CKS Proteins Promote Checkpoint Recovery by Stimulating Phosphorylation of Treslin

ABSTRACT

CKS proteins are small (9-kDa) polypeptides that bind to a subset of the cyclin-dependent kinases. The two paralogs expressed in mammals, Cks1 and Cks2, share an overlapping function that is essential for early development. However, both proteins are frequently overexpressed in human malignancy. It has been shown that CKS protein overexpression overrides the replication stress checkpoint, promoting continued origin firing. This finding has led to the proposal that CKS protein-dependent checkpoint override allows premalignant cells to evade oncogene stress barriers, providing a causal link to oncogenesis. Here, we provide mechanistic insight into how overexpression of CKS proteins promotes override of the replication stress checkpoint. We show that CKS proteins greatly enhance the ability of Cdk2 to phosphorylate the key replication initiation protein treslin in vitro. Furthermore, stimulation of treslin phosphorylation does not occur by the canonical adapter mechanism demonstrated for other substrates, as cyclin-dependent kinase (CDK) binding-defective mutants are capable of stimulating treslin phosphorylation. This effect is recapitulated in vivo, where silencing of Cks1 and Cks2 decreases treslin phosphorylation, and overexpression of wild-type or CDK binding-defective Cks2 prevents checkpoint-dependent dephosphorylation of treslin. Finally, we provide evidence that the role of CKS protein-dependent checkpoint override involves recovery from checkpoint-mediated arrest of DNA replication.

KEYWORDS:

• CKS protein
• treslin
• treslin phosphorylation
• replication stress checkpoint
• checkpoint recovery

ACKNOWLEDGMENTS

We thank Anindya Dutta for plasmids required for E. coli production of active cyclin E/Cdk2.

This work was supported by NIH grants CA074224 and CA078343 to S.I.R., GM043974 and GM070891 to W.G.D., and P41 GM103533 to J.R.Y. We also thank the training grants NCATS 5 TL1 TR001114 and UL1 TR001114 and the Scripps Family Foundation and ARCS Foundation San Diego Chapter predoctoral fellowships for support of J.T.

S.I.R. supervised the research described. R.M., J.T., Y.Z., and S.I.R. designed and carried out experiments. R.Z. carried out experiments. A.K. and W.G.D. provided advice and reagents. S.I.R. wrote the manuscript, with editing help from A.K., W.G.D., J.T., and J.R.Y. J.R.Y. provided experimental resources.