| Inactivation of both copies of retinoblastoma susceptibility gene is linked to the appearance of certain tumors including retinoblastomas and osteosarcomas. The retinoblastoma protein (RB) is a 928 aa phosphorylated nuclear protein which is synthesized in a wide variety of mammalian cells. One of its functions is to negatively regulate cell growth. To date, all naturally occurring RB mutations known to be compatible with stable protein expression map to the T/E1A and cellular protein-binding region (the 'pocket' domain). When full-length RB and certain truncated forms were synthesized in human RB ;As a continued effort to probe the mechanisms of RB action, the transcription factor E2F-1, a suspected target of RB action, was overproduced and tested for its ability to overcome RB-induced phenotypes (G1 arrest and large cell phenotype). A central question was whether RB function only relied upon its ability to sequester E2F-1. Both wt E2F-1 and a mutant unable to bind to RB could overcome an RB induced G1 block. The E2F-1 overriding effect did not result in the hyperphosphorylation of RB. Overproduction of E2F-1 mutants capable of binding to RB, yet unable to bind to DNA due to either deletion or subtle mutations in the DNA-binding domain, reproducibly failed to overcome the RB-mediated G1 arrest. Therefore, stable RB-E2F complexes, instead of the sequestered E2F, are the active principals in delivering a G1 stop signal. Furthermore, it appears that the ability of E2F-1 to bind to DNA was necessary and sufficient to block the formation of large cells by RB. However, to promote S-phase entry, both the ability of E2F-1 to bind to DNA and the ability of E2F-1 to transactivate were required. Taken together, these results are consistent with a model where RB-E2F complexes deliver a growth inhibitor's signal which depends upon the binding to certain E2F responsive promoters. |
