Reconstitution and use of highly active human CDK1:Cyclin-B:CKS1 complexes.
CDK1
CKS1
cell cycle
cyclin
cyclin dependent kinase
cyclin-B
phosphorylation
phostag
processivity
recombinant protein
Journal
Protein science : a publication of the Protein Society
ISSN: 1469-896X
Titre abrégé: Protein Sci
Pays: United States
ID NLM: 9211750
Informations de publication
Date de publication:
02 2022
02 2022
Historique:
revised:
31
10
2021
received:
24
09
2021
accepted:
02
11
2021
pubmed:
19
11
2021
medline:
22
3
2022
entrez:
18
11
2021
Statut:
ppublish
Résumé
As dividing cells transition into mitosis, hundreds of proteins are phosphorylated by a complex of cyclin-dependent kinase 1 (CDK1) and Cyclin-B, often at multiple sites. CDK1:Cyclin-B phosphorylation patterns alter conformations, interaction partners, and enzymatic activities of target proteins and need to be recapitulated in vitro for the structural and functional characterization of the mitotic protein machinery. This requires a pure and active recombinant kinase complex. The kinase activity of CDK1 critically depends on the phosphorylation of a Threonine residue in its activation loop by a CDK1-activating kinase (CAK). We developed protocols to activate CDK1:Cyclin-B either in vitro with purified CAKs or in insect cells through CDK-CAK co-expression. To boost kinase processivity, we reconstituted a ternary complex consisting of CDK1, Cyclin-B, and CKS1. In this work, we provide and compare detailed protocols to obtain and use highly active CDK1:Cyclin-B (CC) and CDK1:Cyclin-B:CKS1 (CCC).
Identifiants
pubmed: 34791727
doi: 10.1002/pro.4233
pmc: PMC8819839
doi:
Substances chimiques
CDC2 Protein Kinase
EC 2.7.11.22
CDK1 protein, human
EC 2.7.11.22
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
528-537Informations de copyright
© 2021 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.
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