Relevant to Numbers 1C7. NIHMS952168-product-1.xlsx (41K) GUID:?860D29FF-5419-46E7-8763-AF26677B4543 2: Supplemental Table S2. Red1 kinase and PARKIN ubiquitin ligase by proteomic counting of ubiquitylation and phosphorylation events. We define the kinetics and site-specificity of PARKIN-dependent target ubiquitylation and demonstrate the power of this approach to quantify pathway modulators and to mechanistically define the part of PARKIN UBL phosphorylation in KI67 antibody pathway activation in induced neurons. Finally, through modulation of pS65-Ub on mitochondria, we demonstrate that Ub hyper-phosphorylation is definitely inhibitory to mitophagy receptor recruitment, indicating that pS65-Ub stoichiometry in vivo is definitely optimized to coordinate PARKIN recruitment via pS65-Ub and mitophagy receptors via unphosphorylated chains. eTOC Blurb The PARKIN ubiquitin ligase is definitely activated on damaged mitochondria via the Red1 kinase, where it ubiquitylates an array of proteins. Ordureau et al. develop a quantitative proteomics approach to measure the dynamics, site-specificity and stoichiometry of PARKIN-dependent substrate ubiquitylation in neuronal cells, providing a quantitative analysis of the pathway. Intro Cellular decisions often involve the coordination of protein kinase and ubiquitin (Ub) ligase-driven signaling networks (Hunter, 2007). While network architecture varies, three features generally apply: 1) signals are propagated in space and time within the cell, often with feedback control, 2) both kinases and Ub ligases often modify multiple target sites on varied proteins within a pathway inside a distributive manner, sometimes including multiple Ub chain linkage types (Kulathu and Komander, 2012), and 3) pathway flux depends upon changes stoichiometry within individual pools of target proteins. However, we hardly ever understand the degree to which complex modifications within a pathway are spatially or kinetically distinguishable, due in part to the absence of antibodies that can reveal site specificity and kinetics. Here, we develop targeted Kgg remnant and phosphoproteomics as a means by which to provide digital snapshots of main site-specificity, kinetics and MK-2206 2HCl stoichiometry of the individual changes events inside a dynamic kinase and Ub ligase driven signaling cascade critical for mitochondrial quality control. Mitochondrial oxidative or proteotoxic stress can promote removal of damaged mitochondria through a form of selective autophagy called mitophagy, requiring the PARKIN RING-Between-RING (RBR) Ub ligase and mitochondrially-localized protein kinase Red1, both found mutated in Parkinsons Disease (examined in (Pickrell and Youle, 2015)). When mitochondria are healthy, PINK1 large quantity in mitochondria is definitely low and PARKIN is definitely localized in the cytosol in an auto-inhibited form (Pickrell and Youle, 2015). In response to mitochondrial damage, Red1 accumulates within the mitochondrial outer membrane (MOM) (Lazarou et al., 2012; Narendra et al., 2010b; Yamano and Youle, 2013) where it promotes PARKIN recruitment to mitochondria and activation of MOM protein ubiquitylation through a complex feed-forward mechanism including: 1) phosphorylation of S65 in Ub chains on the MOM having a stoichiometry of ~0.2 in MK-2206 2HCl the HeLa cell system, 2) phosphorylation of S65 in PARKINs Ub-like (UBL) website to greatly enhance its ligase activity by reversal of auto-inhibition, and 3) binding of MK-2206 2HCl PARKIN to pS65-Ub chains to both retain it on the MOM and promote UBL phosphorylation by Red1 (Kane et al., 2014; Kazlauskaite et al., 2014a; Kazlauskaite et al., 2014b; Kazlauskaite et al., 2015; Koyano et al., 2014; Narendra et al., 2008; Okatsu et al., 2015; Ordureau et al., 2015; Ordureau et al., 2014; Wauer et al., 2015a). Retention of active PARKIN on the MOM results in ubiquitylation of numerous substrates (Bingol et al., 2014; Rose et al., 2016; Sarraf et al., 2013) and the assembly of Ub chains that serve to recruit autophagy receptors and promote downstream methods in mitophagy (Pickrell and Youle, 2015). Despite these improvements, numerous gaps exist in MK-2206 2HCl our understanding of the dynamics and sequence of steps in the process of MOM ubiquitylation by PARKIN (examined in (Harper et al., 2018)). First, we do not understand the degree to which PARKIN functions inside a site-specific manner to ubiquitylate Lys residues in target proteins, nor do we know what part substrate abundance takes on in MOM ubiquitylation. Previous studies identifying PARKIN main ubiquitylation sites used cell lines with assorted PARKIN levels, and a wide range of depoloarization occasions. This, coupled with the stochastic nature of antibody-directed Kgg peptide recognition, greatly limits our understanding of the relative rates of ubiquitylation of individual main sites in PARKIN focuses on. Second, to day, the kinetics and specificity of PARKIN target ubiquitylation has not been examined in neuronal.