The histidine triad protein Hint1 interacts with Pontin and Reptin and inhibits TCF–β-catenin-mediated transcription

Abstract

Pontin and Reptin previously were identified as nuclear β-catenin interaction partners that antagonistically modulate β-catenin transcriptional activity. In this study, Hint1/PKCI, a member of the evolutionary conserved family of histidine triad proteins, was characterised as a new interaction partner of Pontin and Reptin. Pull-down assays and co-immunoprecipitation experiments show that Hint1/PKCI directly binds to Pontin and Reptin. The Hint1/PKCI-binding site was mapped to amino acids 214-295 and 218-289 in Pontin and Reptin, respectively. Conversely, Pontin and Reptin bind to the N-terminus of Hint1/PKCI. Moreover, by its interaction with Pontin and Reptin, Hint1/PKCI is associated with the LEF-1/TCF–β-catenin transcription complex. In this context, Hint1/PKCI acts as a negative regulator of TCF–β-catenin transcriptional activity in Wnt-transfected cells and in SW480 colon carcinoma cells as shown in reporter gene assays. Consistent with these observations, Hint1/PKCI represses expression of the endogenous target genes cyclin D1 and axin2 whereas knockdown of Hint1/PKCI by RNA interference increases their expression. Disruption of the Pontin/Reptin complex appears to mediate this modulatory effect of Hint1/PKCI on TCF–β-catenin-mediated transcription. These data now provide a molecular mechanism to explain the tumor suppressor function of Hint1/PKCI recently suggested from the analysis of Hint1/PKCI knockout mice.