The Role Of Protein Phosphatase 6 In Regulating Necroptosis Signaling Pathway

Necroptosis is a caspase-independent and precisely regulated programmed cell death,which could be induced by tumor necrosis factor receptor(TNFR),toll-like receptor(TLR),or the Z-DNA binding protein-1(ZBP-1).Necroptosis pathway is modulated by the receptor interacting protein kinase 1(RIPK1),receptor interacting protein kinase 3(RIPK3)and executed by mixed lineage kinase domain-like protein(MLKL)through oligomerization,translocation to,and perturbation of,the plasma membrane,ultimately resulting in cell death with necrotic morphological changes such as organelle swelling,plasma membrane rupture,and release of intracellular components.In recent years,an increasing number of researches have shown that necroptosis,acting as an essential alternative pathway of apoptosis,has a wide range of biological functions and is closely associated with a variety of diseases,especially inflammation-related diseases or organ damage such as acute pancreatitis,acute myocardial infarction,terminal ileitis and ischemia-reperfusion injury.Thus,further studies on necroptosis signaling,including identification of new regulators and revelation of its molecular mechanism will hopefully provide a new perspective and theoretical base for the clinical treatment and drug development of necroptosis-related diseases.The reversible phosphorylation modification of proteins is an important approach to regualte their functions,affecting all aspects of life process such as cell proliferation,cell differentiation and cell death.Protein kinases and protein phosphatases are two crucial enzymes that act as switches for reversible phosphorylation modification.They regulate various intracellular signal transductions and decide the cell fate by precisely controlling the phosphorylation or dephosphorylation of their substrate proteins.Previous studies more focused on the protein phosphorylation modification by kinases.However,in recent years,accumulating evidence showed that corresponding to kinases,phosphatases also play an important role in the modulation of various physiological activities through regulating related signaling pathway.This study is devoted to further exploring novel regulators of necroptosis in order to get an enhanced understanding of the necroptosis signaling pathway.We performed a positive-selection screen by using CRISPR/Cas9 gene knockout library against20,611 genes of the mice cells and found a subset of sg RNAs targeting 79 genes significantly enriched,within which we identified PPP6 C,the catalytic subunit of protein phosphatase 6,as a new positive regulator of necroptosis.Our results demonstrated that PPP6 C regulates TNF-induced necroptosis relies on its phosphatase activity and loss of Ppp6 c protects cells from necroptosis.Meanwhile,consistent with previous research findings,we found Ppp6 c deficiency increases TNF-induced NFκB activation but the resitance to TNF-induced necroptosis in Ppp6 c deficient cells is independent of NFκB signaling.Mechanistically,PPP6 C acts as a TGF-β activated kinase 1(TAK1)phosphatase to inactivate its kinase activity.Deletion of Ppp6 c leads to hyperactivation of TAK1 and promotes the activation of TAK1 downstream kinase IKKα/β,which increases IKKα/β-mediated RIPK1 phosphorylation at S25.S25 phosphorylation directly inhibiting RIPK1 kinase activity and preventing TNFmediated necroptosis.Additionally,our results indicated that the regulation of PPP6 C on necroptosis is dependent on TAB2 because depletion of Tab2 restored the the sensitivity of Ppp6 c deficient cells to TNF-induced necroptotic cell death,and further research suggests that Tab2 may regualte RIPK1-dependent cell death in a TAK1-independent manner.Finally,we demonstrated in disease models that partial deletion of Ppp6 c alleviates DSS or TNF induced enteritis in mice,implying that inhibition of PPP6 C may be a potential mean to alleviate necroptosis-associated inflammatory diseases.Taken together,we identified Ppp6 c as a new positive regulator of necroptosis and revealed the regulation mechanism,which not only enhances our further understanding of necroptotic signaling,but also provides a new idea and theoretical support for the treatment of necroptosis-related inflammatory diseases.