Systematic Phosphorylation Analysis of Human Mitotic Protein Complexes

Sci. Signal., 8 November 2011
Vol. 4, Issue 198, p. rs12
DOI: 10.1126/scisignal.2001993

Systematic Phosphorylation Analysis of Human Mitotic Protein Complexes

  1. Björn Hegemann1,*,,
  2. James R. A. Hutchins1,*,,
  3. Otto Hudecz1,2,
  4. Maria Novatchkova1,2,
  5. Jonathan Rameseder3,
  6. Martina M. Sykora1,
  7. Sihan Liu3,§,
  8. Michael Mazanek1,2,
  9. Péter Lénárt1,4,
  10. Jean-Karim Hériché4,
  11. Ina Poser5,
  12. Norbert Kraut6,
  13. Anthony A. Hyman5,
  14. Michael B. Yaffe3,7,
  15. Karl Mechtler1,2, and
  16. Jan-Michael Peters1,
  1. 1Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7, 1030 Vienna, Austria.
  2. 2Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Dr. Bohr-Gasse 3, 1030 Vienna, Austria.
  3. 3Departments of Biology and Biological Engineering, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
  4. 4European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
  5. 5Max Planck Institute for Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany.
  6. 6Boehringer Ingelheim RCV GmbH & Co KG, Dr. Boehringer-Gasse 5-11, 1121 Vienna, Austria.
  7. 7Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Avenue, Boston, MA 02215, USA.
  1. To whom correspondence should be addressed. E-mail: peters{at}
  • * These authors contributed equally to this work.

  • Present address: Institute of Biochemistry, Eidgenössische Technische Hochschule Zürich, Schafmattstrasse 18, CH-8093 Zürich, Switzerland.

  • Present address: CNRS Institute of Human Genetics, 141 rue de la Cardonille, 34396 Montpellier, France.

  • § Present address: Computation and Systems Biology, Singapore-MIT Alliance, 4 Engineering Drive 3, Singapore 117576, Republic of Singapore.


Progression through mitosis depends on a large number of protein complexes that regulate the major structural and physiological changes necessary for faithful chromosome segregation. Most, if not all, of the mitotic processes are regulated by a set of mitotic protein kinases that control protein activity by phosphorylation. Although many mitotic phosphorylation events have been identified in proteome-scale mass spectrometry studies, information on how these phosphorylation sites are distributed within mitotic protein complexes and which kinases generate these phosphorylation sites is largely lacking. We used systematic protein-affinity purification combined with mass spectrometry to identify 1818 phosphorylation sites in more than 100 mitotic protein complexes. In many complexes, the phosphorylation sites were concentrated on a few subunits, suggesting that these subunits serve as “switchboards” to relay the kinase-regulatory signals within the complexes. Consequent bioinformatic analyses identified potential kinase-substrate relationships for most of these sites. In a subsequent in-depth analysis of key mitotic regulatory complexes with the Aurora kinase B (AURKB) inhibitor Hesperadin and a new Polo-like kinase (PLK1) inhibitor, BI 4834, we determined the kinase dependency for 172 phosphorylation sites on 41 proteins. Combination of the results of the cellular studies with Scansite motif prediction enabled us to identify 14 sites on six proteins as direct candidate substrates of AURKB or PLK1.


B. Hegemann, J. R. Hutchins, O. Hudecz, M. Novatchkova, J. Rameseder, M. M. Sykora, S. Liu, M. Mazanek, P. Lénárt, J.-K. Hériché, I. Poser, N. Kraut, A. A. Hyman, M. B. Yaffe, K. Mechtler, and J.-M. Peters, Systematic Phosphorylation Analysis of Human Mitotic Protein Complexes. Sci. Signal. 4, rs12 (2011).

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