Abstract
Receptor-interacting serine/threonine-protein kinase 3 (RIPK3) normally signals to necroptosis by phosphorylating MLKL. We report here that when the cellular RIPK3 chaperone Hsp90/CDC37 level is low, RIPK3 also signals to apoptosis. The apoptotic function of RIPK3 requires phosphorylation of the serine 165/threonine 166 sites on its kinase activation loop, resulting in inactivation of RIPK3 kinase activity while gaining the ability to recruit RIPK1, FADD, and caspase-8 to form a cytosolic caspase-activating complex, thereby triggering apoptosis. We found that PGF2α induces RIPK3 expression in luteal granulosa cells in the ovary to cause luteal regression through this RIPK3-mediated apoptosis pathway. Mice carrying homozygous phosphorylation-resistant RIPK3 S165A/T166A knockin mutations failed to respond to PGF2α but retained pro-necroptotic function, whereas mice with phospho-mimicking S165D/T166E homozygous knock-in mutation underwent spontaneous apoptosis in multiple RIPK3-expressing tissues and died shortly after birth. Thus, RIPK3 signals to either necroptosis or apoptosis depending on its serine 165/threonine 166 phosphorylation status.
Highlights
Regulated cell death with distinctive associated morphological changes happens either in the form of apoptosis or necrosis (Elmore, 2007; Fink and Cookson, 2005; Kroemer et al, 2009; Wallach et al, 2016)
This form of apoptosis did not signal through the classic intrinsic mitochondrial pathway as the loss of cell viability through this pathway could not be blocked by caspase inhibitors nor was the apoptosis triggered by the TNF receptor family, which would be RIPK1 kinase-dependent
dinoprost tromethamine (DT) treatment induced the elevation of phosphorylation-resistant RIPK3(S165A/T166A) protein in the ovaries of Ripk3S165A-T166A/S165A-T166A knock-in mice to the same level as wild-type RIPK3 protein (Figure 7—figure supplement 1D), significantly less active caspase-3 signal was observed in these ovaries, similar to the ovaries of Ripk3-/, Fadd-/-/Mlkl-/, or Ptgfr-/- mice, confirming that the observed DT-induced apoptosis in luteal cells was through the RIPK3/FADD-mediated apoptosis pathway triggered by the phosphorylation of serine 165/threonine 166 sites (Figure 7F, G)
Summary
Regulated cell death with distinctive associated morphological changes happens either in the form of apoptosis or necrosis (Elmore, 2007; Fink and Cookson, 2005; Kroemer et al, 2009; Wallach et al, 2016). Cells that harbor this mutation are sensitive to apoptosis induction when treated with several small-molecule RIPK3 inhibitors that bind to its ATPbinding pocket (Mandal et al, 2014) Whether this apoptosis-inducing activity of RIPK3 is an anomaly of the mutant protein, the small-molecule inhibitor bound form, or a true physiological function of RIPK3 remains to be resolved. Such a process stops the production of progesterone and triggers another cycle of ovulation When mammals age, their follicles and corpora lutea are gradually lost, and the ovary eventually becomes a corpus albicans-filled organ that ceases to produce eggs and hormones (Perheentupa and Huhtaniemi, 2009; Tilly, 2001). We found that the serine 165/threonine 166 phospho-RIPK3 signal appeared in corpora lutea/albicans when mice reached more than 4 months of age and PGF2a can trigger this apoptotic pathway during the process of corpus luteum involution in a mouse hyper-ovulation model
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