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 .The transcription factor LSF (late simian virus 40 factor [20], also named CP2 [24], LBP-1c [53], and SEF [6]) is ubiquitously expressed in all cell types (32, 44). LSF is essential for cell cycle progression at the G 1 /S transition after reentry of quiescent cells into the cell cycle, substantially through its regulation of thymidylate synthase (Tyms) gene expression (31). Disruption of LSF function can lead either to apoptosis in S phase (31) or to cell cycle arrest at the G 1 /S transition (11), both phenotypes being relieved by a source of exogenous thymidine, thereby circumventing the requirement for thymidylate synthase activity.In investigating how LSF activity could be modulated during cell cycle progression, we previously demonstrated that LSF is targeted by the MEK/extracellular signal-regulated kinase (ERK) signaling pathway, which is critical for reentry of quiescent cells into the G 1 phase of the cell cycle (35). ERK phosphorylates LSF at S291 upon mitogenic stimulation of multiple cell types, including mouse fibroblasts and primary peripheral human T lymphocytes (30, 49). Signaling pathways essential for the subsequent progression through G 1 into S phase include the G 1 cyclin-dependent kinases (CDKs) (38). These include the extensively investigated D-type cyclins and their associated kinases CDK4/6 as well as E-type cyclins associated with CDK2 (39, 41). Recently, an additional cyclin/ CDK complex, cyclin C/CDK3, was also shown to facilitate efficient exit of human cells from G 0 into the cell cycle (33, 36). Cyclin C was not previously thought to be a regulator of cell cycle progression but was instead demonstrated...

No abstract