EGFR was immunoprecipitated from EGF treated or untreated HeLa cell lysates, and proteins were separated and immunoblotted (MADD can constitutively bind to TNFR1, and upon TNF binding to the TNFR, MADD facilitates quick recruitment of Grb2 to TNFR1 that leads to the activation of Ras and other downstream MAPK signaling molecules. impact epidermal growth factor-induced MAPK activation thereby demonstrating the specific requirement of MADD for TNF receptor-mediated Imidaprilate MAPK activation. Re-expression of short hairpin RNA-resistant MADD in the absence of endogenous 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 malignancy cells from TNF-induced apoptosis by specifically activating MAPKs through Grb2 Imidaprilate and Sos1/2 Imidaprilate recruitment, and its potential as a novel cancer therapeutic target. Genes in higher organisms generate alternate transcripts that are translated into closely Imidaprilate related proteins with different functions. Perturbations in the tightly regulated alternate splicing of important 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 usually recepteur d’origine nantais receptor tyrosine Imidaprilate kinase) splice variant of RON receptor tyrosine kinase (1). The study of genes that undergo alternative splicing is usually therefore likely to unravel novel therapeutic targets against malignancy (2C4). The (insulinoma-glucagonoma) is usually one such gene previously recognized in our laboratory (4) that is implicated in malignancy cell survival, proliferation, apoptosis, and other regulated functions through alternate splicing (5C20). The gene encodes at least six different splice variants (SVs)3 of which the expression of KIAA0358 and isoforms is restricted to certain neuronal tissues (17), with KIAA acting as a Rab3a-GEP (20C22). The other four, namely are constitutively expressed, whereas the may or may not be expressed. Among the isoforms, MADD is usually overexpressed in malignancy cells and tissues, and by acting as a negative regulator of caspase-8 activation, it contributes to cancer cell survival (18). Abrogation of MADD, but not the other (is the insulinoma-glucagonoma clone 20 splice variant), renders cancer cells more susceptible to spontaneous as well as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis (18, 19). Moreover, expression of an shRNA-resistant MADD, and not the other isoforms of the gene, can rescue cells from Rabbit Polyclonal to ALK (phospho-Tyr1096) undergoing apoptosis upon shRNA-mediated abrogation of expression of all isoforms of the gene (19). Endogenous MADD can prevent caspase-8 activation without directly interacting with caspase-8. The intriguing finding that MADD plays a predominant role in malignancy cell survival and confers resistance to TRAIL-induced apoptosis led us to further examine the role of endogenous MADD in TNF-induced apoptosis and the underlying signaling pathways such as NF-B and MAPKs (ERK, JNK, and p38) in this study. The MAPKs are serine/threonine-specific protein kinases that respond to a variety of extracellular stimuli and regulate several important and crucial cellular functions such as cell cycle progression, expression of cytokines, motility, and adherence. Hence MAPKs influence cell survival, proliferation, differentiation, development, and apoptosis (23C24). The three main members of MAPK family are ERK1/2 or more commonly referred to as MAPK, JNK, and p38. Relatively high levels of MAPK activity are noted in approximately one-third of all human cancers, thereby making MAPK an attractive target in the development of novel cancer therapies (23C25). Activated MAPKs phosphorylate several nuclear and cytoplasmic substrates involved in diverse cellular processes, including regulation of transcription and activation of kinases and phosphatases. One of the key substrates of MAPK that plays an important role in cell growth and proliferation is p90RSK (26C27). In addition, MAPK also activates the transcription factor ELK1 that triggers the transcription of c-Fos (28C29). It is important to note that p90RSK.