Akt-phosphorylated Mitogen-activated Kinase-activating Death Domain Protein (MADD) Inhibits TRAIL-induced Apoptosis by Blocking Fas-associated Death Domain (FADD) Association with Death Receptor 4

Abstract

MADD plays an essential role in cancer cell survival. Abrogation of endogenous MADD expression results in significant spontaneous apoptosis and enhanced susceptibility to tumor necrosis factor ␣-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. However, the regulation of MADD function is largely unknown. Here, we demonstrate that endogenous MADD is phosphorylated at three highly conserved sites by Akt, and only the phosphorylated MADD can directly interact with the TRAIL receptor DR4 thereby preventing Fas-associated death domain recruitment. However, in cells susceptible to TRAIL treatment, TRAIL induces a reduction in MADD phosphorylation levels resulting in MADD dissociation from, and Fas-associated death domain association with DR4, which allows death-inducing signaling complex (DISC) formation leading to apoptosis. Thus, the pro-survival function of MADD is dependent upon its phosphorylation by Akt. Because Akt is active in most cancer cells and phosphorylated MADD confers resistance to TRAIL-induced apoptosis, co-targeting Akt-MADD axis is likely to increase efficacy of TRAIL-based therapies.A fine balance between opposing signaling events that regulate survival and death appears to determine the outcome of cell fate. A complex array of genes regulates the above process, and one such gene we previously identified is IG20 (insulinomaglucagonoma 20) (1). The IG20 can profoundly affect cancer cell survival and death through alternative splicing (2, 3). The IG20 gene encodes six different splice variants (SVs).3 The KIAA and IG20-SV4 isoforms are selectively expressed only in neuronal tissues (4). The KIAA is analogous to rat Rab3a GEP (also referred to as MADD/DENN) and plays an important role in neuronal vesicular trafficking and is required for animal survival (5-7). The IG20pa, MADD, IG20-SV2, and DENN-SV are more ubiquitously expressed. Of these, MADD is physiologically the most important isoform. MADD is expressed at very low levels in a variety of healthy tissues; however, its expression levels are much higher in many types of human tumors and tumor cell lines (1, 8). Knockdown of endogenous MADD or all IG20 SVs results in enhanced spontaneous as well as tumor necrosis factor ␣-related apoptosis-inducing ligand (TRAIL)-induced apoptosis (9 -11). Interestingly, expression of exogenous MADD, and not other SVs, in the absence of endogenous IG20 SVs can rescue the cells from undergoing apoptosis and indicates that only the MADD isoform is required and sufficient to promote cancer cell survival (10, 11).The extrinsic apoptotic pathway is initiated by death ligands such as Fas ligand, TRAIL, or tumor necrosis factor ␣ (12-15). Unlike Fas ligand, TRAIL can induce cancer cell death with little or no effect on most normal cells (16). However, a number of different factors can confer resistance to TRAIL-induced apoptosis in different cells (17). TRAIL binding to death receptors 4 and 5 (DR4/DR5) induces receptor trimerization and recruitment of FADD (16, 18 -20). This facilitates recruitment of procasp...

Knockdown of MADD and c-FLIP overcomes resistance to TRAIL-induced apoptosis in ovarian cancer cells

“…The preparation of anti-MADD exon 13L (anti-13L) specific antibodies has been reported earlier 24 . The goat anti-mouse IgG1 peroxidase-conjugated secondary antibody was obtained from Caltag Laboratories (Burlingame, CA) and the anti-rabbit peroxidase-conjugated polyclonal secondary antibody was purchased from GE Healthcare (Piscataway, NJ).…”

Control of mitochondrial activity by miRNAs

“…The extrinsic pathway is initiated by the binding of death ligands such as Fas ligand (FasL) or tumor necrosis factor α (TNF-α) to their corresponding death receptors, Fas or TNF receptor-1 (TNFR-1), respectively. Upon ligand binding, the death receptors undergo trimerization and recruit Fas-associated death domain protein (FADD) that then associates with procaspase-8 to form death-inducing signaling complex (DISC) [Li et al, 2010b]. However, the intrinsic pathway is initiated through mitochondria.…”

A monoclonal antibody (Mc178-Ab) targeted to the ecto-ATP synthase β-subunit-induced cell apoptosis via a mechanism involving the MAKase and Akt pathways

“…Akt inhibits the activation of pro-apoptotic proteins and induces nuclear translocation of the survival protein, NF-jB. Akt also affects cell cycle machinery and is involved in cell migration and invasion [8,9,19].…”