Recombinant cyclin B-Cdk1-Suc1 capable of multi-site mitotic phosphorylation in vitro.
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 a...
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2024-01-01
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| Series: | PLoS ONE |
| Online Access: | https://doi.org/10.1371/journal.pone.0299003 |
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| author | Keishi Shintomi Yuki Masahara-Negishi Masami Shima Shoji Tane Tatsuya Hirano |
| author_facet | Keishi Shintomi Yuki Masahara-Negishi Masami Shima Shoji Tane Tatsuya Hirano |
| author_sort | Keishi Shintomi |
| collection | DOAJ |
| description | 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 in-vitro 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 in vitro 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. |
| format | Article |
| id | doaj-art-3a2f855061af4b0e88a07d544c2fa978 |
| institution | Kabale University |
| issn | 1932-6203 |
| language | English |
| publishDate | 2024-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj-art-3a2f855061af4b0e88a07d544c2fa9782025-08-20T03:37:58ZengPublic Library of Science (PLoS)PLoS ONE1932-62032024-01-01193e029900310.1371/journal.pone.0299003Recombinant cyclin B-Cdk1-Suc1 capable of multi-site mitotic phosphorylation in vitro.Keishi ShintomiYuki Masahara-NegishiMasami ShimaShoji TaneTatsuya HiranoCyclin-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 in-vitro 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 in vitro 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.https://doi.org/10.1371/journal.pone.0299003 |
| spellingShingle | Keishi Shintomi Yuki Masahara-Negishi Masami Shima Shoji Tane Tatsuya Hirano Recombinant cyclin B-Cdk1-Suc1 capable of multi-site mitotic phosphorylation in vitro. PLoS ONE |
| title | Recombinant cyclin B-Cdk1-Suc1 capable of multi-site mitotic phosphorylation in vitro. |
| title_full | Recombinant cyclin B-Cdk1-Suc1 capable of multi-site mitotic phosphorylation in vitro. |
| title_fullStr | Recombinant cyclin B-Cdk1-Suc1 capable of multi-site mitotic phosphorylation in vitro. |
| title_full_unstemmed | Recombinant cyclin B-Cdk1-Suc1 capable of multi-site mitotic phosphorylation in vitro. |
| title_short | Recombinant cyclin B-Cdk1-Suc1 capable of multi-site mitotic phosphorylation in vitro. |
| title_sort | recombinant cyclin b cdk1 suc1 capable of multi site mitotic phosphorylation in vitro |
| url | https://doi.org/10.1371/journal.pone.0299003 |
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