title: Myomegalin is a novel A-kinase anchoring protein involved in the phosphorylation of cardiac myosin binding protein C.
abstract: BACKGROUND
  Cardiac contractility is regulated by dynamic phosphorylation of sarcomeric proteins by kinases such as cAMP-activated protein kinase A (PKA). Efficient phosphorylation requires that PKA be anchored close to its targets by A-kinase anchoring proteins (AKAPs). Cardiac Myosin Binding Protein-C (cMyBPC) and cardiac troponin I (cTNI) are hypertrophic cardiomyopathy (HCM)-causing sarcomeric proteins which regulate contractility in response to PKA phosphorylation.
  
  
  RESULTS
  During a yeast 2-hybrid (Y2H) library screen using a trisphosphorylation mimic of the C1-C2 region of cMyBPC, we identified isoform 4 of myomegalin (MMGL) as an interactor of this N-terminal cMyBPC region. As MMGL has previously been shown to interact with phosphodiesterase 4D, we speculated that it may be a PKA-anchoring protein (AKAP).To investigate this possibility, we assessed the ability of MMGL isoform 4 to interact with PKA regulatory subunits R1A and R2A using Y2H-based direct protein-protein interaction assays. Additionally, to further elucidate the function of MMGL, we used it as bait to screen a cardiac cDNA library. Other PKA targets, viz. CARP, COMMD4, ENO1, ENO3 and cTNI were identified as putative interactors, with cTNI being the most frequent interactor.We further assessed and confirmed these interactions by fluorescent 3D-co-localization in differentiated H9C2 cells as well as by in vivo co-immunoprecipitation. We also showed that quantitatively more interaction occurs between MMGL and cTNI under β-adrenergic stress. Moreover, siRNA-mediated knockdown of MMGL leads to reduction of cMyBPC levels under conditions of adrenergic stress, indicating that MMGL-assisted phosphorylation is requisite for protection of cMyBPC against proteolytic cleavage.
  
  
  CONCLUSIONS
  This study ascribes a novel function to MMGL isoform 4: it meets all criteria for classification as an AKAP, and we show that is involved in the phosphorylation of cMyBPC as well as cTNI, hence MMGL is an important regulator of cardiac contractility. This has further implications for understanding the patho-aetiology of HCM-causing mutations in the genes encoding cMyBPC and cTNI, and raises the question of whether MMGL might itself be considered a candidate HCM-causing or modifying factor.
authors.affiliation: US/MRC Centre for Molecular and Cellular Biology, Department of Biomedical Sciences, University of Stellenbosch, South Africa.
authors.forename: Gerrida M
authors.initials: GM
authors.lastname: Uys
authors.forename: Amsha
authors.initials: A
authors.lastname: Ramburan
authors.forename: Benjamin
authors.initials: B
authors.lastname: Loos
authors.forename: Craig J
authors.initials: CJ
authors.lastname: Kinnear
authors.forename: Lundi J
authors.initials: LJ
authors.lastname: Korkie
authors.forename: Jomien
authors.initials: J
authors.lastname: Mouton
authors.forename: Johann
authors.initials: J
authors.lastname: Riedemann
authors.forename: Johanna C
authors.initials: JC
authors.lastname: Moolman-Smook
mesh_terms: D054758:A Kinase Anchor Proteins / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000378:metabolism*; D010766:Phosphorylation; D054730:Protein Interaction Domains and Motifs; D020033:Protein Isoforms / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D011943:Receptors, Adrenergic, beta / Q000378:metabolism; D019210:Troponin I / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D000099163:Myosin Binding Protein C
chemical_list: D054758:A Kinase Anchor Proteins; D002352:Carrier Proteins; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D008869:Microtubule-Associated Proteins; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D011943:Receptors, Adrenergic, beta; D019210:Troponin I; D000099163:Myosin Binding Protein C; C426025:Pde4dip protein, rat