In response to TGF-beta stimulation, a complex composed of SMAD2/3:SMAD4 heterotrimer and RBL1 (p107), E2F4/5 and DP1/2 can be detected in the nucleus. Formation of this complex was confirmed by both co-immunoprecipitation of the endogenous complex from the human keratinocyte cell line HaCat and by protein interaction studies using tagged recombinant proteins. It is possible that cells contain pre-assembled cytosolic complexes of SMAD2/3, RBL1 (p107) and E2F4/5, that translocate to the nucleus after TGF-beta stimulation, when phosphorylated SMAD2/3 recruit SMAD4. The MH2 domain of SMAD3 establishes independent contacts with the N-termini of E2F4 (or E2F5) and unphosphorylated RBL1 (p107). RBL2 (p130), RB1 and E2F1 do not interact with SMAD2/3 (Chen et al. 2002).
Complex formed by RBL1 (p107), E2F4/5, DP1/2 and a trimer of phosphorylated R-SMADs (SMAD2/3) and SMAD4 (Co-SMAD) cooperatively binds to TIE (TGF-beta inhibitory element) and E2F sites in the MYC gene promoter (Chen et al. 2002).
Complex formed by RBL1 (p107), E2F4/5, DP1/2 and a trimer of phosphorylated R-SMADs (SMAD2/3) and SMAD4 (Co-SMAD) cooperatively binds to TIE (TGF-beta inhibitory element) and E2F sites in the MYC promoter and promotes cell-cycle independent inhibition of MYC transcription in response to TGF-beta stimulation (Chen et al. 2002).
p130 (RBL2) is able to bind complexes of CDK2 with either cyclin A or cyclin E through the cyclin-binding LFG motif within the pocket domain, which is conserved in p107 (RBL1) and p21/WAF1/Cip1 family of cyclin-dependent kinases. In addition to LFG motif, amino terminal region of p130 (RBL2), conserved in p107 (RBL1), is necessary for inhibition of CDK2 kinase activity. Presence of E2F is not required for this interaction.
The promoter of the CDC25C gene contains p53 response elements as well as E2F binding sites and can bind both TP53 (St Clair et al. 2004) and E2F4 (Benson et al. 2014). E2F4 transcription repressor complex consists of E2F4, a transcriptional co-factor TFDP1 (DP1) or TFDP2 (DP2), and a retinoblastoma family protein RBL1 (p107) or RBL2 (p130). The CDK inhibitor p21 (CDKN1A), induced by TP53, positively affects E2F4 recruitment to the CDC25C promoter, probably by upregulating RBL2 (Helmbold et al. 2009, Benson et al. 2014).