Reconstitution and use of highly active human CDK1:Cyclin-B:CKS1 complexes

Abstract

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 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 CDK1 activating kinases (CAK) or in insect cells through CDK-CAK co-expression. To boost kinase processivity, we reconstituted a tripartite 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).

Conformational transitions of the Spindly adaptor underlie its interaction with Dynein and Dynactin

“…The eluate was pooled, concentrated, snap-frozen, and stored at −80°C until use. Pre-dephosphorylated RZZ and Spindly were phosphorylated with the mitotic kinases CDK1:CyclinB, MPS1, and Aurora B, which were all purified in-house ( Huis In ’t Veld et al, 2021 ; Raisch et al, 2022 ; Sessa et al, 2005 ). Proteins were diluted to 10 µM in Spindly buffer, and kinases were added at a final concentration of 500 nM, and incubated on ice for 15 min.…”

Conformational transitions of the mitotic adaptor Spindly underlie its interaction with Dynein and Dynactin

“…The eluate was pooled, concentrated, snap-frozen and stored at −80° C until use. Pre-dephosphorylated RZZ and Spindly were phosphorylated with the mitotic kinases CDK1:CyclinB, MPS1, and Aurora B, which were all purified in-house (Huis In ’t Veld et al, 2021; Raisch et al, 2021; Sessa et al, 2005). Proteins were diluted to 10 µM in Spindly buffer, and kinases were added at a final concentration of 500 nM, and incubated on ice for 15 minutes.…”

Master mitotic kinases regulate viral genome delivery during papillomavirus cell entry

“…Subsequently, buffer exchange was performed by dialysis into refolding buffer (0.5 M L-Arginine, 10 mM Tris, 200 mM NaCl and 5% glycerol, pH 7.5) at 4 °C for 16 h. Following centrifugation at 24,000 × g at 4 °C for 8 h, refolded L2 amount was quantified by Coomassie Brilliant Blue staining using a standard bovine albumin curve. Purified 6xHis-PLK1 and the CDK1-CyclinB:CKS1 complex were kindly provided by Marion Pesenti (Musacchio Lab) 57 , 93 . A molar kinase-to-substrate ratio of 1:50 was adopted for all reactions using 100 nM of PLK1 and/or CDK1-CyclinB:CKS1 and 5 µM of WT or SSTP212AAAA mutant L2.…”