New insights into the regulation of apoptosis, necroptosis, and pyroptosis by receptor interacting protein kinase 1 and caspase-8

Necroptosis and pyroptosis are inflammatory forms of regulated necrotic cell death as opposed to apoptosis that is generally considered immunologically silent. Recent studies revealed unexpected links in the pathways regulating and executing cell death in response to activation of signaling cascades inducing apoptosis, necroptosis, and pyroptosis. Emerging evidence suggests that receptor interacting protein kinase 1 and caspase-8 control the cross-talk between apoptosis, necroptosis, and pyroptosis and determine the type of cell death induced in response to activation of cell death signaling

Autophosphorylation at serine 166 regulates RIP kinase 1-mediated cell death and inflammation

Receptor interacting protein kinase 1 (RIPK1) regulates cell death and inflammatory responses downstream of TNFR1 and other receptors, and has been implicated in the pathogenesis of inflammatory and degenerative diseases. RIPK1 kinase activity induces apoptosis and necroptosis, however the mechanisms and phosphorylation events regulating RIPK1-dependent cell death signaling remain poorly understood. Here we show that RIPK1 autophosphorylation at serine 166 plays a critical role for the activation of RIPK1 kinase-dependent apoptosis and necroptosis. Moreover, we show that S166 phosphorylation is required for RIPK1 kinase-dependent pathogenesis of inflammatory pathologies in vivo in four relevant mouse models. Mechanistically, we provide evidence that trans autophosphorylation at S166 modulates RIPK1 kinase activation but is not by itself sufficient to induce cell death. These results show that S166 autophosphorylation licenses RIPK1 kinase activity to induce downstream cell death signaling and inflammation, suggesting that S166 phosphorylation can serve as a reliable biomarker for RIPK1 kinase-dependent pathologies

The role of RIPK1 auto-phosphorylation at S166 in cell death and inflammatory signaling

Receptor-Interacting Protein Kinase 1 (RIPK1) functions as a key player downstream of innate immune receptors such as Tumor necrosis factor receptor 1 (TNFR1) and Toll-like receptors(TLRs). In these signaling pathways, RIPK1 promotes cell survival and tissue homeostasis in a manner independent of its catalytic function, whereas its kinase activity mediates cell death. Importantly, RIPK1 kinase activity has emerged as a driver of cell death and inflammation in mouse models and has been implicated in the pathogenesis of human inflammatory and degenerative diseases. RIPK1 auto-phosphorylates at multiple sites, including Serine (S) 14/15, S161, S166 and Threonine (T)169 and to date, auto-phosphorylation is believed to be the main function of its kinase activity. Whereas auto-phosphorylation at S166 is routinely used as a biomarker for RIPK1 activation, its functional importance and physiological relevance have not been investigated. In this work, we aimed to shed light on the role of RIPK1 autophosphorylation at S166 for RIPK1-mediated signaling. To this aim, we generated mice expressing RIPK1 with non-phosphorylatable serine to alanine mutation at position 166 from the endogenous Ripk1 locus (Ripk1S166A/S166A mice). We show that RIPK1S166A mutation does not impair the kinase activity-independent function of RIPK1 to induce pro-inflammatory and pro-survival signaling downstream of TNFR1, TLR 3 and 4. On the other hand, we observed a partial protection from TNFR1-induced, RIPK1 kinase activity-dependent apoptosis and necroptosis as well as from TLR 3- and 4-induced necroptosis in Ripk1S166A/S166A cells, showing that RIPK1 auto-phosphorylation at S166 drives its catalytic activity-dependent function to induce cell death. Strikingly, RIPK1S166A mutation completely protected mice from RIPK1 kinase activity-dependent inflammatory pathologies in the colon, liver and skin and decreased lethality in a model of systemic inflammation, showing that in vivo, RIPK1 auto-phosphorylation at S166 is essential to drive cell death and inflammation. Mechanistically, RIPK1S166A mutation inhibited the formation of cell death-inducing complexes downstream of TNFR1 as well as TLR 3 and 4, demonstrating that auto-phosphorylation at S166 enables engagement of RIPK1 in cytotoxic complexes. Furthermore, our data suggest that RIPK1 autophosphorylation at S166 acts as a driver of kinase activity and facilitates auto-phosphorylation at other sites. Taken together, the data presented in this thesis show that auto-phosphorylation at S166 licenses RIPK1 kinase activity to induce downstream signaling and promote cell death. Therefore, our results warrant the use of S166 phosphorylation as a marker for RIPK1 activation, which can aid in the stratification of human patients that may benefit from treatment with RIPK1 kinase inhibitors

MK2 Phosphorylates RIPK1 to Prevent TNF-Induced Cell Death

TNF is an inflammatory cytokine that upon binding to its receptor, TNFR1, can drive cytokine production, cell survival, or cell death. TNFR1 stimulation causes activation of NF-kappa B, p38 alpha, and its downstream effector kinase MK2, thereby promoting transcription, mRNA stabilization, and translation of target genes. Here we show that TNF-induced activation of MK2 results in global RIPK1 phosphorylation. MK2 directly phosphorylates RIPK1 at residue S321, which inhibits its ability to bind FADD/caspase-8 and induce RIPK1-kinase-dependent apoptosis and necroptosis. Consistently, a phospho-mimetic S321D RIPK1 mutation limits TNF-induced death. Mechanistically, we find that phosphorylation of S321 inhibits RIPK1 kinase activation. We further show that cytosolic RIPK1 contributes to complex-II-mediated cell death, independent of its recruitment to complex-I, suggesting that complex-II originates from both RIPK1 in complex-I and cytosolic RIPK1. Thus, MK2-mediated phosphorylation of RIPK1 serves as a checkpoint within the TNF signaling pathway that integrates cell survival and cytokine production

The Cdkn1a(SuPER) Mouse as a Tool to Study p53-Mediated Tumor Suppression

Cdkn1a, which encodes p21, functions as a major route for p53-mediated cell-cycle arrest. However, the consequence of Cdkn1a gene dosage on tumor suppression has not been systematically investigated. Here, we employed BAC transgenesis to generate a Cdkn1a(SUPER) mouse, which harbors an additional Cdkn1a allele within its natural genomic context. We show that these mice display enhanced cell-cycle arrest and reduced apoptosis in response to genotoxic stress. Furthermore, using a chemically induced skin cancer model and an autochthonous Krasdriven lung adenocarcinoma model, we show that Cdkn1a(SUPER) mice display a cancer protection phenotype that is indistinguishable from that observed in Tp53 suPER animals. Moreover, we demonstrate that Tp53 and Cdkn1a cooperate in mediating cancer resistance, using a chemically induced fibrosarcoma model. Overall, our Cdkn1a(SUPER )allele enabled us to assess the contribution of Cdkn1a to Tp53-mediated tumor suppression