Dpi Dp2

Fig.12.1 The mammalian E2F protein family.The E2F family is composed of E2Fs 1-7, DPI and DP2. All E2Fs have a conserved DNA binding domain (DBD) and (except for E2F 7) a dimerization domain for binding to DP (Dim). The activating E2Fs 1 -3 contain a cyclin/cdk binding domain (cdk) at the N terminus and a strong activation domain at the C terminus. This activation domain overlaps with the Rb binding domain (Rb) so that binding by Rb masks the activation domain. The repressive E2Fs 4 and 5 also bind the pocket proteins but lack the cyclin/cdk binding domain and have weak transactivation domains. E2F 6 shares only the DNA binding domain and dimerization domain with the rest of the family, while E2F 7 has two conserved DNA binding domains, allowing it to function without DP to form a heterodimer. The DP proteins are distantly related members of the family that share the DNA binding domain and dimerization domain, allowing them to bind to E2Fs.

Rb family of proteins during the G0/G1 phases of the cell cycle (Muller et al., 1997; Verona et al, 1997). E2Fs 6 and 7, each representing one of the remaining two subgroups, also function as transcriptional repressors (Attwooll et al, 2004b; de Bruin et al., 2003; Di Stefano et al., 2003; Trimarchi et al., 1998; Trimarchi et al., 2001). However, in contrast to E2Fs 1-5, these E2F proteins do not have the Rb binding sequence at the C terminus, and therefore their function and regulation are independent of the Rb family of proteins.

The Rb Family of Proteins

Rb, pl07, and pl30 are members of a family of closely related proteins (Fig. 12.2). Together, these proteins are often referred to as the "pocket proteins" because their main sequence similarity resides in a domain, the pocket domain, which mediates interactions with viral oncoproteins such as El a. A spacer region that is not conserved separates the pocket domain into the A and B pockets. The spacers of pl07 and pl30 but not Rb contain binding sites for cyclin/cdk complexes. There are many phosphorylation sites on Rb which play critical roles in regulating the function of Rb (see below). Currently, over 100 proteins have been reported to interact with the Rb protein (Morris and Dyson, 2001), and most, if not all, of these interactions also involve the pocket domain. Interestingly, despite the sequence similarities between the Rb family members, specific members of the Rb family preferentially interact with specific members of the E2F family. As shown in Fig. 12.3, while Rb preferentially binds to E2Fs 1-4, pl07 and pl30 predominantly bind to E2F 4 and E2F 5 (Classon and Harlow, 2002). The preferential binding of activating E2Fs by Rb but not by pi07 or pi30 potentially underlies the observation that only Rb mutations are detected in cancers.

RB and E2F Proteins in Drosophila

There are two E2F (dE2Fl and dE2F2), one DP (dDP), and two Rb family (RBF and RBF2) genes in the Drosophila genome (Du et al, 1996a; Dynlacht et al, 1994; Ohtani and Nevins, 1994; Sawado et al, 1998; Stevaux et al, 2002). The Drosophila E2F proteins share many of the functional properties of their mammalian counterparts: they can dimerize with dDP and bind to E2F binding sites to activate or repress transcription. Similar to their mammalian counterparts, Drosophila E2F proteins regulate a set of genes coordinately expressed during S phase such as RNR2, PCNA, cyclin E, and DNA pola (Duronio and O'Farrell, 1994; Duronio et al, 1995). Interestingly, the two Drosophila E2F proteins behave like the first two subgroups of the mammalian E2F proteins: dE2Fl mainly functions as a transcription activator (Du, 2000), comparable to the mammalian activating E2Fs 1-3, while dE2F2 mainly functions to mediate active repression, similar to the mammalian repressive E2Fs 4 and 5 (Frolov et al., 2001). Furthermore, similar to the mammalian Rb protein that can bind to both the activating and the repressive E2F proteins, RBF can bind to both dE2Fl and dE2F2 proteins in Drosophila (see Fig. 12.3). In contrast, RBF2 can only bind dE2F2 (Stevaux et al., 2002) analogous to the mammalian pl07/pl30 proteins that bind specifically to the repressive E2F proteins (see Fig. 12.3). Thus the Rb-E2F pathway is well conserved and is much simpler in Drosophila than in the mammalian systems. The advantages of this simplified model system have been exploited in examining the relative importance of the E2F and Rb family members as seen in many of the experiments described below.

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