PROTEIN-KINASE-A ACTIVATION OF THE SURFACTANT PROTEIN-B GENE IS MEDIATED BY PHOSPHORYLATION OF THYROID TRANSCRIPTION FACTOR-1

Thyroid transcription factor 1 (TTF-1) is a homeodomain-containing nuc lear transcription factor expressed in epithelial cells of the lung an d thyroid. TTF-1 binds to and activates the transcription of genes exp ressed selectively in the respiratory epithelium including pulmonary s urfactant A, B, C and Clara cell secretory protein. Transfection with a plasmid encoding the cyclic AMP-dependent protein kinase (protein ki nase A; PKA) catalytic subunit, Cat-beta, stimulated the phosphorylati on of a TTF-l-flag fusion protein 6-7-fold in H441 pulmonary adenocarc inoma cells. Recombinant TTF-1 was phosphorylated by purified PKA cata lytic subunit in the presence of [gamma-P-32]ATP. PKA catalytic subuni t family members, Cat-alpha and Cat-beta markedly enhanced the transcr iptional activation of surfactant B gene promoters by TTF-1 in vitro. Peptide mapping was used to identify a PKA phosphorylation site at the NH2 terminus of TTF-1. A 17-amino acid synthetic peptide comprising t his site completely inhibited the PKA-dependent phosphorylation of TTF -1 in vitro. A substitution mutation of TTF-1 (Thr(9) --> Ala) abolish ed phosphorylation by PKA and reduced transactivation of the surfactan t B gene promoter. Transfection with a plasmid encoding the cAMP regul atory element binding factor inhibited transcriptional activity of the surfactant protein B gene promoter. Phosphorylation of TTF-1 mediates PKA-dependent activation of surfactant protein B gene transcription.