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Detection of cardiac myosin binding protein-C (cMyBP-C) by a phospho-specific PKD antibody in contracting rat cardiomyocytes

¹ Department of Molecular Genetics, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands
² Department of Human Biology, Nutrition and Toxicology Research Institute Maastricht (NUTRIM), Maastricht University, Maastricht, The Netherlands
³ Protein Phosphorylation Unit, Université Catholique de Louvain, Brussels, Belgium
⁴ Department of Human Biology, Nutrition and Toxicology Research Institute Maastricht (NUTRIM), Maastricht University, Maastricht, The Netherlands
⁵ Department of Cell and Molecular Physiology, Stritch School of Medicine, Loyola University, Chicago, USA
⁶ Department of Molecular Genetics, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands
⁷ Department of Molecular Genetics, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands
⁸ Department of Molecular Genetics, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands

Advances in Bioscience and Biotechnology·Volume 04 (2013)·Pages 1–6·Published 29 April 2013·DOI10.4236/abb.2013.44A001

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 observ ed for a regular cMyBP-C antibody. To our knowl edge there are no antibodies commercially availa ble for phosphorylated cMyBP-C. Thus, the p-PKD- Ser744/748 antibody can accelerate research into th e role of cMyBP-C phosphorylation in cardiomyo cytes.

KeywordsProtein Kinase DCardiac Myosin Binding Protein-CPhospho-Specific AntibodyProtein Phosphorylation

Cohen, P. (2002) The origins of protein phosphorylation. Nature Cell Biology, 4, E127-E130. doi:10.1038/ncb0502-e127
Decker, R.S., Decker, M.L., Kulikovskaya, I., Nakamura, S., Lee, D.C., Harris, K., Klocke, F.J. and Winegrad, S. (2005) Myosin-binding protein c phosphorylation, myofibril structure and contractile function during low-flow ischemia. Circulation, 111, 906-912. doi:10.1161/01.CIR.0000155609.95618.75
Jacques, A.M., Copeland, O., Messer, A.E., Gallon, C.E., King, K., McKenna, W.J., Tsang, V.T. and Marston, S.B. (2008) Myosin-binding protein c phosphorylation in normal, hypertrophic and failing human heart muscle. Journal of Molecular and Cellular Cardiology, 45, 209- 216. doi:10.1016/j.yjmcc.2008.05.020
Sadayappan, S., Osinska, H., Klevitsky, R., Lorenz, J.N., Sargent, M., Molkentin, J.D., Seidman, C.E., Seidman, J. G. and Robbins, J. (2006) Cardiac myosin-binding protein c phosphorylation is cardioprotective. Proceedings of the National Academy of Sciences of the United States of America, 103, 16918-16923. doi:10.1073/pnas.0607069103
Barefield, D. and Sadayappan, S. (2010) Phosphorylation and function of cardiac myosin-binding protein c in health and disease. Journal of Molecular and Cellular Cardiology, 48, 866-875. doi:10.1016/j.yjmcc.2009.11.014
Schlossarek, S., Mearini, G. and Carrier, L. (2011) Cardiac myosin-binding protein c in hypertrophic cardiomyopathy: Mechanisms and therapeutic opportunities. Journal of Molecular and Cellular Cardiology, 50, 613- 620. doi:10.1016/j.yjmcc.2011.01.014
Rykx, A., De Kimpe, L., Mikhalap, S., Vantus, T., Seufferlein, T., Vandenheede, J.R. and Van Lint, J. (2003) Protein kinase D: A family affair. FEBS Letters, 546, 81- 86. doi:10.1016/S0014-5793(03)00487-3
Luiken, J.J., Vertommen, D., Coort, S.L., Habets, D.D., El Hasnaoui, M., Pelsers, M.M., Viollet, B., Bonen, A., Hue, L., Rider, M.H. and Glatz, J.F. (2008) Identification of protein kinase D as a novel contraction-activated kinase linked to GLUT4-mediated glucose uptake, independent of AMPK. Cell Signal, 20, 543-556. doi:10.1016/j.cellsig.2007.11.007
Fielitz, J., Kim, M.S., Shelton, J.M., Qi, X., Hill, J.A., Richardson, J.A., Bassel-Duby, R. and Olson, E.N. (2008) Requirements of protein kinase d1 for pathological remodeling. Proceedings of the National Academy of Sciences of the United States of America, 105, 3059-3063. doi:10.1073/pnas.0712265105
Bardswell, S.C., Cuello, F., Rowland, A.J., Sadayappan, S., Robbins, J., Gautel, M., Walker, J.W., Kentish, J.C. and Avkiran, M. (2010) Distinct sarcomeric substrates are responsible for protein kinase D-mediated regulation of cardiac myofilament Ca2+ sensitivity and cross-bridge cycling. The Journal of Biological Chemistry, 285, 5674- 5682. doi:10.1074/jbc.M109.066456

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Detection of cardiac myosin binding protein-C (cMyBP-C) by a phospho-specific PKD antibody in contracting rat cardiomyocytes

Abstract

References (17)