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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

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

  AUTHORS: Ellen Dirkx, Freek G. Bouwman, Didier Vertommen, Edwin C. Mariman, Sakthivel Sadayappan, Jan F. C. Glatz, Joost J. F. P. Luiken, Guillaume J. van Eys

  KEYWORDS: Protein Kinase D; Cardiac Myosin Binding Protein-C; Phospho-Specific Antibody; Protein Phosphorylation

  JOURNAL NAME: Advances in Bioscience and Biotechnology, Vol.4 No.4A, April 29, 2013

  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.