Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Structural basis for activation of the titin kinase domain during myofibrillogenesis

Nature volume 395, pages 863–869 (1998)Cite this article

An Erratum to this article was published on 25 February 1999

Abstract

The giant muscle protein titin (connectin) is essential in the temporal and spatial control of the assembly of the highly ordered sarcomeres (contractile units) of striated muscle. Here we present the crystal structure of titin's only catalytic domain, an autoregulated serine kinase (titin kinase). The structure shows how the active site is inhibited by a tyrosine of the kinase domain. We describe a dual mechanism of activation of titin kinase that consists of phosphorylation of this tyrosine and binding of calcium/calmodulin to the regulatory tail. The serine kinase domain of titin is the first known non-arginine–aspartate kinase to be activated by phosphorylation. The phosphorylated tyrosine is not located in the activation segment, as in other kinases, but in the P+ 1 loop, indicating that this tyrosine is a binding partner of the titinkinase substrate. Titin kinase phosphorylates the muscle protein telethonin in early differentiating myocytes, indicating that this kinase may act in myofibrillogenesis.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Sequence alignment of active-site regions.
Figure 2: Titin kinase constructs used in this study.
Figure 3: The three-dimensional structure of the autoinhibited form of titin kinase.
Figure 4: Active-site conformation of the autoinhibited forms of titin kinase, twitchin and IRK.
Figure 5: Titin kinase is tyrosine-phosphorylated on Y170 in differentiating C2C12 cells.
Figure 6: Titin kinase phosphorylates the muscle protein telethonin.
Figure 7: Full activation of titin kinase requires both tyrosine phosphorylation and Ca2+/calmodulin.

Similar content being viewed by others

References

  1. Taylor, S. S., Radzio-Andelzelm, E. & Hunter, T. How do protein kinases discriminate between serine/threonine and tyrosine? Structural insights from the insulin receptor tyrosine kinase. FASEB J. 9, 1255–1266 (1995).

    Article  CAS  Google Scholar 

  2. Johnson, L. N., Noble, M. E. M. & Owen, D. J. Active and inactive protein kinases: structural basis for regulation. Cell 85, 149–158 (1996).

    Article  CAS  Google Scholar 

  3. Johnson, L. N., Lowe, E. D., Noble, M. E. M. & Owen, D. J. The structural basis for substrate recognition and control by protein kinases. FEBS Lett. 430, 1–11 (1998).

    Article  CAS  Google Scholar 

  4. Trinick, J. Titin as a scaffold and spring. Curr. Biol. 6, 258–260 (1996).

    Article  CAS  Google Scholar 

  5. Maruyama, K. Connectin/titin, giant elastic protein of muscle. FASEB J. 11, 341–345 (1997).

    Article  CAS  Google Scholar 

  6. Heierhorst, J.et al. Autophosphorylation of molluscan twitchin and interaction of its kinase domain with calcium/calmodulin. J. Biol. Chem. 269, 21086–21093 (1994).

    CAS  PubMed  Google Scholar 

  7. Vibert, P., Edelstein, S. M., Castellani, L. & Elliot, B. W. Mini-titins in striated and smooth molluscan muscles: structure, location and immunological crossreactivity. J. Muscle Res. Cell Motil. 14, 598–607 (1993).

    Article  CAS  Google Scholar 

  8. Obermann, W. M. J.et al. The structure of the sarcomeric M band: localization of defined domains of myomesin, M.-protein and the 250 kD carboxy-terminal region of titin by immunoelectron microscopy. J. Cell Biol. 134, 1441–1453 (1996).

    Article  CAS  Google Scholar 

  9. Gautel, M.et al. Acalmodulin-binding sequence in the C-terminus of human cardiac titin kinase. Eur. J. Biochem. 230, 752–759 (1995).

    Article  CAS  Google Scholar 

  10. Kobe, B.et al. Giant protein kinases: domain interactions and structural basis of autoregulation. EMBO J. 15, 6810–6821 (1996).

    Article  CAS  Google Scholar 

  11. Sebestyén, M. G., Fritz, J. D., Wolff, J. A. & Greaser, M. L. Primary structure of the kinase domain region of rabbit skeletal and cardiac titin. J. Muscle Res. Cell Motil. 17, 343–348 (1996).

    Article  Google Scholar 

  12. Heierhorst, J.et al. Ca2+/S100 regulation of giant protein kinases. Nature 380, 636–639 (1996).

    Article  ADS  CAS  Google Scholar 

  13. Goldberg, J., Nairn, A. C. & Kuriyan, J. Structural basis for the auto-inhibition of calcium/calmodulin-dependent protein kinase I. Cell 84, 875–887 (1996).

    Article  CAS  Google Scholar 

  14. Crivici, A. & Ikura, M. Modular and structural basis of target recognition by calmodulin. Annu. Rev. Biomol. Struct. 24, 85–116 (1995).

    Article  CAS  Google Scholar 

  15. Heierhorst, J.et al. Phosphorylation of myosin regulatory light chains by the molluscan twitchin kinase. Eur. J. Biochem. 233, 426–431 (1995).

    Article  CAS  Google Scholar 

  16. Chin, D., Winkler, K. E. & Means, A. R. Characterisation of substrate phosphorylation and use of calmodulin mutants to address implications from the enzyme crystal structure of calmodulin-dependent protein kinase I. J. Biol. Chem. 272, 31235–31240 (1997).

    Article  CAS  Google Scholar 

  17. Hubbard, S. R. Crystal structure of the activated insulin receptor tyrosine kinase in complex with peptide substrate and ATP analog. EMBO J. 16, 5572–5581 (1997).

    Article  CAS  Google Scholar 

  18. Zhang, F.et al. Atomic structure of the MAP kinase ERK2 at 2.3 å resolution. Nature 367, 704–711 (1994).

    Article  ADS  CAS  Google Scholar 

  19. Songyang, Z.et al. Astructural basis for substrate specificities of protein Ser/Thr kinases: primary sequence preference of casein kinase I and II, phosphorylatse kinase, calmodulin-dependent kinase II, CDK5 and Erk1. Mol. Cell. Biol. 16, 6486–6493 (1996).

    Article  CAS  Google Scholar 

  20. Lowe, E. D.et al. The crystal structure of a phosphorylase inase peptide substrate complex: kinase substrate recognition. EMBO J. 16, 6646–6658 (1997).

    Article  CAS  Google Scholar 

  21. Taylor, S. S.et al. Atemplate for the protein kinase family. Trends Biochem. Sci. 18, 84–89 (1993).

    Article  CAS  Google Scholar 

  22. Valle, G.et al. Telethonin, a novel sarcomeric protein of heart and skeletal muscle. FEBS Lett. 415, 163–168 (1997).

    Article  CAS  Google Scholar 

  23. Mues, A.et al. Two immunoglobulin-like domains of the Z-disk portion of titin interact in a conformation-dependent way with telethonin. FEBS Lett. 428, 111–114 (1998).

    Article  CAS  Google Scholar 

  24. Engelkamp, D., Schäfer, B. W., Erne, P. & Heizmann, C. W. S100 alpha, CAPL, and CACY: molecular cloning and expression analysis of three calcium-binding proteins from human heart. Biochemistry 31, 10258–10264 (1992).

    Article  CAS  Google Scholar 

  25. Hubbard, S. R., Wei, L., Ellis, L. & Hendrickson, W. A. Crystal structure of the tyrosine kinase domain of the human insulin receptor. Nature 372, 746–754 (1994).

    Article  ADS  CAS  Google Scholar 

  26. Heierhorst, J.et al. Substrate specificity and inhibitor sensitivity of Ca2+/S100-dependent protein kinases. Eur. J. Biochem. 242, 454–459 (1996).

    Article  CAS  Google Scholar 

  27. Songyang, Z.et al. Use of an oriented peptide library to determine the optimal substrates of protein kinases. Curr. Biol. 4, 973–982 (1994).

    Article  CAS  Google Scholar 

  28. Konishi, H.et al. Activation of protein kinase C by tyrosine phosphorylation in response to H2O2. Proc. Natl Acad. Sci. USA 94, 11233–11237 (1997).

    Article  ADS  CAS  Google Scholar 

  29. Andersson, S.et al. Cloning, structure, and expression of the mitochondrial cytochrome P-450 sterol 26-hydroxylase, a bile acid biosynthetic enzyme. J. Biol. Chem. 264, 8222–8229 (1989).

    CAS  PubMed  Google Scholar 

  30. Young, P., Ferguson, C., Bañuelos, S. & Gautel, M. Molecular structure of the sarcomeric Z-disk: two types of titin interactions lead to an asymmetrical sorting of α-actinin. EMBO J. 17, 1614–1624 (1998).

    Article  CAS  Google Scholar 

  31. Cohen, O., Feinstein, E. & Kimchi, A. DAP-kinase is a Ca2+/calmodulin-dependent, cytoskeletal-associated protein kinase, with cell-death inducing functions that depend on its catalytic activity. EMBO J. 16, 998–1008 (1997).

    Article  CAS  Google Scholar 

  32. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685 (1970).

    Article  ADS  CAS  Google Scholar 

  33. Weijland, A.et al. The purification and characterization of the catalytic domain of Src expressed in Schizosaccharomyces pombe. Eur. J. Biochem. 240, 756–764 (1996).

    Article  CAS  Google Scholar 

  34. Wilm, M. & Mann, M. Analytical properties of the nanoelectrospray ion source. Anal. Chem. 68, 1–8 (1996).

    Article  CAS  Google Scholar 

  35. Dedman, J. R. & Kaetzel, M. A. Calmodulin purification and fluorescence labeling. Methods Enzymol. 102, 1–8 (1983).

    Article  CAS  Google Scholar 

  36. Chayen, N. E. Anovel technique to control the rate of vapour diffusion, giving larger protein crystals. J. Appl. Crystallogr. 30, 198–202 (1997).

    Article  CAS  Google Scholar 

  37. Otkinowski, Z. in Proceedings of the CCP4 Study Weekend (eds Sawyer, L., Isaacs, N. & Bailey, S.) 56–62 (SERC Daresbury Lab., (1993)).

    Google Scholar 

  38. Navaza, J. AMoRe: an automated package for molecular replacement. Acta Crystallogr. A 50, 577–587 (1994).

    Article  Google Scholar 

  39. Collaborative Computaitonal Project 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–767 (1994).

  40. Kleywegt, G. T. & Jones, T. A. in Proceedings of the CCP4 Study Weekend (eds Sawyer, L., Isaacs, N. & Bailey, S.) 56–62 (SERC, Daresbury Lab., (1994)).

    Google Scholar 

  41. Brünger, A. T., Kuriyan, J. & Karplus, M. Crystallographic R factor refinement by molecular dynamics. Science 235, 458–466 (1987).

    Article  ADS  Google Scholar 

  42. Tan, J. L. & Spudich, J. Characterization and bacterial expression of the Dictyostelium myosin light chain kinase cDNA. J. Biol. Chem. 266, 16044–16049 (1991).

    CAS  PubMed  Google Scholar 

  43. Cohen, G. E. Aprogram to superimpose protein coordinates, accounting for insertions and deletions. J. Appl. Crystallogr. 30, 1160–1161 (1997).

    Article  ADS  CAS  Google Scholar 

  44. Kraulis, P. J. MOLSCRIPT: a program to produce both detailed and schematic plots of protein structure. J. Appl. Crystallogr. 24, 251–259 (1991).

    Article  Google Scholar 

  45. Nicholls, A., Sharp, K. A. & Honig, B. Protein folding and association: insight from the interfacial and thermodynamic properties of hydrocarbons. Proteins 11, 281–296 (1991).

    Article  CAS  Google Scholar 

  46. Jones, T. A. Diffraction methods for biological macromolecules. Interactive computer graphics: FRODO. Methods Enzymol. 115, 157–171 (1985).

    Article  CAS  Google Scholar 

  47. Fürst, D. O., Osborn, M., Nave, R. & Weber, K. The organization of titin filaments in the half-sarcomere revealed by monoclonal antibodies in immunoelectron microscopy; a map of ten non-repetitive epitopes starting at the Z line extends close to the M line. J. Cell Biol. 106, 1563–1572 (1988).

    Article  Google Scholar 

Download references

Acknowledgements

We thank M. Saraste for support in the early stages of this project and J. Heierhorst for stimulating and fruitful discussions.

Author information

Authors and Affiliations

  1. European Molecular Biology Laboratory, Hamburg Outstation c/o DESY, Notkestrasse 85, D-22603, Hamburg, Germany

    Olga Mayans & Matthias Wilmanns

  2. Department for Cell Biology, University of Potsdam, Lenneéstrasse, D-14471 7a, Potsdam, Germany

    Peter F. M. van der Ven & Dieter O. Fürst

  3. European Molecular Biology Laboratory Heidelberg, Meyerhofstrasse 1, Postfach 10 22 09, Heidelberg, D-69012, Germany

    Matthias Wilm, Alexander Mues, Paul Young & Mathias Gautel

Authors
  1. Olga Mayans
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Peter F. M. van der Ven
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Matthias Wilm
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Alexander Mues
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Paul Young
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Dieter O. Fürst
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Matthias Wilmanns
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Mathias Gautel
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Matthias Wilmanns or Mathias Gautel.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Mayans, O., van der Ven, P., Wilm, M. et al. Structural basis for activation of the titin kinase domain during myofibrillogenesis. Nature 395, 863–869 (1998). https://doi.org/10.1038/27603

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/27603

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing