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Identification of novel protein kinase A phosphorylation sites in the M-domain of human and murine cardiac myosin binding protein-C using mass spectrometry analysis.

ABSTRACT: Cardiac myosin binding protein-C (cMyBP-C) is a large multidomain accessory protein bound to myosin thick filaments in striated muscle sarcomeres. It plays an important role in the regulation of muscle contraction, and mutations in the gene encoding cMyBP-C are a common cause of familial hypertrophic cardiomyopathy, the leading cause of sudden cardiac death in young people. (1) The N-terminal domains including the C0, C1, cMyBP-C motif, and C2 domains play a crucial role in maintaining and modulating actomyosin interactions (keeping normal cardiac function) in a phosphorylation-dependent manner. The cMyBP-C motif or "M-domain" is a highly conserved linker domain in the N-terminus of cMyBP-C that contains three to five protein kinase A (PKA) phosphorylation sites, depending on species. For the human isoform, three PKA sites were previously identified (Ser(275), Ser(284), and Ser(304)), while three homologous sites exist in the murine isoform (Ser(273), Ser(282), and Ser(302)). The murine cMyBP-C isoform contains an additional conserved consensus site, Ser(307) that is not present in the human isoform. In this study, we investigated sites of PKA phosphorylation of murine and human cMyBP-C by treating the recombinant protein C0C2 ( approximately 50 KDa, which contains the N-terminal C0, C1, M, and C2 domains) and C1C2 (approximately 35 KDa, contains C1, M, and C2 domains) with PKA and assessing the phosphorylation states using SDS-PAGE with ProQ Diamond staining, and powerful hybrid mass spectrometric analyses. Both high-accuracy bottom-up and measurements of intact proteins mass spectrometric approaches were used to determine the phosphorylation states of C0C2 and C1C2 proteins with or without PKA treatment. Herein, we report for the first time that there are four PKA phosphorylation sites in both murine and human M-domains; both murine Ser(307) and a novel human Ser(311) can be phosphorylated in vitro by PKA. Future studies are needed to investigate the phosphorylation state of murine and human cMyBP-C in vivo.

SUBMITTER: Jia W

PROVIDER: S-EPMC3000796 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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Identification of novel protein kinase A phosphorylation sites in the M-domain of human and murine cardiac myosin binding protein-C using mass spectrometry analysis.

Jia Weitao W Shaffer Justin F JF Harris Samantha P SP Leary Julie A JA

Journal of proteome research 20100401 4

Cardiac myosin binding protein-C (cMyBP-C) is a large multidomain accessory protein bound to myosin thick filaments in striated muscle sarcomeres. It plays an important role in the regulation of muscle contraction, and mutations in the gene encoding cMyBP-C are a common cause of familial hypertrophic cardiomyopathy, the leading cause of sudden cardiac death in young people. (1) The N-terminal domains including the C0, C1, cMyBP-C motif, and C2 domains play a crucial role in maintaining and modul ...[more]

PMID: 20151718

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Project description:BackgroundCardiac 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.ResultsDuring 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.ConclusionsThis 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.

Dataset Information

Identification of novel protein kinase A phosphorylation sites in the M-domain of human and murine cardiac myosin binding protein-C using mass spectrometry analysis.

Publications

Identification of novel protein kinase A phosphorylation sites in the M-domain of human and murine cardiac myosin binding protein-C using mass spectrometry analysis.

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