Journal: Open Biology
Article Title: PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65
Figure Lengend Snippet: Human Parkin Ser 65 is a substrate of human PINK1 upon CCCP stimulation. ( a ) Confirmation by mass spectrometry that Ser 65 of human Parkin is phosphorylated by CCCP-induced activation of human wild-type PINK1-FLAG. Flp-In T-Rex HEK293 cells expressing FLAG-empty, wild-type PINK1-FLAG, and kinase-inactive PINK1-FLAG (D384A) were co-transfected with HA-Parkin, induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. Whole-cell extracts were obtained following lysis with 1% Triton and approximately 30 mg of whole-cell extract were subjected to immunoprecipitation with anti-HA-agarose and run on 10% SDS-PAGE and stained with colloidal Coomassie blue. Coomassie-stained bands migrating with the expected molecular mass of HA-Parkin were excised from the gel, digested with trypsin, and subjected to high performance liquid chromatography with tandem mass spectrometry (LC-MS-MS) on an LTQ-Orbitrap mass spectrometer. Extracted ion chromatogram analysis of Ser 131 and Ser 65 phosphopeptide (3 + R.NDWTVQNCDLDQQ S IVHIVQRPWR.K+P). The total signal intensity of the phosphopeptide is plotted on the y -axis and retention time is plotted on the x -axis. The m / z value corresponding to the Ser 131 phosphopeptide was detected in all conditions whilst that of the Ser 65 phosphopeptide was only detected in samples from wild-type PINK1-FLAG-expressing cells following CCCP treatment. ( b ) Characterization of Parkin phospho-Ser 65 antibody. Flp-In T-Rex HEK293 cells expressing FLAG-empty, wild-type PINK1-FLAG, and kinase-inactive PINK1-FLAG were co-transfected with untagged wild-type (WT) or Ser 65 Ala (S65A) mutant Parkin, induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. 0.25 mg of 1% Triton whole-cell lysate were subjected to immunoprecipitation with anti-Parkin antibody (S966C) covalently coupled to protein G Sepharose and then immunoblotted with anti-phospho-Ser 65 antibody in the presence of dephosphorylated peptide. Ten per cent of the immunoprecipitate (IP) was immunoblotted with total anti-Parkin antibody. Twenty five micrograms of whole cell lysate was immunoblotted with total PINK1 antibody.
Article Snippet: Isolated phosphopeptides were analysed by LC-MS-MS on a proxeon Easy-nLC nano liquid chromatography system coupled to a Thermo LTQ-orbitrap mass spectrometer.
Techniques: Mass Spectrometry, Activation Assay, Expressing, Transfection, Lysis, Immunoprecipitation, SDS Page, Staining, High Performance Liquid Chromatography, Liquid Chromatography with Mass Spectroscopy, Mutagenesis