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

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Deciphering the interplay among multisite phosphorylation, interaction dynamics, and conformational transitions in a tripartite protein system. / Lössl, Philip; Brunner, Andrea M.; Liu, Fan; Leney, Aneika C.; Yamashita, Masami; Scheltema, Richard A.; Heck, Albert J.R.

In: ACS Central Science, Vol. 2, No. 7, 27.07.2016, p. 445-455.

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Lössl, Philip ; Brunner, Andrea M. ; Liu, Fan ; Leney, Aneika C. ; Yamashita, Masami ; Scheltema, Richard A. ; Heck, Albert J.R. / Deciphering the interplay among multisite phosphorylation, interaction dynamics, and conformational transitions in a tripartite protein system. In: ACS Central Science. 2016 ; Vol. 2, No. 7. pp. 445-455.

Bibtex

@article{2ef49a4247614dd8abc6659cfa10f027,
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.",
author = "Philip L{\"o}ssl and Brunner, {Andrea M.} and Fan Liu and Leney, {Aneika C.} and Masami Yamashita and Scheltema, {Richard A.} and Heck, {Albert J.R.}",
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.",
year = "2016",
month = jul,
day = "27",
doi = "10.1021/acscentsci.6b00053",
language = "English",
volume = "2",
pages = "445--455",
journal = "ACS Central Science",
issn = "2374-7951",
publisher = "American Chemical Society",
number = "7",

}

RIS

TY - JOUR

T1 - Deciphering the interplay among multisite phosphorylation, interaction dynamics, and conformational transitions in a tripartite protein system

AU - Lössl, Philip

AU - Brunner, Andrea M.

AU - Liu, Fan

AU - Leney, Aneika C.

AU - Yamashita, Masami

AU - Scheltema, Richard A.

AU - Heck, Albert J.R.

N1 - 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.

PY - 2016/7/27

Y1 - 2016/7/27

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=85010313292&partnerID=8YFLogxK

U2 - 10.1021/acscentsci.6b00053

DO - 10.1021/acscentsci.6b00053

M3 - Article

AN - SCOPUS:85010313292

VL - 2

SP - 445

EP - 455

JO - ACS Central Science

JF - ACS Central Science

SN - 2374-7951

IS - 7

ER -