Deciphering the interplay among multisite phosphorylation, interaction dynamics, and conformational transitions in a tripartite protein system

Philip Lössl, Andrea M. Brunner, Fan Liu, Aneika C. Leney, Masami Yamashita, Richard A. Scheltema, Albert J.R. Heck*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

20 Citations (Scopus)
216 Downloads (Pure)


Multisite phosphorylation is a common pathway to regulate protein function, activity, and interaction pattern in vivo, but routine biochemical analysis is often insufficient to identify the number and order of individual phosphorylation reactions and their mechanistic impact on the protein behavior. Here, we integrate complementary mass spectrometry (MS)-based approaches to characterize a multisite phosphorylation-regulated protein system comprising Polo-like kinase 1 (Plk1) and its coactivators Aurora kinase A (Aur-A) and Bora, the interplay of which is essential for mitotic entry after DNA damage-induced cell cycle arrest. Native MS and cross-linking-MS revealed that Aur-A/Bora-mediated Plk1 activation is accompanied by the formation of Aur-A/Bora and Plk1/Bora heterodimers. We found that the Aur-A/Bora interaction is independent of the Bora phosphorylation state, whereas the Plk1/ Bora interaction is dependent on extensive Bora multisite phosphorylation. Bottom-up and top-down proteomics analyses showed that Bora multisite phosphorylation proceeds via a well-ordered sequence of site-specific phosphorylation reactions, whereby we could reveal the involvement of up to 16 phosphorylated Bora residues. Ion mobility spectrometry-MS demonstrated that this multisite phosphorylation primes a substantial structural rearrangement of Bora, explaining the interdependence between extensive Bora multisite phosphorylation and Plk1/Bora complex formation. These results represent a first benchmark of our multipronged MS strategy, highlighting its potential to elucidate the mechanistic and structural implications of multisite protein phosphorylation.

Original languageEnglish
Pages (from-to)445-455
Number of pages11
JournalACS Central Science
Issue number7
Early online date10 Jun 2016
Publication statusPublished - 27 Jul 2016

Bibliographical note

ACS AuthorChoice - This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.

ASJC Scopus subject areas

  • Chemistry(all)
  • Chemical Engineering(all)


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