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