Investigation of the Phosphorylation of theC-terminal domains of the cardiac MyosinBinding Protein C by the 5-AMP-activatedProtein Kinase
The existence of MyBP-C in striated muscle has been known for over 35 years and about 150 mutations in the gene encoding cMyBP-C have been found to be a common cause of hypertrophic cardiomyopathy. Despite this, the structure and function of MyBP-C remains less well understood than most other sarc...
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|Summary:||The existence of MyBP-C in striated muscle has been known for over 35 years and
about 150 mutations in the gene encoding cMyBP-C have been found to be a common
cause of hypertrophic cardiomyopathy. Despite this, the structure and function of MyBP-C remains less well understood than most other sarcomeric proteins, with roles in both regulation of contraction and thick filament formation/stability being proposed. In addition to the well known interactions of MyBP-C with other proteins of the sarcomeric apparatus (LMM, titin, actin) and with PKA, CaMKK and PKC at the N-terminal end of the protein, the aim of this study was to investigate interactions of MyBP-Cs C-terminus with the 5-AMP-activated protein kinase. This enzyme came in the focus of research during the last decade as it appears to function in a plethora of cell processes. Further, it has been elucidated that mutations in PRKAG2, encoding for the γ2 subunit of AMPK, causes left ventricular hypertrophy associated with conduction system diseases (e.g. Wolf-Parkinson-White syndrome). Important questions that have to be answered for a better understanding of this issue are, beside others, the identification of the full repertoire of cardiac protein targets.
My project aimed at identifying the site or sites of AMPK phosphorylation within the C-terminal three domains of cMyBP-C as suggested by earlier yeast-two-hybridscreen data and biochemical work. The latter hinted that the C8 domain was most likely the target, and it is this fragment that my work began with. Having optimised the expression and purification of recombinant wild type MyBP-C C8 domain and a number of mutated C8 domains as discussed in Chapter 3, it was possible to disprove the hypothesis of phosphorylatable residues being in this domain. In contrast, it was revealed that a phosphorylatable serine moiety was present in the N-terminal leader of the recombinant protein, encoded by the vector pET-28a. This serine lies in the thrombin recognition sequence itself and its phosphorylation inhibits cleavage. However, it was shown in vitro that a phosphorylatable serine residue is located in the C10 domain of the protein and this further confirms the association of the C8-C10 fragment of MyBP-C with AMPK, first observed in the yeast two-hybrid assay. The hypotheses that arise from these results will be discussed in this chapter. Additionally, I showed that the N-terminal domains of cMyBP-C (C0-C2), which contain the well characterized PKA and CaMII sites, are not a good substrate for AMPK in vitro.|