Cks proteins associate with cyclin-dependent kinases and have therefore been assumed to play a direct role in cell cycle regulation. Mammals have two paralogs, Cks1 and Cks2, and individually deleting the gene encoding either in the mouse has previously been shown not to impact viability. In this study we show that simultaneously disrupting CKS1 and CKS2 leads to embryonic lethality, with embryos dying at or before the morula stage after only two to four cell division cycles. RNA interference (RNAi)-mediated silencing of CKS genes in mouse embryonic fibroblasts (MEFs) or HeLa cells causes cessation of proliferation. In MEFs CKS silencing leads to cell cycle arrest in G 2 , followed by rereplication and polyploidy. This phenotype can be attributed to impaired transcription of the CCNB1, CCNA2, and CDK1 genes, encoding cyclin B1, cyclin A, and Cdk1, respectively. Restoration of cyclin B1 expression rescues the cell cycle arrest phenotype conferred by RNAi-mediated Cks protein depletion. Consistent with a direct role in transcription, Cks2 is recruited to chromatin in general and to the promoter regions and open reading frames of genes requiring Cks function with a cell cycle periodicity that correlates with their transcription.

Cyclin-dependent kinase subunit (Cks) proteins are small cyclin-dependent kinase-interacting proteins that are frequently overexpressed in breast cancer, as well as in a broad spectrum of other human malignancies. However, the mechanistic link between Cks protein overexpression and oncogenesis is still unknown. In this work, we show that overexpression of Cks1 or Cks2 in human mammary epithelial and breast cancer-derived cells, as well as in other cell types, leads to override of the intra–S-phase checkpoint that blocks DNA replication in response to replication stress. Specifically, binding of Cks1 or Cks2 to cyclin-dependent kinase 2 confers partial resistance to the effects of inhibitory tyrosine phosphorylation mediated by the intra–S-phase checkpoint, allowing cells to continue replicating DNA even under conditions of replicative stress. Because many activated oncoproteins trigger a DNA damage checkpoint response, which serves as a barrier to proliferation and clonal expansion, Cks protein overexpression likely constitutes one mechanism whereby premalignant cells can circumvent this DNA damage response barrier, conferring a proliferative advantage under stress conditions, and therefore contributing to tumor development.