A mutant E2F-1 transcription factor that affects the phenotype of NIH3T3 fibroblasts inefficiency associates with cyclin A-cdk2.
The amino-terminal domain of the E2F1 transcription factor is the site of association with cyclin A-cdk2, mapping to residues 87-94. A mutant of E2F1 lacking the first 87 amino acids (termed E2F1d87) has a number of potent effects on cellular phenotype when constitutively expressed in NIH3T3 fibroblasts. For example, in these fibroblasts the duration of S phase and the sensitivity to S phase chemotherapeutic agents are both increased. Since E2F1d87 only partially truncates the cyclin A-cdk2 binding domain, it was important to determine the level of cyclin A-cdk2 association with this mutant to correlate any reduction in association with the observed effects on the cell cycle. It was found that cyclin A-cdk2 binds E2F1d87 in an in vitro assay but that this binding is reduced approximately 8 fold compared with binding to full-length E2F1, whereas no detectable binding was seen to a mutant E2F1 that lacks the first 117 amino acids. Correspondingly, H1 kinase activity in E2F1d87 immunoprecipitates from E2F1d87-expressing cells was significantly reduced compared with that seen for full-length E2F1. From these data it appears that E2F1 with reduced cyclin A-cdk2 binding activity mediates the alteration in cell cycle parameters seen in these cells.
Department of Biochemistry and Molecular Pharmacology, Thomas Jefferson University, Philadelphia, PA 19107, USA.
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Jordan-Sciutto
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Hall
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D042846:CDC2-CDC28 Kinases; D002352:Carrier Proteins; D002453:Cell Cycle / Q000502:physiology; D018797:Cell Cycle Proteins; D019925:Cyclin A / Q000378:metabolism*; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism
C533431:Arid4a protein, mouse; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D019925:Cyclin A; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493754:E2f1 protein, mouse; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495893:Cdk2 protein, mouse; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases