Ha-ras Interference with Thyroid Cell Differentiation Is Associated with a Down-Regulation of Thyroid Transcription Factor-1 Phosphorylation

7 pages, 6 figures.Mechanisms responsible for the lack of thyroid-specific differentiation markers in Ha-ras transformed FRTL-5 cells have been investigated. In vivo cell labeling and immunoprecipitation demonstrate that phosphorylation of the thyroid transcription factor-1 (TTF-1) is clearly reduced in thyroid cells transformed with the Ha-ras oncogene. Fingerprinting analysis of phosphotryptic peptides from FRTL-5 and Ha-ras-FRTL-5 cells also reveals a heterogeneous pattern of TTF-1 phosphorylation in the transformed cell line. This heterogeneity is localized in the amino terminal cluster of phosphoserines, as determined by transfection of HeLa cells with TTF-1 mutants in which serine residues have been replaced by alanines. Amplification and nucleotide sequence of the 5'-coding region of the TTF-1 gene in Ha-ras-FRTL-5 cells rule out the possibility that differences in phosphorylation were the consequence of any mutational event affecting residues within the N-terminal protein sequence. Hypophosphorylated TTF-1 is still able to bind its DNA consensus sequence within the thyroglobulin promoter, although a reporter construct whose expression is exclusively dependent on TTF-1 is not transactivated. Transfection of Ha-ras-FRTL-5 cells with an expression vector encoding the cAMP dependent protein kinase A (PKA) catalytic subunit partially reestablishes TTF-1 transcriptional activity. Taken together, these results indicate that the lack of specific thyroid gene expression in Ha-ras-FRTL-5 cells could be a direct consequence of the inability of TTF-1 to promote transcription.This work was supported by Grants from Dirección General de Investigación Científica y Técnica (PM97–0065, PM96–0074), Comunidad Autónoma de Madrid (AE00310, AC117), and Fundación Salud\ud2000 (Spain), and by grants from the Progetto Finalizzato Applicazioni Cliniche della Ricerca Oncologica of Consiglio Nazionale delle Ricerche,\udthe Associazione Italiana per la Ricerca sul Cancro (Italy).Peer reviewe