Phosphoproteomics study based on in vivo inhibition reveals sites of calmodulin-dependent protein kinase II regulation in the heart.
BACKGROUND
The multifunctional Ca(2+)- and calmodulin-dependent 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 large-scale analysis of phosphorylation events by mass spectrometry. However, current large-scale phosphoproteomics approaches have proved inadequate for high-fidelity identification of kinase-specific roles. The purpose of this study was to develop a phosphoproteomics approach to specifically identify CaMKII's downstream effects in cardiac tissue.


RESULTS
To identify putative downstream CaMKII targets in cardiac tissue, animals with myocardial-delimited expression of the specific peptide inhibitor of CaMKII (AC3-I) or an inactive control (AC3-C) were compared using quantitative phosphoproteomics. The hearts were isolated after isoproterenol injection to induce CaMKII activation downstream of β-adrenergic receptor agonist stimulation. Enriched phosphopeptides from AC3-I and AC3-C mice were differentially quantified using stable isotope dimethyl labeling, strong cation exchange chromatography and high-resolution LC-MS/MS. Phosphorylation levels of several hundred sites could be profiled, including 39 phosphoproteins noticeably affected by AC3-I-mediated 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.
Biomolecular Mass Spectrometry & Proteomics, Utrecht Institute for Pharmaceutical Sciences and Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands.
Arjen
A
Scholten
Christian
C
Preisinger
Eleonora
E
Corradini
Vincent J
VJ
Bourgonje
Marco L
ML
Hennrich
Toon A B
TA
van Veen
Paari D
PD
Swaminathan
Mei-Ling
ML
Joiner
Marc A
MA
Vos
Mark E
ME
Anderson
Albert J R
AJ
Heck
D000595:Amino Acid Sequence; D000818:Animals; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002411:Cation Exchange Resins; D002852:Chromatography, Ion Exchange; D004789:Enzyme Activation; D007553:Isotope Labeling; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology*; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods; D015398:Signal Transduction; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry
C527819:AC3-I peptide; D002411:Cation Exchange Resins; D010455:Peptides; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2
CaMKII; mass spectrometry; phosphorylation; proteomics; transgenic mouse model