We have generated rat monoclonal antibodies that specifically recognise caspase-2 from many species, including mouse, rat and humans. Using these antibodies, we have investigated caspase-2 expression, subcellular localisation and processing. We demonstrate that caspase-2 is expressed in most tissues and cell types. Cell fractionation and immunohistochemistry experiments show that caspase-2 is found in the nuclear and cytosolic fractions, including a significant portion present in the Golgi complex. We found that caspase-2 is processed in response to many apoptotic stimuli but experiments with caspase-2 deficient mice demonstrated that it is not required for apoptosis of thymocytes or dorsal root ganglia (DRG) neurons in response to a variety of cytotoxic stimuli. Caspase-2 processing does not occur in thymocytes lacking Apaf-1 or caspase-9, suggesting that in this cell type, activation of caspase-2 occurs downstream of apoptosome formation.

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The tumour necrosis factor receptor (TNFR)/nerve growth factor receptor (NGFR) family of molecules regulate a number of biological functions, such as growth, differentiation and apoptosis in multiple cell types. In the immune system, members of this receptor family are involved in the development of peripheral lymphoid organs, regulation of induced inflammatory responses and removal of cells at the end of an immune response.The TNFR family consists of more than 15 different molecules. Most are type I membrane proteins which resemble each other largely in their extracellular regions, which all contain 2-6 characteristic cysteine-rich domains. 1 The TNF family receptors are activated upon binding of their cognate ligands, most of which are trimers with a structure similar to TNF. Sometimes the ligands are cell bound type II membrane proteins, but several are cleaved off and appear as soluble trimers. Induction of trimers or higher order complexes of the TNF family of receptors allows their cytoplasmic domains to aggregate intracytoplasmic signalling molecules. Signalling pathways controlled by TNF receptorsThe cytoplasmic domains of the TNFR family, which are more diverse than the extracellular portions, do not have any intrinsic enzymatic activity, hence they signal by inducing aggregation of intracellular adaptor molecules (Fig. 1). Death domainsThe cytoplasmic domains of TNFR1 (p55), CD95 (Fas/ APO-1), NGFR (p75), death receptor (DR) 3, TRAIL-R1 and TRAIL-R2 all bear a motif termed a 'death domain' (DD), so-called because it is required for these receptors to transmit apoptotic signals. The DD is a protein-protein interaction motif consisting of six alpha helices that allow two proteins with DD to bind to each other. Structurally the DD is related to two other homotypic interaction domains, the death effector domain (DED), and the caspase recruitment domain (CARD). 2 Death domain adaptors: TRADD, FADD, RIP and RAIDDBinding of TNF to TNFR1 induces recruitment of the DDcontaining protein TRADD to the DD of TNFR1. 3 Overexpression of TRADD alone also induces the TNF-regulated responses apoptosis and activation of the transcription factors NF-κB and Jun kinase (JNK), presumably because TRADD provides docking sites for downstream signalling proteins to the receptor complex. 4 Two of the proteins that TRADD recruits to the signalling complex also bear death domains. One of these, RIP, has an N-terminal DD and a C-terminal kinase domain. Knockout studies have shown that RIP is required for induction of NFκB by TNF. 5 The other, Fas-associated protein with death domain (FADD), has a C-terminal DD, and an N-terminal DED. The FADD is required for cell death signalling by TNFR1 and also by CD95, to which it binds directly via its death domain. [6][7][8] The DED of FADD allows it to bind to DED in the pro-domain of caspase 8.Through these interactions, ligation of TNFR1 or CD95 can result in the formation of a death-inducing signalling complex, which leads to activation of caspase 8, a cell death effector protease. Once ac...

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