Proteomic Analysis Reveals a PLK1-Dependent G2/M Degradation Program and Links PKA-AKAP2 to Cell Cycle Control.

Targeted protein degradation by the ubiquitin-proteasome system is an essential mechanism regulating cellular division. The kinase PLK1 coordinates protein degradation at the G2/M phase of the cell cycle by promoting the binding of substrates to the E3 ubiquitin ligase SCFßTrCP. However, the magnitude to which PLK1 shapes the mitotic proteome has not been characterized. Combining deep, quantitative proteomics with pharmacologic PLK1 inhibition (PLK1i), we identified more than 200 proteins whose abundances were increased by PLK1i at G2/M. We validate many new PLK1-regulated proteins, including several substrates of the cell cycle E3 SCFCyclin F, demonstrating that PLK1 promotes proteolysis through at least two distinct SCF-family E3 ligases. Further, we found that the protein kinase A anchoring protein AKAP2 is cell cycle regulated and that its mitotic degradation is dependent on the PLK1/ßTrCP-signaling axis. Interactome analysis revealed that the strongest interactors of AKAP2 function in signaling networks regulating proliferation, including MAPK, AKT, and Hippo. Altogether, our data demonstrate that PLK1 coordinates a widespread program of protein breakdown at G2/M. We propose that dynamic proteolytic changes mediated by PLK1 integrate proliferative signals with the core cell cycle machinery during cell division. This has potential implications in malignancies where PLK1 is aberrantly regulated.

Coordinating gene expression during the cell cycle.

Cell cycle-dependent gene transcription is tightly controlled by the retinoblastoma (RB):E2F and DREAM complexes, which repress all cell cycle genes during quiescence. Cyclin-dependent kinase (CDK) phosphorylation of RB and DREAM allows for the expression of two gene sets. The first set of genes, with peak expression in G1/S, is activated by E2F transcription factors (TFs) and is required for DNA synthesis. The second set, with maximum expression during G2/M, is required for mitosis and is coordinated by the MuvB complex, together with B-MYB and Forkhead box M1 (FOXM1). In this review, we summarize the key findings that established the distinct control mechanisms regulating G1/S and G2/M gene expression in mammals and discuss recent advances in the understanding of the temporal control of these genes.

MMB-FOXM1-driven premature mitosis is required for CHK1 inhibitor sensitivity.

To identify genes whose loss confers resistance to CHK1 inhibitors, we perform genome-wide CRISPR-Cas9 screens in non-small-cell lung cancer (NSCLC) cell lines treated with the CHK1 inhibitor prexasertib (CHK1i). Five of the top six hits of the screens, MYBL2 (B-MYB), LIN54, FOXM1, cyclin A2 (CCNA2), and CDC25B, are cell-cycle-regulated genes that contribute to entry into mitosis. Knockout of MMB-FOXM1 complex components LIN54 and FOXM1 reduce CHK1i-induced DNA replication stress markers and premature mitosis during Late S phase. Activation of a feedback loop between the MMB-FOXM1 complex and CDK1 is required for CHK1i-induced premature mitosis in Late S phase and subsequent replication catastrophe, indicating that dysregulation of the S to M transition is necessary for CHK1 inhibitor sensitivity. These findings provide mechanistic insights into small molecule inhibitors currently studied in clinical trials and provide rationale for combination therapies.