RIPK1 interaction with RIPK3 further potentiates their kinase activation through autophosphorylation and/or cross-phosphorylation (Cho YS et al. 2009). The kinase function of RIPK1 and RIPK3 is thought to stabilize RIPK1:RIPK3 association within the pronecrotic complex.

Reconstitution of RIPK1-deficient human Jurkat cells with mutated kinase-inactive RIPK1 or RIPK1 lacking the N-terminal serine/threonine kinase domain did not trigger FASL-induced necrotic cell death (Holler N et al. 2000). Similarly, mutations in the kinase domain and RIP homotypic interaction motif (RHIM) of RIPK1 also abolished the RIPK1-mediated rescue of tumor necrosis factor (TNF)/zVAD-fmk-induced regulated necrosis in RIPK1-deficient Jurkat cells (Cho YS et al. 2009). Furthermore, the results of structural and mutagenesis studies using necrostatins, which inhibit RIPK1 kinase activity by targeting the kinase domain, revealed that the N-terminal kinase domain of RIPK1 is required for propagating the pronecrotic signal (Degterev A et al. 2008; Cho YS et al. 2009; Xie T et al. 2013). Mass spectroscopy showed that human RIPK1 is phosphorylated within the kinase domain at multiple serine residues, such as Ser14/15, Ser20, Ser161 and Ser166, suggesting that the phosphorylation might regulate RIPK1 kinase activity (Degterev A et al. 2008). Using in vitro cellular systems, two independent studies reported that alanine substitution at Ser161 (S161A) leads to a reduction in RIPK1 kinase activity (Degterev A et al. 2008; McQuade T et al. 2013). RIPK1 autophosphorylation at Ser166 was found to modulate RIPK1 kinase activation (Laurien L et al. 2020). Studies with Ripk1 S166A/S166A knock-in mice revealed that abolishing phosphorylation at S166 prevented the development of RIPK1-mediated inflammatory conditions in vivo in four relevant mouse models of inflammation. Further, abolishing phosphorylation at S166 considerably inhibited RIPK1 kinase activity-dependent cell death downstream of tumor necrosis factor receptor 1 (TNFR1), toll-like receptor 3 (TLR3) and TLR4 in mouse cells isolated from Ripk1 S166A/S166A mice (Laurien L et al. 2020). Phosphorylation of S166 RIPK1 has been established as a biomarker of RIPK1 target engagement (Degterev A et al. 2008; Ofengeim D et al. 2015). The biological role of phosphorylation of individual serine residues in the kinase domain of RIPK1 remains to be further characterized (McQuade T et al. 2013).

RIPK1 is subjected to complex phosphorylation including several events possibly mediated by other kinases such as MAPK-activated protein kinase 2 (MK2) (Dondelinger Y et al. 2016; Jaco I et al. 2017; Delanghe T et al. 2020). S320 and S335 on human RIPK1 (S321 and S336 in mouse RIPK1) were identified as MK2 phosphorylation sites (Jaco I et al. 2017; Menon NB et al. 2017; Dondelinger Y et al. 2017). Transforming growth factor β-activated kinase 1 (TAK1) was also shown to phosphorylate RIPK1 along with TANK binding kinase 1 (TBK1) and I-kappa-B kinase epsilon (IKKε) to prevent TNF-induced necroptosis or to dictate the multiple cell death pathways in mammalian cells (Lafont E et al. 2018; Xu D et al. 2018). In addition, IKKα/IKKβ is also able to phosphorylate RIPK1 in order to block RIPK1-dependent cell death in mouse models of infection and inflammation (Dondelinger Y et al. 2015, 2019). RIPK3 might also regulate RIPK1 phosphorylation in mammalian cells. For instance, RIPK3 was shown to directly phosphorylate RIPK1 when kinase-dead RIPK1 and RIPK3 were co-expressed in human embryonic kidney HEK293 cells, immunoprecipitated, and subjected to an in vitro kinase assay (Sun X et al. 2002; Cho et al. 2009). Importantly, mutation within RHIM motif of RIPK3 abrogated RIPK1 phosphorylation by RIPK3, suggesting that RIPK1 phosphorylation by RIPK3 is dependent on the formation of the RIPK1:RIPK3 complex (Sun X et al. 2002).

Several FDA-approved anticancer drugs, including sorafenib, pazopanib and ponatinib showed anti-necroptotic activity (Fauster A et al. 2015; Martens S et al. 2017; Fulda S 2018). RIPK1 has been identified as the main functional target of pazopanib, while sorafenib and ponatinib directly targeted both RIPK1 and RIPK3 (Fauster A et al. 2015; Najjar M et al. 2015; Martens S et al. 2017).

TNF-alpha signaling leads to the assembly of a large receptor-bound signaling complex, which includes multiple adaptors (TRADD, TRAF2, and RIPK1), and E3 ubiquitin ligases (cIAP1/2, LUBAC complex). RIPK1 thus plays a critical role in mediating both normal and pathological responses downstream of TNFR1 and RIPK1 inhibitors, including ponatrinib, have progressed successfully passed human phase I clinical studies (Degterev et al. 2020, Najjar et al. 2015).

The binding of TNF-α to TNF receptor 1 (TNFR1) results in the sequential formation of several signaling complexes (Walczak H 2011). The rapidly forming complex-I (TNFR1 signaling complex) is assembled at the receptor’s cytoplasmic tail and consists of TNFR1, TRADD (TNFR1-associated death domain), TRAF2 (TNF receptor associated factor-2), RIPK1 (receptor-interacting serine/threonine protein kinase 1), and E3 ubiquitin (Ub) ligases BIRC2, BIRC3 (cIAP1/2, cellular inhibitor of apoptosis) and LUBAC (linear ubiquitin chain assembly complex) (Micheau O and Tschopp J 2003; Yuan J et al. 2019). Within this complex, RIPK1 and other proteins are rapidly conjugated with Ub chains by various E3 ligases (Micheau O and Tschopp J 2003; Yuan J et al. 2019). The ubiquitination status of RIPK1 determines cell fate downstream of the TNFR1 signaling complex (Yuan J et al. 2019). The conjugation of K63-linked Ub chains by BIRC2/3 or Met1-linked Ub chains by LUBAC, have been shown to promote RIPK1-dependent pro-survival NF-kappa-B signaling while inhibiting RIPK1 kinase-mediated apoptosis and necroptosis. In addition, RIPK1 also interacts with FADD/Caspase-8 or RIPK3/MLKL to form complex IIa or IIb, which activate apoptosis or necroptosis respectively (Yuan J et al. 2019). In these cell death-inducing complexes, RIPK1 function is also regulated by ubiquitination (Amin P et al. 2018; de Almagro MC et al. 2015). The protein stability of RIPK1 is negatively regulated by the carboxyl-terminus of HSC70-interacting protein (CHIP, also known as STIP1 homology and U-Box containing protein 1, STUB1) (Seo J et al. 2016). STUB1 (CHIP), as an E3 ligase, mediates K48-linked ubiquitination of RIPK1 at K571, K604, and K627 and targets it to lysosomal degradation (Seo J et al. 2016). Co-immunoprecipitation analysis using overexpressed proteins revealed interactions between STUB1 (CHIP) and RIPK1 in human embryonic kidney 293T (HEK293T) cells (Seo J et al. 2016). Interaction of endogenous STUB1 and RIPK1 proteins was detected in mouse fibroblast L929 cells. Direct interaction between recombinant proteins was confirmed by GST-pull-down assay (Seo J et al. 2016). Domain mapping revealed that the RHIM domain of RIPK1 interacts with the U-Box domain of STUB1 (Seo J et al. 2016). Similarly, STUB1 interacts with and ubiquitinates RIPK3 inducing lysosome-dependent destabilization of RIPK3 (Seo J et al. 2016). These data suggest that the E3 ligase activity of STUB1 negatively regulates TNF-α–induced cell death by conjugating K48-linked ubiquitin chains to RIPK1 and RIPK3, thereby promoting their degradation.

This Reactome event describes STUB1 (CHIP) binding to RIPK1.

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) can be a part of cell death and survival signaling complexes. Whether RIPK1 functions in apoptosis, necroptosis or NFκB signaling is dependent on autocrine/paracrine signals, on the cellular context and tightly regulated by posttranslational modifications of RIP1 itself. The pro-survival function of RIPK1 is achieved by polyubiquitination which is required for recruitment of signaling molecules/complexes such as the IKK complex and the TAB2:TAK1 complex to mediate activation of NFκB signaling (Ea CK et al. 2006). CYLD-mediated deubiquitination of RIPK1 switches its pro-survival function to caspase-mediated pro-apoptotic signaling (Fujikura D et al. 2012; Moquin DM et al. 2013). Caspase-8 (CASP8) in human and rodent cells facilitates the cleavage of kinases RIPK1 and RIPK3 and prevents RIPK1/RIPK3-dependent necroptosis (Lin Y et al. 1999; Hopkins-Donaldson S et al. 2000; Newton K et al. 2019; Zhang X et al. 2019; Lalaoui N et al. 2020). CASP8-mediated cleavage of human RIPK1 after D324 (D325 in mice) separates the amino-terminal kinase domain from the carboxy-terminal part of the molecule preventing RIPK1 kinase activation through dimerization via the carboxy-terminal death domain and leads to the dissociation of the complex TRADD:TRAF2:RIP1:FADD:CASP8 (Lin Y et al. 1999; Meng H et al. 2018). The lack of CASP8 proteolytic activity in the presence of viral (e.g. CrmA and vICA) or pharmacological caspase inhibitors results in necroptosis induction via RIPK1 and RIPK3 (Tewari M & Dixit VM 1995; Fliss PM & Brune W 2012; Hopkins-Donaldson S et al. 2000). Cellular FLICE-like inhibitory protein (cFLIP), which is an NF-κB target gene, form heterodimer with procaspase-8 and inhibits activation of CASP8 within the the TRADD:TRAF2:RIP1:FADD:CASP8:FLIP complex (Yu JW et al. 2009; Pop C et al. 2011). The presence of cFLIP (long form) limits CASP8 to cleave CASP3/7 but allow cleavage of RIPK1 to cause the dissociation of the TRADD:TRAF2:RIP1:FADD:CASP8, thereby inhibiting both apoptosis and necroptosis (Boatright KM et al. 2004; Yu JW et al. 2009; Pop C et al. 2011; Feoktistova M et al. 2011). Mice that lack CASP8 or knock-in mice that express catalytically inactive CASP8 (C362A) die in a RIPK3- and MLKL-dependent manner during embryogenesis (Kaiser WJ et al. 2011; Newton K et al. 2019). Studies using mice that express RIPK1(D325A), in which the CASP8 cleavage site Asp325 had been mutated, further confirmed that cleavage of RIPK1 by CASP8 is a mechanism for dismantling death-inducing complexes for limiting aberrant cell death in response to stimuli (Newton K et al. 2019; Lalaoui N et al. 2020). Disrupted cleavage of RIPK1 variants with mutations at D324 by CASP8 in humans leads to an autoinflammatory response by promoting the activation of RIPK1 (Tao P et al. 2020; Lalaoui N et al. 2020).

Receptor-interacting serine/threonine-kinase protein 1 (RIPK1) and RIPK3-dependent necrosis is called necroptosis or programmed necrosis. The kinase activities of RIPK1 and RIPK3 are essential for the necroptotic cell death in human, mouse cell lines and genetic mice models (Cho YS et al. 2009; He S et al. 2009, 2011; Zhang DW et al. 2009; McQuade T et al. 2013; Newton et al. 2014). The initiation of necroptosis can be stimulated by the same death ligands that activate extrinsic apoptotic signaling pathway, such as tumor necrosis factor (TNF) alpha, Fas ligand (FasL), and TRAIL (TNF-related apoptosis-inducing ligand) or toll like receptors 3 and 4 ligands (Holler N et al. 2000; He S et al. 2009; Feoktistova M et al. 2011; Voigt S et al. 2014). In contrast to apoptosis, necroptosis represents a form of cell death that is optimally induced when caspases are inhibited (Holler N et al. 2000; Hopkins-Donaldson S et al. 2000; Sawai H 2014). Specific inhibitors of caspase-independent necrosis, necrostatins, have recently been identified (Degterev A et al. 2005, 2008). Necrostatins have been shown to inhibit the kinase activity of RIPK1 (Degterev A et al. 2008). Importantly, cell death of apoptotic morphology can be shifted to a necrotic phenotype when caspase 8 activity is compromised, otherwise active caspase 8 blocks necroptosis by the proteolytic cleavage of RIPK1 and RIPK3 (Kalai M et al. 2002; Degterev A et al. 2008; Lin Y et al. 1999; Feng S et al. 2007). When caspase activity is inhibited under certain pathophysiological conditions or by pharmacological agents, deubiquitinated RIPK1 is engaged in physical and functional interactions with the cognate kinase RIPK3 leading to formation of necrosome, a necroptosis-inducing complex consisting of RIPK1 and RIPK3 (Sawai H 2013; Moquin DM et al. 2013; Kalai M et al. 2002; Cho YS et al. 2009, He S et al. 2009, Zhang DW et al. 2009). Within the necrosome RIPK1 and RIPK3 bind to each other through their RIP homotypic interaction motif (RHIM) domains. The RHIMs can facilitate RIPK1:RIPK3 oligomerization, allowing them to form amyloid-like fibrillar structures (Li J et al. 2012; Mompean M et al. 2018). RIPK3 in turn interacts with mixed lineage kinase domain-like protein (MLKL) (Sun L et al. 2012; Zhao J et al. 2012; Murphy JM et al. 2013; Chen W et al. 2013). The precise mechanism of MLKL activation by RIPK3 is incompletely understood and may vary across species (Davies KA et al. 2020). Mouse MLKL activation relies on transient engagement of RIPK3 to facilitate phosphorylation of the pseudokinase domain (Murphy JM et al. 2013; Petrie EJ et al. 2019a), while it appears that stable recruitment of human MLKL by necrosomal RIPK3 is an additional crucial step in human MLKL activation (Davies KA et al. 2018; Petrie EJ et al. 2018, 2019b). RIPK3-mediated phosphorylation is thought to initiate MLKL oligomerization, membrane translocation and membrane disruption (Sun L et al. 2012; Wang H et al. 2014; Petrie EJ et al. 2020; Samson AL et al. 2020). Studies in human cell lines suggest that upon induction of necroptosis MLKL shifts to the plasma membrane and membranous organelles such as mitochondria, lysosome, endosome and ER (Wang H et al. 2014), but it is trafficking via a Golgi-microtubule-actin-dependent mechanism that facilitates plasma membrane translocation, where membrane disruption causes death (Samson AL et al. 2020). The mechanisms of necroptosis regulation and execution downstream of MLKL remain elusive. The precise oligomeric form of MLKL that mediates plasma membrane disruption has been highly debated (Cai Z et al. 2014; Chen X et al. 2014; Dondelinger Y et al. 2014; Wang H et al. 2014; Petrie EJ et al. 2017, 2018; Samson AL et al. 2020 ). However, microscopy data revealed that MLKL assembles into higher molecular weight species upon cytoplasmic necrosomes within human cells, and upon phosphorylation by RIPK3, MLKL is trafficked to the plasma membrane (Samson AL et al. 2020). At the plasma membrane, phospho-MLKL forms heterogeneous higher order assemblies, which are thought to permeabilize cells, leading to release of DAMPs to invoke inflammatory responses. MLKL also exerts non-necroptotic functions such as regulation of endosomal trafficking or MLKL-induced activation of the NLRP3 inflammasome (Yoon S et al. 2017; Shlomovitz I et al. 2020; Yoon S et al. 2022). While RIPK1, RIPK3 and MLKL are the core signaling components in the necroptosis pathway, many additional molecules have been proposed to positively and negatively tune the signaling pathway. Currently, this picture is evolving rapidly as new modulators continue to be discovered.

The Reactome module describes MLKL-mediated necroptotic events on the plasma membrane.

Receptor Interacting Serine/Threonine Kinase 1 (RIPK1)-mediated regulated necrosis also called necroptosis is an important type of programmed cell death in addition to apoptosis. Necroptosis eventually leads to cell lysis and release of cytoplasmic content into the extracellular region. Necroptosis must be tightly controlled. Disregulated or defective necroptotic cell death is often associated with a tissue damage resulting in an intense inflammatory response. Defects of necroptosis may contribute to various pathological processes, including autoimmune disease, neurodegeneration, multiple cancers, and kidney injury.

Activation of receptor-interacting serine/threonine protein (RIP) kinases RIPK1 and RIPK3 coordinate an immunogenic form of programmed cell death known as regulated necrosis or necroptosis (Upton JW et al. 2017). This form of necrosis leads to anti-viral inflammation in host through cell death-associated release of damage-associated molecular patterns (DAMPs) (Nailwal H & Ka-Ming Chan F 2019; Upton JW et al. 2017). Microbial pathogens are able to modulate host regulated necrosis through different triggers and pathways. The promotion and inhibition of host cell death vary and depend on the microbe types, virulence, and phenotypes (Upton JW et al. 2010, 2012, 2017; Jaclyn S Pearson JS et al. 2017; Petrie EJ et al. 2019; Fletcher-Etherington A et al. 2020; Nailwal H & Ka-Ming Chan F 2019; ).

The inflammatory cytokine tumor necrosis factor alpha (TNF-α) is expressed in immune and nonimmune cell types including macrophages, T cells, mast cells, granulocytes, natural killer (NK) cells, fibroblasts, neurons, keratinocytes and smooth muscle cells as a response to tissue injury or upon immune responses to pathogenic stimuli (Köck A. et al. 1990; Dubravec DB et al. 1990; Walsh LJ et al. 1991; te Velde AA et al. 1990; Imaizumi T et al. 2000). TNF-α interacts with two receptors, namely TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2). Activation of TNFR1 can trigger multiple signal transduction pathways inducing inflammation, proliferation, survival or cell death (Ward C et al. 1999; Micheau O and Tschopp J 2003; Widera D et al. 2006). Whether a TNF-α-stimulated cell will survive or die is dependent on autocrine/paracrine signals, and on the cellular context.

TNF binding to TNFR1 results initially in the formation of complex I that consists of TNFR1, TRADD (TNFR1-associated death domain), TRAF2 (TNF receptor associated factor-2), RIPK1 (receptor-interacting serin/threonine protein kinase 1), and E3 ubiquitin ligases BIRC2,BIRC3 (cIAP1/2,cellular inhibitor of apoptosis) and LUBAC (Micheau O and Tschopp J 2003). The conjugation of ubiquitin chains by BIRC2/3 and LUBAC (composed of HOIP, HOIL-1 and SHARPIN ) to RIPK1 allows further recruitment and activation of the TAK1 (also known as mitogen-activated protein kinase kinase kinase 7 (MAP3K7)) complex and IκB kinase (IKK) complex. TAK1 and IKK phosphorylate RIPK1 to limit its cytotoxic activity and activate both nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NFkappaB) and mitogen‐activated protein (MAP) kinase signaling pathways promoting cell survival by induction of anti-apoptotic proteins such as BIRC, cellular FLICE (FADD-like IL-1β-converting enzyme)-like inhibitory protein (cFLIP) and secretion of pro-inflammatory cytokines (TNF and IL-6). When the survival pathway is inhibited, the TRADD:TRAF2:RIPK1 detaches from the membrane-bound TNFR1 signaling complex and recruits Fas-associated death domain-containing protein (FADD) and procaspase-8 (also known as complex II). Once recruited to FADD, multiple procaspase-8 molecules interact via their tandem death-effector domains (DED), thereby facilitating both proximity-induced dimerization and proteolytic cleavage of procaspase-8, which are required for initiation of apoptotic cell death (Hughes MA et al. 2009; Oberst A et al. 2010). When caspase activity is inhibited under certain pathophysiological conditions (e.g., expression of caspase-8 inhibitory proteins such as CrmA and vICA after infection with cowpox virus or CMV) or by pharmacological agents, deubiquitinated RIPK1 is physically and functionally engaged by its homolog RIPK3 leading to formation of the necrosome, a necroptosis-inducing complex consisting of RIPK1 and RIPK3 (Tewari M & Dixit VM 1995; Fliss PM & Brune W 2012; Sawai H 2013; Moquin DM et al. 2013; Kalai M et al. 2002; Cho YS et al. 2009, He S et al. 2009, Zhang DW et al., 2009). Within the complex II procaspase-8 can also form heterodimers with cFLIP isoforms, FLIP long (L) and FLIP short (S), which are encoded by the NFkappaB target gene CFLAR (Irmler M et al. 1997; Boatright KM et al. 2004; Yu JW et al. 2009; Pop C et al. 2011). FLIP(S) appears to act purely as an antagonist of caspase-8 activity blocking apoptotic but promoting necroptotic cell death (Feoktistova et al. 2011). The regulatory function of FLIP(L) has been found to differ depending on its expression levels. FLIP(L) was shown to inhibit death receptor (DR)-mediated apoptosis only when expressed at high levels, while low cell levels of FLIP(L) enhanced DR signaling to apoptosis (Boatright KM et al. 2004; Okano H et al. 2003; Yerbes R et al. 2011; Yu JW et al. 2009; Hughes MA et al. 2016). In addition, caspase-8:FLIP(L) heterodimer activity within the TRADD:TRAF2:RIPK1:FADD:CASP8:FLIP(L) complex allowed cleavage of RIPK1 to cause the dissociation of the TRADD:TRAF2:RIP1:FADD:CASP8, thereby inhibiting RIPK1-mediated necroptosis (Feoktistova et al. 2011, 2012). TNF-α can also activate sphingomyelinase (SMASE, such as SMPD2,3) proteins to catalyze hydrolysis of sphingomyeline into ceramide (Adam D et al.1996; Adam-Klages S et al. 1998; Ségui B et al. 2001). Activation of neutral SMPD2,3 leads to an accumulation of ceramide at the cell surface and has proinflammatory effects. However, TNF can also activate the pro-apoptotic acidic SMASE via caspase-8 mediated activation of caspase-7 which in turn proteolytically cleaves and activates the 72kDa pro-A-SMase form (Edelmann B et al. 2011). Ceramide induces anti-proliferative and pro-apoptotic responses. Further, ceramide can be converted by ceramidase into sphingosine, which in turn is phosphorylated by sphingosine kinase into sphingosine-1-phosphate (S1P). S1P exerts the opposite biological effects to ceramide by activating cytoprotective signaling to promote cell growth counteracting the apoptotic stimuli (Cuvillier O et al. 1996). Thus, TNF-α-induced TNFR1 activation leads to divergent intracellular signaling networks with extensive cross-talk between the pro-apoptotic/necroptotic pathway, and the other NFkappaB, and MAPK pathways providing highly specific cell responses initiated by various types of stimuli.

Receptor-interacting serine/threonine-protein kinase 1