Phosphorylation by cyclin C/cyclin-dependent kinase 2 following mitogenic stimulation of murine fibroblasts inhibits transcriptional activity of LSF during G1 progression.
Transcription factor LSF is required for progression from quiescence through the cell cycle, regulating thymidylate synthase (Tyms) expression at the G(1)/S boundary. Given the constant level of LSF protein from G(0) through S, we investigated whether LSF is regulated by phosphorylation in G(1). 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 G(1) 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 G(1) 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 G(1)/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 G(1). Unexpectedly, this phosphorylation prevents induction of LSF target genes until late G(1).
Saxena
Utsav H
UH
Department of Biology, Boston University, 5 Cummington Street, Boston, MA 02215, USA.
Powell
Christina M H
CM
Fecko
Jill K
JK
Cacioppo
Roxanne
R
Chou
Hubert S
HS
Cooper
Geoffrey M
GM
Hansen
Ulla
U
D000818:Animals; D056745:Cyclin C; D019927:Cyclin E / Q000235:genetics / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2 / Q000235:genetics / Q000378:metabolism*; D056849:Cyclin-Dependent Kinase 3; D018844:Cyclin-Dependent Kinases / Q000235:genetics / Q000378:metabolism; D016213:Cyclins / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004790:Enzyme Induction; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology*; D016193:G1 Phase / Q000502:physiology*; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D010766:Phosphorylation; D012694:Serine / Q000378:metabolism; D013940:Thymidylate Synthase / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
C503675:Ccnc protein, mouse; D056745:Cyclin C; D019927:Cyclin E; D016213:Cyclins; D004268:DNA-Binding Proteins; C501905:Tfcp2 protein, mouse; D014157:Transcription Factors; D012694:Serine; D013940:Thymidylate Synthase; C495893:Cdk2 protein, mouse; C490914:Cdk3 protein, mouse; D051357:Cyclin-Dependent Kinase 2; D056849:Cyclin-Dependent Kinase 3; D018844:Cyclin-Dependent Kinases