Caspase-8 may be the initiator caspase of the extrinsic apoptosis pathway

Caspase-8 may be the initiator caspase of the extrinsic apoptosis pathway and also has a role in non-apoptotic physiologies. cell we could unequivocally assign the cleavage activity to caspase-8. Importantly cleavage of HDAC7 alters its subcellular localization and abrogates its Nur77 repressor function. Thus we demonstrate a direct role for initiator caspase-mediated proteolysis in promoting gene PF 573228 transcription. During apoptosis cells are systematically dismantled and packaged into small membrane-bound particles PPP3CB ready for removal by professional phagocytes by a process that is driven by members of the caspase family of proteases. PF 573228 Members of the caspase family have generally been separated into two groups as follows: those involved in apoptosis (caspase-2 -3 and -6-10) and those involved in non-apoptotic processes such as inflammation and differentiation (caspase-1 -4 -5 and -14) (1). However this simple demarcation is complicated by evidence suggesting that some apoptotic caspases may have functions in non-apoptotic physiologies including but not limited to cell differentiation migration proliferation T and B cell activation and nuclear factor-κB (NF-κB)3 activation (2). Of the initiator apoptotic caspases the strongest evidence for alternative non-apoptotic roles is for caspase-8. The proteolytic activity of the caspase may be dispensable for some of these processes. For instance tumor necrosis factor (TNF)-mediated NF-κB activation in T cells fibroblasts and epithelial cells is dependent on caspase-8 protein but not its proteolytic activity. In contrast NF-κB activation in response to T cell receptor ligation in T cells does require caspase-8 activity (3). Regarding differentiation caspase-8 is required for maturation of monocytes into macrophages and pan-caspase-inhibitors block this process (4 5 In addition a requirement for caspase-8 activity has been demonstrated during differentiation of placental villous trophoblasts (6). Targeted deletion reveals that caspase-8 protein is also required for T cell activation formation of blood vessels and maintenance of hemopoietic progenitor cells in mice (4 7 8 More significantly caspase-8 null mice and humans manifest a complex condition including immunodeficiency early in their life and autoimmunity as the individuals age (9 10 If the cell utilizes lethal pro-apoptotic proteases such as caspase-8 to perform other cellular functions an immediate challenge it must overcome is how to survive while harboring active caspase-8. One possibility is to sequester the “apoptotic” substrates (procaspase-3 procaspase-7 and Bid) from active caspase-8 while leaving non-apoptotic substrates available for proteolysis. To test this hypothesis we need to identify the elusive non-apoptotic caspase-8 substrates. To this end we employed a bioinformatic approach. Searching the human proteome with a matrix model based on the well defined substrate specificity of caspase-8 revealed a number of potential substrates. We formally tested three of these and confirmed that one HDAC7 is very efficiently cleaved by caspase-8 both and protein assay was from Bio-Rad. promoter (Nur77-luc) was described previously (18). Human Bid was PCR-amplified with primers containing flanking EcoRI sites and cloned into the EcoRI site of pGEX-4T-1. HDAC7 D375A mutant was generated by site-directed mutagenesis using QuickChange (Stratagene). and purified by nickel-affinity chromatography as described PF 573228 previously (19). Caspases were titrated with Z-VAD-fmk to determine the concentration of catalytic sites as described previously (20). GST-Bid was expressed and purified as described previously (14). Recombinant HDAC7 and TRIM3 were purified from transfected HEK293. Cells were lysed with modified radioimmunoprecipitation buffer containing 200 μm phenylmethylsulfonyl fluoride 1 μg/ml aprotinin 2 μg/ml leupeptin 1 μg/ml pepstatin and 2 μm E-64 as described previously (21). Lysates were clarified by centrifugation and combined with 50 mm Tris-Cl pH 7.4 150 mm NaCl 5 mm EDTA 0.05% (v/v) Nonidet P-40 0.25% (w/v) gelatin at a 1:1 ratio. For every 1 ml of lysate 20 μl of anti-FLAG M2 affinity gel beads (Sigma) were added and immunoprecipitated for up to 4 h at 4 °C and PF 573228 the beads were washed three times in PBS. Beads were either used directly in caspase cleavage assays or resuspended in 50 mm Tris-Cl 150 mm.