Fig 2: Crystal structure of the talin-CDK1 complex.A and B, cartoon representation of the X-ray structure of the talin R7R8 fragment complex with CDK1(206–223). Talin R8 (white) and CDK1 (magenta) are indicated. A, face on and B, side on views. Key residues are highlighted. C and D, structure of talin R8-LD motif complexes. C, talin-DLC1 (PDB ID: 5FZT (32)) and D, talin-RIAM (PDB ID: 4W8P (39)), orientated as in (A). E and F, structural model of a talin-CDK1-cyclin complex. E, modeled structure of talin (gray) bound to the complex of CDK1 (blue) and cyclin B1 (orange) using (PDB ID: 4YC3 (41)). F, structure of the tripartite complex of CDK1 (blue), cyclin B1 (orange), and CKS2 (gray) (PDB ID: 4YC3). Talin R8 and CKS2 both bind CDK1 via the same region on the opposite face to the cyclin-binding surface.

Fig 3: The CDK1 LD motif is required for CDK1 binding to talin-1.A, CDK1 structure (PDB ID: 4YC3 (41)) with the talin-binding LD motif highlighted in magenta. The LD motif consensus residues (E209, I210, D211, F214, and F217) and the I210A and D211A, 2A mutation are highlighted. B, aligned sequences of CDK1 WT and CDK1-2A mutant peptides. C, binding of BODIPY-labeled CDK1 WT and 2A 206 to 223 peptide with talin-1 R7R8 domains using fluorescence polarization. Dissociation constants ±SE (μM) for the interactions are indicated. All measurements were performed in triplicate. D, 1H,15N HSQC spectra of 70 μM 15N-labeled talin-1 R8 (residues 1461–1580) (black) with 420 μM CDK1 peptide (green), 420 μM CDK1-2A peptide (red) added. E, mapping of the CDK1-binding site on R8 as detected by NMR using weighted chemical shift differences (red)—mapped onto the R7R8 structure. F, GFP-Trap pull-downs from cells expressing GFP alone, GFP-WT-talin-1, or GFP-ΔR8-talin-1 along with HA-tagged WT or 2A-CDK1, followed by western blotting for GFP and HA.

Fig 4: WG-391D down-regulates CDC25B and inactivates CDC2 and AKT. (A) Molecular structure of WG-391D. (B) RNA sequencing analysis of CDC25A, CDC25B, and CDC25C mRNA expression in SKOV3 cells treated with 5 μM WG-391D for 24 h. (C) qRT-PCR detection of CDC25B mRNA expression in SKOV3 cells treated with/without 5 μM WG-391D for 24 h. (D) CDC2 and AKT expression and/or activation was evaluated in SKOV3 cells treated with a series of concentrations of inhibitor for 24 h. (E) CDC2 and AKT expression and/or activation was evaluated in GFY004 cells treated with a series of concentrations of inhibitor for 24 h. Expression levels of CDC25B, CDC2, AKT, and GAPDH were detected, as indicated. Phosphorylation of CDC2 and AKT were detected (pCDC2 and pAKT, respectively). GAPDH served as a house-keeping control. Total proteins (20 μg per sample) were analyzed by SDS-PAGE. (F) Effect of WG-391D (10 μM) on CDC25B dephosphorylation catalytic activity was determined by in vitro enzyme assay. NC, DMSO served as the negative control. PC, NSC663284 served as the positive control. NS, not significant. ***p < 0.001.

Fig 5: Phosphorylation of ATF2 (at Thr69/Thr71) and ATF7 (at Thr51/Thr53) by Cdk1-cyclin B1.(A) Amino acid sequence of ATF2 and ATF7 with the common phosphorylation sites. pATF2, phosphorylated ATF2; pATF7, phosphorylated ATF7; ATF2-TA and ATF7-TA, T→A mutants. (B–E Cells that were transiently transfected with the indicated constructs were cultured for 24 h, and whole cell lysates were analyzed by WB. (B) Cells were cotransfected with ATF2-wt together with control vector, cyclin B1, Cdk1 or cyclin B1 plus Cdk1. (C) Cells were cotransfected with ATF7-wt together with control vector, cyclin B1, Cdk1, or cyclin B1 plus Cdk1. (D) Cells were cotransfected with control vector together with GFP-ATF2-wt or GFP-ATF2-TA, or cells were cotransfected with cyclin B1 plus Cdk1 together with GFP-ATF2-wt or GFP-ATF2-TA. (E) Cells were cotransfected with control vector together with ATF7-wt or ATF7-TA, or cells were cotransfected with cyclin B1 plus Cdk1 together with ATF7-wt or ATF7-TA. Full-length blots are presented in S10 Fig.