HA-RAS INTERFERENCE WITH THYROID-CELL DIFFERENTIATION IS ASSOCIATED WITH A DOWN-REGULATION OF THYROID TRANSCRIPTION FACTOR-I PHOSPHORYLATION

Mechanisms responsible for the lack of thyroid-specific differentiatio n markers in Ha-ras transformed FRTL-5 cells have been investigated. I n vivo cell labeling and immunoprecipitation demonstrate that phosphor ylation of the thyroid transcription factor-1 (TTF-1) is clearly reduc ed in thyroid cells transformed with the Ha-ras oncogene. Fingerprinti ng analysis of phosphotryptic peptides from FRTL-5 and Ha-ras-FRTL-5 c ells also reveals a heterogeneous pattern of TTF-1 phosphorylation in the transformed cell line. This heterogeneity is localized in the amin o terminal cluster of phosphoserines, as determined by transfection of HeLa cells with TTF-1 mutants in which serine residues have been repl aced by alanines. Amplification and nucleotide sequence of the 5'-codi ng region of the TTF-1 gene in Ha-ras-FRTL-5 cells rule out the possib ility that differences in phosphorylation were the consequence of any mutational event affecting residues within the N-terminal protein sequ ence. Hypophosphorylated TTF-1 is still able to bind its DNA consensus sequence within the thyroglobulin promoter, although a reporter const ruct whose expression is exclusively dependent on TTF-1 is not transac tivated. 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 expres sion in Ha-ras-FRTL-5 cells could be a direct consequence of the inabi lity of TTF-1 to promote transcription.