Phosphorylation of the mitochondrial autophagy receptor Nix enhances its interaction with LC3 proteins.
Vladimir V RogovHironori SuzukiMija MarinkovićVerena LangRyuichi KatoMasato KawasakiMaja BuljubašićMatilda ŠprungNatalia RogovaSoichi WakatsukiAnne Hamacher-BradyVolker DötschIvan ĐikićNathan R BradyIvana NovakPublished in: Scientific reports (2017)
The mitophagy receptor Nix interacts with LC3/GABARAP proteins, targeting mitochondria into autophagosomes for degradation. Here we present evidence for phosphorylation-driven regulation of the Nix:LC3B interaction. Isothermal titration calorimetry and NMR indicate a ~100 fold enhanced affinity of the serine 34/35-phosphorylated Nix LC3-interacting region (LIR) to LC3B and formation of a very rigid complex compared to the non-phosphorylated sequence. Moreover, the crystal structure of LC3B in complex with the Nix LIR peptide containing glutamic acids as phosphomimetic residues and NMR experiments revealed that LIR phosphorylation stabilizes the Nix:LC3B complex via formation of two additional hydrogen bonds between phosphorylated serines of Nix LIR and Arg11, Lys49 and Lys51 in LC3B. Substitution of Lys51 to Ala in LC3B abrogates binding of a phosphomimetic Nix mutant. Functionally, serine 34/35 phosphorylation enhances autophagosome recruitment to mitochondria in HeLa cells. Together, this study provides cellular, biochemical and biophysical evidence that phosphorylation of the LIR domain of Nix enhances mitophagy receptor engagement.
Keyphrases
- simultaneous determination
- protein kinase
- mass spectrometry
- liquid chromatography
- cell death
- magnetic resonance
- solid phase extraction
- binding protein
- induced apoptosis
- tandem mass spectrometry
- cell cycle arrest
- cell proliferation
- drug delivery
- endoplasmic reticulum stress
- transcription factor
- high resolution mass spectrometry