25 research outputs found
Phosphoproteomics study based on in vivo inhibition reveals sites of calmodulin-dependent protein kinase II regulation in the heart
Background The multifunctional Ca2+â and calmodulinâdependent protein kinase II (CaMKII) is a crucial mediator of cardiac physiology and pathology. Increased expression and activation of CaMKII has been linked to elevated risk for arrhythmic events and is a hallmark of human heart failure. A useful approach to determining CaMKII's role therein is largeâscale analysis of phosphorylation events by mass spectrometry. However, current largeâscale phosphoproteomics approaches have proved inadequate for highâfidelity identification of kinaseâspecific roles. The purpose of this study was to develop a phosphoproteomics approach to specifically identify CaMKII's downstream effects in cardiac tissue. Methods and Results To identify putative downstream CaMKII targets in cardiac tissue, animals with myocardialâdelimited expression of the specific peptide inhibitor of CaMKII (AC3âI) or an inactive control (AC3âC) were compared using quantitative phosphoproteomics. The hearts were isolated after isoproterenol injection to induce CaMKII activation downstream of ÎČâadrenergic receptor agonist stimulation. Enriched phosphopeptides from AC3âI and AC3âC mice were differentially quantified using stable isotope dimethyl labeling, strong cation exchange chromatography and highâresolution LCâMS/MS. Phosphorylation levels of several hundred sites could be profiled, including 39 phosphoproteins noticeably affected by AC3âIâmediated CaMKII inhibition. Conclusions Our data set included known CaMKII substrates, as well as several new candidate proteins involved in functions not previously implicated in CaMKII signaling
Phosphoproteomics study based on in vivo inhibition reveals sites of calmodulin-dependent protein kinase II regulation in the heart
Background The multifunctional Ca2+â and calmodulinâdependent protein kinase II (CaMKII) is a crucial mediator of cardiac physiology and pathology. Increased expression and activation of CaMKII has been linked to elevated risk for arrhythmic events and is a hallmark of human heart failure. A useful approach to determining CaMKII's role therein is largeâscale analysis of phosphorylation events by mass spectrometry. However, current largeâscale phosphoproteomics approaches have proved inadequate for highâfidelity identification of kinaseâspecific roles. The purpose of this study was to develop a phosphoproteomics approach to specifically identify CaMKII's downstream effects in cardiac tissue. Methods and Results To identify putative downstream CaMKII targets in cardiac tissue, animals with myocardialâdelimited expression of the specific peptide inhibitor of CaMKII (AC3âI) or an inactive control (AC3âC) were compared using quantitative phosphoproteomics. The hearts were isolated after isoproterenol injection to induce CaMKII activation downstream of ÎČâadrenergic receptor agonist stimulation. Enriched phosphopeptides from AC3âI and AC3âC mice were differentially quantified using stable isotope dimethyl labeling, strong cation exchange chromatography and highâresolution LCâMS/MS. Phosphorylation levels of several hundred sites could be profiled, including 39 phosphoproteins noticeably affected by AC3âIâmediated CaMKII inhibition. Conclusions Our data set included known CaMKII substrates, as well as several new candidate proteins involved in functions not previously implicated in CaMKII signaling