Abstract T-cell factor (TCF) family proteins play a crucial role in regulating the activation of Wnt/β-catenin responsive target genes. Among the family proteins, TCF-4 is predominantly expressed in human HCC tissues. We have recently cloned 14 TCF-4 isoforms from human hepatoma cell lines, and naming them in alphabetical order from A to M. A TCF-4J and K pair has been identified based on the presence (K) or absence (J) of the SxxSS motif at the beginning of exon 9. This motif may modulate transcriptional activity of TCF-4 through phosphorylation/dephosphorylation of serine residues (Pukrop et al., Cancer Res 2001). However, cell-based functional analysis of this motif has yet to be performed. The AIM of this study was to investigate whether TCF-4J and K have distinct cellular phenotypes due to SxxSS motif expression. Methods: The human hepatoma cell line Huh-7 was used in this study. The cells were transfected with TCF-4J and K expression plasmids, and protein levels in cell lysates and nuclear extracts were examined by Western blot analysis. TCF-4K mutants with substitution of serine (S) to alanine (A) were generated by site-directed mutagenesis. Results: Protein expression of all cloned TCF-4 isoforms was confirmed by Western blot analysis. Expression levels of cyclin D1, which is a direct target of TCF-4, was increased in both cell lysates and the nuclear extracts of the TCF-4K, but not in TCF-4J-transfected cells. The TCF-4K isoform with a S269A mutation revealed a decrease in the expression level of cyclin D1, while the mutant with either a S272A or S273A substitution did not, which suggests that phosphorylation of serine 269 was critical to up-regulating cyclin D1 gene expression. Conclusion: The findings from functional analysis following mutagenesis of the SxxSS motif suggest that serine phosphorylation of this motif resulted in the altered target genes expression, including cyclin D1. Therefore, TCF-4 isoforms regulate the phenotype of HCC cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4067.