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J Biol Chem. 2009 May 22;284(21):14126-35. doi: 10.1074/jbc.M900687200. Epub 2009 Mar 25.

Coordinated activation of the origin licensing factor CDC6 and CDK2 in resting human fibroblasts expressing SV40 small T antigen and cyclin E.

Author information

  • 1Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.

Abstract

We have previously shown that SV40 small t antigen (st) cooperates with deregulated cyclin E to activate CDK2 and bypass quiescence in normal human fibroblasts (NHF). Here we show that st expression in serum-starved and density-arrested NHF specifically induces up-regulation and loading of CDC6 onto chromatin. Coexpression of cyclin E results in further accumulation of CDC6 onto chromatin concomitantly with phosphorylation of CDK2 on Thr-160 and CDC6 on Ser-54. Investigation of the mechanism leading to CDC6 accumulation and chromatin loading indicates that st is a potent inducer of cdc6 mRNA expression and increases CDC6 protein stability. We also show that CDC6 expression in quiescent NHF efficiently promotes cyclin E loading onto chromatin, but it is not sufficient to activate CDK2. Moreover, we show that CDC6 expression is linked to phosphorylation of the activating T loop of CDK2 in serum-starved NHF stimulated with mitogens or ectopically expressing cyclin E and st. Our data suggest a model where the combination of st and deregulated cyclin E result in cooperative and coordinated activation of both an essential origin licensing factor, CDC6, and an activity required for origin firing, CDK2, resulting in progression from quiescence to S phase.

PMID:
19321444
[PubMed - indexed for MEDLINE]
PMCID:
PMC2682861
Free PMC Article

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