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 divers e set of genes. Previous studies implicated protein kinase C (PKC)-mediated signal transduction in modulating TEF function. We demonstrate that in hum an choriocarcinoma BeWo cells, the PKC activator 12-O-tetradecanoyl phorbol 13-acetate and PHC inhibitor bisindolylmaleimide reciprocally down- and up -regulate, respectively, TEF-mediated GGAATG core enhancer activity. In vit ro TEF-1 phosphorylation with several PKC isozymes and phosphoamino acid an alysis confirmed that TEF-1 is a potential PKC substrate. TEF-1(.)DNA compl exes 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. T EF-1 phosphorylation significantly reduced its DNA binding activity both in vitro and in vivo, providing a possible mechanism for the inhibitory actio n 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-acetat e treatment. These data strongly suggest that PKC-mediated phosphorylation is a key factor controlling TEF function.