Raf-1: a novel cardiac troponin T kinase

  • Paul Pfleiderer
  • Marius P. Sumandea
  • Vitalyi O. Rybin
  • Chaojian Wang
  • Susan F. Steinberg
Original Paper

Abstract

Phosphorylation of cardiac troponin is a key mechanism involved in regulation of contractile function. In vitro kinase assays revealed that lysates prepared from resting cardiomyocytes contain cardiac troponin I (cTnI) and cTnT kinase activity. cTnI phosphorylation is inhibited by pharmacologic inhibitors of PKA, PKC, Rho kinase and PKC effectors such as RSK and PKD; these kinase inhibitors do not inhibit phosphorylation of cTnT. Rather, cTnT phosphorylation is decreased by the Raf inhibitor GW5074. In vitro kinase assays show that recombinant Raf phosphorylates cTnT, and that Raf-dependent cTnT phosphorylation is abrogated by a T206E substitution; Raf does not phosphorylate cTnI. These studies identify Raf-dependent cTnT-Thr206 phosphorylation as a novel mechanism that would link growth factor-dependent signaling pathways to dynamic changes in cardiac contractile function.

Keywords

Cardiac troponin T Raf-1 GW5074 Cardiomyocytes Phosphorylation 

Abbreviations

PKA

Protein kinase A

PKC

Protein kinase C

PKD

Protein kinase D

PDK

Phosphoinositide-dependent protein kinase

ASK-1

Apoptosis signal-regulating kinase-1

RSK

Ribosomal S6 kinase

ROCK

Rho-A-dependent kinase

Notes

Acknowledgments

This work was supported by United States Public Health Service—National Heart, Lung and Blood Institute grants HL-77860, HL-74161, and AHA-SDG 0335199 N.

References

  1. Bain J, Plater L, Elliott M, Shpiro N, Hastie CJ, McLauchlan H et al (2007) The selectivity of protein kinase inhibitors: a further update. Biochem J 408:297–315. doi: 10.1042/BJ20070797 PubMedCrossRefGoogle Scholar
  2. Guo J, Sabri A, Elouardighi H, Rybin V, Steinberg SF (2006) Alpha1-adrenergic receptors activate AKT via a Pyk2/PDK-1 pathway that is tonically inhibited by novel protein kinase C isoforms in cardiomyocytes. Circ Res 99:1367–1375. doi: 10.1161/01.RES.0000252830.01581.fd PubMedCrossRefGoogle Scholar
  3. Harris IS, Zhang S, Treskov I, Kovacs A, Weinheimer C, Muslin AJ (2004) Raf-1 kinase is required for cardiac hypertrophy and cardiomyocyte survival in response to pressure overload. Circulation 110:718–723. doi: 10.1161/01.CIR.0000138190.50127.6A PubMedCrossRefGoogle Scholar
  4. Haworth RS, Cuello F, Herron TJ, Franzen G, Kentish JC, Gautel M et al (2004) Protein kinase D is a novel mediator of cardiac troponin I phosphorylation and regulates myofilament function. Circ Res 95:1091–1099. doi: 10.1161/01.RES.0000149299.34793.3c PubMedCrossRefGoogle Scholar
  5. He X, Liu Y, Sharma V, Dirksen RT, Waugh R, Sheu SS et al (2003) ASK1 associates with troponin T and induces troponin T phosphorylation and contractile dysfunction in cardiomyocytes. Am J Pathol 163:243–251PubMedGoogle Scholar
  6. Itoh S, Ding B, Bains CP, Wang N, Takeishi Y, Jalili T et al (2005) Role of p90 ribosomal S6 kinase (p90RSK) in reactive oxygen species and protein kinase C beta (PKC-beta)-mediated cardiac troponin I phosphorylation. J Biol Chem 280:24135–24142. doi: 10.1074/jbc.M413015200 PubMedCrossRefGoogle Scholar
  7. Kobayashi T, Solaro RJ (2005) Calcium, thin filaments, and the integrative biology of cardiac contractility. Annu Rev Physiol 67:39–67. doi: 10.1146/annurev.physiol.67.040403.114025 PubMedCrossRefGoogle Scholar
  8. Komander D, Kular GS, Bain J, Elliott M, Alessi DR, Van Aalten DM (2003) Structural basis for UCN-01 (7-hydroxystaurosporine) specificity and PDK1 (3-phosphoinositide-dependent protein kinase-1) inhibition. Biochem J 375:255–262. doi: 10.1042/BJ20031119 PubMedCrossRefGoogle Scholar
  9. Layland J, Solaro RJ, Shah AM (2005) Regulation of cardiac contractile function by troponin I phosphorylation. Cardiovasc Res 66:12–21. doi: 10.1016/j.cardiores.2004.12.022 PubMedCrossRefGoogle Scholar
  10. Montgomery DE, Chandra M, Huang Q, Jin J, Solaro RJ (2001) Transgenic incorporation of skeletal TnT into cardiac myofilaments blunts PKC-mediated depression of force. Am J Physiol Heart Circ Physiol 280:H1011–H1018PubMedGoogle Scholar
  11. Montgomery DE, Wolska BM, Pyle WG, Roman BB, Dowell JC, Buttrick PM et al (2002) alpha-Adrenergic response and myofilament activity in mouse hearts lacking PKC phosphorylation sites on cardiac TnI. Am J Physiol Heart Circ Physiol 282:H2397–H2405PubMedGoogle Scholar
  12. Morimoto S (2008) Sarcomeric proteins and inherited cardiomyopathies. Cardiovasc Res 77:659–666. doi: 10.1093/cvr/cvm084 PubMedCrossRefGoogle Scholar
  13. Noland TA Jr, Raynor RL, Jideama NM, Guo X, Kazanietz MG, Blumberg PM et al (1996) Differential regulation of cardiac actomyosin S-1 MgATPase by protein kinase C isozyme-specific phosphorylation of specific sites in cardiac troponin I and its phosphorylation site mutants. Biochemistry 35:14923–14931. doi: 10.1021/bi9616357 PubMedCrossRefGoogle Scholar
  14. Rohde S, Sabri A, Kamasamudran R, Steinberg SF (2000) The alpha(1)-adrenoceptor subtype- and protein kinase C isoform-dependence of norepinephrine’s actions in cardiomyocytes. J Mol Cell Cardiol 32:1193–1209. doi: 10.1006/jmcc.2000.1153 PubMedCrossRefGoogle Scholar
  15. Sumandea MP, Pyle WG, Kobayashi T, de Tombe PP, Solaro RJ (2003) Identification of a functionally critical protein kinase C phosphorylation residue of cardiac troponin T. J Biol Chem 278:35135–35144. doi: 10.1074/jbc.M306325200 PubMedCrossRefGoogle Scholar
  16. Sumandea MP, Rybin VO, Hinken AC, Wang C, Kobayashi T, Harleton E et al (2008) Tyrosine phosphorylation modifies protein kinase c {delta}-dependent phosphorylation of cardiac troponin I. J Biol Chem 283:22680–22689. doi: 10.1074/jbc.M802396200 PubMedCrossRefGoogle Scholar
  17. Vahebi S, Kobayashi T, Warren CM, de Tombe PP, Solaro RJ (2005) Functional effects of rho-kinase-dependent phosphorylation of specific sites on cardiac troponin. Circ Res 96:740–747. doi: 10.1161/01.RES.0000162457.56568.7d PubMedCrossRefGoogle Scholar
  18. Yamaguchi O, Watanabe T, Nishida K, Kashiwase K, Higuchi Y, Takeda T et al (2004) Cardiac-specific disruption of the c-raf-1 gene induces cardiac dysfunction and apoptosis. J Clin Invest 114:937–943PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • Paul Pfleiderer
    • 1
  • Marius P. Sumandea
    • 2
  • Vitalyi O. Rybin
    • 1
  • Chaojian Wang
    • 1
  • Susan F. Steinberg
    • 1
  1. 1.Department of Pharmacology, College of Physicians and SurgeonsColumbia UniversityNew YorkUSA
  2. 2.Department of PhysiologyUniversity of KentuckyLexingtonUSA

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