Abstract
Protein phosphorylation plays an important role in physiological processes, such as muscle contraction. Phospho-specific antibodies have become powerful tools to study these processes. Cardiac myosin binding protein-C (cMyBP-C) is one of the proteins that make up the contractile apparatus of cardiomyocytes. Phosphorylation of cMyBP-C is essential for normal cardiac function, since dephosphorylation of this protein leads to its degradation and has been associated with cardiomyopathy. One of the upstream kinases, which phosphorylate cMyBP-C, is protein kinase D (PKD). While studying the role of PKD in cMyBP-C phosphorylation, we tried to analyze phosphorylation of PKD with a phospho-specific PKD-Ser744/748 antibody. Contrary to the expected 115 kDa, a signal was found for a 150-kDa protein. By MALDI-TOF mass spectrometry, we identified this protein to be cMyBP-C. These data were confirmed by immunostaining using the p-PKD-Ser744/748 antibody, which displayed a striated pattern similar to the one observed for a regular cMyBP-C antibody. To our knowledge there are no antibodies commercially available for phosphorylated cMyBP-C. Thus, the p-PKD-Ser744/748 antibody can accelerate research into the role of cMyBP-C phosphorylation in cardiomyocytes.
Highlights
Reversible phosphorylation changes kinetic properties of proteins
This is in agreement with previous observations which showed that oligomycin-treatment of cardiomyocytes did not result in translocation of various PKCs [8] and showed that this type of protein kinase D (PKD) activation is independent of Ser744/748 phosphorylation
Lysates from control and oligomycin-treated cardiomyocytes were dissolved in urea buffer and subjected to western blotting and probed with p-PKD-Ser744/748 antibody to confirm PKD-Ser744/748 phosphorylation and its location on the gel (PE treatment was used as a control) (Figure 2(a))
Summary
Reversible phosphorylation changes kinetic properties of proteins. As a result, protein kinases can modify the function of a protein in almost every conceivable way. A decrease in cMyBP-C interferes with crossbridge formation that has been demonstrated to depend on cMyBP-C phosphorylation through enzymes such as Ca2+-calmodulin-activated kinase II, protein kinase A and protein kinase C [5]. E. Dirkx et al / Advances in Bioscience and Biotechnology 4 (2013) 1-6 important because cMyBP-C phosphorylation has been suggested as a biomarker for diagnosing myocardial infarction and as a potential target for therapeutic intervention [5,6]. Dirkx et al / Advances in Bioscience and Biotechnology 4 (2013) 1-6 important because cMyBP-C phosphorylation has been suggested as a biomarker for diagnosing myocardial infarction and as a potential target for therapeutic intervention [5,6] Such medical applications await an improved insight in the role of cMyBP-C phosphorylation in cardiac contraction. Further investigations showed that this phospho-PKD “specific” antibody recognized cMyBP-C phosphorylation, making this antibody an important tool in further research into the function of cMyBP-C
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