RIPK1 is phosphorylated

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R-HSA-5213464
Type
Reaction [transition]
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Homo sapiens
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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).

Literature References
PubMed ID Title Journal Year
32269263 Autophosphorylation at serine 166 regulates RIP kinase 1-mediated cell death and inflammation

Laurien, L, Nagata, M, Schünke, H, Delanghe, T, Wiederstein, JL, Kumari, S, Schwarzer, R, Corona, T, Krüger, M, Bertrand, MJM, Kondylis, V, Pasparakis, M

Nat Commun 2020
24059293 Positive and negative phosphorylation regulates RIP1- and RIP3-induced programmed necrosis

McQuade, T, Cho, Y, Chan, FK

Biochem. J. 2013
19524513 Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation

Cho, YS, Challa, S, Moquin, D, Genga, R, Ray, TD, Guildford, M, Chan, FK

Cell 2009
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Title
protein serine/threonine kinase activity of RIPK1:RIPK3 [cytosol]
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