title: Phosphoproteome analysis reveals regulatory sites in major pathways of cardiac mitochondria.
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.
authors.affiliation: Department of Physiology, Division of Cardiology, David Geffen School of Medicine at UCLA, Los Angeles, California 90095, USA.
authors.forename: Ning
authors.initials: N
authors.lastname: Deng
authors.forename: Jun
authors.initials: J
authors.lastname: Zhang
authors.forename: Chenggong
authors.initials: C
authors.lastname: Zong
authors.forename: Yueju
authors.initials: Y
authors.lastname: Wang
authors.forename: Haojie
authors.initials: H
authors.lastname: Lu
authors.forename: Pengyuan
authors.initials: P
authors.lastname: Yang
authors.forename: Wenhai
authors.initials: W
authors.lastname: Wang
authors.forename: Glen W
authors.initials: GW
authors.lastname: Young
authors.forename: Yibin
authors.initials: Y
authors.lastname: Wang
authors.forename: Paavo
authors.initials: P
authors.lastname: Korge
authors.forename: Christopher
authors.initials: C
authors.lastname: Lotz
authors.forename: Philip
authors.initials: P
authors.lastname: Doran
authors.forename: David A
authors.initials: DA
authors.lastname: Liem
authors.forename: Rolf
authors.initials: R
authors.lastname: Apweiler
authors.forename: James N
authors.initials: JN
authors.lastname: Weiss
authors.forename: Huilong
authors.initials: H
authors.lastname: Duan
authors.forename: Peipei
authors.initials: P
authors.lastname: Ping
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods; D004579:Electron Transport; D006321:Heart / Q000502:physiology; D008297:Male; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D008813:Mice, Inbred ICR; D008928:Mitochondria / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry; D010641:Phenotype; D010766:Phosphorylation; D010770:Phosphotransferases / Q000737:chemistry; D040901:Proteomics / Q000379:methods*
chemical_list: D010455:Peptides; D010770:Phosphotransferases