c-fos transcriptional activation and repression correlate temporally with the phosphorylation status of TCF.

Abstract

EGF-induction of human astrocytoma and A431 cells leads to c-fos transcriptional activation and then repression. This could be correlated with changes in the DNA binding characteristics of the c-fos regulatory protein ternary complex factor (TCF) present in nuclear extracts from these cells. Band shifts showed the appearance of induction-related slowly migrating protein -DNA complexes, detected as ternary complexes on the c-fos SRE using a truncated SRF molecule and by direct binding to the Drosophila E74 Ets-protein recognition sequence. By several criteria both types of complexes represented TCF. The appearance of the slow ternary and direct complexes correlated with c-fos transcriptional activation, and their disappearance coincided with the ensuing c-fos shut-off. Blocking c-fos transcriptional repression with the phosphatase inhibitor okadaic acid led to their continued presence. They were sensitive to protein phosphatase 2A but not 1α, and similar slow complexes were formed by partially purified p62 TCF phosphorylated by a copurifying kinase activity. Thus the phosphorylation state of TCF correlated strongly with C-fos promoter activity. Since ternary complex formation mediated by full-sized SRF was only slightly affected under comparable conditions, we propose a model for c-fos regulation involving modification of constitutively bound TCF.