title: Focused Proteomics Revealed a Novel Rho-kinase Signaling Pathway in the Heart.
abstract: Protein phosphorylation plays an important role in the physiological regulation of cardiac function. Myocardial contraction and pathogenesis of cardiac diseases have been reported to be associated with adaptive or maladaptive protein phosphorylation; however, phosphorylation signaling in the heart is not fully elucidated. We recently developed a novel kinase-interacting substrate screening (KISS) method for exhaustive screening of protein kinase substrates, using mass spectrometry and affinity chromatography. First, we examined protein phosphorylation by extracellular signal-regulated kinase (ERK) and protein kinase A (PKA), which has been relatively well studied in cardiomyocytes. The KISS method showed that ERK and PKA mediated the phosphorylation of known cardiac-substrates of each kinase such as Rps6ka1 and cTnI, respectively. Using this method, we found about 330 proteins as Rho-kinase-mediated substrates, whose substrate in cardiomyocytes is unknown. Among them, CARP/Ankrd1, a muscle ankyrin repeat protein, was confirmed as a novel Rho-kinase-mediated substrate. We also found that non-phosphorylatable form of CARP repressed cardiac hypertrophy-related gene Myosin light chain-2v (MLC-2v) promoter activity, and decreased cell size of heart derived H9c2 myoblasts more efficiently than wild type-CARP. Thus, focused proteomics enable us to reveal a novel signaling pathway in the heart.
authors.affiliation: Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University.
authors.forename: Yoshimitsu
authors.initials: Y
authors.lastname: Yura
authors.forename: Mutsuki
authors.initials: M
authors.lastname: Amano
authors.forename: Mikito
authors.initials: M
authors.lastname: Takefuji
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Bando
authors.forename: Kou
authors.initials: K
authors.lastname: Suzuki
authors.forename: Katsuhiro
authors.initials: K
authors.lastname: Kato
authors.forename: Tomonari
authors.initials: T
authors.lastname: Hamaguchi
authors.forename: Md
authors.initials: M
authors.lastname: Hasanuzzaman Shohag
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Takano
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Funahashi
authors.forename: Shinichi
authors.initials: S
authors.lastname: Nakamuta
authors.forename: Keisuke
authors.initials: K
authors.lastname: Kuroda
authors.forename: Tomoki
authors.initials: T
authors.lastname: Nishioka
authors.forename: Toyoaki
authors.initials: T
authors.lastname: Murohara
authors.forename: Kozo
authors.initials: K
authors.lastname: Kaibuchi
mesh_terms: D048948:14-3-3 Proteins / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000378:metabolism; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D007150:Immunohistochemistry; D013058:Mass Spectrometry; D008856:Microscopy, Fluorescence; D009124:Muscle Proteins / Q000737:chemistry / Q000378:metabolism; D009206:Myocardium / Q000201:enzymology*; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D040901:Proteomics; D051381:Rats; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction; D013379:Substrate Specificity; D054460:rho-Associated Kinases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D048948:14-3-3 Proteins; C491303:Ankrd1 protein, rat; D009124:Muscle Proteins; D009687:Nuclear Proteins; D012097:Repressor Proteins; D054460:rho-Associated Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D048049:Extracellular Signal-Regulated MAP Kinases