## 7 Dephosphorylation of RIPK1  
Protein phosphatases play a complementary role in regulating phosphorylation homeostasis. These enzymes are classified into three main families based on the sequence similarity of the catalytic domain and substrate specificity: PTPs (protein tyrosine phosphatases), PPPs (phosphoprotein phosphatases), and PPMs (protein phosphatase metal-dependent). PTPs specifically dephosphorylate phospho-tyrosine residues, while PPPs and PPMs dephosphorylate phospho-serine and phospho-threonine residues. In addition, a subfamily of PTPs, called the dual-specificity phosphatases, dephosphorylate all three phospho-amino acids. PPPs and PPMs differ in that PPMs require metal ions, such as magnesium or manganese, for their activity and function as single-subunit enzymes, while PPPs require regulatory subunits [93]. PP1 (protein phosphatase 1) is an important subfamily of PPPs. Its catalytic subunits (PPIc), including PP1\(\alpha\), PP1\(\beta\), and PP1\(\gamma\), are responsible for dephosphorylation of the majority phospho-serine and phospho-threonine sites in mammalian cells, regulating a broad range of cellular processes. Each PP1 catalytic subunit is obligatorily complexed with one or two regulatory subunits to form distinct PP1 holoenzymes. The regulatory subunits, also known as PP1-interacting proteins (PIPs) or regulatory interactors of protein phosphatase one (RIPPOs), determine substrate specificity by directing PP1c to the subcellular locations of its substrates and modulating its activity toward different substrates. There are approximately 200 validated PIPs, which assemble into more than 650 different PP1 holoenzymes in mammalian cells, enabling the dephosphorylation of diverse substrates [94]. While numerous kinases have been identified to phosphorylate RIPK1, only a limited number of phosphatases are found to dephosphorylate RIPK1 or RIPK3. For example, PPm1b, a metal-ion-dependent phosphatase, dephosphorylates and inactivates RIPK3 to prevent the recruitment of MLKL into the necrosome, thus inhibiting subsequent necroptotiss. Moreover, _PPm1b-/-_ mice exhibited heightened sensitivity to TNF-induced SIRS compared to WT mice, confirming its role in inhibiting necroptosis in vivo [95]. A sensitized CRISPR whole-genome knockout screen revealed that PPP1R3G (protein phosphatase 1 regulator subunit 3G) is essential for necroptosis [96]. Specifically, PPP1R3G forms a holoenzyme with PP1\(\gamma\) to directly dephosphorylate the inhibitory phosphorylation sites of human RIPK1, including S25, S320, and S335, thereby activating RIPK1-dependent apoptosis and necroptosis (Figure 4). An interesting note is that the holoenzyme does not remove the activating phosphorylation of S166 in vitro. In this context, upon treatment with TNF/Smac-mimetic/Z-VAD-FMK (T/S/Z), TRAF interacts with PPP1R3G to recruit the PPP1R3G/PP1\(\gamma\) holoenzyme to complex I, where PP1\(\gamma\) dephosphorylates the inhibitory phosphorylation sites of RIPK1, activating RIPK1 kinase. Loss of PPP1R3G leads to loss of RIPK1 autophosphorylation at S166 and subsequent failure to form complex IIb to induce cell death. Like many other PP1 regulatory subunits, PPP1R3G interacts with PP1\(\gamma\) through an RVXF motif (X stands for any amino acids) [97]. Mutation of RVQF in PPP1R3G to RAQA disrupts the interaction with PP1\(\gamma\). Importantly, the RAQA mutant fails to rescue RIPK1 activation and cell death in PPP1R3G knockout cells. Furthermore, prevention of RIPK1 inhibitory phosphorylation with p38 or IKK inhibitors or mutation of serine 25 of RIPK1 to alanine largely restores cell death in PPP1R3G-knockout cells. Finally, _PPP1r3g\({}^{-/-}\)_ mice are protected from TNF-induced SIRS, confirming the important role of PPP1R3G in regulating apoptosis and necroptosis in vivo. Due to experimental sensitivity limitations, the authors were unable to determine if PPP1R3G/PP1\(\gamma\) removes the inhibitory phosphorylation of T189. This warrants further analysis in the future.