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Molecular and Cellular Biology, May 2009, p. 2335-2345, Vol. 29, No. 9
0270-7306/09/$08.00+0     doi:10.1128/MCB.00687-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Phosphorylation by Cyclin C/Cyclin-Dependent Kinase 2 following Mitogenic Stimulation of Murine Fibroblasts Inhibits Transcriptional Activity of LSF during G1 Progression{triangledown}

Utsav H. Saxena,1,{dagger} Christina M. H. Powell,1,{dagger} Jill K. Fecko,1 Roxanne Cacioppo,1,{ddagger} Hubert S. Chou,2,§ Geoffrey M. Cooper,1 and Ulla Hansen1*

Department of Biology, Boston University, Boston, Massachusetts 02215,1 Harvard Medical School and Massachusetts General Hospital Cancer Center, Charlestown, Massachusetts 021292

Received 27 April 2008/ Returned for modification 19 May 2008/ Accepted 10 February 2009

Transcription factor LSF is required for progression from quiescence through the cell cycle, regulating thymidylate synthase (Tyms) expression at the G1/S boundary. Given the constant level of LSF protein from G0 through S, we investigated whether LSF is regulated by phosphorylation in G1. In vitro, LSF is phosphorylated by cyclin E/cyclin-dependent kinase 2 (CDK2), cyclin C/CDK2, and cyclin C/CDK3, predominantly on S309. Phosphorylation of LSF on S309 is maximal 1 to 2 h after mitogenic stimulation of quiescent mouse fibroblasts. This phosphorylation is mediated by cyclin C-dependent kinases, as shown by coimmunoprecipitation of LSF and cyclin C in early G1 and by abrogation of LSF S309 phosphorylation upon suppression of cyclin C with short interfering RNA. Although mouse fibroblasts lack functional CDK3 (the partner of cyclin C in early G1 in human cells), CDK2 compensates for this absence. By transient transfection assays, phosphorylation at S309, mediated by cyclin C overexpression, inhibits LSF transactivation. Moreover, overexpression of cyclin C and CDK3 inhibits induction of endogenous Tyms expression at the G1/S transition. These results identify LSF as only the second known target (in addition to pRb) of cyclin C/CDK activity during progression from quiescence to early G1. Unexpectedly, this phosphorylation prevents induction of LSF target genes until late G1.

* Corresponding author. Mailing address: Department of Biology, Boston University, 5 Cummington Street, Boston, MA 02215. Phone: (617) 353-8730. Fax: (617) 353-8484. E-mail: uhansen{at}bu.edu

{triangledown} Published ahead of print on 23 February 2009.

{dagger} These authors contributed equally to this work.

{ddagger} Present address: Millennium Pharmaceuticals, Inc., 40 Landsdowne Street, Cambridge, MA 02139.

§ Present address: Daiichi-Sankyo Pharma Development, 339 Thornall Street, Edison, NJ 08837.

Molecular and Cellular Biology, May 2009, p. 2335-2345, Vol. 29, No. 9
0270-7306/09/$08.00+0     doi:10.1128/MCB.00687-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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