pRB-related Proteins Research Articles

Activation of cyclin A gene expression by the cyclin encoded by human herpesvirus-8.

Human herpesvirus-8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus, encodes a protein, referred to as HHV8-Vcyc, with sequence similarity to human G1 cyclins, in particular of the D type. HHV8-Vcyc is expressed in Kaposi's sarcoma and functional analysis suggests that it can activate cyclin-dependent kinases (cdk) and thereby trigger inactivation of the retinoblastoma protein (pRb), indicating that HHV8-Vcyc may contribute to the oncogenic potential of HHV-8. We show here that HHV8-Vcyc can activate transcription of the human cyclin A gene in quiescent cells, a property shared with known transforming oncogenes. Transcriptional activation by HHV8-Vcyc depends on an E2F-binding site in the cyclin A promoter, and cdk6 kinase activity is required. The ability of HHV8-Vcyc to activate cyclin A gene expression is shared by D-type cyclins and cyclin E. Unlike D-type cyclins, HHV8-Vcyc is unable to trigger phosphorylation of the pRb-related protein p107 and fails to induce dissociation of p107 from E2F. Unlike cyclin E, HHV8-Vcyc fails to interact physically with E2F complexes on the cyclin A promoter. These results provide additional evidence for the notion that the HHV-8-encoded cyclin differs in several properties from cellular G1 cyclins.

The Role of the J domain of SV40 Large T in Cellular Transformation

SV40 large T antigen (TAg)-mediated transformation is dependent on binding to p53 and the retinoblastoma tumor suppressor protein (pRB) and inactivating their growth suppressive functions. Transformation minimally requires three regions of TAg: a C-terminal domain that mediates binding to p53; the LXCXE motif (residues 103–107), necessary for binding to pRB and the related proteins p107 and p130; and an N-terminal domain (residues 1–82) that contains homology to the J domain found in cellular DnaJ/Hsp40 molecular chaperone proteins. We have found that the N-terminal J domain of T Ag cooperates with the LXCXE motif to inactivate the growth suppressive functions of the pRB-related proteins.

An unexpected role for p53 in augmenting SV40 large T antigen-mediated tumorigenesis.

Simian virus 40 large T antigen transforms cells by sequestration and inactivation of the tumor suppressor proteins p53, retinoblastoma gene product (pRb), and the pRb-related proteins p107 and p130. Thus, the absence of functional p53 is expected to promote T antigen-mediated tumorigenesis. However, in a transgenic mouse model of T antigen-mediated beta cell carcinogenesis (Rip1Tag2), tumor volumes are significantly diminished when these mice are intercrossed with p53-deficient mice. Whereas the incidence of beta tumor cell apoptosis is unaffected, their proliferation rate is reduced in p53-deficient beta cell tumors in vivo and in cell lines established from these tumors in vitro. Biochemical analyses reveal higher levels of T antigen in wild-type tumor cells as compared to p53-deficient tumor cells. The data indicate that p53 stabilizes SV40 large T antigen, thereby augmenting its oncogenic potential as manifested by increased proliferation rates in wild-type beta tumor cells as compared to p53-deficient beta tumor cells.

PRB plays an essential role in cell cycle arrest induced by DNA damage.

To maintain genome stability, cells with damaged DNA must arrest to allow repair of mutations before replication. Although several key components required to elicit this arrest have been discovered, much of the pathway remains elusive. Here we report that pRB acts as a central mediator of the proliferative block induced by a diverse range of DNA damaging stimuli. Rb-/- mouse embryo fibroblasts are defective in arrest after gamma-irradiation, UV irradiation, and treatment with a variety of chemotherapeutic drugs. In contrast, the pRB related proteins p107 and p130 do not play an essential part in the DNA damage response. pRB is required specifically for the G1/S phase checkpoint induced by gamma-irradiation. Despite a defect in G1/S phase arrest, levels of p53 and p21 are increased normally in Rb-/- cells in response to gamma-irradiation. These results lead us to propose a model in which pRB acts as an essential downstream target of the DNA damage-induced arrest pathway. The ability of pRB to prevent replication of damaged DNA is likely to inhibit the propagation of carcinogenic mutations and may therefore contribute to its role as a tumor suppressor. Furthermore, because many cancer therapies act by damaging DNA, these findings also have implications for the treatment of tumors in which pRB is inactivated.

Inactivation of pRB-related proteins p130 and p107 mediated by the J domain of simian virus 40 large T antigen.

Inactivation of the retinoblastoma tumor suppressor protein (pRB) contributes to tumorigenesis in a wide variety of cancers. In contrast, the role of the two pRB-related proteins, p130 and p107, in oncogenic transformation is unclear. The LXCXE domain of simian virus 40 large T antigen (TAg) specifically binds to pRB, p107, and p130. We have previously shown that the N terminus and the LXCXE domain of TAg cooperate to alter the phosphorylation state of p130 and p107. Here, we demonstrate that TAg promotes the degradation of p130 and that the N terminus of TAg is required for this activity. The N terminus of TAg has homology to the J domain of the DnaJ family of molecular chaperone proteins. Mutants with mutations in the J-domain homology region of TAg are defective for altering p130 and p107 phosphorylation and for p130 degradation. A heterologous J-domain from a human DnaJ protein can functionally substitute for the N terminus of TAg in the effect on p107 and p130 phosphorylation and p130 stability. We further demonstrate that the J-domain homology region of TAg confers a growth advantage to wild-type mouse embryo fibroblasts (MEFs) but is dispensable in the case of MEFs lacking both p130 and p107. This indicates that p107 and p130 have overlapping growth-suppressing activities whose inactivation is mediated by the J domain of TAg.

Characterization of an E2F-p130 complex formed during growth arrest.

Electrophoretic mobility shift assays were used to analyse the pattern of E2F transcription factor complexes containing pRB and related 'pocket' proteins associated with changes in growth of monkey CV-1 cells. Little change was noted in pRB/E2F complexes following growth arrest or serum stimulation. Serum starvation induced the formation of a novel slowly-migrating p130/E2F complex, termed C7, which was comparable to one reported previously in terminally differentiated C2C12 mouse cells (Corbeil et al., 1995, Oncogene 11, 909-920) and thought to contain one or more additional unidentified proteins. After serum stimulation, C7 complex disappeared in S-phase but returned during mitosis. A major E2F complex containing p107 appeared during S-phase but was undetectable at other times. It appeared likely that regulation of pRB, p107 and p130 occurs by several mechanisms. First, phosphorylation has previously been proposed to be involved. All pRB-related proteins were found to be highly phosphorylated during S-phase when E2F activity was highest, and hypophosphorylated in arrested cells when E2F activity was low. Second, the absolute levels of some members of the RB family varied during the cell cycle. Levels of pRB protein and pRB/E2F complexes changed little; however, the amounts of both p107/E2F and p130/E2F complexes varied according to the overall levels of p107 and p130 proteins. Formation of the novel C7 complex in serum arrested cells suggested a third regulatory mechanism mediated by interactions with additional proteins. Our studies indicated that binding of such proteins with p130/E2F occurred via a site in the 'pocket' similar to that utilized by adenovirus E1A proteins. Such proteins could function as additional regulators of E2F-driven transcription.

Sp1 Is Required for the Early Response of α2(I) Collagen to Transforming Growth Factor-β1

It is currently debated whether AP1 or Sp1 is the factor that mediates transforming growth factor beta1 (TGF-beta) stimulation of the human alpha2(I) collagen (COL1A2) gene by binding to an upstream promoter element (TbRE). The present study was designed to resolve this controversy by correlating expression of COL1A2, AP1, and Sp1 in the same cell line and under different experimental conditions. The results strongly indicate that Sp1 is required for the immediate early response of COL1A2 to TGF-beta and AP1 is not. The Sp1 inhibitor mithramycin blocked stimulation of alpha2(I) collagen mRNA accumulation by TGF-beta, whereas the AP1 inhibitor curcumin had no effect. Furthermore, antibodies against Jun-B and c-Jun failed to identify immunologically related proteins in the TbRE-bound complex, irrespective of whether they were purified from untreated or TGF-beta-treated cells. AP1 did bind to the TbRE probe in vitro, but only in the absence of the upstream Sp1 recognition sequence. Based on this finding and DNA transfection results, we conclude that the AP1 sequence of the TbRE represents a cryptic site used under experimental conditions that either eliminate the more favorable Sp1 binding site or force the balance toward the less probable. Finally, a combination of cell transfections and DNA-binding assays excluded that COL1A2 transactivation involves the retinoblastoma gene product (pRb), an activator of Sp1, the pRb-related protein p107, an inhibitor of Sp1, or the Sp1-related repressor, Sp3.

P130 and p107 use a conserved domain to inhibit cellular cyclin-dependent kinase activity.

The pRB-related proteins p107 and p130 are thought to suppress growth in part through their associations with two important cell cycle kinases, cyclin A-cdk2 and cyclin E-cdk2, and transcription factor E2F. Although each protein plays a critical role in cell proliferation, the functional consequences of the association among growth suppressor, cyclin-dependent kinase, and transcription factor have remained elusive. In an attempt to understand the biochemical properties of such complexes, we reconstituted each of the p130-cyclin-cdk2 and p107-cyclin-cdk2 complexes found in vivo with purified, recombinant proteins. Strikingly, stoichiometric association of p107 or p130 with either cyclin E-cdk2 or cyclin A-cdk2 negated the activities of these kinases. The results of our experiments suggest that inhibition does not result from substrate competition or loss of cdk2 activation. Kinase inhibitory activity was dependent upon an amino-terminal region of p107 that is highly conserved with p130. Further, a role for this amino-terminal region in growth suppression was uncovered by using p107 mutants unable to bind E2F. To determine whether cellular complexes might display similar regulatory properties, we purified p130-cyclin A-cdk2 complexes from human cells and found that such complexes exist in two forms, one that contains E2F-4-DP-1 and one that lacks the heterodimer. These endogenous complexes behaved like the in vitro-reconstituted complexes, exhibiting low levels of associated kinase activity that could be significantly augmented by dissociation of p130. The results of these experiments suggest a mechanism whereby p130 and p107 suppress growth by inhibiting important cell cycle kinases.

Polyomavirus large T-antigen binds the ‘pRb related’ protein p130 through sequences in conserved region 2

The transforming potential of DNA tumor viruses derives mainly from the ability of their encoded oncogene products to interact with cellular proteins. Many of these viral oncoproteins share regions of sequence similarity, designated conserved region 1 and 2, which have been implicated in complex formation with pRb, the product of the retinoblastoma tumor suppressor gene, and related p107 and p130 species. It has now been shown that the E1A protein of adenovirus is able to bind to all three pRb-related proteins through sequences in conserved region 1 and 2. We have shown previously that polyomavirus large T-antigen also interacts with pRb and p107 in vitro. The pRb and p107 binding domains reside between residues 141–158, which include conserved region 2. In the present study, we demonstrate that polyomavirus large T-antigen also interacted with p130 in vitro through the same sequences in conserved region 2.

CDP/cut is the DNA-binding subunit of histone gene transcription factor HiNF-D: a mechanism for gene regulation at the G1/S phase cell cycle transition point independent of transcription factor E2F.

Transcription of the genes for the human histone proteins H4, H3, H2A, H2B, and H1 is activated at the G1/S phase transition of the cell cycle. We have previously shown that the promoter complex HiNF-D, which interacts with cell cycle control elements in multiple histone genes, contains the key cell cycle factors cyclin A, CDC2, and a retinoblastoma (pRB) protein-related protein. However, an intrinsic DNA-binding subunit for HiNF-D was not identified. Many genes that are up-regulated at the G1/S phase boundary are controlled by E2F, a transcription factor that associates with cyclin-, cyclin-dependent kinase-, and pRB-related proteins. Using gel-shift immunoassays, DNase I protection, and oligonucleotide competition analyses, we show that the homeodomain protein CDP/cut, not E2F, is the DNA-binding subunit of the HiNF-D complex. The HiNF-D (CDP/cut) complex with the H4 promoter is immunoreactive with antibodies against CDP/cut and pRB but not p107, whereas the CDP/cut complex with a nonhistone promoter (gp91-phox) reacts only with CDP and p107 antibodies. Thus, CDP/cut complexes at different gene promoters can associate with distinct pRB-related proteins. Transient coexpression assays show that CDP/cut modulates H4 promoter activity via the HiNF-D-binding site. Hence, DNA replication-dependent histone H4 genes are regulated by an E2F-independent mechanism involving a complex of CDP/cut with cyclin A/CDC2/ RB-related proteins.

Skeletal muscle cells lacking the retinoblastoma protein display defects in muscle gene expression and accumulate in S and G2 phases of the cell cycle.

Viral oncoproteins that inactivate the retinoblastoma tumor suppressor protein (pRb) family both block skeletal muscle differentiation and promote cell cycle progression. To clarify the dependence of terminal differentiation on the presence of the different pRb-related proteins, we have studied myogenesis using isogenic primary fibroblasts derived from mouse embryos individually deficient for pRb, p107, or p130. When ectopically expressed in fibroblasts lacking pRb, MyoD induces an aberrant skeletal muscle differentiation program characterized by normal expression of early differentiation markers such as myogenin and p21, but attenuated expression of late differentiation markers such as myosin heavy chain (MHC). Similar defects in MHC expression were not observed in cells lacking either p107 or p130, indicating that the defect is specific to the loss of pRb. In contrast to wild-type, p107-deficient, or p130-deficient differentiated myocytes that are permanently withdrawn from the cell cycle, differentiated myocytes lacking pRb accumulate in S and G2 phases and express extremely high levels of cyclins A and B, cyclin-dependent kinase (Cdk2), and Cdc2, but fail to readily proceed to mitosis. Administration of caffeine, an agent that removes inhibitory phosphorylations on inactive Cdc2/cyclin B complexes, specifically induced mitotic catastrophe in pRb-deficient myocytes, consistent with the observation that the majority of pRb-deficient myocytes arrest in S and G2. Together, these findings indicate that pRb is required for the expression of late skeletal muscle differentiation markers and for the inhibition of DNA synthesis, but that a pRb-independent mechanism restricts entry of differentiated myocytes into mitosis.

Interaction of Mouse Adenovirus Type 1 Early Region 1A Protein with Cellular Proteins pRb and p107

We demonstrated functional associations between mouse adenovirus type 1 (MAV-1) early region 1A (E1A) protein and both the mouse retinoblastoma protein (pRb) and the mouse pRb-related protein, p107. Interactions between MAV-1 E1A and mouse pRb or mouse p107 proteins were examined in infected cell lysates using a mouse embryonic fibroblast cell line infected with wild-type and mutant MAV-1 viruses. Using a polyclonal antibody to MAV-1 E1A, exogenously added mouse pRb or mouse p107 was coimmunoprecipitated from wild-type-, dlE105 (CR1Δ)-, and dlE106 (CR3Δ)-infected cell lysates. No coimmunoprecipitation was seen with cell lysates from dlE102 (CR2Δ) or pmE109, a mutant virus that produces no detectable E1A protein due to an ATG to TTG point mutation in the initiator methionine. Introduction of mouse pRb into SAOS-2 cells resulted in a flat and enlarged cell phenotype, whereas cotransfection of mouse pRb and MAV-1 E1A resulted in a significant reduction of flat cells, presumably due to E1A binding pRb. CR1Δ and CR2Δ E1A proteins were less effective at reducing the number of flat, enlarged cells induced by pRb expression than were the CR3Δ or wild-type E1A proteins. The reduced ability of these mutants to inactivate pRb relative to wild-type E1A correlated with their reduced ability to bind pRb in thein vitrocoimmunoprecipitation experiments. As a measure of p107/MAV-1 E1A complex formation in MAV-1-infected cells, we used mobility shift assays to examine cell extracts for the presence of p107-containing E2F protein–DNA complexes. Mock-, dlE102-, and pmE109-infected cell extracts formed a p107-containing complex, whereas wild-type-infected cell extracts did not. Thus the formation of a p107–E2F complex in wild-type- or these mutant-infected extracts inversely correlated with the presence of E1A–p107 complexes identified in thein vitrocoimmunoprecipitation experiments. This is consistent with E1A–p107 complexes forming in wild-type MAV-1-infected cells.

Importance of the Ser-132 phosphorylation site in cell transformation and apoptosis induced by the adenovirus type 5 E1A protein.

The 289-residue (289R) and 243R early region 1A (E1A) proteins of human adenovirus type 5 induce cell transformation in cooperation with either E1B or activated ras. Here we report that Ser-132 in both E1A products is a site of phosphorylation in vivo and is the only site phosphorylated in vitro by purified casein kinase II. Ser-132 is located in conserved region 2 near the primary binding site for the pRB tumor suppressor and, in 289R, just upstream of the conserved region 3 transactivation domain involved in regulation of early viral gene expression. Mutants containing alanine or glycine in place of Ser-132 interacted with pRB-related proteins at somewhat reduced efficiency; however, all Ser-132 mutants transformed primary rat cells in cooperation with E1B as well as or better than the wild type when both major E1A proteins were expressed. Such was not the case with mutants expressing only 289R. In cooperation with E1B, the Asp-132 and Gly-132 mutants yielded reduced numbers of smaller transformed foci. With activated ras, all Ser-132 mutants were significantly defective for transformation and the rare foci produced were small and contained extensive areas populated by low densities of flat cells. In the absence of E1B, all Ser-132 mutants induced p53-independent cell death more readily than virus expressing wild-type 289R. These results suggested that phosphorylation at Ser-132 may enhance the binding of pRB and related proteins and also reduce the toxicity of E1A 289R, thus increasing transforming activity.

The Epstein-Barr virus bZIP transcription factor Zta causes G0/G1 cell cycle arrest through induction of cyclin-dependent kinase inhibitors.

While oncoproteins encoded by small DNA tumor viruses and Epstein-Barr virus (EBV) latent antigens facilitate G1/S progression, the EBV lytic switch transactivator Zta was found to inhibit growth by causing cell cycle arrest in G0/G1 in several epithelial tumor cell lines. Expression of Zta results in induction of the tumor suppressor protein, p53, and the cyclin-dependent kinase inhibitors, p21 and p27, as well as accumulation of hypophosphorylated pRb. Up-regulation of p53 and p27 occurs by post-transcriptional mechanisms while expression of p21 is induced at the RNA level in a p53-dependent manner. Inactivation of pRb by transient overexpression of the human papillomavirus E7 oncoprotein indicates that pRb or pRb-related proteins are key mediators of the growth-inhibitory function of Zta. These findings suggest that EBV plays an active role in redirecting epithelial cell physiology to facilitate the viral replicative program through a Zta-mediated growth arrest function.

Id2 specifically alters regulation of the cell cycle by tumor suppressor proteins.

Cells which are highly proliferative typically lack expression of differentiated, lineage-specific characteristics. Id2, a member of the helix-loop-helix (HLH) protein family known to inhibit cell differentiation, binds to the retinoblastoma protein (pRb) and abolishes its growth-suppressing activity. We found that Id2 but not Id1 or Id3 was able to bind in vitro not only pRb but also the related proteins p107 and p130. Also, an association between Id2 and p107 or p130 was observed in vivo in transiently transfected Saos-2 cells. In agreement with these results, expression of Id1 or Id3 did not affect the block of cell cycle progression mediated by pRb. Conversely, expression of Id2 specifically reversed the cell cycle arrest induced by each of the three members of the pRb family. Furthermore, the growth-suppressive activities of cyclin-dependent kinase inhibitors p16 and p21 were efficiently antagonized by high levels of Id2 but not by Id1 Id3. Consistent with the role of p16 as a selective inhibitor of pRb and pRb-related protein kinase activity, p16-imposed cell cycle arrest was completely abolished by Id2. Only a partial reversal of p21-induced growth suppression was observed, which correlated with the presence of a functional pRb. We also documented decreased levels of cyclin D1 protein and mRNA and the loss of cyclin D1-cdk4 complexes in cells constitutively expressing Id2. These data provide evidence for important Id2-mediated alterations in cell cycle components normally involved in the regulatory events of cell cycle progression, and they highlight a specific role for Id2 as an antagonist of multiple tumor suppressor proteins.

E2F-1 but not E2F-4 can overcome p16-induced G1 cell-cycle arrest

Background: The transition from G1 to S phase is the key regulatory step in the mammalian cell cycle. This transition is regulated positively by G1-specific cyclin-dependent kinases (cdks) and negatively by the product of the retinoblastoma tumour suppressor gene, pRb. Hypophosphorylated pRb binds to and inactivates the E2F transcription factor, which controls the expression of genes required for S-phase progression. Hyperphosphorylation of pRb in late G1 phase results in the accumulation of active E2F, a critical event in the progression to S phase. The E2F factor is not a single entity, but rather represents a family of highly related molecules, all of which bind to pRb or the pRb-related proteins p107 and p130.Results In this study, we have used specific inhibitors of cdks to explore the requirements for cell-cycle progression from G1 to S phase. Expression of p16Ink4, which specifically inhibits cyclin D-directed cdks, blocks cells in G1 phase; this block can be overcome by expression of the viral proteins that inactivate pRb or by E2F-1. Importantly however, the G1 arrest is not overcome by overexpression of E2F-4. By using chimeric E2F proteins, containing amino-acid sequences from E2F-1 and E2F-4, we have shown that their differential abilities to overcome a p16-imposed arrest is determined by their respective amino-terminal regions. We also demonstrate that E2F-1 can promote entry into S phase without concomitant phosphorylation of pRb. In contrast to the p16-mediated G1 block, G1 arrest mediated by the cdk inhibitors p21Cip1or p27Kip1 cannot be bypassed either by inactivation of pRb or overexpression of E2F family members.Conclusion These data demonstrate that the role of the cyclin D-directed cdks in promoting the progression of cells from G1 into S phase is wholly to activate an E2F-1-like activity through phosphorylation, thus preventing the formation of the E2F–pRb complex. The cyclin E–cdk2 complex is also required for the G1/S transition but has a different and as yet undefined role. We also provide evidence for a functional difference between E2F-1 and E2F-4, dependant upon the region that contains the DNA-binding and dimerization domains. These results indicate that these two E2F family members are likely to regulate the expression of different subsets of E2F-responsive promoters.

The helix-loop-helix protein Id-1 and a retinoblastoma protein binding mutant of SV40 T antigen synergize to reactivate DNA synthesis in senescent human fibroblasts.

Normal somatic cells of higher organisms do not divide indefinitely. After a finite number of divisions, normal cells irreversibly cease proliferation by a process termed replicative or cellular senescence. Replicative senescence is controlled by multiple, dominant-acting genes about which very little is known. The only genes known to reactivate DNA synthesis in senescent cells are viral oncogenes encoding proteins that bind and inactivate the p53 and retinoblastoma (pRb) tumor suppressor proteins. SV40 T antigen is the best studied of these viral oncoproteins. T[K1] is a T antigen point mutant that selectively is defective in binding pRb and the pRb-related proteins p107 and p130. We show that T[K1] stimulated quiescent human fibroblasts to synthesize DNA nearly as well as wild-type T but was incapable of stimulating senescent cells. We tested several growth regulatory genes that are repressed in senescent cells for ability to restore activity to T[K1]. These included c-fos, c-jun, Id-1, Id-2, E2F-1, and cdc2. Only the helix-loop-helix (HLH) protein, Id-1, restored the ability of T[K1] to reactivate DNA synthesis in senescent cells. This activity of Id-1 was not shared by Id-2, a related protein, and depended on an intact HLH domain. It did not appear that Id-1 interacted directly with pRb or p107. Constitutive Id-1 expression failed to rescue proliferating cells from growth inhibition by pRb, p107, or p130, and failed to interact with pRb in the yeast two hybrid system. Because Id proteins negatively regulate basic-HLH (bHLH) transcription factors, we suggest that senescent cells express one or more bHLH factor that cooperates with pRb, or pRb-related proteins, to suppress proliferation.

E1A promotes association between p300 and pRB in multimeric complexes required for normal biological activity.

The oncogenes of the small DNA tumor viruses encode transforming proteins with multiple domains that influence the cell cycle and aspects of the transformed phenotype. Like other gene products of this type, the adenovirus E1A proteins influence the cell by binding to specific cell growth control proteins. These include members of the retinoblastoma gene product (pRB) family, which are bound by the E1A region 2-specific site, and p300, which is bound at the E1A amino terminus. Binding at these two sites is largely independent, and discrete transcription-regulating functions remain intact in E1A products when only one or the other binding site is functional. In this report, immunoprecipitation with p300 antibodies reveals the presence of the pRB family proteins in p300 complexes when E1A is expressed in host cells, indicating that E1A can mediate physical contact between p300 and the pRB-related proteins. The ability of E1A to induce proliferation efficiently in quiescent primary cells correlates closely with the ability to bind p300 and individual members of the pRB family simultaneously in multimeric complexes, even though the E1A active sites can bind their target proteins efficiently when separated on different molecules. Conservation of a spacer region between the two binding sites that is required for simultaneous binding and efficient induction of proliferation supports the concept that the E1A protein structure has evolved to facilitate simultaneous binding. These results indicate that the E1A proteins are designed not merely to sequester these cellular products, but also to bring them into proximal association with each other in biologically significant complexes.

E2F-1:DP-1 induces p53 and overrides survival factors to trigger apoptosis.

The E2F DNA binding activity consists of a heterodimer between E2F and DP family proteins, and these interactions are required for association of E2F proteins with pRb and the pRb-related proteins p107 and p130, which modulate E2F transcriptional activities. E2F-1 expression is sufficient to release fibroblasts from G0 and induce entry into S phase, yet it also initiates apoptosis. To investigate the mechanisms of E2F-induced apoptosis, we utilized interleukin-3 (IL-3)-dependent 32D.3 myeloid cells, a model of hematopoietic progenitor programmed cell death. In the absence of IL-3, E2F-1 alone was sufficient to induce apoptosis, and p53 levels were diminished. DP-1 alone was not sufficient to induce cell cycle progression or alter rates of death following IL-3 withdrawal. However, overexpression of both E2F-1 and DP-1 led to the rapid death of cells even in the presence of survival factors. In the presence of IL-3, levels of endogenous wild-type p53 increased in response to E2F-1, and coexpression of DP-1 further augmented p53 levels. These results provide evidence that E2F is a functional link between the tumor suppressors p53 and pRb. However, induction of p53 alone was not sufficient to trigger apoptosis, suggesting that the ability of E2F to override survival factors involves additional effectors.

Role of pRb-Related Proteins in Simian Virus 40 Large-T-Antigen-Mediated Transformation

Simian virus 40 large T-antigen (TAg) transformation is thought to be mediated, at least in part, by binding to and modulating the function of certain cellular proteins, including the retinoblastoma tumor suppressor gene product, pRb. TAg can disrupt the inhibitory complexes formed by pRb with the oncogenic transcription factor E2F, and this mechanism has been suggested to be important for TAg-mediated transformation. Residues 102 to 114 of TAg (including the LXCXE motif) are required for binding to pRb. Mutations within this LXCXE motif abolish the ability of TAg to bind to pRb as well as to transform certain cell types. TAg can also bind to at least two other cellular proteins, p107 and p130, that are related to pRb by sequence homology and share the ability to bind E2F. However, whether p107 and p130 are also targets in TAg-mediated transformation is less clear. To assess the relative contribution of the inactivation of pRb, p107, and p130 to transformation by TAg, fibroblasts were prepared from embryos derived from matings of mice heterozygous for an Rb knockout allele. The ability of TAg to transform fibroblasts homozygous for either wild-type or knockout Rb alleles was evaluated. It is demonstrated that the integrity of the LXCXE motif provides a growth advantage in Rb+/+ and Rb-/- cells. Furthermore, wild-type TAg, but not the LXCXE mutants, could bind to p107 and p130 and disrupt p107-E2F and p130-E2F binding complexes. These results suggest that p107 and p130 participate in TAg-mediated transformation and that they may behave as tumor suppressors.