Prolyl isomerase Pin1 regulates transcription factor LSF (TFCP2) by facilitating dephosphorylation at two serine-proline motifs.

Transcription factor LSF is essential for cell cycle progression, being required for activating expression of the thymidylate synthase (Tyms) gene at the G1/S transition. We previously established that phosphorylation of LSF in early G1 at Ser-291 and Ser-309 inhibits its transcriptional activity and that dephosphorylation later in G1 is required for its reactivation. Here we reveal the role of prolyl cis-trans isomerase Pin1 in activating LSF, by facilitating dephosphorylation at both Ser-291 and Ser-309. We demonstrate that Pin1 binds LSF both in vitro and in vivo. Using coimmunoprecipitation assays, we identify three SP/TP motifs in LSF (at residues Ser-291, Ser-309, and Thr-329) that are required and sufficient for association with Pin1. Co-expression of Pin1 enhances LSF transactivation potential in reporter assays. The Pin1-dependent enhancement of LSF activity requires residue Thr-329 in LSF, requires both the WW and PPiase domains of Pin1, and correlates with hypophosphorylation of LSF at Ser-291 and Ser-309. These findings support a model in which the binding of Pin1 at the Thr-329-Pro-330 motif in LSF permits isomerization by Pin1 of the peptide bonds at the nearby phosphorylated SP motifs (Ser-291 and Ser-309) to the trans configuration, thereby facilitating their dephosphorylation.

Transcription factor Late SV40 Factor (LSF) functions as an oncogene in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a highly aggressive cancer with no currently available effective treatment. Understanding of the molecular mechanism of HCC development and progression is imperative for developing novel, effective, and targeted therapies for this lethal disease. In this article, we document that the cellular transcription factor Late SV40 Factor (LSF) plays an important role in HCC pathogenesis. LSF protein was significantly overexpressed in human HCC cells compared to normal hepatocytes. In 109 HCC patients, LSF protein was overexpressed in >90% cases, compared to normal liver, and LSF expression level showed significant correlation with the stages and grades of the disease. Forced overexpression of LSF in less aggressive HCC cells resulted in highly aggressive, angiogenic, and multiorgan metastatic tumors in nude mice. Conversely, inhibition of LSF significantly abrogated growth and metastasis of highly aggressive HCC cells in nude mice. Microarray studies revealed that as a transcription factor, LSF modulated specific genes regulating invasion, angiogenesis, chemoresistance, and senescence. The expression of osteopontin (OPN), a gene regulating every step in tumor progression and metastasis, was robustly up-regulated by LSF. It was documented that LSF transcriptionally up-regulates OPN, and loss-of-function studies demonstrated that OPN plays an important role in mediating the oncogenic functions of LSF. Together, these data establish a regulatory role of LSF in cancer, particularly HCC pathogenesis, and validate LSF as a viable target for therapeutic intervention.

Transcription factors LSF and E2Fs: tandem cyclists driving G0 to S?

Cell cycle progression in mammalian cells from G(1) into S phase requires sensing and integration of multiple inputs, in order to determine whether to continue to cellular DNA replication and subsequently, to cell division. Passage to S requires transition through the restriction point, which at a molecular level consists of a bistable switch involving E2Fs and pRb family members. At the G(1)/S boundary, a number of genes essential for DNA replication and cell cycle progression are upregulated, promoting entry into S phase. Although the activating E2Fs are the most extensively characterized transcription factors driving G(1)/S expression, LSF is also a transcription factor essential for stimulating G(1)/S gene expression. A critical LSF target gene at this stage, Tyms, encodes thymidylate synthetase. In investigating how LSF is activated in a cell cycle-dependent manner, we recently identified a novel time delay mechanism for regulating its activity during G(1) progression, which is apparently independent of the E2F/pRb axis. This involves inhibition of LSF in early G(1) by two major proliferative signaling pathways: ERK and cyclin C/CDK, followed by gradual dephosphorylation during mid- to late-G(1). Whether LSF and E2F act independently or in concert to promote G(1)/S progression remains to be determined.

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).