Accumulation of Fra-1 in ras-Transformed Cells Depends on Both Transcriptional Autoregulation and MEK-Dependent Posttranslational Stabilization

Abstract

The AP-1 transcription factor plays an essential role in cell proliferation and tumorigenesis. It was previously shown that the fra-1 gene product is upregulated by various oncogenes and is involved in the in vitro and in vivo transformation of thyroid cells. Here we show that the ras oncogene-dependent accumulation of Fra-1 is mediated by a positive feedback mechanism which requires both transcriptional autoregulation and posttranslational stabilization of the protein. The oncogene-dependent transcriptional activation involves the cooperation between both Raf-dependent and Raf-independent pathways and is mediated by an AP-1 site within the fra-1 first intron, which becomes stably occupied by a transcriptionally active Fra-1-containing complex in ras-transformed cells. The posttranslational stabilization results in a drastic increase in the Fra-1 half-life in ras-transformed cells and is totally dependent on the activity of the MEK/ERK phosphorylation pathway. The analysis of the Fra-1 transactivation potential shows that the protein is able to stimulate a heterologous promoter in a ras-dependent manner, but the transactivating activity requires the recruitment of a heterodimeric partner. These data show that the alteration of multiple regulatory mechanisms is required for the constitutive activation of Fra-1 as a nuclear target of ras transformation.

ACKNOWLEDGMENTS

We thank Julian Downward for providing the plasmids expressing the Ras effector mutants and Meinrad Busslinger for fra-1/β-globin reporter constructs and the Gal4/Fra-1 fusion derivatives. We are grateful to Maurizio D'Esposito and Maria R. Matarazzo for help with ChIp and to Rita Vito for technical assistance. We thank Roberto Di Lauro for the generous gift of the FRTL-5-derived cell clones. We also thank Alfredo Fusco and Massimo Santoro for cell lines and helpful discussions.

This work was supported by grants from Associazione Italiana per la Ricerca sul Cancro, from Regione Campania (L.R. 41/94), and from MURST-CNR Program Legge 488/92 (cluster 02).