DNA binding of TEA/ATTS domain factors is regulated by protein kinase C phosphorylation in human choriocarcinoma cells.
Transcription enhancer factor 1 (TEF-1) controls the expression of a diverse set of genes. Previous studies implicated protein kinase C (PKC)-mediated signal transduction in modulating TEF function. We demonstrate that in human choriocarcinoma BeWo cells, the PKC activator 12-O-tetradecanoyl phorbol 13-acetate and PKC inhibitor bisindolylmaleimide reciprocally down- and up-regulate, respectively, TEF-mediated GGAATG core enhancer activity. In vitro TEF-1 phosphorylation with several PKC isozymes and phosphoamino acid analysis confirmed that TEF-1 is a potential PKC substrate. TEF-1.DNA complexes formed by BeWo nuclear extracts are supershifted by phosphoserine- and phosphothreonine- but not phosphotyrosine-specific antibodies, indicating that TEF-1 is phosphorylated in vivo at serine and threonine residues. The TEF-1 phosphorylation domain was localized to the third alpha-helix of the DNA binding domain and adjacent hinge region by phosphopeptide analysis. TEF-1 phosphorylation significantly reduced its DNA binding activity both in vitro and in vivo, providing a possible mechanism for the inhibitory action of PKC. Finally, BeWo cells contained abundant levels of gamma and delta PKC isoforms, and their overexpression resulted in even greater inhibition of GGAATG core enhancer activity after 12-O-tetradecanoyl phorbol 13-acetate treatment. These data strongly suggest that PKC-mediated phosphorylation is a key factor controlling TEF function.
Jiang
S W
SW
Department of Medicine, Mayo Clinic, Rochester, Minnesota 55905, USA.
Dong
M
M
Trujillo
M A
MA
Miller
L J
LJ
Eberhardt
N L
NL
D000595:Amino Acid Sequence; D002822:Choriocarcinoma; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D015536:Down-Regulation; D004742:Enhancer Elements, Genetic; D006801:Humans; D007527:Isoenzymes / Q000502:physiology; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D010769:Phosphothreonine / Q000378:metabolism; D011493:Protein Kinase C / Q000502:physiology*; D051745:Protein Kinase C-delta; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D000091262:TEA Domain Transcription Factors; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D007527:Isoenzymes; D009687:Nuclear Proteins; D000091262:TEA Domain Transcription Factors; C057182:TEAD1 protein, human; D014157:Transcription Factors; D010769:Phosphothreonine; D010768:Phosphoserine; D004247:DNA; C087857:protein kinase C gamma; C497258:PRKCD protein, human; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D013755:Tetradecanoylphorbol Acetate