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Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
Published on: May 3, 2018
A critical function for Ser-282 in cardiac Myosin binding protein-C phosphorylation and cardiac function
Sakthivel Sadayappan1, James Gulick, Hanna Osinska
1Department of Cell and Molecular Physiology, Stritch School of Medicine, Loyola University Chicago, IL, USA.
Circulation Research
|May 21, 2011
Summary
Phosphorylation of cardiac myosin-binding protein-C (cMyBP-C) at Ser-282 is crucial for Ser-302 phosphorylation and normal cardiac function during beta-adrenergic stimulation. This study reveals Ser-282
Area of Science:
- Molecular and Cellular Cardiology
- Cardiac Muscle Physiology
- Protein Phosphorylation
Background:
- Cardiac myosin-binding protein-C (cMyBP-C) phosphorylation regulates myocardial contractility.
- Specific phosphorylation sites (Ser-273, Ser-282, Ser-302) have suggested nonequivalence.
- Ser-282 phosphorylation's role in overall cMyBP-C phosphorylation and cardiac function remains to be fully defined.
Purpose of the Study:
- To investigate if complete cMyBP-C phosphorylation is dependent on Ser-282 phosphorylation.
- To determine the specific role of Ser-282 phosphorylation in regulating myocardial function, particularly during beta-adrenergic stimulation.
- To test the hypothesis that Ser-282 regulates Ser-302 phosphorylation and cardiac function.
Main Methods:
- In vitro phosphorylation assays using recombinant cMyBP-C peptides with various kinases (PKA, PKC, CaMKII).
- Generation of transgenic mouse lines expressing cMyBP-C with specific serine-to-alanine or serine-to-aspartate substitutions (cMyBP-C(SAS), cMyBP-C(ADA), cMyBP-C(DAD)).
- Breeding of transgenic lines into a cMyBP-C null background (t/t) for complete protein substitution.
- In vivo hemodynamic studies and beta-agonist infusion to assess cardiac function.
Main Results:
- In vitro, PKA phosphorylated Ser-273, Ser-282, and Ser-302; PKC phosphorylated Ser-273 and Ser-302; CaMKII phosphorylated Ser-302, and Ser-282 at high calcium.
- CaMKII-mediated Ser-302 phosphorylation was abolished by ablating Ser-282 phosphorylation (Ser-282-Ala substitution).
- Transgenic mice (cMyBP-C(SAS(t/t)), cMyBP-C(ADA(t/t)), cMyBP-C(DAD(t/t)))) showed partially rescued cMyBP-C null phenotype without increased morbidity.
- Loss of Ser-282 phosphorylation altered the beta-adrenergic response, decreasing cardiac function augmentation.
- Diastolic function was reduced at baseline in cMyBP-C(SAS(t/t)) mice.
Conclusions:
- Ser-282 phosphorylation is critical for subsequent Ser-302 phosphorylation, highlighting its unique regulatory role.
- While all studied residues contribute to beta-agonist response regulation, Ser-282 plays a pivotal role in the phosphorylation cascade.
- Complete cMyBP-C phosphorylation at multiple sites is essential for normal beta-adrenergic responsiveness in the heart.
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