Serine 25 phosphorylation inhibits RIPK1 kinase-dependent cell death in models of infection and inflammation

Abstract

RIPK1 regulates cell death and inflammation through kinase-dependent and -independent mechanisms. As a scaffold, RIPK1 inhibits caspase-8-dependent apoptosis and RIPK3/MLKL-dependent necroptosis. As a kinase, RIPK1 paradoxically induces these cell death modalities. The molecular switch between RIPK1 pro-survival and pro-death functions remains poorly understood. We identify phosphorylation of RIPK1 on Ser25 by IKKs as a key mechanism directly inhibiting RIPK1 kinase activity and preventing TNF-mediated RIPK1-dependent cell death. Mimicking Ser25 phosphorylation (S > D mutation) protects cells and mice from the cytotoxic effect of TNF in conditions of IKK inhibition. In line with their roles in IKK activation, TNF-induced Ser25 phosphorylation of RIPK1 is defective in TAK1- or SHARPIN-deficient cells and restoring phosphorylation protects these cells from TNF-induced death. Importantly, mimicking Ser25 phosphorylation compromises the in vivo cell death-dependent immune control of Yersinia infection, a physiological model of TAK1/IKK inhibition, and rescues the cell death-induced multi-organ inflammatory phenotype of the SHARPIN-deficient mice.

OTULIN prevents liver inflammation and hepatocellular carcinoma by inhibiting FADD- and RIPK1 kinase-mediated hepatocyte apoptosis

“…On the one hand, it promotes the transcriptional upregulation of anti-apoptotic proteins by NF-κB, which protect cells from RIPK1 kinase-independent apoptosis (Wang et al, 2008). On the other hand, it allows the phosphorylation of RIPK1 by IKKα/β- and TBK1/IKKε, which was shown to protect cells from RIPK1 kinase-dependent apoptosis (Dondelinger et al, 2015, 2019; Lafont et al, 2018; Xu et al, 2018; Ting and Bertrand, 2016). While several studies have reported the importance of proper TNF signaling for the maintenance of liver homeostasis and to prevent liver inflammation and inflammation-induced HCC (Luedde et al, 2014), our study did not identify TNF as a driving cytokine in the liver pathology of OTULIN LPC-KO mice.…”

“…Indeed, apart from pro-inflammatory molecules, activation of NF-κB also leads to the transcriptional upregulation of anti-apoptotic proteins, which protect cells from RIPK1 kinase-independent apoptosis (Wang et al, 2008). In addition, the linear ubiquitin-dependent phosphorylation of RIPK1 by IKKα/β- and TBK1/IKKε was shown to protect cells from RIPK1 kinase-dependent apoptosis (Dondelinger et al, 2015, 2019; Lafont et al, 2018; Xu et al, 2018; Ting and Bertrand, 2016). Consequently, when these protective brakes are compromised, such as following LUBAC deficiency (Peltzer et al, 2014, 2018) (Priem et al CDDis 2019), activated TRADD and RIPK1 dissociate from complex I, and respectively induce RIPK1 kinase-independent and -dependent apoptotic cascades through association with Fas-associated death domain (FADD) protein and procaspase-8 to form the cytosolic death inducing complex II.…”

3‐O‐acetylrubianol C (3AR‐C) induces RIPK1‐dependent programmed cell death by selective inhibition of IKKβ

“…Consistent with this possibility, genetic deletion or pharmacological inhibition of TAK1 and IKKα/β resulted in further sensitization of cell death by activating RIPK1, independently of NF‐κB activation . Subsequent biochemical analyses revealed that RIPK1 phosphorylation on Ser 321 or Ser 25 by TAK1 or IKKα/β in complex‐I prevented the RIPK1‐mediated assembly of the complex‐II/necrosome and then inhibited apoptosis/necroptosis in an NF‐κB‐independent manner . Furthermore, it has been proposed that MAPK‐activated protein kinase 2 (MK2) also induces RIPK1 phosphorylation in complex‐I and protects against complex‐II‐mediated cell death .…”

“…18,19 Subsequent biochemical analyses revealed that RIPK1 phosphorylation on Ser 321 or Ser 25 by TAK1 or IKKα/β in complex-I prevented the RIPK1-mediated assembly of the complex-II/necrosome and then inhibited apoptosis/necroptosis in an NF-κBindependent manner. 20,21 Furthermore, it has been proposed that MAPK-activated protein kinase 2 (MK2) also induces RIPK1 phosphorylation in complex-I and protects against complex-II-mediated cell death. [22][23][24] It is now believed that RIPK1 phosphorylation by upstream kinases acts as an early cell death checkpoint to control the cytotoxic potential of TNF.…”

The involvement of regulated cell death forms in modulating the bacterial and viral pathogenesis

“…However, as a deubiquitinated protein, RIPK1 can promote the stimulation of both cell death signaling pathways (Varfolomeev and Vucic, 2018). Apart from the decisive role of ubiquitination, phosphorylation at Ser89 by protein kinase A, C, and phosphorylation at several sites by IKKs have been found to be important to attenuate the cytotoxic potential of this signaling protein (Dondelinger et al, 2019;McQuade et al, 2013).…”