Phosphorylation-dependent activation of the adenovirus-inducible E2F transcription factor in a cell-free system

Abstract

Adenovirus infection induces a large increase in the DNA binding activity of a cellular transcription factor that is utilized by the viral E2 promoter and termed E2F. Using cell-free extracts, we have developed an assay for the in vitro activation of DNA binding activity of E2F. E2F activity is undetectable in HeLa extracts but upon incubation with a fraction from adenovirus-infected cells, there is an ATP-dependent increase in E2F DNA binding activity. This increase does not occur using an equivalent fraction from dl312 (E1A-)-infected cells. Incubation of E2F with phosphatase inactivates E2F binding activity. Incubation of the phosphatase-inactivated E2F with an infected cell fraction restores E2F activity as does incubation with a known protein kinase. In contrast, incubation with an extract from mock-infected cells does not restore activity. We conclude that the DNA binding activity of E2F is regulated by phosphorylation in an E1A-dependent manner.