course=”kwd-title”>Keywords: in vivo phosphoproteomics cardiac signaling beta-adrenergic signaling quantitative phosphoproteomics ion channel phosphorylation cardiac kinase regulation Kv7. in mass spectrometric instrumentation sample preparation and computational proteomics developments BTZ038 that now make it possible to analyze proteins1 or post-translational modifications (PTMs) of proteins2 on a global scale and compare their relative abundance between different cell states. As the proteomics technology rapidly advances it is becoming increasingly popular in cell biology where it has especially proven a powerful tool to characterize cellular responses by analyzing global protein phosphorylation changes in a stimulus- and time-resolved manner.3 Protein phosphorylation is tightly regulated in the cell by the action of kinases and phosphatases and it is involved in regulating essentially all cellular processes where site-specific phosphorylation events often function as molecular switches that modification or fine-tune the action of focus on protein either by altering their enzymatic activity or by affecting interaction companions or subcellular localization. Inside our group we’ve within the last few years concentrated our initiatives on developing solid and reproducible solutions to analyze PTMs such as for example phosphorylation and acetylation on proteins extracted from tissues examples.4 5 That is an important stage for the impact of proteomics in biology since it opens new avenues for investigating cell signaling systems in vivo. There is absolutely no question that quantitative phosphoproteomics provides revolutionized the investigations of cell signaling systems in a BTZ038 CORO2A global and unbiased manner but the investigations have so far largely been limited to cell culture models. However for many physiological processes it is not sufficient to investigate the responses elicited by a given stimulus in cell culture as immortalized cell lines lack many tissue-specific proteins. One such example is proteins involved in excitation-contraction coupling of the heart. In a recent study for the first time we investigated cardiac β-adrenergic signaling on a global scale by analyzing the phosphorylation site changes of proteins extracted from murine hearts that were treated with β-blockers and activators 6 thus performing quantitative phosphoproteomics in vivo. Adrenaline stimulates β-adrenergic receptors (βAR) as an essential component of the “fight-or-flight” response in human physiology resulting in increased cardiac output mediated by increased contractile force and heart rate. Activation of the βARs initiates protein phosphorylation-dependent signaling cascades that increase myocardial contractility and relaxation rate. β-blockers that inhibit βARs are widely used in the clinic to prevent cardiac arrhythmias and treat hypertension but knowledge of their downstream molecular targets remains limited. Therefore delineating the cardiac signaling pathways regulated by phosphorylation as a result of βAR stimulation bears important etiological and therapeutic implications in diseases such BTZ038 as hypertension and heart failure. In our published work 6 we treated a control group of mice with specific βAR inhibitors and another group of mice with βAR activators (Fig. 1). BTZ038 To delineate the downstream effectors of βAR activation we excised the hearts of the mice and subjected them to phosphoproteomics investigation. We identified more than 600 phosphorylation sites on 300 proteins that are significantly regulated by the stimulus. Our data set covers all previously described regulatory phosphoproteins in this response but importantly it expands our knowledge of βAR-regulated phosphorylation sites from tens to hundreds. Our data supports the notion of important roles of the PKA and CamKIID kinases in the response but we further provide evidence for involvement of the AMPK and AKT kinases. We also show that 6 ion channels and transporters that are important regulators of cardiac excitability have increased phosphorylation levels. For the Kv7.1 voltage-gated potassium channel which controls cardiac repolarization we demonstrated that βAR induced phosphorylation of S92 occurs on channels residing at the plasma membrane and that phosphorylation increases the current conduction of the channel. In a physiological context this obtaining provides mechanistic insight into how a faster repolarization of cardiomyocytes is usually supported by the Kv7.1 channel upon βAR stimulation which is required for a faster heart rate. By providing molecular details of.