title: Phosphoproteome analysis of cardiomyocytes subjected to beta-adrenergic stimulation: identification and characterization of a cardiac heat shock protein p20.
abstract: Posttranslational modification of target substrates underlies biological processes through activation/inactivation of signaling cascades. To concurrently identify the phosphoprotein substrates associated with cardiac beta-adrenergic signaling, the mouse myocyte phosphoproteome was analyzed using 2-D gel electrophoresis in combination with 32P autoradiography. Phosphoprotein spots, detected by silver staining, were identified using MALDI-TOF mass spectrometry in conjunction with computer-assisted protein spot matching. Stimulation with isoproterenol (1 micromol/L for 5 minutes) was associated with maximal increases in myocyte contractile parameters, and significant stimulation of the phosphorylation of troponin I (190+/-23%) and succinyl CoA synthetase (160+/-16%), whereas the phosphorylation of pyruvate dehydrogenase (48+/-10%), NADH-ubiquinone oxidoreductase (46+/-6%), heat shock protein 27 (18+/-3%), alphaB-crystallin (20+/-3%), and an unidentified 26-kDa protein (29+/-7%) was significantly decreased, compared with unstimulated cells (100%). After sustained (30 minutes) stimulation with isoproterenol, only the alterations in the phosphorylation levels of troponin I and NADH-ubiquinone oxidoreductase were maintained and de novo phosphorylation of a phosphoprotein (approximately 20 kDa and pI 5.5) was observed. The tryptic peptide fragments of this phosphoprotein were sequenced using postsource decay mass spectrometry, and the protein was subsequently cloned and designated as p20, based on its high sequence homology with rat and human skeletal p20. The mouse cardiac p20 contains the conserved domain sequences for heat shock proteins, and the RRAS consensus sequence for cAMP-PKA substrates. LC-MS/MS phosphorylation mapping confirmed phosphorylation of Ser16 in p20 on beta-agonist stimulation. Adenoviral gene transfer of p20 was associated with significant increases in contractility and Ca transient peak in adult rat cardiomyocytes, suggesting an important role of p20 in cardiac function. These findings suggest that cardiomyocytes undergo significant posttranslational modification via phosphorylation in a multitude of proteins to dynamically fine-tune cardiac responses to beta-adrenergic signaling.
authors: Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0575, USA.
authors: Guoxiang
authors: G
authors: Chu
authors: Gregory F
authors: GF
authors: Egnaczyk
authors: Wen
authors: W
authors: Zhao
authors: Su-Hyun
authors: SH
authors: Jo
authors: Guo-Chang
authors: GC
authors: Fan
authors: John E
authors: JE
authors: Maggio
authors: Rui-Ping
authors: RP
authors: Xiao
authors: Evangelia G
authors: EG
authors: Kranias
mesh_terms: D000318:Adrenergic beta-Agonists / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000818:Animals; D020013:Calcium Signaling; D048429:Cell Size / Q000187:drug effects; D003001:Cloning, Molecular; D015180:Electrophoresis, Gel, Two-Dimensional; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology*; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D009200:Myocardial Contraction; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D010446:Peptide Fragments / Q000737:chemistry; D010766:Phosphorylation / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D040901:Proteomics; D051381:Rats; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D000318:Adrenergic beta-Agonists; D006360:Heat-Shock Proteins; D009124:Muscle Proteins; D010446:Peptide Fragments; C483911:p20 protein, mouse; D007545:Isoproterenol