We investigated the physiological role of endogenous MAPKactivating death domain-containing protein (MADD), a splice variant of the IG20 gene, that can interact with TNFR1 in tumor necrosis factor-␣ (TNF␣)-induced activation of NF-B, MAPK, ERK1/2, JNK, and p38. Using exon-specific short hairpin RNAs expressing lentiviruses, we knocked down the expression of all IG20 splice variants or MADD, which is overexpressed in cancer cells. Abrogation of MADD expression rendered cells highly susceptible to TNF␣-induced apoptosis in the absence of cycloheximide. It also resulted in a dramatic loss in TNF␣-induced activation of MAPK without any apparent effect on NF-B activation. This observation was substantiated by an accompanying loss in the activation of p90RSK, a key downstream target of MAPK, whereas the NF-B-regulated interleukin 6 levels remained unaffected. Endogenous MADD knockdown, however, did not affect epidermal growth factor-induced MAPK activation thereby demonstrating the specific requirement of MADD for TNF receptor-mediated MAPK activation. Re-expression of short hairpin RNA-resistant MADD in the absence of endogenous IG20 expression rescued the cells from TNF␣-induced apoptosis. The requirement for MADD was highly specific for TNF␣-induced activation of MAPK but not the related JNK and p38 kinases. Loss of MADD expression resulted in reduced Grb2 and Sos1/2 recruitment to the TNFR1 complex and decreased Ras and MEKK1/2 activation. These results demonstrate the essential role of MADD in protecting cancer cells from TNF␣-induced apoptosis by specifically activating MAPKs through Grb2 and Sos1/2 recruitment, and its potential as a novel cancer therapeutic target.Genes in higher organisms generate alternate transcripts that are translated into closely related proteins with different functions. Perturbations in the tightly regulated alternate splicing of key genes in cancers can result in the accumulation of select splice variants of a particular gene or suppression of others. For instance, some cancers are known to preferentially express the more oncogenic and constitutively active RON⌬ (where RON is recepteur d'origine nantais receptor tyrosine kinase) splice variant of RON receptor tyrosine kinase (1). The study of genes that undergo alternative splicing is therefore likely to unravel novel therapeutic targets against cancer (2-4). The IG20 (insulinoma-glucagonoma) is one such gene previously identified in our laboratory (4) that is implicated in cancer cell survival, proliferation, apoptosis, and other regulated functions through alternative splicing (5-20). The IG20 gene encodes at least six different splice variants (SVs) 3 of which the expression of KIAA0358 and IG20-SV4 isoforms is restricted to certain neuronal tissues (17), with KIAA acting as a Rab3a-GEP (20 -22). The other four, namely IG20pa, MADD, IG20-SV2, and DENN-SV, are expressed more ubiquitously (4). Of these, MADD and DENN-SV are constitutively expressed, whereas the IG20pa and IG20-SV2 may or may not be expressed.Among the IG20 isoforms, ...