E2F-4, a new member of the E2F gene family, has oncogenic activity and associates with p107 in vivo.

Abstract

The E2F family of transcription factors controls the expression of genes that are involved in cell cycle regulation. E2F DNA-binding activity is found in complex with the retinoblastoma protein, pRb, and with the pRb-related pl07 and pl30. To date, cDNAs for three members of the E2F gene family have been isolated. However, all three E2Fs associate in vivo exclusively with pRb. We report here the cloning and functional analysis of a fourth E2F family member. E2F-4 encodes a 413-amino-acid protein with significant homology to E2F-1. E2F-4 antibodies recognize a 60-kD protein in anti-pl07 immunoprecipitates, indicating that E2F-4 associates with pi07 in vivo. Like the other E2Fs, E2F-4 requires DP-1 for efficient DNA binding and transcriptional activation of E2F site-containing promoters. Increased expression of E2F-4 and DP-1 in SaoS-2 osteosarcoma cells causes a shift from Gj-phase cells to S and G2/M-phase cells, suggesting a role for E2F-4 in regulation of cell-cycle progression. We show that expression of E2F-4 and DP-1 together with an activated ras oncogene in rat embryo fibroblasts, causes transformation, indicating that E2F-4 has oncogenic activity.

Functional interaction between a novel protein phosphatase 2A regulatory subunit, PR59, and the retinoblastoma-related p107 protein

“…HA tagged PR65 was subcloned into the mammalian expression vector pRC/CMV (Invitrogen). pCMV-pRb, pCMV-p107, pCMV-HAp107 and expression vectors for Cyclin D1, cdk4 and cdk4 dominant negative have been described previously (Hinds et al, 1992;van den Heuvel and Harlow, 1993;Zhu et al, 1993;Beijersbergen et al, 1994). pECE-HApRb was a gift from K Alevizopoulos.…”

“…Immunoprecipitations from transfected U2-OS or untransfected B104 cells were performed as described previously (Beijersbergen et al, 1994). In short, the cells were scraped in ELB (250 mM NaCl, 0.1% NP40, 50 mM HEPES pH 7.0, 5 mM EDTA) supplemented with protease inhibitors (Complete, Boehringer Mannheim and 1 mM phenylmethylsulfonyl¯uoride) and incubated on ice for 25 min.…”

“…Transfected cells were immunoprecipitated as described previously (Beijersbergen et al, 1994). The immunoprecipitate or the total cell lysate were separated on a SDS 10% or 7% polyacrylamide gel and transferred to nitrocellulose.…”

Identification of a p130 domain mediating interactions with cyclin A/cdk 2 and cyclin E/cdk 2 complexes

“…The E2F transcription factors are thought to control expression of a number of genes involved in cell cycle progression (reviewed in Cobrinik, 1996). The interactions between the Rb family members and E2F members e ectively inhibit E2F-mediated transactivation (Hamel et al, 1992;Heibert et al, 1992;Weintraub et al, 1992;Zamanian and LaThangue, 1992;Flemington et al, 1993;Helin et al, 1993;Schwartz et al, 1993;Zamanian and LaThangue, 1993;Zhu et al, 1993;Beijersbergen et al, 1994;Ginsberg et al, 1994;Johnson, 1995;Smith and Nevins, 1995;Vairo et al, 1995). Interactions involving pRB or p107 and various other cellular proteins, including other transcription factors, also have been described (reviewed in Whyte, 1995).…”

Granulocytic differentiation of myeloid progenitor cells by p130, the retinoblastoma tumor suppressor homologue

“…Through this interaction, p107 and p130 are capable of inhibiting the kinase activity of cyclin ± Cdk complexes (Zhu et al, 1995;Woo et al, 1997;Castano et al, 1998). In addition, p107 and p130 selectively bind E2F-4 and E2F-5 (Beijersbergen et al, 1994;Hijmans et al, 1995;Sardet et al, 1995;Vairo et al, 1995), whereas pRB preferentially binds E2F-1, E2F-2 and E2F-3 (Ikeda et al, 1996;Moberg et al, 1996). Together with the di erential regulation of protein levels during cell cycle (Garriga et al, 1998), each member of the pRB family appears to perform shared as well as unique cell cycle-regulatory roles within a single cell.…”