Expression cloning of a cDNA encoding a retinoblastoma-binding protein with E2F-like properties

Abstract

An expression vector was modified to permit the rapid synthesis of purified, 32P-labeled, glutathione S-transferase (GST)-retinoblastoma (RB) fusion proteins. The products were used to screen λgt11 expression libraries, from which we cloned a cDNA encoding a polypeptide (RBAP-1) capable of binding directly to a putative functional domain (the pocket) of the retinoblastoma gene product (RB). The RB “pocket” is known to bind, directly or indirectly, to the cellular transcription factor, E2F, implicated in cell growth control. We have found that RBAP-1 copurifies with E2F, interacts specifically with the adenovirus E4 ORF 6 7 protein, binds specifically and directly to a known E2F DNA recognition sequence, and contains a functional transactivation domain. Therefore, RBAP-1 is a species of E2F and can bind specifically to the RB pocket.