## 6 Phosphorylation of RIPK1
### Inhibitory Phosphorylation  
The kinase activity of RIPK1 is tightly controlled at multiple levels to prevent spontaneous activation. Various post-translational modifications on RIPK1, such as ubiquitination and inhibitory phosphorylation, are intricately connected to keep RIPK1 kinase activity in check. For instance, in complex I, RIPK1 undergoes K63 ubiquitination by cIAP1/2 and M1 linear ubiquitination by LUBAC. These modifications stabilize complex I, inhibiting its dissociation and formation of cell-death-promoting complex II. In addition to activating the MAPK pathway and the NF-kB pathway to activate gene expression that promotes cell survival and inflammation, TAK1 and IKK kinases further suppress cell death by performing inhibitory phosphorylation on RIPK1 to block its kinase activity (Figure 3). For instance, TAK1 activates MK2, which directly phosphorylates S320 and S335 of human RIPK1, or S321 and S336 of mouse RIPK1, to inhibit RIPK1 kinase activity and subsequent apoptosis or necroptosis [85; 86; 87]. Interestingly, TAK1 is also reported to directly phosphorylate mouse RIPK1 at S321 [29]. In addition, TAK1 activates IKK\(\alpha/\beta\), which in turn phosphorylates S25 in the kinase domain of RIPK1. Phosphorylation of S25 prevents ATP binding and inhibits RIPK1 kinase activation [88]. Furthermore, TBK1/IKK\(\epsilon\) phosphorylates T189 in the kinase domain to inhibit RIPK1 kinase activity [31; 32]. It is important to note that MK2 phosphorylates cytosolic RIPK1, while IKK\(\alpha/\beta\), TBK1/IKK\(\epsilon\), and TAK1 phosphorylate ubiquitinated RIPK1 in complex I. Recently, kinases outside of the TNF pathway have also been found to directly phosphorylate RIPK1 to inhibit its kinase activity and cell death. For example, glucose starvation activates AMPK (adenosine monophosphate-activated protein kinase), which phosphorylates S416 of human RIPK1 (or S415 of mouse RIPK1) to inhibit RIPK1 kinase activity and cell death [89]. In addition to serine/threonine phosphorylation, tyrosine phosphorylation has also been found to inhibit RIPK1 activity. Studies have shown that JAK1 (Janus Kinase 1) and Src kinases phosphorylate Y384 of human RIPK1 (or Y383 of mouse RIPK1) to inhibit RIPK1 kinase activity and subsequent cell death [90]. Inhibitory phosphorylation of RIPK1 plays a pivotal role in host defense against pathogens and modulates inflammatory responses. For example, the Gram-negative bacterial pathogen _Yersinia_ counters the host defense by inhibiting NF-kB- and MAPK-mediated pro-inflammatory cytokines' expression, while promoting RIPK1 activation-dependent cell death [91]. Its effector protein, acetyltransferase YopJ, elicits multiple functions in the process. First, it inactivates IKK\(\alpha/\beta\) and TAK1 to block NF-kB and MAPK activation [92]. Second, it blocks the inhibitory phosphorylation of S25 of RIPK1 by IKK\(\alpha/\beta\) to promote  
Figure 3: Phosphorylation events impacting RIPK1 kinase activity. Schematic representation of RIPK1, showing key phosphorylation sites. Green characters denote auto-activating phosphorylation events, while red characters denote inhibitory phosphorylation events. Multiple kinases catalyze inhibitory phosphorylation events, which serve as critical checkpoints for cell death activation. Notably, the kinase responsible for S89 phosphorylation has not yet been reported. Abbreviations: TBK1, TANK-binding kinase 1; IKK\(\epsilon\), IxB kinase \(\epsilon\); MK2, MAPK-activated protein kinase 2; JAK1, Janus kinase 1; AMPK, AMP-activated protein kinase. RIPK1-dependent macrophage cell death [88]. Lastly, it inactivates MK2, preventing inhibitory phosphorylation of S321 and S335 on RIPK1, thereby activating RIPK1-dependent cell death [85; 86; 87]. As a consequence, mice expressing the S25D-RIPK1 mutant fail to activate RIPK1-dependent cell death and are defective in defending against _Yersinia_ infection, similar to the mice expressing the RIPK1 kinase-dead mutant K45A [88].