Copper is essential for cyclin B1-mediated CDK1 activation

Abstract

Cyclin-dependent kinase 1 (CDK1) is the pivotal kinase responsible for initiating cell division. Its activation is dependent on binding to regulatory cyclins, such as CCNB1. Our research demonstrates that copper binding to both CDK1 and CCNB1 is essential for activating CDK1 in cells. Mutations in the copper-binding amino acids of either CDK1 or CCNB1 do not disrupt their interaction but are unable to activate CDK1. We also reveal that CCNB1 facilitates the transfer of copper from ATOX1 to CDK1, consequently activating its kinase function. Disruption of copper transfer through the ATOX1-CCNB1-CDK1 pathway can impede CDK1 activation and halt cell cycle progression. In summary, our findings elucidate a mechanism through which copper promotes CDK1 activation and the G2/M transition in the cell cycle. These results could provide insight into the acquisition of proliferative properties associated with increased copper levels in cancer and offer targets for cancer therapy.

Fig. 1. The influence of copper on cell cycle G2/M progression.

a Flow cytometric analysis of the impact of copper on cell cycle progression in CTR1 knocked-down HepG2 cells. After synchronization to the S phase by 2.5 mM TdR, cells were released and cultured for 12 h. The percentages of different cell cycle stages are quantified on the right. The same treatment condition was used hereafter, if not specifically noted. b The schematic representation of FUCCI reporter system. Magenta: G1 phase; bright green: G2/M phase. c FUCCI reporter assay assessing the cell cycle progression of CTR1 knocked-down HepG2 cells. Scale bars are 50 μm. d, e Examination of the effect of copper-binding deficiencies of CTR1 on cell cycle progression using flow cytometry and FUCCI assay. Scale bars are 50 μm. f, g Flow cytometry (f) and FUCCI reporter assay (g) analyzing copper’s impact on cell cycle progression of HepG2 cells under different treatments. The cells were synchronized to the S phase using TdR and cultured in metal-deprived medium without or with copper (-Metal or Cu, respectively), or cultured in regular medium containing 20 μM TEPA or 50 μM TTM for 12 h. Scale bars represent 50 μm. In (a, d, f), data are representative of three biologically independent experiments. Source data are provided as a Source Data file.

Fig. 2. Role of copper binding in CDK1 activation and G2/M progression.

a Intersection analysis of copper-binding proteins (CBPs) from G2-stage HepG2 cells and G2/M progression regulatory proteins compiled from the AmiGO database. Lysates from the cytoplasm or nuclei of HepG2 cells synchronized to the G2 phase were examined using mass spectrometry (MS) after copper-bead pulldown. b The filtered results are charted for copper-beads enriched proteins. c Pulldown assay to verify copper binding to CDK1 within the nuclear and cytoplasmic compartments of HepG2 cells. d In vitro pulldown assay to assess the copper-binding affinity of purified GST-CDK1, using GST-ATOX1 and GST as positive or negative controls, respectively. e Pulldown assay gauging copper binding to GST-CDK1 wild-type (WT) or its copper-binding deficient mutants. f ICP-OES-quantified copper-binding capacities of 10 μg purified recombinant CDK1^WT and CDK1^CBM proteins. (P-values indicated on top). g, h Examination of the effects of copper-binding deficiencies of CDK1 on cell cycle progression using flow cytometry and FUCCI assay after transfecting cells with CDK1 ^WT or CDK1^CBM expression plasmids. The cells were synchronized to the S phase and then treated with 20 μM copper in the CBM groups, followed by flow cytometric analysis (g) or FUCCI-based assays (h). The same treatment condition was used hereafter, if not specifically noted. Scale bars represent 50 μm. i Analysis of H1.4 and p53 phosphorylation in cells with inducible CDK1 knockdown and transfected by HA-CDK1^WT or HA-CDK1^CBM. j Molecular modeling analysis of CDK1 protein structures to visualize the differences in conformation between WT and CBM mutants. k IP assay to examine interactions of transfected CDK1 WT or CBM mutant with endogenous CCNB1. In (f), unpaired two-tailed Student’s t-test was used for statistical analysis. In (f, g), data are representative of three biologically independent experiments. Source data are provided as a Source Data file.

Fig. 3. Effect of copper on CDK1 activity.

a Analysis of H1.4 phosphorylation by incubation with CDK1 and CCNB1 alone or in combination, with or without 5 μM copper. The densitometric analysis of protein phosphorylation levels is shown on the blot. b Analysis of H1.4 phosphorylation with the same conditions as (a) using apo-CCNB1 and apo-CDK1, with densitometric analysis depicted on the blot. c, d Assessment of H1.4 phosphorylation using in vitro kinase assay, with H1.4 incubated with a gradient concentration of copper (c) or TTM (d). e Examination of CDK1^WT and CDK1^CBM’s ability to phosphorylate H1.4 and p53 in vitro in the presence or absence of copper through western blot analysis. f Evaluation of H1.4 and p53 phosphorylation in CCNB1-silenced cells treated with increasing concentrations of copper. g Co-IP analysis of CDK1 and CCNB1 interaction in lysates of HepG2 cells with CDK7 knockdown by CRISPR-Cas9. h Analysis of H1.4, p53, and CDK1-T161 phosphorylation in sgCDK7 cells treated with 20 μM copper. In (a–d), data are representative of three biologically independent experiments. Source data are provided as a Source Data file.

Fig. 4. Role of CCNB1 in copper-Activated CDK1 during G2/M Stage.

a Pulldown assay assessing copper binding to CCNB1 in cell nucleus and cytoplasm lysates. b Pulldown assay examining copper binding to purified GST-CCNB1; using GST-ATOX1 and GST as positive and negative controls, respectively. c Immunoblot detection of copper binding to GST-CCNB1 WT and its CBM mutants of GST-CCNB1-F2. d Pulldown assay testing copper binding to WT or its CBM of CCNB1 FL with putative copper-binding residues replaced by alanines. e ICP-OES-quantified copper-binding capacities of 10 μg purified recombinant CCNB1^WT and CCNB1^CBM proteins (P-values indicated on top). f Molecular modeling analysis of CCNB1 protein structures, with blue and red ribbons representing WT and CBM mutants, respectively. g Analysis of H1.4 and p53 phosphorylation in CCNB1 knockdown cells transfected with WT or its CBM type of HA-CCNB1 plasmids. All cells were synchronized to the S phase and treated with copper. h, i Effects of copper-binding deficiencies of CCNB1 on cell cycle progression evaluated using flow cytometry and FUCCI assay. Scale bars represent 50 μm. j ICP-OES-quantified copper contents of CCNB1 and CDK1 samples alone and after their incubation. Apo- and Cu-states of purified CCNB1 and CDK1 alone were directly tested. Upon incubation, when Cu-CCNB1 and apo-CDK1 were separated by SEC and analyzed for copper contents. k Western blot analysis of in vitro GST-p53 phosphorylation when incubated with CCNB1 and CDK1 in their apo- or Cu-states, or in the indicated combinations. In (e), unpaired two-tailed Student’s t-test was used for statistical analysis. In (e, f), data are representative of three biologically independent experiments. Source data are provided as a Source Data file.

Fig. 5. CCNB1 transfers copper from ATOX1 to CDK1, thereby activating its kinase activity and promoting G2/M transition.

a IP analysis examining interactions of HA-tagged copper chaperone proteins with Flag-CCNB1 in HepG2 cells. In vitro pulldown assay using Ni agarose (b) and co-IP analysis (c) to evaluate CCNB1 and ATOX1 interaction. d BiFC analysis of CCNB1 and ATOX1 interaction. Scale bars represent 100 μm. e ICP-OES-quantified copper contents of purified proteins alone or after their incubations to assess the copper transfer. f Analysis of p53 phosphorylation after incubation with apo- or Cu-states of ATOX1, CCNB1, CDK1, or their different combinations as indicated. g, h Analysis of H1.4 and p53 phosphorylation in cells expressing shATOX1 (g) or expressing both shATOX1 and HA-ATOX1 WT or CBM (h), synchronized to the S phase and treated with copper. i, j Flow cytometry (i) and FUCCI (j) assay to assess the cell cycle progression of HepG2 cells with ATOX1 knockdown. k Mechanistic model of copper-promoting cell cycle progression by activating CDK1. In (i), data are representative of three biologically independent experiments. Source data are provided as a Source Data file.