Recombinant cyclin B-Cdk1-Suc1 capable of multi-site mitotic phosphorylation in vitro

DOI DOI Web Site Web Site 被引用文献1件 参考文献42件 オープンアクセス

書誌事項

公開日
2024-03-25
資源種別
journal article
権利情報
  • http://creativecommons.org/licenses/by/4.0/
DOI
  • 10.1371/journal.pone.0299003
  • 10.1101/2023.09.14.557837
公開者
Public Library of Science (PLoS)

説明

<jats:p>Cyclin-dependent kinase 1 (Cdk1) complexed with cyclin B phosphorylates multiple sites on hundreds of proteins during mitosis. However, it is not fully understood how multi-site mitotic phosphorylation by cyclin B-Cdk1 controls the structures and functions of individual substrates. Here we develop an easy-to-use protocol to express recombinant vertebrate cyclin B and Cdk1 in insect cells from a single baculovirus vector and to purify their complexes with excellent homogeneity. A series of <jats:italic>in-vitro</jats:italic> assays demonstrate that the recombinant cyclin B-Cdk1 can efficiently and specifically phosphorylate the SP and TP motifs in substrates. The addition of Suc1 (a Cks1 homolog in fission yeast) accelerates multi-site phosphorylation of an artificial substrate containing TP motifs. Importantly, we show that mitosis-specific multi-subunit and multi-site phosphorylation of the condensin I complex can be recapitulated <jats:italic>in vitro</jats:italic> using recombinant cyclin B-Cdk1-Suc1. The materials and protocols described here will pave the way for dissecting the biochemical basis of critical mitotic processes that accompany Cdk1-mediated large-scale phosphorylation.</jats:p>

収録刊行物

  • PLOS ONE

    PLOS ONE 19 (3), e0299003-, 2024-03-25

    Public Library of Science (PLoS)

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