Ha-ras interference with thyroid cell differentiation is associated with a down-regulation of thyroid transcription factor-1 phosphorylation.
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cluster of phosphoserines, as determined by transfection of HeLa cells with TTF-1 mutants in which serine residues have been replaced by alanines.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.
Velasco
J A
JA
Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Cientificas, Madrid, Spain.
Acebrón
A
A
Zannini
M
M
Martín-Pérez
J
J
Di Lauro
R
R
Santisteban
P
P
D000595:Amino Acid Sequence; D002454:Cell Differentiation / Q000502:physiology; D002461:Cell Line, Transformed; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D011905:Genes, ras / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000502:physiology; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D012694:Serine / Q000378:metabolism; D013961:Thyroid Gland / Q000166:cytology* / Q000378:metabolism*; D000074482:Thyroid Nuclear Factor 1; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
C000614412:NKX2-1 protein, human; D009687:Nuclear Proteins; D000074482:Thyroid Nuclear Factor 1; D014157:Transcription Factors; D012694:Serine; D017868:Cyclic AMP-Dependent Protein Kinases