Phosphorylation-dependent activation of the adenovirus-inducible E2F transcription factor in a cell-free system.
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.
Bagchi
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S
Howard Hughes Medical Institute, Duke University Medical Center, Department of Microbiology/Immunology, Durham, NC 27710.
Raychaudhuri
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P
Nevins
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JR
D017456:Adenovirus Early Proteins; D000260:Adenoviruses, Human / Q000235:genetics*; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D002474:Cell-Free System; D005814:Genes, Viral; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000235:genetics; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000378:metabolism*; D010766:Phosphorylation; D011401:Promoter Regions, Genetic
D017456:Adenovirus Early Proteins; D009856:Oncogene Proteins, Viral; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors