title: Deciphering the Interplay among Multisite Phosphorylation, Interaction Dynamics, and Conformational Transitions in a Tripartite Protein System.
abstract: 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.
authors: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Padualaan 8, 3584CH Utrecht, The Netherlands; Netherlands Proteomics Center, Padualaan 8, 3584CH Utrecht, The Netherlands.
authors: Philip
authors: P
authors: Lössl
authors: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Padualaan 8, 3584CH Utrecht, The Netherlands; Netherlands Proteomics Center, Padualaan 8, 3584CH Utrecht, The Netherlands.
authors: Andrea M
authors: AM
authors: Brunner
authors: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Padualaan 8, 3584CH Utrecht, The Netherlands; Netherlands Proteomics Center, Padualaan 8, 3584CH Utrecht, The Netherlands.
authors: Fan
authors: F
authors: Liu
authors: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Padualaan 8, 3584CH Utrecht, The Netherlands; Netherlands Proteomics Center, Padualaan 8, 3584CH Utrecht, The Netherlands.
authors: Aneika C
authors: AC
authors: Leney
authors: Department of Structural Cell Biology, Max Planck Institute of Biochemistry , Am Klopferspitz 18, 82152 Martinsried, Germany.
authors: Masami
authors: M
authors: Yamashita
authors: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Padualaan 8, 3584CH Utrecht, The Netherlands; Netherlands Proteomics Center, Padualaan 8, 3584CH Utrecht, The Netherlands.
authors: Richard A
authors: RA
authors: Scheltema
authors: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Padualaan 8, 3584CH Utrecht, The Netherlands; Netherlands Proteomics Center, Padualaan 8, 3584CH Utrecht, The Netherlands.
authors: Albert J R
authors: AJ
authors: Heck