, SLAP is a novel candidate downstream signaling intermediate and the first member of the Src family that resembles an adapter molecule.
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