A phosphorylation of RIPK3 kinase initiates an intracellular apoptotic pathway that promotes prostaglandin2α-induced corpus luteum regression
- National Institute of Biological Sciences, China
- Tsinghua Institute of Multidisciplinary Biomedical Research, Tsinghua University, China
- Institute of Neuroscience, State Key Laboratory of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Research Center for Brain Science and Brain-Inspired Intelligence, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, China
Figures
Figure 1 with 1 supplement
RIPK3-induced apoptosis in MCF7 and KGN cells.
(A) Cultured MCF7/TO-RIPK3 and HeLa/TO-RIPK3 cells were treated with DMSO or Dox (1 μg/ml) induction for 36 hr. Cell viability was determined by measuring cellular ATP levels (upper panel). The data …
Figure 1—figure supplement 1
RIPK3-induced apoptosis in human granulosa lutein cells (KGN).
Cultured KGN/TO-RIPK3 cells were treated with DMSO or Dox (1 μg/ml) induction for 36 hr. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the …
Figure 2
RIPK3-induced apoptosis was dependent on RIPK1, FADD, and caspase-8.
(A) Cultured MCF7/TO-RIPK3 cells were treated with DMSO or Dox plus the indicated agent for 24 hr. The cells were then harvested, and RIPK3 was immunoprecipitated from the cell lysates using …
Figure 3 with 2 supplements
RIPK3-mediated apoptosis is dependent on the auto-phosphorylation of the S164/T165 sites.
(A) Cultured MCF7/TO-RIPK3 and HeLa/TO-RIPK3 cells were treated with Dox plus z-VAD for 24 hr. RIPK3 was immunoprecipitated from the cell lysates using anti-Flag resin. The RIPK3 bands were excised …
Figure 3—figure supplement 1
Characterization of RIPK3 auto-phosphorylation sites.
(A) Cultured MCF7/TO-RIPK3 and MCF7/TO-RIPK3(S164A/T165A) cells were treated with DMSO(-) or Dox plus z-VAD for 24 hr. The lysates were analyzed by western blotting using antibodies against the …
Figure 3—figure supplement 2
The phosphorylation site of RIPK3 is conserved among different mammalian species.
(A) Alignment of amino acid sequences of RIPK3 orthologs in five mammalian species. Amino acid residues conserved in 80% or more of the sequences are shaded in black. The putative phosphorylation …
Figure 4 with 2 supplements
Hsp90/CDC37 chaperone determined the apoptotic and necroptotic function of RIPK3 kinase.
(A) The cell lysates from cultured HT29, HeLa, MCF7, and KGN cells were analyzed by western blotting using antibodies as indicated. (B, C) Cultured HeLa-RIPK3, MCF7/TO-RIPK3, and KGN/TO-RIPK3 cells …
Figure 4—figure supplement 1
Hsp90/CDC37 chaperone determines the necroptotic or apoptotic function of RIPK3 kinase.
(A) HeLa/TO-RIPK3 and HeLa/TO-RIPK3-shRNA-HSP90 cells were treated with the indicated stimuli for 36 hr. Cell viability was determined by measuring cellular ATP levels. The data are represented as …
Figure 4—figure supplement 2
RIPK3 form amyloid-like structure in MCF7 and KGN cells.
(A) Cultured HeLa/TO-RIPK3 and MCF7/TO-RIPK3 cells were treated with DMSO or Dox plus TSZ for 24 hr. Immunofluorescence of the cells with Flag-RIPK3 (green) antibody. Counterstaining with DAPI …
Figure 5 with 2 supplements
Ripk3S165D-T166D/S165D-T166D (Ripk3ST-DE/ST-DE) mice die within 1 month after birth.
(A, B) Macroscopic features (A) and body weights (B) of Ripk3+/+ and Ripk3ST-DE/ST-DE littermate mice at 14 days of age (n ≥ 9). The result from each individual animal is presented as an indicated …
Figure 5—figure supplement 1
Generation of Ripk3ST-DE/ST-DE and Ripk3ST-AA/ST-AA mice.
(A) Two guide RNA and donate oligo sequences of Ripk3(S165D/T166E) and Ripk3(S165A/T166A) knock-in mice. (B) Schematic of CRISPER-Cas9 strategy for the generation for Ripk3(S165D/T166E) and Ripk3(S16…
Figure 5—figure supplement 2
Hsp90/CDC37 chaperone protein levels were low in corpus luteum and corpus albicans.
(A, B) Immunofluorescence of ovary from wild-type mice (8 months; n = 3) with RIPK3 (red) and HSP90 (green) antibody in (A). Higher-power views of selected areas were acquired in right panel. The …
Figure 6 with 1 supplement
Phospho-serine 164/threonine 165-RIPK3 signals were present in aged ovary corpus albican.
(A) Western blot analysis of RIPK1, RIPK3, and MLKL levels in perfused mouse ovary extracts of different ages. Each group is representative of at least three mice. (B) H&E and immunofluorescence …
Figure 6—figure supplement 1
Generation of RIPK3 C-terminus HA-3xFlag knock-in and Ptgfr-/- mice.
(A) Schematic of CRISPER-Cas9 strategy for RIPK3 C-terminus HA-3xFlag knock-in mice. The gene structure of RIPK3 and guide RNA sequences targeting the Ripk3 is shown with the PAM sequences …
Figure 7 with 1 supplement
Prostaglandin F2alpha (PGF2α) induces ovarian RIPK3 expression for corpus luteum involution.
(A–C) Primary granulosal lutein cells (WT, Ripk3-/-) were isolated from 3-month-old mice ovaries. The cells were treated with dinoprost tromethamine (DT) at the indicated concentration for 36 hr in …
Figure 7—figure supplement 1
Prostaglandin F2alpha (PGF2α) stimulates RIPK3 expression through the MAPK pathway.
(A) Primary granulosal lutein cells were isolated from the 3-month-old mice ovary. The cells were then treated with 1 μM dinoprost tromethamine (DT) or plus MAPK inhibitors PD-98059 (5 μM) and U0126 …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Homo sapiens) | HEK293T | ATCC | CRL-11268 | Female |
| Cell line (Homo sapiens) | HeLa | ATCC | CCL-2 | Female |
| Cell line (Homo sapiens) | HT-29 | ATCC | HTB-38 | Female |
| Cell line (Homo sapiens) | MCF7 | ATCC | HTB-22 | Female |
| Cell line (Homo sapiens) | KGN | Nishi et al., 2001 | N/A | Female |
| Cell line (Homo sapiens) | L929(Ripk3-/-) | Dr. Xiaodong Wang lab at National Institute of Biological Sciences, Beijing | N/A | |
| Cell line (Homo sapiens) | KGN/TO-RIPK3 | Dr. Xiaodong Wang lab at National Institute of Biological Sciences, Beijing | N/A | |
| Cell line (Homo sapiens) | KGN/TO- RIPK3(S164A/T165A) | Dr. Xiaodong Wang lab at National Institute of Biological Sciences, Beijing | N/A | |
| Cell line (Homo sapiens) | MCF7/TO-RIPK3 | Dr. Xiaodong Wang lab at National Institute of Biological Sciences, Beijing | N/A | |
| Cell line (Homo sapiens) | MCF7/TO- RIPK3(D160N) | Dr. Xiaodong Wang lab at National Institute of Biological Sciences, Beijing | N/A | |
| Cell line (Homo sapiens) | MCF7/TO- RIPK3(K50A) | Dr. Xiaodong Wang lab at National Institute of Biological Sciences, Beijing | N/A | |
| Cell line (Homo sapiens) | MCF7/TO- RIPK3(S164A/T165A) | Dr. Xiaodong Wang lab at National Institute of Biological Sciences, Beijing | N/A | |
| Cell line (Homo sapiens) | MCF7/TO-RIPK3 (RIPK1-/-/Caspase-8-/-/FADD-/-/cFLIP-/-) | Dr. Xiaodong Wang lab at National Institute of Biological Sciences, Beijing | N/A | |
| Antibody | Anti-RIPK3 (Rabbit polyclonal) | ProSci | Cat# 2283; RRID:AB_203256 | WB (1:1000) |
| Antibody | Anti-p-S164/T165-RIPK3 (Rabbit monoclonal) | Abcam | Cat# Ab255705; | Firstly described in this paper; WB (1:1000) IF (1:100) |
| Antibody | Anti-RIPK3 (Mouse monoclonal) | LSBio | Cat# LS-C336804 | WB (1:1000) |
| Antibody | Anti-GAPDH-HRP (Mouse monoclonal) | MBL | Cat# M171-1; RRID:AB_10699462 | WB (1:20,000) |
| Antibody | Anti-β-actin-HRP (Rabbit polyclonal) | MBL | Cat# PM053-7; RRID:AB_10697035 | WB (1:20,000) |
| Antibody | Anti-Tubulin-HRP (Rabbit polyclonal) | MBL | Cat# PM054-7; RRID:AB_10695326 | WB (1:20,000) |
| Antibody | Anti-Flag-HRP (Mouse monoclonal) | Sigma-Aldrich | Cat# A8592; RRID:AB_439702 | WB (1:10,000) |
| Antibody | Anti-RIPK1 (Rabbit polyclonal) | Cell Signaling | Cat# 3493S; RRID:AB_2305314 | WB (1:1000) |
| Antibody | Anti-cleaved-caspase3 (Rabbit polyclonal) | Cell Signaling | Cat# 9661; RRID:AB_2341188 | WB (1:1000) IF(1:100) |
| Antibody | Anti-Mouse-MLKL (Rabbit polyclonal) | ABGENT | Cat# AP14272b; RRID:AB_11134649 | WB (1:1000) |
| Antibody | Anti-Human-MLKL (Rabbit monoclonal) | Abcam | Cat# ab184718; RRID:AB_2755030 | WB (1:1000) |
| Antibody | Anti-caspase-8 (Mouse monoclonal) | Cell Signaling | Cat# 9746; RRID:AB_2275120 | WB (1:1000) |
| Antibody | anti-FADD (Rabbit polyclonal) | Cell Signaling | Cat# 2782; RRID:AB_2100484 | WB (1:1000) |
| Antibody | Anti-p-S227-RIP3 (Rabbit monoclonal) | Abcam | Cat# ab209384; RRID:AB_2714035 | WB (1:1000) |
| Antibody | Anti-p-S232-RIP3 (Rabbit monoclonal) | Abcam | Cat# ab222302 | WB (1:1000) |
| Antibody | Anti-cFLIP (Rabbit polyclonal) | Abcam | Cat# ab6144; RRID:AB_305314 | WB (1:1000) |
| Antibody | Anti-cleaved-caspase-3 (Mouse monoclonal) | St John's Laboratory | Cat# STJ97448 | IHC (1:100) |
| Antibody | Anti-prostaglandin F2 alpha (PTGFR) (Rabbit polyclonal) | Abcam | Cat# ab203342 | WB (1:1000) IF (1:100) |
| Antibody | Anti-HSP90 (Rabbit polyclonal) | Proteintech | Cat# 13171-1-AP;RRID:AB_2120924 | WB (1:1000) IF (1:100) |
| Antibody | Anti-CDC37 (Rabbit monoclonal) | Abcam | Cat# ab108305; RRID:AB_10861724 | WB (1:1000) IF (1:100) |
| Antibody | anti-FLAG M2 (Mouse monoclonal) | Sigma-Aldrich | Cat# F1840 | IHC (1:100) |
| Antibody | Anti-phospho-p44/42 MAPK (Rabbit monoclonal) | Cell Signaling | Cat# 4370S; RRID:AB_2315112 | WB (1:1000) |
| Antibody | Anti-p44/42 MAPK (Rabbit monoclonal) | Cell Signaling | Cat# 4965S | WB (1:1000) |
| Antibody | Anti-phospho-MEK1/2 (Rabbit monoclonal) | Cell Signaling | Cat# 9154S; RRID:AB_2138017 | WB (1:1000) |
| Antibody | Anti-MEK1/2 (Rabbit monoclonal) | Abcam | Cat# ab178876 | WB (1:1000) |
| Antibody | Donkey anti-Mouse, Alexa Fluor 488 (Mouse polyclonal) | Thermo Fisher | Cat# A-21202; RRID:AB_141607 | IF (1:500) |
| Antibody | Donkey anti-Mouse, Alexa Fluor 555 (Mouse polyclonal) | Thermo Fisher | Cat# A-31570; RRID:AB_2536180 | IF (1:500) |
| Antibody | Donkey anti-Rabbit, Alexa Fluor 488 (Rabbit polyclonal) | Thermo Fisher | Cat# A-21206; RRID:AB_141708 | IF (1:500) |
| Antibody | Donkey anti-Rabbit, Alexa Fluor 555 (Rabbit polyclonal) | Thermo Fisher | Cat# A-31572; RRID:AB_162543 | IF (1:500) |
| Antibody | Anti-Flag M2 affinity gel | Sigma-Aldrich | A2220 | |
| Recombinant DNA reagent | pWPI-HA-3xFlag-RIPK3 | This paper | N/A | Described in Materials and methods; available upon request |
| Recombinant DNA reagent | pWPI-HA-3xFlag- RIPK3(D160N) | This paper | N/A | Described in Materials and methods; available upon request |
| Recombinant DNA reagent | pWPI-HA-3xFlag- RIPK3(K50A) | This paper | N/A | Described in Materials and methods; available upon request |
| Recombinant DNA reagent | pWPI-HA-3xFlag- RIPK3(AAAA) | This paper | N/A | Described in Materials and methods; available upon request |
| Recombinant DNA reagent | pWPI-HA-3xFlag-mRIPK3 | This paper | N/A | Described in Materials and methods; available upon request |
| Recombinant DNA reagent | pWPI-HA-3xFlag-RIPK3(S164D/T165E) | This paper | N/A | Described in Materials and methods; available upon request |
| Recombinant DNA reagent | pWPI-HA-3xFlag- RIPK3(S164E) | This paper | N/A | Described in Materials and methods; available upon request |
| Recombinant DNA reagent | pWPI-HA-3xFlag- RIPK3(T165E) | This paper | N/A | Described in Materials and methods; available upon request |
| Recombinant DNA reagent | pWPI-HA-3xFlag- RIPK3(S164A) | This paper | N/A | Described in Materials and methods; available upon request |
| Recombinant DNA reagent | pWPI-HA-3xFlag- RIPK3(T165A) | This paper | N/A | Described in Materials and methods; available upon request |
| Recombinant DNA reagent | pWPI-HA-3xFlag- RIPK3(S164A/T165A) | This paper | N/A | Described in Materials and methods; available upon request |
| Recombinant DNA reagent | pWPI-HA-3xFlag-mRIPK3(D161N) | This paper | N/A | Described in Materials and methods; available upon request |
| Recombinant DNA reagent | pWPI-HA-3xFlag- mRIPK3(S165D/T166E) | This paper | N/A | Described in Materials and methods; available upon request |
| Recombinant DNA reagent | pLVX-Tight-HA-3xFlag-RIPK3 | This paper | N/A | Described in Materials and methods; available upon request |
| Recombinant DNA reagent | pLVX-Tight-HA-3xFlag- RIPK3(D160N) | This paper | N/A | Described in Materials and methods; available upon request |
| Recombinant DNA reagent | pLVX-Tight-HA-3xFlag- RIPK3(K50A) | This paper | N/A | Described in Materials and methods; available upon request |
| Recombinant DNA reagent | pLVX-Tight-HA-3xFlag- RIPK3(S164A/T165A) | This paper | N/A | Described in Materials and methods; available upon request |
| Recombinant DNA reagent | pX458-GFP-RIPK1 | This paper | N/A | Described in Materials and methods; available upon request |
| Recombinant DNA reagent | pX458-GFP-caspase-8 | This paper | N/A | Described in Materials and methods; available upon request |
| Recombinant DNA reagent | pX458-GFP-FADD | This paper | N/A | Described in Materials and methods; available upon request |
| Recombinant DNA reagent | pX458-GFP-cFLIP | This paper | N/A | Described in Materials and methods; available upon request |
| Peptide, recombinant protein | 3xFlag peptide | ChinaPeptides | DYKDHDGDYKDHDIDYKDDDDK | 1 mg/ml |
| Software, algorithm | ImageJ | NIH | N/A | |
| Software, algorithm | Photoshop | Adobe | N/A | |
| Software, algorithm | Lasergene | DNASTAR | N/A | |
| Software, algorithm | GraphPad | GraphPad Software | N/A | |
| Software, algorithm | Prism | GraphPad Software | N/A | |
| Software, algorithm | Nikon A1-R | Nikon | https://www.nikoninstruments.com/Products/Confocal-Microscopes/A1R-HD |
