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

Weitao Jia, Justin F. Shaffer, Samantha P. Harris, Julie A. Leary

Research output: Contribution to journalArticle

44 Citations (Scopus)

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 (Ser275, Ser284, and Ser304), while three homologous sites exist in the murine isoform (Ser273, Ser282, and Ser302). 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 (∼50 KDa, which contains the N-terminal C0, C1, M, and C2 domains) and C1C2 (∼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 Ser307 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.

Original languageEnglish (US)
Pages (from-to)1843-1853
Number of pages11
JournalJournal of Proteome Research
Volume9
Issue number4
DOIs
StatePublished - Apr 5 2010
Externally publishedYes

Fingerprint

Cardiac Myosins
Phosphorylation
Protein Kinases
Cyclic AMP-Dependent Protein Kinases
Mass spectrometry
Mass Spectrometry
Protein Isoforms
Amino Acid Motifs
Muscle
Familial Hypertrophic Cardiomyopathy
Actomyosin
Sarcomeres
Proteins
Diamond
Gene encoding
Striated Muscle
myosin-binding protein C
Sudden Cardiac Death
Accessories
Myosins

Keywords

  • Bottom-up
  • Cardiac myosin binding protein-C
  • LTQ-Orbitrap
  • Mass spectrometry
  • Measurement of intact protein
  • Phosphorylation

ASJC Scopus subject areas

  • Biochemistry
  • Chemistry(all)

Cite this

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; Shaffer, Justin F.; Harris, Samantha P.; Leary, Julie A.

In: Journal of Proteome Research, Vol. 9, No. 4, 05.04.2010, p. 1843-1853.

Research output: Contribution to journalArticle

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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 (Ser275, Ser284, and Ser304), while three homologous sites exist in the murine isoform (Ser273, Ser282, and Ser302). 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 (∼50 KDa, which contains the N-terminal C0, C1, M, and C2 domains) and C1C2 (∼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 Ser307 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.",
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T1 - 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

AU - Jia, Weitao

AU - Shaffer, Justin F.

AU - Harris, Samantha P.

AU - Leary, Julie A.

PY - 2010/4/5

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N2 - 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 (Ser275, Ser284, and Ser304), while three homologous sites exist in the murine isoform (Ser273, Ser282, and Ser302). 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 (∼50 KDa, which contains the N-terminal C0, C1, M, and C2 domains) and C1C2 (∼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 Ser307 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.

AB - 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 (Ser275, Ser284, and Ser304), while three homologous sites exist in the murine isoform (Ser273, Ser282, and Ser302). 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 (∼50 KDa, which contains the N-terminal C0, C1, M, and C2 domains) and C1C2 (∼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 Ser307 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.

KW - Bottom-up

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