A functionally distinct member of the DP family of E2F subunits.

E2F transcription factors regulate genes involved in cell-cycle progression. In mammalian cells, physiological E2F exists as an E2F/DP heterodimer. Currently, eight E2F and two DP subunits have been characterized. We report here the characterization of a new member of the DP family, DP-4. While DP-4 exhibits certain similarities with members of the DP family, it also possesses a number of significant differences. Thus, DP-4 forms a heterodimer with E2F subunits, binds to the E2F site and associates with pocket proteins including pRb. In contrast to DP-1, however, DP-4/E2F-1 complexes exhibit reduced DNA binding activity. Furthermore, DP-4 interferes with E2F-1-dependent transcription and delays cell-cycle progression. These results highlight an emerging complexity in the DP family of E2F subunits, and suggest that DP-4 may endow E2F heterodimers with distinct transcription properties.

A genetic screen to identify genes that rescue the slow growth phenotype of c-myc null fibroblasts.

The c-myc gene is frequently over-expressed in human cancers and is involved in regulation of proliferation, differentiation and apoptosis. c-Myc is a transcription factor that acts primarily by regulating the expression of other genes. However, it has been very difficult to identify bona fide c-Myc target genes that explain its diverse biological activities. The recent generation of c-myc deficient Rat1A fibroblasts with a profound and stable growth defect provides a new system to search for genes that can substitute for c-myc in proliferation. In this study, we have attempted to identify genes that rescue the slow growth phenotype of c-myc null cells through introduction of a series of potent cell cycle regulatory genes and several retroviral cDNA expression libraries. None of the candidate genes tested, including SV40 T-antigen and adenovirus E1A, caused reversal of the c-myc null growth defect. Furthermore, extensive screens with high-complexity retroviral cDNA libraries from three different tissue sources revealed that only c-myc and N-myc rescued the c-myc null slow-growth phenotype. Our data support the notion that there are no functional equivalents of the myc family of proto-oncogenes and also suggest that there are no c-Myc-activated genes that alone can substitute for c-Myc in control of cell proliferation.

Functional interaction between a novel protein phosphatase 2A regulatory subunit, PR59, and the retinoblastoma-related p107 protein.

The proteins of the retinoblastoma family are potent inhibitors of cell cycle progression. It is well documented that their growth-inhibitory activity can be abolished by phosphorylation on serine and threonine residues by cyclin dependent kinases. In contrast, very little is known about the dephosphorylation of retinoblastoma-family proteins. We report here the isolation, by virtue of its ability to associate with p107, of a novel Protein Phosphatase 2A (PP2A) regulatory subunit, named PR59. PR59 shares sequence homology with a known regulatory subunit of PP2A, PR72, but differs from PR72 in its expression pattern and its functional properties. We show that PR59 co-immunoprecipitates with the PP2A catalytic subunit, indicating that PR59 is a genuine component of PP2A holo-enzymes. In vivo, PR59 associates specifically with p107, but not with pRb. Elevated expression of PR59 results in dephosphorylation of p107, but not of pRb, and inhibits cell proliferation by causing cells to accumulate in G1. These data support a model in which the distinct PP2A regulatory subunits act to target the PP2A catalytic subunit to specific substrates and suggest a role for PP2A in regulation of p107.

Ligand-independent recruitment of steroid receptor coactivators to estrogen receptor by cyclin D1.

The estrogen receptor (ER) is an important regulator of growth and differentiation of breast epithelium. Transactivation by ER depends on a leucine-rich motif, which constitutes a ligand-regulated binding site for steroid receptor coactivators (SRCs). Cyclin D1 is frequently amplified in breast cancer and can activate ER through direct binding. We show here that cyclin D1 also interacts in a ligand-independent fashion with coactivators of the SRC-1 family through a motif that resembles the leucine-rich coactivator binding motif of nuclear receptors. By acting as a bridging factor between ER and SRCs, cyclin D1 can recruit SRC-family coactivators to ER in the absence of ligand. A cyclin D1 mutant that binds to ER but fails to recruit coactivators preferentially interferes with ER activation in breast cancer cells that have high levels of cyclin D1. These data support that cyclin D1 contributes significantly to ER activation in breast cancers in which the protein is overexpressed. Our present results reveal a novel route of coactivator recruitment to ER and establish a direct role for cyclin D1 in regulation of transcription.

Repression of c-Myc responsive genes in cycling cells causes G1 arrest through reduction of cyclin E/CDK2 kinase activity.

The c-myc gene encodes a sequence-specific DNA binding protein involved in proliferation and oncogenesis. Activation of c-myc expression in quiescent cells is sufficient to mediate cell cycle entry, whereas inhibition of c-myc expression causes cycling cells to withdraw from the cell cycle. To search for components of the cell cycle machinery that are targets of c-Myc, we have made a mutant c-Myc protein, named MadMyc, that actively represses c-myc target genes. Expression of MadMyc in cycling NIH3T3 cells causes a significant accumulation of cells in G1. The MadMyc-induced G1 arrest is rescued by ectopic expression of cyclin E/CDK2 and cyclin D1/ CDK4, but not by Cdc25A, a known cell cycle target of c-Myc. The MadMyc G1 arrest does not require the presence of a functional retinoblastoma protein and is associated with a strong reduction in cyclin E/CDK2 kinase activity in arrested cells. MadMyc does not cause alterations in the expression levels of cyclin E, CDK2, p27kip1, cyclin D1 or CDK4 in G1-arrested cells. These data indicate that inhibition of c-Myc activity in exponentially growing cells leads to G1 arrest through loss of cyclin E-associated kinase activity.

mUBC9, a novel adenovirus E1A-interacting protein that complements a yeast cell cycle defect.

Adenovirus E1A encodes two nuclear phosphoproteins that can transform primary rodent fibroblasts in culture. Transformation by E1A is mediated at least in part through binding to several cellular proteins, including the three members of the retinoblastoma family of growth inhibitory proteins. We report here the cloning of a novel murine cDNA whose encoded protein interacts with both adenovirus type 5 and type 12 E1A proteins. The novel E1A-interacting protein shares significant sequence homology with ubiquitin-conjugating enzymes, a family of related proteins that is involved in the proteasome-mediated proteolysis of short-lived proteins. Highest homology was seen with a Saccharomyces cerevisiae protein named UBC9. Importantly, the murine E1A-interacting protein complements a cell cycle defect of a S. cerevisiae mutant which harbors a temperature-sensitive mutation in UBC9. We therefore named this novel E1A-interacting protein mUBC9. We mapped the region of E1A that is required for mUBC9 binding and found that the transformation-relevant conserved region 2 of E1A is required for interaction.

Functional analysis of Burkitt's lymphoma mutant c-Myc proteins.

The c-myc gene encodes a sequence-specific DNA binding protein that activates transcription of cellular genes. Transcription activation by Myc proteins is regulated by phosphorylation of serine and threonine residues within the transactivation domain and by complex formation with the retinoblastoma-related protein p107. In Burkitt's lymphoma, missense mutations within the c-Myc transactivation domain have been found with high frequency. It has been reported that mutant c-Myc proteins derived from Burkitt's lymphoma cell lines are resistant to inhibition by p107, thus providing a rationale for the increased oncogenic activity of these mutant c-Myc proteins. It has been suggested that these mutant c-Myc proteins resist down-modulation by p107 because they lack cyclin A-cdk2-dependent phosphorylation. Here, we have examined three different Burkitt's lymphoma mutant c-Myc proteins found in primary Burkitt's lymphomas and one mutant c-Myc protein detected in a Burkitt's lymphoma cell line. All four have an unaltered ability to activate transcription and are sensitive to inhibition of transactivation by p107. Furthermore, we provide evidence that down-modulation of c-Myc transactivation by p107 does not require phosphorylation of the c-Myc transactivation domain by cyclin A-cdk2. Our data indicate that escape from p107-induced suppression is not a general consequence of all Burkitt's lymphoma-associated c-Myc mutations, suggesting that other mechanisms exist to deregulate c-Myc function.

Molecular and functional characterisation of E2F-5, a new member of the E2F family.

The transcription factor DRTF1/E2F is implicated in the control of cellular proliferation due to its interaction with key regulators of cell cycle progression, such as the retinoblastoma tumour suppressor gene product and related pocket proteins, cyclins and cyclin-dependent kinases. DRTF1/E2F DNA binding activity arises when a member of two distinct families of proteins, DP and E2F, interact as DP/E2F heterodimers. Here, we report the isolation and characterisation of a new member of the E2F family of proteins, called E2F-5. E2F-5 was isolated through a yeast two hybrid assay in which a 14.5 d.p.c. mouse embryo library was screened for molecules capable of binding to murine DP-1, but also interacts with all known members of the DP family of proteins. E2F-5 exists as a physiological heterodimer with DP-1 in the generic DRTF1/E2F DNA binding activity present in mammalian cell extracts, an interaction which results in co-operative DNA binding activity and transcriptional activation through the E2F site. A potent transcriptional activation domain, which functions in both yeast and mammalian cells and resides in the C-terminal region of E2F-5, is specifically inactivated upon pocket protein binding. Comparison of the sequence with other members of the family indicates that E2F-5 shows a greater level of similarity with E2F-4 than to E2F-1, -2 and -3. The structural and functional similarity of E2F-5 and E2F-4 defines a subfamily of E2F proteins.

E2F-5, a new E2F family member that interacts with p130 in vivo.

E2F DNA binding sites are found in a number of genes whose expression is tightly regulated during the cell cycle. The activity of E2F transcription factors is regulated by association with specific repressor molecules that can bind and inhibit the E2F transactivation domain. For E2F-1, E2F-2, and E2F-3, the repressor is the product of the retinoblastoma gene, pRb. E2f-4 interacts with pRb-related p107 and not with pRb itself. Recently, a cDNA encoding a third member of the retinoblastoma gene family, p130, was isolated. p130 also interacts with E2F DNA binding activity, primarily in the G0 phase of the cell cycle. We report here the cloning of a fifth member of the E2F gene family. The human E2F-5 cDNA encodes a 346-amino-acid protein with a predicted molecular mass of 38 kDa. E2F-5 is more closely related to E2F-4 (78% similarity) than to E2F-1 (57% similarity). E2F-5 resembles the other E2Fs in that it binds to a consensus E2F site in a cooperative fashion with DP-1. By using a specific E2F-5 antiserum, we found that under physiological conditions, E2F-5 interacts preferentially with p130.

Interaction of c-Myc with the pRb-related protein p107 results in inhibition of c-Myc-mediated transactivation.

The product of the c-myc proto-oncogene, c-Myc, is a sequence-specific DNA binding protein with an N-terminal transactivation domain and a C-terminal DNA binding domain. Several lines of evidence indicate that c-Myc activity is essential for normal cell cycle progression. Since the abundance of c-Myc during the cell cycle is constant, c-Myc's activity may be regulated at a post-translational level. We have shown previously that the N-terminus of c-Myc can form a specific complex with the product of the retinoblastoma gene, pRb, in vitro. These data suggested a model in which pRb, or pRb-related proteins, regulate c-Myc activity through direct binding. We show here that the pRb-related protein p107, but not pRb itself, forms a specific complex with the N-terminal transactivation domain of c-Myc in vivo. Binding of p107 to c-Myc causes a significant inhibition of c-Myc transactivation. Expression of c-Myc releases cells from a p107-induced growth arrest, but not from pRb-induced growth arrest. Our data suggest that p107 can control c-Myc activity through direct binding to the transactivation domain and that c-Myc is a target for p107-mediated growth suppression.