E2F-1 binding affinity for pRb is not the only determinant of the E2F-1 activity.
E2F-1 is the major cellular target of pRB and is regulated by pRB during cell proliferation. Interaction between pRB and E2F-1 is dependent on the phosphorylation status of pRB. Despite the fact that E2F-1 and pRB have antagonistic activities when they are overexpressed, the role of the E2F-1-pRB interaction in cell growth largely remains unknown. Ideally, it would be better to study the properties of a pRB mutant that fails to bind to E2F, but retains all other activities. To date, no pRB mutation has been characterized in sufficient detail to show that it specifically eliminates E2F binding but leaves other interactions intact. An alternative approach to this issue is to ask whether mutations that change E2F proteins binding affinity to pRB are sufficient to change cell growth in aspect of cell cycle and tumor formation. Therefore, we used the E2F-1 mutants including E2F-1/S332-7A, E2F-1/S375A, E2F-1/S403A, E2F-1/Y411A and E2F-1/L132Q that have different binding affinities for pRB to better understand the roles of the E2F-1 phosphorylation and E2F-1-pRB interaction in the cell cycle, as well as in transformation and gene expression. Data presented in this study suggests that in vivo phosphorylation at amino acids 332-337, 375 and 403 is important for the E2F-1 and pRB interaction in vivo. However, although E2F-1 mutants 332-7, 375 and 403 showed similar binding affinity to pRB, they showed different characteristics in transformation efficiency, G(0) accumulation, and target gene experiments.
Sahin
Fikret
F
Department of Microbiology and Immunology, Finch University of Health Sciences/Chicago Medical School (now Rosalind Franklin University), North Chicago, Illinois 60064-3095, USA. fsahin29@hotmail.com
Sladek
Todd L
TL
D000818:Animals; D002453:Cell Cycle; D002460:Cell Line; D050687:E2F1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D005434:Flow Cytometry; D015151:Immunoblotting; D047468:Immunoprecipitation; D051379:Mice; D010766:Phosphorylation; D011485:Protein Binding; D016160:Retinoblastoma Protein / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D050687:E2F1 Transcription Factor; C493754:E2f1 protein, mouse; D016160:Retinoblastoma Protein