Abstract
Mitochondrial functions are dynamically regulated in the heart. In particular, protein phosphorylation has been shown to be a key mechanism modulating mitochondrial function in diverse cardiovascular phenotypes. However, site-specific phosphorylation information remains scarce for this organ. Accordingly, we performed a comprehensive characterization of murine cardiac mitochondrial phosphoproteome in the context of mitochondrial functional pathways. A platform using the complementary fragmentation technologies of collision-induced dissociation (CID) and electron transfer dissociation (ETD) demonstrated successful identification of a total of 236 phosphorylation sites in the murine heart; 210 of these sites were novel. These 236 sites were mapped to 181 phosphoproteins and 203 phosphopeptides. Among those identified, 45 phosphorylation sites were captured only by CID, whereas 185 phosphorylation sites, including a novel modification on ubiquinol-cytochrome c reductase protein 1 (Ser-212), were identified only by ETD, underscoring the advantage of a combined CID and ETD approach. The biological significance of the cardiac mitochondrial phosphoproteome was evaluated. Our investigations illustrated key regulatory sites in murine cardiac mitochondrial pathways as targets of phosphorylation regulation, including components of the electron transport chain (ETC) complexes and enzymes involved in metabolic pathways (e.g. tricarboxylic acid cycle). Furthermore, calcium overload injured cardiac mitochondrial ETC function, whereas enhanced phosphorylation of ETC via application of phosphatase inhibitors restored calcium-attenuated ETC complex I and complex III activities, demonstrating positive regulation of ETC function by phosphorylation. Moreover, in silico analyses of the identified phosphopeptide motifs illuminated the molecular nature of participating kinases, which included several known mitochondrial kinases (e.g. pyruvate dehydrogenase kinase) as well as kinases whose mitochondrial location was not previously appreciated (e.g. Src). In conclusion, the phosphorylation events defined herein advance our understanding of cardiac mitochondrial biology, facilitating the integration of the still fragmentary knowledge about mitochondrial signaling networks, metabolic pathways, and intrinsic mechanisms of functional regulation in the heart.
Highlights
From the ‡Departments of Physiology and Medicine, Division of Cardiology, David Geffen School of Medicine at UCLA, Los Angeles, California 90095, §Department of Biomedical Engineering, Key Laboratory for Biomedical Engineering of Ministry of Education, Zhejiang University, Hangzhou 310027, China, ʈDepartment of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, China, and **European Molecular Biology Laboratory/European Bioinformatics Institute, Hinxton, Cambridge CB10f 1SD, United Kingdom
A total of 236 phosphorylation sites were identified in 203 unique peptides that were mapped to 181 mitochondrial proteins
The delineation of regulatory sites in diverse mitochondrial pathways was supported by the combination of phosphopeptide enrichment procedures and complementary proteomics technologies of electron transfer dissociation (ETD) and collision-induced dissociation (CID)
Summary
Preparative SDS-PAGE and In-gel Digestion—Purified cardiac mitochondria were lysed with 0.5% N-dodecyl -D-maltoside in the isolation buffer on ice. After centrifugation at 13,000 ϫ g for 30 min, mitochondrial proteins were collected from the supernatant and incubated with Laemmli sample buffer for 30 min. The following search parameters were set: partial enzymatic digestion (trypsin) permitting three missed cleavages; fixed modification of cysteine carboxyamidomethylation (ϩ57 Da); and dynamic modifications of methionine oxidation (ϩ16 Da) and serine, threonine, and tyrosine phosphorylation (ϩ80 Da). Manual inspection was conducted on all potential phosphopeptides to remove false positives This two-step screening procedure for both ETD and CID analyses was implemented to minimize the bias of a particular search engine as well as to improve the confidence of identification. The phosphoproteins that do not associate with a GO term via g:Profiler were manually annotated using the following knowledgebases: Gene Ontology Annotation database (http://www.ebi.ac.uk/GOA/) [31], UniProt Knowledgebase (www.uniport.org) [32], and LOCATE database (www. scivee.tv/node/1413) [33]
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