Molecular cloning of cellular genes encoding retinoblastoma-associated proteins: identification of a gene with properties of the transcription factor E2F.

Abstract

The retinoblastoma protein interacts with a number of cellular proteins to form complexes which are probably crucial for its normal physiological function. To identify these proteins, we isolated nine distinct clones by direct screening of cDNA expression libraries using purified RB protein as a probe. One of these clones, Apl2, is expressed predominantly at the G1-S boundary and in the S phase of the cell cycle. The nucleotide sequence of Apl2 has features characteristic of transcription factors. The C-terminal region binds to unphosphorylated RB in regions similar to those to which T antigen binds and contains a transactivation domain. A region containing a potential leucine zipper flanked by basic residues is able to bind an E2F recognition sequence specifically. Expression ofApl2 in mammalian cells significantly enhances E2F-dependent transcriptional activity. These results suggest that Apl2 encodes a protein with properties known to be characteristic of transcription factor E2F.The retinoblastoma gene (RB), the first tumor suppressor gene identified, encodes a nuclear phosphoprotein which is ubiquitously expressed in vertebrates (19,20,42,44 (45,47), fluctuates with the cell cycle (7, 11, 13); (ii) the unphosphorylated form of RB is present predominantly in the Go-G1 stage (11, 13); (iii) microinjection of unphosphorylated RB into cells at early G1 inhibits their progression into the S phase (22). These data suggest that RB serves as a critical regulator of entry into the cell cycle and that its inactivation in normal cells leads to deregulated growth.How RB functions is the subject of intense inquiry. Two known biochemical properties of the RB protein have been described; one is its intrinsic DNA-binding activity, which was mapped to its C-terminal 300 amino acid residues (44, 66); another is its ability to interact with several oncoproteins of DNA tumor viruses (12,16,67). This interaction was mapped to two discontinuous regions at amino acids 394 to 571 and 649 to 773, designated as the T-binding domains (29,33). Interestingly, mutations of RB protein in tumors were *

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

“…Several other cellular proteins which have been shown to stably interact with cyclin/cdk complexes also contain a region that is homologous to the p130 cyclin binding region. Included among these are the highly related p107 protein, the cdk inhibitors p21 cip1 , p27 kip1 , p57 kip2 (ElDeiry et al, 1993;Gu et al, 1993;Harper et al, 1993;Xiong et al, 1993;Polyak et al, 1994;Toyoshima and Hunter, 1994;Lee et al, 1995;Matsuoka et al, 1995) and the transcription factor E2F-1 (Helin et al, 1992;Kaelin et al, 1992;Shan et al, 1992). A similar sequence is found in E2F-2 and -3; however, these proteins have not been demonstrated to interact with the cyclin/cdk complexes (Ivey-Hoyle et al, 1993;Lees et al, 1993).…”

“…An examination of the sequences of various other cyclin/cdk interacting proteins revealed sequences similar to the p130 RRRLFVE motif in several other proteins including E2F-1, 2 and 3, p21 cip1 , p27 kip1 and p57 kip2 (Helin et al, 1992;Kaelin et al, 1992;Shan et al, 1992;El-Deiry et al, 1993;Gu et al, 1993;Harper et al, 1993;Ivey-Hoyle et al, 1993;Lees et al, 1993;Xiong et al, 1993;Polyak et al, 1994;Toyoshima and Hunter, 1994;Lee et al, 1995;Matsuoka et al, 1995) (Figure 5a). As discussed below, each of these proteins has been shown to be present in complexes containing cyclin A or E. In some cases, this particular region has been implicated in the cyclin interactions.…”

“…Particularly noteworthy among these are the interactions with the E2F/DP family of transcription factors (Cao et al, 1992;Devoto et al, 1992;Helin et al, 1992Helin et al, , 1993Kaelin et al, 1992;Shan et al, 1992;Shirodkar et al, 1992;Cobrinik et al, 1993;Ivey-Hoyle et al, 1993;Lees et al, 1993;Bandara et al, 1994;Bejersbergen et al, 1994;Ginsberg et al, 1994;Buck et al, 1995;Hijmans et al, 1995;Ormondroyd et al, 1995;Sardet et al, 1995;Wu et al, 1995;Zhang and Chellappan, 1995). The E2F transcription factors are thought to control expression of a number of genes involved in cell cycle progression (reviewed in Cobrinik, 1996).…”

“…In almost all cases involving interactions with pRB, phosphorylation of pRB abrogates the interaction. For example, phosphorylation of pRB results in its dissociation from E2F transcription factors (Chellappan et al, 1991;Helin et al, 1992;Shan et al, 1992;Ewen et al, 1993;Dynlacht et al, 1994;Bremner et al, 1995;Suzuki-Takahashi et al, 1995) and from E1A, T antigen and E7 (Ludlow et al, 1990;Imai et al, 1991;Templeton and Weinberg, 1991). Phosphorylation of p107 appears to regulate its association with E2F proteins in a manner analogous to pRB (Bejersbergen et al, 1995;Xiao et al, 1996).…”

E2F1 controls germ cell apoptosis during the first wave of spermatogenesis

“…E2F1 is a member of the eight‐member E2F‐protein‐family that controls cell cycle, apoptosis, DNA damage response, and cell differentiation (for review see (Chen et al ., 2009)). Since its discovery, E2F1 has been implicated as a direct target of the retinoblastoma tumor suppressor protein (Helin et al ., 1992; Kaelin et al ., 1992; Shan et al ., 1992). In quiescent cells hypophosphorylated RB binds E2F1 and inhibits E2F‐mediated transcription.…”