Molecular Mechanisms Of RIP3-induced Apoptosis In Tumor Cells

Cell apoptosis is an active cell death process controlled by gene regulation and is vital for maintaining cell homeostasis. RIP3(receptor-interacting protein 3), a member of the RIP Ser/Thr kinase family, is involved in autophosphorylation, apoptosis induction and NF-κB activation. Moreover, RIP3 is recruited to the TNFR1 signaling complex through interaction with RIP via its C-terminal segment. However, its subcellular localization and the molecular mechanisms underlying RIP3-induced apoptosis remain unclear.The previous study has demonstrated that the kinase domain in the N-terminus of RIP3 is required for its kinase activity and autophosphorylation, while the unique domain in the C-terminus is very critical for apoptosis induction and NF-κB activation. In order to understand the detail mechanisms of RIP3's pro-apoptotic activity, we focus on the C-terminus of RIP3. We found that a truncated form of RIP3 (tRIP3) containing only the unique C-terminal region (aa 224-518) induced significant apoptosis in human hepatocellular carcinoma cells QGY-7703. FADD is a crucial component in death receptor pathway. A FADD dominant negative mutant FADD-DN can block TNF-a, FasL, TRAIL-induced apoptosis in many cell lines. To explore the correlation between RIP3 and FADD in TNFR-1 signaling pathway, our study showed that a FADD-dominant negative (FADD-DN) was able to significantly block apoptosis induced by tRIP3. In contrast, the RIP3 dominant negative (RIP3-DN) was found unable to block FADD-induced apoptosis. Thus, we conclude that tRIP3 may function upstream of FADD in the apoptosis pathway mediated by TNFR1. Unexpectedly, FADD-DN was shown unable to inhibit RIP3-induced apoptosis. This discrepancy suggests that full-length RIP3 and tRIP3 may induce apoptosis through different pathways.Considering the previous reports that two of RIP family members, RIP and RIP4 are cleaved by caspase during death receptor-mediated apoptosis, we supposed RIP3 might undergo similar process. As expected, we show that RIP3 is cleaved at Asp328 by Caspase-8 under apoptotic stimuli, which is blocked by general caspase inhibitor Z-VAD-FMK but not Caspase-8 specific inhibitor Z-IETD-FMK, implying other possible executors for RIP3 cleavage. In addition, full-length RIP3 induces both caspase-dependent and-independent apoptosis, as well as activates NF-κB. After cleavage, the N-terminal fragment contains intact kinase domain(RIP3-N, aa 1-328) and the C-terminal fragment contains main of unique domain(RIP3-C, aa 329-518). Full-length RIP3 locates mostly in the cytoplasm in a diffused pattern; RIP3-N localizes evenly throughout the cell; RIP3-C keeps RIP3's localization pattern under low expression level and localized evenly in the cytoplasm with punctuate or filament-like structures under high expression level. RIP3-N loses some physiological activities of RIP3, such as apoptosis and NF-κB activation, whereas RIP3-C keeps both of these two activities with some alteration. RIP3-C induces only caspase-dependent apoptosis and exhibits a markedly higher NF-κB-activating activity than full-length RIP3. Notably, ectopically expressed RIP3 is a short-lived protein, after cleavage, both N-terminal and C-terminal fragments of RIP3 display the increasing stability compared to wild-type RIP3, suggesting that both kinase domain and unique domain might contribute to the instability of RIP3.We also observed the effect of RIP3 point-mutations on its functions. The cleavage-resistant mutant of RIP3, RIP3(D328A) showed similar localization pattern and pro-apoptotic activity to RIP3. The K50 of RIP3 is critical for enzymatic activity and ATP binding, and is quite conserved in RIP family. RIP3(KSOA), a kinase-dead RIP3 mutant, also induces only caspase-dependent apoptosis as RIP3-C lack of the kinase domain does. These data suggest that the kinase activity is essential for RIP3-induced caspase-independent apoptosis. Our previous work has demonstrated that RIP3 acts a nucleo-cytoplasmic shuttling protein, and our research reveals that RIP3(D328A) is also able to shuttling between nucleus and cytoplasm. Interestingly, RIP3(K50A) and RIP3-N both lose their nucleo-cytoplasmic shuttling activities. RIP3-C keeps this activity when it located evenly throughout the cytoplasm, but it will lose this activity when RIP3-C forms punctuate or filament-like structures. These results indicate that the nucleo-cytoplasmic shuttling activity of RIP3 is closely related to caspase-independent apoptosis induced by RIP3. In addition, RIP3 and RIP3(D328A) were shown to translocate from cytosol to nucleus during apoptosis, suggesting that RIP3 might induce caspase-independent apoptosis in the nucleus.