Parkin regulates NF-κB by mediating site-specific ubiquitination of RIPK1

Abstract

Parkin (Park2), a RING-between-RING-type E3 ubiquitin ligase, has been implicated in regulating NF-κB. Mutations in Parkin are associated with Parkinson's disease. Here we investigated the interaction of Parkin with Receptor-interacting protein kinase 1 (RIPK1) kinase, a key mediator of multiple signaling pathways activated by TNFR1 including NF-κB pathway. We report that Parkin interacts with RIPK1 and mediates K63 ubiquitination of RIPK1 on K376 in TNFR1-signaling pathway. The expression of Parkin promotes the recruitment of transforming growth factor β (TGF-β)-activated kinase 1 (TAK1), nuclear factor-κB (NF-κB) essential molecule (NEMO), Sharpin and A20 in complex I associated with TNFR1 upon TNFα stimulation. Ubiquitination of RIPK1 by Parkin increases the activation of NF-κB and mitogen-activated protein kinases (MAPKs) by promoting the phosphorylation of inhibitor of kappa B kinase (IKK)α/β and IκBα and nuclear translocation of p65. Thus, we conclude that Parkin modulates the K63 ubiquitination status of RIPK1 to promote the activation of NF-κB and MAPKs.

Fig. 1. Parkin interacts with RIPK1.

a 293 T cells were transfected with expression vectors of FLAG-tagged Parkin and HA-tagged RIPK1 for 24 h and then lysed with NP40 buffer and protease inhibitors with no EDTA. HA-tagged RIPK1 was immunoprecipitated with anti-HA antibody conjugated agarose and analyzed by mass spectrometry and quantified with LFQ module implemented in MaxQuant. The table listed the number of peptides from RIPK1 and Parkin identified in mass spectrometry analysis. b The expression vectors of HA-tagged full-length RIPK1, RIPK1- ΔKD, RIPK1- ΔC, and RIPK1- ΔDD were constructed. c The expression vectors of FLAG-tagged Parkin and HA-tagged full-length RIPK1, RIPK1-ΔKD, RIPK1-ΔC, and RIPK1-ΔDD were transfected into 293 T cells for 24 h. FLAG-Parkin was immunoprecipitated with anti-FLAG. The immunoprecipitates and cell lysates were analyzed by western blot with indicated antibodies

Fig. 2. Parkin mediates K63 ubiquitination of RIPK1.

a 293 T cells were transfected with expression vectors of RIPK1 and His-Ub or His-K63 ubiquitin with or without that of Parkin for 24 h and then lysed with 6 M urea. His-tagged proteins were pulled down with Ni-NTA. The pulled-down proteins and cell lysates were analyzed by western blotting with indicated antibodies. b 293 T cells were transfected with expression vectors of His-K63 ubiquitin, RIPK1 and Parkin or K150E Parkin for 24 h and then lysed with 6 M urea. His-tagged proteins were pulled down with Ni-NTA. The pulled-down proteins and cell lysates were analyzed by western blotting with indicated antibodies. c 293 T cells were transfected with expression vectors of His-K63 ubiquitin, Flag-RIPK1 with or without that of Parkin in 293 T cells for 24 h. Flag-tagged RIPK1 was isolated by immunoprecipitation with anti-FLAG agarose beads and analyzed for ubiquitination sites by mass spectrometry. Ubiquitinated Lys residues of RIPK1 identified and quantified by mass spectrometry are listed in the Table. d 293 T cells were transfected with expression vectors of His-K63 ubiquitin, K376R mRIPK1, K550R mRIPK1, K20R mRIPK1, K105R RIPK1 with or without that of Parkin as indicated for 24 h and then lysed with 6 M urea. His-tagged proteins were pulled down with Ni-NTA. The pulled-down proteins and cell lysates were analyzed by western blotting with indicated antibodies. e 293 T cells were transfected with expression vectors of His-K63 ubiquitin and WT RIPK1 or K376R RIPK1 with or without that of Parkin for 24 h and then treated with SM164 (200 nM) or DMSO for 4 h before lysed with 6 M urea. His-tagged proteins were pulled down with Ni-NTA. The pulled-down proteins and cell lysates were analyzed by western blotting with indicated antibodies. f 0.1 mM E1 UBE1, 1 mM E2 (UbcH7), 0.1 mM ubiquitin (Boston Biochem), 5 mM MgCl₂, 2 mM ATP in 50 mM Tris-HCl (pH 7.5) mixed with Flag-Parkin or Flag-K150E Parkin immunoprecipitated with anti-FLAG, and HA-RIPK1 or HA-K376R RIPK1 immunoprecipitated with anti-HA from HEK 293 cells and incubated in a total volume of 50 μL for 30 min at 37 °C

Fig. 3. Parkin is recruited into TNF-RSC in RIPK1-dependent manner.

a WT and Ripk1^−/− MEFs stably expressing Parkin were treated with TNFα 10 ng/mL for 0, 5, and 15 min. The cell lysates were immunoprecipitated with anti-TNFR1. The immunoprecipitates and cell lysates were analyzed by western blotting with indicated antibodies. b 661 W cells stably expressing GFP, Parkin or K150E Parkin were treated with TNFα10 ng/mL for 0, 5, and 15 min. The cell lysates were immunoprecipitated with anti-RIPK1. The immunoprecipitates and cell lysates were analyzed by western blotting with indicated antibodies. c 661 W cells stably expressing GFP, WT Parkin or K150E mutant Parkin were treated with TNFα 10 ng/mL for 0, 5, and 15 min. Complex I was immunoprecipitated with anti-TNFR1. The immunoprecipitates and cell lysates were analyzed by western blotting with indicated antibodies

Fig. 4. Interaction between Parkin and RIPK1 promotes the recruitment of components in complex I under TNFα stimulation.

a The expression vectors of HA-tagged RIPK1 with or without FLAG-tagged Parkin were transfected into 293 T cells for 24 h. HA-RIPK1 was immunoprecipitated with anti-HA. The binding proteins of RIPK1 were identified by mass spectrometry and quantified with LFQ module implemented in MaxQuant. b 661 W cells stably expressing GFP or Parkin were treated with TNFα 10 ng/mL for 0, 5, 15 and 30 min. The cell lysates were immunoprecipitated with anti-TNFR1. The immunoprecipitates and cell lysates were analyzed by western blotting with indicated antibodies. c 661 W cells stably expressing Parkin or K150E Parkin were treated with TNFα 10 ng/mL for 0, 5 and 15 min. The cell lysates were immunoprecipitated with anti-TNFR1. The immunoprecipitates and cell lysates were analyzed by western blotting with indicated antibodies

Fig. 5. Interaction between Parkin and RIPK1 activate MAPK and NF-κB signaling under TNFα stimulation.

a, b 661 W cells stably expressing GFP or Parkin (a) or Parkin or K150E Parkin (b) were treated with TNFα 10 ng/mL for 0, 5, 15, 30, and 60 min and then lysed with 6 M urea for western blotting with indicated antibodies. c Ripk1^−/− MEFs cells stably expressing Parkin were transfected with expression vectors of RIPK1 or K376 RIPK1, and treated with TNFα 10 ng/mL for 0, 5, 15, and 30 min. The cells were then lysed with 6 M urea for western blotting with indicated antibodies

Fig. 6. Interaction between Parkin and RIPK1 promotes NF-κB p65 nuclear translocation.

a Nuclear translocation of NF-κB p65 was analyzed by indirect immunostaining in 661 W cell stably expressing vector, Parkin or K150E Parkin. These cells were transfected with control shRNA or RIPK1 shRNA for 24 h as indicated then treated with TNFα (10 ng/ml) for 0, 5, 15, and 30 min. Scale bars, 50 μm. b Colocalizations of p65 and DAPI were quantified in three independent experiments performed in duplicate. Data were represented as mean ± SEM. c 661 W cells stably expressing vector, Parkin or K150E Parkin were transfected with NF-κB luciferase reporter vector, CMV-Renilla (sea pansy) luciferase vector and control shRNA or RIPK1 shRNA as indicated for 16 h and then treated with or without TNFα (10 ng/ml) for 8 h as indicated. Luciferase activity in cell lysates was determined 24 h after transfection. The knockdown efficiency and expression levels were determined by western blotting. Data were represented as mean ± SEM of triplicates

Fig. 7

In response to TNFα, Parkin is recruited into complex I by interacting with RIPK1 to mediate its K63 ubiquitination on K376, which in turn promotes the recruitment of other complex I components, including TAK1, Sharpin, NEMO and A20, etc., to mediate the activation of NF-κB and MAPK.