The B-domain lysine patch of pRB is required for binding to large T antigen and release of E2F by phosphorylation

Cell cycle-dependent, site-specific phosphorylation of the retinoblastoma protein, pRB, is mediated by cyclin-dependent kinases (CDKs) and regulates the binding of pRB to many proteins. We previously showed that the interaction of pRB with E2F on DNA was regulated by the accumulation of phosphate groups on pRB. Here we show that positively charged lysine residues in the B domain of pRB are necessary for the release of pRB from E2F on DNA following phosphorylation by cyclin E-cdk2 kinase. These lysine residues are also important in the binding of the simian virus 40 large T antigen (TAg) to pRB, and mutation of these lysines to arginines alters the dependency of the pRB-TAg interaction on phosphorylation of pRB.     

Cyclin-dependent kinases phosphorylate the adenovirus E1A protein, enhancing its ability to bind pRb and disrupt pRb-E2F complexes.

The adenovirus E1A protein of 243 amino acids has been shown to affect a variety of cellular functions, most notably the immortalization of primary cells and the promotion of quiescent cells into S phase. The activity of E1A is derived, in part, from its association with various cellular proteins, many of which play important roles in regulating cell cycle progression. E1A is known to have multiple sites of phosphorylation. It has been suggested that cell cycle-dependent phosphorylation may also control some of E1A's functions. We find now that immune complexes of cyclin-dependent kinases such as cdk4, cdk2, and cdc2 are all capable of phosphorylating E1A in vitro. Additionally, the sites on E1A phosphorylated by these kinases in vitro are similar to the E1A sites phosphorylated in vivo. We have also found that a phosphorylated E1A is far more efficient than an unphosphorylated E1A in associating with pRB and in disrupting E2F/DP-pRB complexes as well. On the basis of our findings and the differences in timing and expression levels of the various cyclins regulating cdks, we suggest that E1A functions at different control points in the cell cycle and that phosphorylation controls, to some extent, its biological functions.     

Human papillomavirus type 16 E7 protein inhibits DNA binding by the retinoblastoma gene product.

The human papillomavirus E7 gene can transform murine fibroblasts and cooperate with other viral oncogenes in transforming primary cell cultures. One biochemical property associated with the E7 protein is binding to the retinoblastoma tumor suppressor gene product (pRB). Biochemical properties associated with pRB include binding to viral transforming proteins (E1A, large T, and E7), binding to cellular proteins (E2F and Myc), and binding to DNA. The mechanism by which E7 stimulates cell growth is uncertain. However, E7 binding to pRB inhibits binding of cellular proteins to pRB and appears to block the growth-suppressive activity of pRB. We have found that E7 also inhibits binding of pRB to DNA. A 60-kDa version of pRB (pRB60) produced in reticulocyte translation reactions or in bacteria bound quantitatively to DNA-cellulose. Recombinant E7 protein used at a 1:1 or 10:1 molar ratio with pRB60 blocked 50 or greater than 95% of pRB60 DNA-binding activity, respectively. A mutant E7 protein (E7-Ala-24) with reduced pRB60-binding activity exhibited a parallel reduction in its blocking of pRB60 binding to DNA. An E7(20-29) peptide that blocks binding of E7 protein to pRB60 restored the DNA-binding activity of pRB60 in the presence of E7. Peptide E7(2-32) did not block pRB60 binding to DNA, while peptide E7(20-57) and an E7 fragment containing residues 1 to 60 partially blocked DNA binding. E7 species containing residues 3 to 75 were fully effective at blocking pRB60 binding to DNA. These studies indicate that E7 protein specifically blocks pRB60 binding to DNA and suggest that the E7 region responsible for this property lies between residues 32 and 75. The functional significance of these observations is unclear. However, we have found that a point mutation in pRB60 that impairs DNA-binding activity also blocks the ability of pRB60 to inhibit cell growth. This correlation suggests that the DNA-binding activity of retinoblastoma proteins contributes to their biological properties.     

Collective inhibition of pRB family proteins by phosphorylation in cells with p16INK4a loss or cyclin E overexpression

The activity of the retinoblastoma protein pRB is regulated by phosphorylation that is mediated by G(1) cyclin-associated cyclin-dependent kinases (CDKs). Since the pRB-related pocket proteins p107 and p130 share general structures and biological functions with pRB, their activity is also considered to be regulated by phosphorylation. In this work, we generated phosphorylation-resistant p107 and p130 molecules by replacing potential cyclin-CDK phosphorylation sites with non-phosphorylatable alanine residues. These phosphorylation-resistant mutants retained the ability to bind E2F and cyclin. Upon introduction into p16(INK4a)-deficient U2-OS osteosarcoma cells, in which cyclin D-CDK4/6 is dysregulated, the phosphorylation-resistant mutants, but not wild-type p107 or p130, were capable of inhibiting cell proliferation. Furthermore, when ectopically expressed in pRB-deficient SAOS-2 osteosarcoma cells, the wild-type as well as the phosphorylation-resistant pRB family proteins were capable of inducing large flat cells. The flat cell-inducing activity of the wild-type proteins, but not that of the phosphorylation-resistant mutants, was abolished by coexpressing cyclin E. Our results indicate that the elevated cyclin D- or cyclin E-associated kinase leads to systemic inactivation of the pRB family proteins and suggest that dysregulation of the pRB kinase provokes an aberrant cell cycle in a broader range of cell types than those induced by genetic inactivation of the RB gene.     

E2F-1 binding affinity for pRb is not the only determinant of the E2F-1 activity

E2F-1 is the major cellular target of pRB and is regulated by pRB during cell proliferation. Interaction between pRB and E2F-1 is dependent on the phosphorylation status of pRB. Despite the fact that E2F-1 and pRB have antagonistic activities when they are overexpressed, the role of the E2F-1-pRB interaction in cell growth largely remains unknown. Ideally, it would be better to study the properties of a pRB mutant that fails to bind to E2F, but retains all other activities. To date, no pRB mutation has been characterized in sufficient detail to show that it specifically eliminates E2F binding but leaves other interactions intact. An alternative approach to this issue is to ask whether mutations that change E2F proteins binding affinity to pRB are sufficient to change cell growth in aspect of cell cycle and tumor formation. Therefore, we used the E2F-1 mutants including E2F-1/S332-7A, E2F-1/S375A, E2F-1/S403A, E2F-1/Y411A and E2F-1/L132Q that have different binding affinities for pRB to better understand the roles of the E2F-1 phosphorylation and E2F-1-pRB interaction in the cell cycle, as well as in transformation and gene expression. Data presented in this study suggests that in vivo phosphorylation at amino acids 332-337, 375 and 403 is important for the E2F-1 and pRB interaction in vivo. However, although E2F-1 mutants 332-7, 375 and 403 showed similar binding affinity to pRB, they showed different characteristics in transformation efficiency, G₀ accumulation, and target gene experiments.     

p55, the Drosophila ortholog of RbAp46/RbAp48, is required for the repression of dE2F2/RBF-regulated genes

Many proteins have been proposed to be involved in retinoblastoma protein (pRB)-mediated repression, but it is largely uncertain which cofactors are essential for pRB to repress endogenous E2F-regulated promoters. Here we have taken advantage of the stream-lined Drosophila dE2F/RBF pathway, which has only two E2Fs (dE2F1 and dE2F2), and two pRB family members (RBF1 and RBF2). With RNA interference (RNAi), we depleted potential corepressors and looked for the elevated expression of groups of E2F target genes that are known to be directly regulated by RBF1 and RBF2. Previous studies have implicated histone deacetylase (HDAC) and SWI/SNF chromatin-modifying complexes in pRB-mediated repression. However, our results fail to support the idea that the SWI/SNF proteins are required for RBF-mediated repression and suggest that a requirement for HDAC activities is likely to be limited to a subset of targets. We found that the chromatin assembly factor p55/dCAF-1 is essential for the repression of dE2F2-regulated targets. The removal of p55 deregulated the expression of E2F targets that are normally repressed by dE2F2/RBF1 and dE2F2/RBF2 complexes in a cell cycle-independent manner but had no effect on the expression of E2F targets that are normally coupled with cell proliferation. The results indicate that the mechanisms of RBF regulation at these two types of E2F targets are different and suggest that p55, and perhaps p55's mammalian orthologs RbAp46 and RbAp48, have a conserved function in repression by pRB-related proteins.