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 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).

Keywords: CDK1; CKS1; cell cycle; cyclin; cyclin dependent kinase; cyclin-B; phosphorylation; phostag; processivity; recombinant protein.

FIGURE 1

Reconstitution of stoichiometric CDK1:Cyclin‐B and CDK1:Cyclin‐B:CKS1 complexes. (a) Surface view of a complex between CDK1 (green) and Cyclin‐B (purple) (PDB 4YC3). The CDK1^162‐173 activation loop is shown in gold and Threonine 161 in orange. Within the closed activation loop, residues CDK1^162‐164 (HEV) are not visible and replaced with a dashed line. (b) Surface view of CDK2:Cyclin‐A with bound ATP (PDB 1JST). The side chains of Arginines 50, 126, and 150 coordinate the activation loop with a phosphorylated Threonine 160. (c) Surface view of a tripartite CDK1:Cyclin‐B:CKS1 complex (PDB 4YC3). Compared to Panel (a), the structure is rotated 45° along the x‐axis. (d, e) Analysis of purified CDK1, Cyclin‐B, CKS1, CDK1:Cyclin‐B (CC), and CDK1:Cyclin‐B:CKS1 (CCC) by SDS‐PAGE followed by Coomassie staining and size exclusion chromatography using a Superdex 200 increase 5/150 column

FIGURE 2

Phosphorylation of CDK1 in insect cells and in vitro by a CDK activating kinase. (a) Reaction scheme for the phosphorylation of CDK1 with scCAK1. (b, c) SDS‐PAGE analysis of CDK1, Cyclin‐B, and scCAK1. The presence of phostag‐acrylamide (Panel c) slows the migration of phosphorylated CDK1. (d) Quantification of the phosphorylated and non‐phosphorylated forms of CDK1 from the phostag gel shown in Panel c

FIGURE 3

Phosphorylation of CDK1 Threonine 161 activates recombinant CDK1:Cyclin‐B. (a, b) Preparation and SDS‐PAGE analysis of CDK1:Cyclin‐B complexes that were either in vitro assembled from purified components or pre‐assembled into dimeric or trimeric complexes. Reactions (a) and (e) are technical replicates. (c) CDK1:Cyclin‐B complexes were analyzed by phostag SDS‐PAGE. The effect of scCAK1 or hsCDK7 co‐expression on the phostag‐migration of CDK1 (reactions b, c, d) is shown in Figure 2c, Lanes 1–3. (d) Six phosphorylated residues were detected by mass spectrometry in the CCC complex. Andromeda scores of the best‐identified phosphopeptides and the ratio of the intensities of phosphorylated (mod) over non‐modified (base) peptides are shown. (e) Five different peptides containing CDK1 Threonine 161 were detected after trypsin digestion. From three technical replicates, the average summed intensities (AU) were combined for peptides with phosphorylated Thr 161 and for peptides with non‐phosphorylated Thr 161. The intensity of phosphopeptides as a fraction of the total peptide intensity is shown in the last column. The CCC sample that was exposed to lambda‐phosphatase is marked CCC‐λ. (f, g) Fluorescently labeled CENP‐T was exposed to different kinase complexes and analyzed for multisite phosphorylation using phostag SDS‐PAGE. Samples were analyzed after 15 min (upper gels) and after 90 min (lower gels). In‐gel fluorescence (CENP‐T) was recorded before Coomassie staining the same gels. Reactions in Lanes 1 and 4 as well as 8 and 11 are technical replicates

FIGURE 4

CKS1 boosts the processivity of CDK1:Cyclin‐B. (a) Preparation and phostag SDS‐PAGE analysis of CDK1:Cyclin‐B and CDK1:Cyclin‐B:CKS1 complexes that were either assembled from purified components or pre‐assembled into dimeric or trimeric complexes. (b, c) CENP‐T was exposed to different kinase complexes and analyzed for multi‐site phosphorylation using phostag SDS‐PAGE and in‐gel fluorescence (CENP‐T^TMR). (d) Quantification of phosphorylated/non‐phosphorylated signals from the gels shown in Panel (c). (e) SDS‐PAGE analysis of samples CC (3) and CCC (4) as in Panel (c) in the presence of 10 μM phostag acrylamide (all other gels contain 50 μM phostag‐acrylamide)