MADD, a splice variant of IG20, is indispensable for MAPK activation and protection against apoptosis upon tumor necrosis factor-alpha treatment.
We investigated the physiological role of endogenous MAPK-activating death domain-containing protein (MADD), a splice variant of the IG20 gene, that can interact with TNFR1 in tumor necrosis factor-alpha (TNFalpha)-induced activation of NF-kappaB, 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 TNFalpha-induced apoptosis in the absence of cycloheximide. It also resulted in a dramatic loss in TNFalpha-induced activation of MAPK without any apparent effect on NF-kappaB activation. This observation was substantiated by an accompanying loss in the activation of p90RSK, a key downstream target of MAPK, whereas the NF-kappaB-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 TNFalpha-induced apoptosis. The requirement for MADD was highly specific for TNFalpha-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 TNFalpha-induced apoptosis by specifically activating MAPKs through Grb2 and Sos1/2 recruitment, and its potential as a novel cancer therapeutic target.
Kurada
Bapi Raju V V S N
BRVVSN
Department of Microbiology and Immunology, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612.
Li
Liang Cheng
LC
Department of Microbiology and Immunology, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612.
Mulherkar
Nirupama
N
Department of Microbiology and Immunology, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612.
Subramanian
Mahesh
M
Department of Microbiology and Immunology, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612.
Prasad
Kanteti V
KV
Department of Microbiology and Immunology, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612.
Prabhakar
Bellur S
BS
Department of Microbiology and Immunology, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612. Electronic address: bprabhak@uic.edu.
D017398:Alternative Splicing / Q000235:genetics; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D003513:Cycloheximide / Q000494:pharmacology; D053418:Death Domain Receptor Signaling Adaptor Proteins / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000235:genetics / Q000378:metabolism*; D051380:GRB2 Adaptor Protein / Q000235:genetics / Q000378:metabolism; D020868:Gene Silencing; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D015850:Interleukin-6 / Q000494:pharmacology; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D009369:Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000628:therapy; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000235:genetics / Q000378:metabolism; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000235:genetics / Q000378:metabolism; D020837:SOS1 Protein / Q000235:genetics / Q000378:metabolism; D020664:Son of Sevenless Proteins / Q000235:genetics / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D053418:Death Domain Receptor Signaling Adaptor Proteins; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D020662:Guanine Nucleotide Exchange Factors; C508600:IL6 protein, human; D015850:Interleukin-6; C105852:MADD protein, human; D016328:NF-kappa B; D009363:Neoplasm Proteins; D011500:Protein Synthesis Inhibitors; D047888:Receptors, Tumor Necrosis Factor, Type I; D020837:SOS1 Protein; C485509:SOS2 protein, human; D020664:Son of Sevenless Proteins; D014409:Tumor Necrosis Factor-alpha; D003513:Cycloheximide; D038744:Ribosomal Protein S6 Kinases, 90-kDa; D048049:Extracellular Signal-Regulated MAP Kinases