RIPK3 is phosphorylated

Necroptosis is a form of regulated necrotic cell death mediated by interaction of receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3 via a RIP homotypic interaction motif (RHIM) domain. RIPK1:RIPK3 complex formation further potentiates kinase activation through autophosphorylation and/or transphosphorylation, propagating the pronecrotic signal. RIPK1, RIPK3 and their kinase activities were shown to be essential for necroptosis (Degterev A et al. 2008; Cho YS et al. 2009). A RIPK3 kinase-dead mutant (K50A) was found to function as a dominant negative mutant, which blocked tumor necrosis factor alpha (TNFα)-induced necrotic pathway in human colorectal adenocarcinoma HT-29 cells (He S et al. 2009). Studies in mice expressing catalytically inactive RIPK3 showed that RIPK3 D161N stimulated RIPK1-dependent apoptosis and embryonic lethality in RIPK3 D161N homozygous mice, while K51A knock in mice developed into fertile and immunocompetent adults, suggesting that the kinase activity of RIPK3 determines whether cells die by necroptosis or caspase-8-dependent apoptosis (Mandal P et al. 2014; Newton K et al. 2014; Raju S et al. 2018). Further, differentially tagged constructs of RIPK3 kinase domain (KD) were found to form dimers after their co-expression in human embryonic kidney (HEK) 293T cells, and mutation of residues at the dimer interface impaired dimerization (Raju S et al. 2018). Phosphorylation on the serine residue 227 (S227) of human RIPK3 (S231 and S232 on mouse RIPK3) is thought to mediate recruitment and activation of mixed-lineage kinase domain-like (MLKL), a crucial downstream substrate of RIPK3 in the necrosis pathway (Sun et al. 2012; Chen et al. 2013). The phosphorylation occurs in the αG helix in the C-lobe of the RIPK3 kinase, not the activation loop (Petrie EJ et al. 2019;. Consequently it remains unclear why this would be an activating event and how this would lead to MLKL interaction Although RIPK1 activation is associated with phosphorylation of the RIPK3 activation loop, most studies, however, suggest that RIPK1 does not phosphorylate RIPK3 (Cho YS et al. 2009). Rather, it is thought that active RIPK1 serves as a scaffold to enable RIPK3 to assemble into homooligomers. The precise mechanism of MLKL activation by RIPK3 is incompletely understood and may vary across species (Davies KA et al. 2020). The underlying mechanism is still debated, but the point is that RIPK3 transphosphorylation is crucial for MLKL activation (Cook WD et al. 2014; Orozco S et al. 2014; Mompean M et al. 2018).<p>FDA-approved anticancer drugs, including sorafenib and ponatinib, showed anti-necroptotic activity (Fauster A et al. 2015; Martens S et al. 2017; Fulda S 2018). These compounds are tyrosine kinase inhibitors (TKI) that directly targeted RIPK3 and RIPK1 and blocked their kinase activity (Fauster A et al. 2015; Martens S et al. 2017; Fulda S 2018). Pazopanib, another multi-targeting TKI, was shown to suppress necroptosis preferentially by targeting RIPK1 (Fauster A et al. 2015).

细胞凋亡性坏死是一种由受体相互作用丝氨酸/苏氨酸蛋白激酶1（RIPK1）与RIPK3通过RIP同源相互作用基序（RHIM）域相互作用介导的调控性坏死细胞死亡形式。RIPK1:RIPK3复合物的形成进一步通过自磷酸化和/或转磷酸化增强激酶的激活，从而传播倾向坏死信号。研究表明，RIPK1、RIPK3及其激酶活性对于细胞凋亡性坏死至关重要（Degterev A 等，2008；Cho YS 等，2009）。研究发现，RIPK3激酶失活突变体（K50A）作为一种显性负突变体，能够阻断人结直肠腺癌HT-29细胞中肿瘤坏死因子α（TNFα）诱导的坏死途径（He S 等，2009）。在表达催化性非活性RIPK3的小鼠研究中，发现RIPK3 D161N可刺激RIPK1依赖性细胞凋亡和胚胎致死性，而K51A敲入小鼠则发育为可育且免疫竞争的成年个体，这表明RIPK3的激酶活性决定了细胞是通过细胞凋亡性坏死还是通过caspase-8依赖性细胞凋亡死亡（Mandal P 等，2014；Newton K 等，2014；Raju S 等，2018）。此外，在共表达于人类胚胎肾（HEK）293T细胞后的RIPK3激酶结构域（KD）的不同标记构建体被发现形成二聚体，而二聚界面残基的突变则损害了二聚化（Raju S 等，2018）。认为人类RIPK3的丝氨酸残基227（S227）（小鼠RIPK3上的S231和S232）的磷酸化介导了混合谱系激酶结构域样（MLKL）的募集和激活，MLKL是RIPK3在坏死途径中的关键下游底物（Sun 等，2012；Chen 等，2013）。磷酸化发生在RIPK3激酶C-lobe中的αG螺旋上，而不是激活环（Petrie EJ 等，2019）。因此，尚不清楚这为何是一种激活事件以及它如何导致MLKL相互作用。尽管RIPK1的激活与RIPK3激活环的磷酸化相关，但大多数研究表明RIPK1并不磷酸化RIPK3（Cho YS 等，2009）。相反，认为活化的RIPK1作为支架，使RIPK3能够组装成同源寡聚体。MLKL由RIPK3激活的确切机制尚不完全清楚，并且可能在不同物种间存在差异（Davies KA 等，2020）。尽管其潜在机制仍存在争议，但RIPK3的跨磷酸化对于MLKL的激活至关重要（Cook WD 等，2014；Orozco S 等，2014；Mompean M 等，2018）。美国食品药品监督管理局（FDA）批准的抗肿瘤药物，包括索拉非尼和帕唑帕尼，表现出抗细胞凋亡性坏死活性（Fauster A 等，2015；Martens S 等，2017；Fulda S，2018）。这些化合物是酪氨酸激酶抑制剂（TKI），直接靶向RIPK3和RIPK1，并阻断其激酶活性（Fauster A 等，2015；Martens S 等，2017；Fulda S，2018）。帕佐帕尼作为一种多靶点TKI，已被证明通过靶向RIPK1优先抑制细胞凋亡性坏死（Fauster A 等，2015）。