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At least 19 recordsLinked to original sources
PubMed1998

Differential activities of E2F family members: unique functions in regulating transcription.

Several regulators of E2F transcriptional activity, including the retinoblastoma tumor suppressor (Rb) protein, p16Ink4a, cyclin D1, and cyclin-dependent kinase 4, have been shown to be targets for genetic alterations that underlie the development of human cancers. Deregulation of E2F transcription factors as a result of these genetic alterations is believed to contribute to tumor development. This hypothesis is supported by the finding that at least some members of the E2F gene family can contribute to oncogenic transformation when overexpressed. Each E2F family member can dimerize with DP proteins, bind consensus E2F sites, and activate transcription. Several pieces of evidence suggest, however, that the various E2F species have unique functions in regulating transcription. We compared the abilities of E2F1, E2F4, and E2F5 to activate transcription from a variety of gene promoters and found that in all cases E2F1 was the most potent activator, followed by E2F4 and then by E2F5. Construction of chimeric proteins between E2F1 and E2F4 demonstrated that either the carboxy terminus or the amino terminus of E2F1 could make E2F4 a more potent activator. In contrast, neither the carboxy terminus nor the amino terminus of E2F1 could significantly increase the activity of E2F5. We found that, consistent with a role for E2F5 in transcriptional repression, E2F5's binding partner p130, like Rb, could also actively repress transcription when directly bound to a target promoter.

Base Sequence↗
PubMed2005

Cloning and characterization of mouse E2F8, a novel mammalian E2F family member capable of blocking cellular proliferation.

The E2F transcription factor family plays a crucial and well established role in cell cycle progression. Deregulation of E2F activities in vivo leads to developmental defects and cancer. Based on current evidence in the field, mammalian E2Fs can be functionally categorized into either transcriptional activators (E2F1, E2F2, and E2F3a) or repressors (E2F3b, E2F4, E2F5, E2F6, and E2F7). We have identified a novel E2F family member, E2F8, which is conserved in mice and humans and has its counterpart in Arabidopsis thaliana (E2Ls). Interestingly, E2F7 and E2F8 share unique structural features that distinguish them from other mammalian E2F repressor members, including the presence of two distinct DNA-binding domains and the absence of DP-dimerization, retinoblastoma-binding, and transcriptional activation domains. Similar to E2F7, overexpression of E2F8 significantly slows down the proliferation of primary mouse embryonic fibroblasts. These observations, together with the fact that E2F7 and E2F8 can homodimerize and are expressed in the same adult tissues, suggest that they may have overlapping and perhaps synergistic roles in the control of cellular proliferation.

Amino Acid Sequence↗
PubMed1999

Regulation of the G1/S transition phase in mesangial cells by E2F1.

It has been established that E2F transcription factors are essential for the regulation of the cell cycle. The E2Fs play an important role in G1/S transition phase, as they regulate the activation of several genes whose products are required for DNA synthesis. E2Fs bind to the retinoblastoma protein family and their transcriptional activities are suppressed in the G0 and early G1 phases. The E2F family consists of a group of five closely related proteins (E2F1 through E2F5). Proliferation of the mesangial cell is a common feature of many glomerular diseases, but the regulation of mesangial cell cycle has not been clarified, nor has the participation of the E2F family in mesangial cells. To elucidate the mechanisms of G1/S transition phase in mesangial cells, we investigated the roles of the E2F family in the mesangial cell cycle. In primary cultured mesangial cells, the protein expression of E2F1 through E2F3 was induced by fetal calf serum (FCS) stimulation. E2F1 especially was strongly induced by mitogenic stimulation. The E2F4 protein was abundantly expressed in the quiescent state and was slightly increased by FCS stimulation. We considered E2F1 to be representative of the E2F family, and used adenovirus-mediated gene transfer to investigate the function of E2F1 to show that overexpression of E2F1 promoted cell cycle progression as measured by a flow cytometer. Furthermore, we investigated the effect of E2F1 overexpression to cyclin D1 and cyclin E expression. Because we previously reported that the regulation of G1 cyclins is a key factor in the G1/S transition phase in mesangial cells, we showed that overexpression of E2F1 induced protein expression of cyclin D1 and cyclin E and increased promoter activity. Thus, we conclude that E2F1 plays an important role in the G1/S transition phase and acts on the mesangial cell cycle through two distinct pathways: (1) E2F1 directly transcribes an S-phase gene, and (2) E2F1 promotes cell cycle progression via the induction of cyclin D1 and cyclin E.

Animals↗
PubMed1997

Position-dependent transcriptional regulation of the murine dihydrofolate reductase promoter by the E2F transactivation domain.

Activity of the dihydrofolate reductase (dhfr) promoter increases at the G1-S-phase boundary of the cell cycle. Mutations that abolish protein binding to an E2F element in the dhfr promoter also abolish the G1-S-phase increase in dhfr transcription, indicating that transcriptional regulation is mediated by the E2F family of proteins. To investigate the mechanism by which E2F regulates dhfr transcription, we moved the E2F element upstream and downstream of its natural position in the promoter. We found that the E2F element confers growth regulation to the dhfr promoter only when it is proximal to the transcription start site. Using a heterologous E2F element, we showed that position-dependent regulation is a property that is promoter specific, not E2F element specific. We demonstrated that E2F-mediated growth regulation of dhfr transcription requires activation of the dhfr promoter in S phase and that the C-terminal activation domains of E2F1, E2F4, and E2F5, when fused to the Gal4 DNA binding domain, are sufficient to specify position-dependent activation. To further investigate the role of activation in dhfr regulation, we tested other transactivation domains for their ability to activate the dhfr promoter. We found that the N-terminal transactivation domain of VP16 cannot activate the dhfr promoter. We propose that, unlike other E2F-regulated promoters, robust transcription from the dhfr promoter requires an E2F transactivation domain close to the transcription start site.

3T3 Cells↗
PubMed1996

Regulation of dihydrofolate reductase gene expression and E2F components in human diploid fibroblasts during growth and senescence.

The induction of dihydrofolate reductase (DHFR), a key enzyme in DNA biosynthesis that is induced just before the onset of S phase, is markedly attenuated in senescent human fibroblasts (Pang and Chen, 1994, J. Cell. Physiol., 160:531-538). Footprinting analysis of the 365 bp promoter region of the human DHFR gene (-381 to -17) indicated that nuclear proteins bind to a cluster of cis-elements, including two overlapping E2F binding sequences, two Sp1 sites, and one Yi sequence. Gel mobility shift assays were performed to assess the role of each cis-element in the regulation of DHFR gene expression. We found that 1) Sp1 binding activity was constitutively expressed throughout the cell cycle in early passage and senescent cells; 2) Yi binding activity was undetectable in both early passage and senescent cells; and 3) E2F binding activity was serum-inducible, senescence-dependent, and prominent in presenescent cells but strikingly diminished in senescent cells. Northern blot analysis of the expression of E2F and DP family members showed that the E2F-1, E2F-4, and E2F-5 mRNA was growth- and senescence-dependent, whereas E2F-3, DP-1, and DP-2 expression was constitutive and senescence-independent. In contrast, E2F-2 mRNA was not detectable in IMR-90 or WI-38 human fibroblasts. Western blot analysis showed that among the E2F-associated proteins, the expression of E2F-1, cyclin A, and cyclin B but not p107 was cell cycle- and senescence-dependent. A nuclear extract mixing experiment suggested that an inhibitory factor may further reduce E2F binding activity in senescent cells.

Base Sequence↗
PubMed1998

The molecular and functional characterization of E2F-5 transcription factor.

The E2F activity plays a critical role in the control of cell cycle and action of tumor suppressor proteins and is also a target of the transforming proteins of small DNA tumor viruses. We describe here molecular cloning and functional characterization of a fifth member of the E2F family of transcription factors. E2F-5 protein is more homologous to E2F-4 (72% amino acid identity) than to E2F-1, E2F-2, and E2F-3 (35% amino acid identity). Based on structural and functional criteria, the E2F family appears to comprise two distinct sub-families, one composed of E2F-1, E2F-2, and E2F-3 and the other composed of E2F-4 and E2F-5, E2F-5 mRNA is expressed in a wide variety of human tissues. The protein is expressed as multiple species ranging in size from 46 to 54 kDa as a result of differential phosphorylation. The expression of a reporter gene containing E2F binding sites in the promoter is transcriptionally activated by E2F-5 in a cooperative manner with the DP-1 protein. The interaction between E2F-5 and DP-1 is demonstrated using a two-hybrid system in mammalian cells. We have also demonstrated the presence of a strong transactivation domain at the carboxy terminus (273-346 amino acid residues) of E2F-5 protein.

Amino Acid Sequence↗
PubMed2002

Role of pRB-family/E2F complex in the inhibition of IL-3-dependent lymphoid cell proliferation.

Interleukin 3 (IL-3)-dependent proliferation of haematopoietic cells is specifically inhibited by p130, a member of the pRB-family proteins. p130 interacts with the cell-cycle regulatory E2F transcription factors, notably E2F-4 and E2F-5, and affects promoters containing E2F-binding sites through two distinct mechanisms. First, upon complex formation with E2F, it blocks transcriptional activation by E2F. Second, the formed p130-E2F complex binds to E2F sites and actively represses transcription by inhibiting the activity of surrounding enhancer elements on the promoter. To pursue the relative contributions of each mechanism in the p130-mediated inhibition of IL-3-dependent cell proliferation, we employed a dominant-negative DP-1, which suppresses both E2F-dependent transactivation and the formation of active transcriptional repressors. Ectopic expression of the dominant negative DP-1 in the IL-3-dependent BaF3 lymphoid cells gave rise to an inhibition of cell proliferation, which was concomitantly associated with a decrease in levels of cyclin E, an indispensable molecule for G1 to S-phase cell-cycle progression. Our results indicate that blocking E2F-dependent transactivation, but not the formation of p130-E2F transcriptional repressor complexes, is responsible for the inhibition of IL-3-dependent cell growth by p130.

Animals↗
PubMed2002

SU9516, a cyclin-dependent kinase 2 inhibitor, promotes accumulation of high molecular weight E2F complexes in human colon carcinoma cells.

The E2F family plays a critical role in the expression of genes required for entry into and progression through S phase. E2F-mediated transcription is repressed by the tumor suppressor retinoblastoma protein (pRb), which results in sequestration of E2F in a multiprotein complex that includes pRb. Derepression of E2F results from a series of complex phosphorylation events mediated by cyclin D/cdk4 and cyclin E/cdk2. We have employed a novel 3-substituted indolinone compound, 3-[1-(3H-imidazol-4-yl)-meth-(Z)-ylidene]-5-methoxy-1,3-dihydro-indol-2-one (SU9516), which selectively inhibits cdk2 activity (Lane et al., Cancer Res 2001;61:6170-7) to investigate these events. Electrophoretic mobility gel shift assays were performed on SU9516-treated and -untreated HT-29, SW480, and RKO human colon cancer cell extracts. Treatment with 5 microM SU9516 prevented dissociation of pRb from E2F1 in all cell lines (HT-29>RKO>SW480). Treatment effects were time-dependent, demonstrating greater inhibition at 48 hr versus 24hr in HT-29 cells. Furthermore, E2F species were sequestered in complexes with p107, p130, DP-1, and cyclins A and E. After a 24-hr treatment with 5 microM SU9516, cyclin D1 and cdk2 levels decreased by 10-60%. These findings delineate a previously undescribed mechanism for SU9516-mediated cell growth arrest through down-regulation of cyclin D1, inhibition of cdk2 levels and activity, and pan-sequestration of E2F.

CDC2-CDC28 Kinases↗
PubMed2002

E2F4/5 and p107 as Smad cofactors linking the TGFbeta receptor to c-myc repression.

Smad3 is a direct mediator of transcriptional activation by the TGFbeta receptor. Its target genes in epithelial cells include cyclin-dependent kinase inhibitors that generate a cytostatic reponse. We defined how, in the same context, Smad3 can also mediate transcriptional repression of the growth-promoting gene c-myc. A complex containing Smad3, the transcription factors E2F4/5 and DP1, and the corepressor p107 preexists in the cytoplasm. In response to TGFbeta, this complex moves into the nucleus and associates with Smad4, recognizing a composite Smad-E2F site on c-myc for repression. Previously known as the ultimate recipients of cdk regulatory signals, E2F4/5 and p107 act here as transducers of TGFbeta receptor signals upstream of cdk. Smad proteins therefore mediate transcriptional activation or repression depending on their associated partners.

Active Transport, Cell Nucleus↗
PubMed2001

Expression of the E2F and retinoblastoma families of proteins during neural differentiation.

Development of the brain is determined by a strictly orchestrated program of proliferation, migration, apoptosis, differentiation, synaptogenesis, tract formation, and myelination. The E2F family of transcription factors, whose activity and functions are regulated in large part through interactions with the retinoblastoma (Rb) family of tumor suppressor proteins, has been implicated as a key regulator of proliferation, differentiation, and apoptosis in a variety of tissues. We have examined levels of the E2F and Rb families of proteins during both brain development and neural differentiation of P19 cells, and found the expression profiles during these two processes of neural development and maturation to be quite similar, i.e., strong up-regulation of p130, pronounced down-regulation of p107, moderate up-regulation of pRb, and significant down-regulation of most species of E2F and dimerization protein (DP). However, several specific isoforms, namely a 30 kDa form of DP-2, a 57 kDa species of E2F-3, a 59 kDa form of E2F-5 and the isoforms of E2F-1 recognized by the E2F-1 (KH-95) antibody were up-regulated suggesting that these particular isoforms of E2F and DP play a tissue-specific function in differentiation and maturation of nervous tissue. The potential role of the E2F/DP family of transcription factors in aspects of neural development and differentiation are considered.

Animals↗
PubMed2000

E2F transcription factors are differentially expressed in murine gametes and early embryos.

We have examined the murine genes encoding transcription factors E2F1, -3, -5 and -6 in gametes and early embryos. All genes are expressed as maternal transcripts and all are efficiently transcribed after the blastocyst stage. Between those two stages, each E2F mRNA is transcribed with a distinctive and unique pattern. E2F proteins are also differentially expressed and compartmentalized in pre-implantation embryos.

3T3 Cells↗
PubMed1997

Expression patterns of the E2F family of transcription factors during mouse nervous system development.

The E2F family of transcription factors consists of two subgroups termed E2F and DP. E2F is required for cell proliferation, and is necessary for fruit fly development. E2F activity is a target for regulation by the retinoblastoma gene family, which includes pRB, p107 and p130. Mutant RB-/-, RB-/-:p107-/- and p107-/-:p130-/- mice develop abnormally, probably as a result of dysregulation in the activity of E2F, indicating the importance of E2F in mammalian development. To investigate the role of E2F in murine development, we have examined the patterns of expression of E2F-1 through E2F-5, and DP-1 in the developing nervous system by in situ hybridization. E2F-1, E2F-2 and E2F-5 are first detected in the 9.5 days post-coitus (dpc) forebrain. Expression of these E2F forms extends caudally thereafter and includes the developing brain and the upper half of the 10.5 dpc spinal cord. By 11.5 dpc, these E2F factors are expressed throughout the central nervous system. In 12.5 dpc embryos, E2F-1, E2F-2 and E2F-5 are highly expressed in proliferating, undifferentiated neuronal precursors. As neurons differentiate and migrate to the outer marginal zones in the nervous system, expression of these E2F members is extinguished. In the developing retina, another neuronal tissue, E2F-1 expression is also confined to the proliferating, undifferentiated retinoblastic layer. In contrast, E2F-3 expression is up-regulated as retinoblasts differentiate into the ganglion cell layer. In non-neuronal tissues, high E2F-4 transcript levels are present in regions corresponding to proliferative chondrocytes, whereas E2F-2 and E2F-4 transcripts are very abundant in the thymic cortex, which contains immature thymocytes. We conclude that individual E2F forms are differentially regulated during the development of distinct tissues, and especially during neuronal development.

3T3 Cells↗
PubMed2000

Regulation of E2F transcription by cyclin E-Cdk2 kinase mediated through p300/CBP co-activators.

The E2F proteins form a family of transcription factors that regulate the transition from the G1 to the S phase in the cell cycle. E2F activity is regulated by members of the retinoblastoma protein (pRb) family, ensuring the tight control of E2F-responsive genes. During the G1 phase, phosphorylation of pRb by cyclin-dependent kinases (CDKs), most notably cyclin D-CDK complexes, releases pRb from E2F, facilitating cell-cycle progression by the timely induction of E2F-targeted genes such as cyclin E. However, it is not known whether E2F proteins are directly targeted by CDKs. Here we show that E2F-5 is phosphorylated by the cyclin E-Cdk2 complex, which functions in the late G1 phase, but not by the early-G1-phase-acting cyclin D-CDK complex. A phosphorylation site in the trans-activation domain of E2F-5 stimulates transcription and cell-cycle progression by the recruitment of the p300/CBP family of co-activators, whose binding to E2F-5 is stabilized upon phosphorylation by cyclin E-Cdk2. These results indicate that E2F activity may be directly regulated by cyclin E-Cdk2, and imply an autoregulatory mechanism for cell-cycle-dependent transcription through the CDK-stimulated interaction of E2F with p300/CBP co-activators.

Animals↗
PubMed2006

14-3-3 proteins integrate E2F activity with the DNA damage response.

The E2F family is composed of at least eight E2F and two DP subunits, which in cells exist as E2F/DP heterodimers that bind to and regulate E2F target genes. While DP-1 is an essential and widespread component of E2F, much less is known about the DP-3 subunit, which exists as a number of distinct protein isoforms that differ in several respects including the presence of a nuclear localisation signal (NLS). We show here that the NLS region of DP-3 harbours a binding site for 14-3-3epsilon, and that binding of 14-3-3epsilon alters the cell cycle and apoptotic properties of E2F. DP-3 responds to DNA damage, and the interaction between DP-3 and 14-3-3epsilon is under DNA damage-responsive control. Further, 14-3-3epsilon is present in the promoter region of certain E2F target genes, and reducing 14-3-3epsilon levels induces apoptosis. These results identify a new level of control on E2F activity and, at a more general level, suggest that 14-3-3 proteins integrate E2F activity with the DNA damage response.

14-3-3 Proteins↗
PubMed1997

BRCA1 proteins are transported to the nucleus in the absence of serum and splice variants BRCA1a, BRCA1b are tyrosine phosphoproteins that associate with E2F, cyclins and cyclin dependent kinases.

BRCA1, a familial breast and ovarian cancer susceptibility gene encodes nuclear phosphoproteins that function as tumor suppressors in human breast cancer cells. Previously, we have shown that overexpression of a BRCA1 splice variant BRCA1a accelerates apoptosis in human breast cancer cells. In an attempt to determine whether the subcellular localization of BRCA1 is cell cycle regulated, we have studied the subcellular distribution of BRCA1 in asynchronous and growth arrested normal, breast and ovarian cancer cells using different BRCA1 antibodies by immunofluorescence and immunohistochemical staining. Upon serum starvation of NIH3T3, some breast and ovarian cancer cells, most of the BRCA1 protein redistributed to the nucleus revealing a new type of regulation that may modulate the activity of BRCA1 gene. We have also characterized two new variant BRCA1 proteins (BRCA1a/p110 and BRCA1b/ p100) which are phosphoproteins containing phosphotyrosine. Immunofluorescence and Western blotting analysis indicate cytoplasmic and nuclear localization of BRCA1a and BRCA1b proteins. To elucidate the biological function of BRCA1, we created a bacterial fusion protein of glutathione-transferase (GST) and BRCA1 zinc finger domain and detected two cellular proteins with molecular weights of approximately 32 and 65 kD, one of which contains phosphotyrosine designated p32 and p65 BRCA1 interacting proteins (BIP) that specifically interact with BRCA1. Western blot analysis of BIP with cyclins/CDKs and E2F antisera indicated association with cdc2, cdk2, cdk4, cyclin B, cyclin D, cyclin A and E2F-4 but not with cdk3, cdk5, cdk6, E2F-1, E2F-2, E2F-3, E2F-5 and cyclin E. Furthermore, we have also demonstrated a direct interaction of in vitro translated BRCA1a and BRCA1b proteins with recombinant cyclin A, cyclin B1, cyclin D1, cdc2, cdk2 and E2F fusion proteins in vitro. Taken together these results seem to suggest that BRCA1 could be an important negative regulator of cell cycle that functions through interaction with E2F transcriptional factors and phosphorylation by cyclins/cdk complexes with the zinc ring finger functioning as a major protein-protein interaction domain. If the interactions we observe in vitro is also seen in vivo then it may be possible that lack or impaired binding of the disrupted BRCA1 proteins to E2F, cyclins/CDKs in patients with mutations in the zinc finger domain could deprive the cell of an important mechanism for braking cell proliferation leading to the development of breast and ovarian cancers.

3T3 Cells↗
PubMed1998

pRb and p107 regulate E2F activity during lens fiber cell differentiation.

During growth arrest and differentiation, activity of the E2F family of transcription factors is inhibited by interactions with pRb and the related proteins, p107 and p130. To determine which members of the E2F and pRb families may contribute to growth arrest as lens epithelial cells differentiate into fiber cells, we examined the expression of individual E2F species and characterized the E2F protein complexes formed in rat lens epithelia and fibers. RT/PCR detected all five known members of the E2F family in lens epithelial cells, but only E2F-1, E2F-3, and E2F-5 in fiber cells. Proteins extracted from lens epithelia of newborn rats formed at least two specific complexes with an E2F consensus oligonucleotide. Proteins from lens fiber cells formed three specific complexes, one of which comigrated with an epithelial cell complex. Incubation of epithelial and fiber cell extracts with an antibody specific for p107 demonstrated that two fiber cell complexes and one epithelial cell complex contained p107. Although the remaining fiber cell complex did not react with antibodies to pRb or p130 in this assay, a strong reaction with pRb antibody was observed when the electromobility shifted complexes were subsequently immunoblotted (shift/Western assay). Immunocytochemistry confirmed that pRb protein is present in the nuclei of both epithelial cells and fiber cells. Immunoblotting of whole cell extracts with pRb antibody showed multiple, phosphorylated forms of pRb in the epithelial cells, but predominantly hypophosphorylated pRb in the fiber cells. None of the complexes formed with E2F were recognized exclusively by the p130 antibody, although the previously identified p107 complexes reacted weakly. The absence of p130/E2F complexes was correlated with the presence of multiple ubiquitinated forms of p130, especially in the fiber cells. Thus, although p130/E2F complexes are implicated in the terminal differentiation of many cell types, in differentiating lens fiber cells pRb and p107 seem to be the primary regulators of E2F activity.

Animals↗
PubMed1998

The E2F-family proteins induce distinct cell cycle regulatory factors in p16-arrested, U343 astrocytoma cells.

We previously demonstrated that P16Ink4a (p16) expression in p16-deficient U343 astrocytoma cells causes a G1 cell cycle arrest, profound changes in cytoskeletal proteins and alterations in expression and activity of the pRB and E2F family proteins. We examine here the effects of expressing wild type or mutant versions of the downstream targets of p16 in U343 astrocytomas. We first attempted to block proliferation of U343 cells using the dominant mutant of pRB, deltap34. Expression of this mutant in the human osteosarcoma, SAOS-2, potently blocked proliferation but did not affect the cell cycle of U343 cells. We next showed that expression of E2F-1, E2F-2, E2F-3 and E2F-4 are each able to overcome this p16-dependent cell cycle arrest but exhibit distinct biological activities. Adenoviral-mediated expression of E2F-1, E2F-2, E2F-3, or E2F-4 overcame the p16-dependent cell cycle block and induced alterations in cell morphology. E2F-5, only in conjunction with DP1, promoted cell cycle progression. For both E2F-1 and E2F-2, but not E2F-3 or E2F-5/DP1, cell cycle re-entry was associated with almost quantitative cell death. Only small numbers of dying cells were observed in E2F-4-expressing cultures. Expression of the different E2F's altered the expression of distinct sets of cell cycle regulatory proteins. E2F-1 induced endogenous E2F-4 expression and also caused an increase in pRB, p107 and cyclin E levels. Expression of E2F-4 caused a weak increase in E2F-1 levels but also strongly induced pRB, p107, p130 and cyclin E. However, E2F-1 and E2F-4 clearly regulate expression of distinct genes, demonstrated when E2F-4 caused a threefold increase in the levels of cdk2 whereas E2F-1 failed to increase in this cyclin dependent kinase. Similarly, expression of E2F-1 or E2F-2 were shown to have distinct effects on the expression of cdk2, cyclin E and pRB despite both of these closely related E2F-family members potently inducing cell death. Thus, E2F-1, E2F-2, E2F-3 and E2F-4 are able to overcome the p16-dependent proliferative block in U343 astrocytoma cells. While overcoming this cell cycle block, each of the E2F's uniquely affect the expression of a number of cell cycle regulatory proteins and have distinct abilities to promote cell death.

Adenoviruses, Human↗
PubMed1999

Association with E2F-1 governs intracellular trafficking and polyubiquitination of DP-1.

The cell cycle-regulated transcription factor E2F is a family of heterodimers composed of E2F and DP protein subunits. While DP proteins stabilize DNA binding of E2F proteins, and influence the entry of E2F-4 and E2F-5 into the nucleus, the role of DP proteins in E2F-dependent gene expression is not well understood. Using immunolocalization, immunoprecipitation, and cell fractionation experiments, here we show association with E2F subunits governs intracellular trafficking and ubiquitination of DP-1. In transient transfection experiments, DP-1 polypeptides that stably bound E2F-1 entered the nucleus. DP-1 proteins that failed to associate with E2F subunits accumulated in the cell cytoplasm as polyubiquitinated DP-1. A Chinese hamster cell line that conditionally expresses HA-DP-1 was used to examine the effect of DP-1 on cell cycle progression. In serum response experiments, moderate increases in HA-DP-1 led to a threefold increase in E2F DNA binding activity in vitro, a corresponding increase in dhfr gene expression during transition of G1, and higher rates of S phase entry. However, flow cytometry showed cells expressing very high levels of HA-DP-1 failed to enter the S phase. Inhibition of cell cycle progression by high levels of HA-DP-1 was associated with the accumulation of other ubiquitinated cellular proteins, including c-jun and the cyclin-dependent kinase inhibitor p21, indicating that degradation of ubiquitinated proteins is required for progression from G0 to S phase even in the presence of activated E2F. Under similar conditions, expression of E2F-1 reduced the levels of ubiquitinated cellular proteins and accelerated cell cycle progression. Our studies indicate association with E2F subunits prevents ubiquitin-dependent degradation of DP-1 in the cytoplasm by promoting nuclear entry of E2F/DP heterodimers.

Animals↗