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Biomedical subjects

W D Cress

Publications and source records attributed to W D Cress.

12 recordsLinked to original sources
PubMed1998

The role of cyclin D3-dependent kinase in the phosphorylation of p130 in mouse BALB/c 3T3 fibroblasts.

We have observed that cyclin D3-dependent kinase activity is increased in the late G1 phase in BALB/c 3T3 fibroblasts. The profile of cyclin D3-associated activity closely parallels that of cyclin D1, which is also induced after mitogenic stimulation of quiescent cells. These activities correlate with the appearance of hyperphosphorylated p130, an Rb family member important in regulating E2F-4 and E2F-5 activity in fibroblastic cells. We demonstrated, however, that only the cyclin D3 activity efficiently phosphorylated p130 in an in vitro kinase assay. This apparent specificity was further demonstrated by experiments which demonstrated that cyclin D3 was physically associated with p130 at the times when D3-dependent kinase activity and p130 hyperphosphorylation were observed. Examination of E2F by electrophoretic mobility shift assay revealed that E2F-4 DNA binding activity existed in a p130.E2F complex at times before D3-dependent kinase activity was apparent and in a free E2F-4 complex after D3 activity developed. Thus, our data suggest that cyclin D3 preferentially phosphorylates p130 and is thereby specifically targeted to overcoming growth-suppressive control mediated through p130 pathways.

3T3 Cells↗
PubMed1998

E2F-3 accumulation is regulated by polypeptide stability.

E2F is a complex family of transcription factors which appears to regulate the transcription of genes required for the S phase of the mammalian cell cycle. In the present work, we have examined the mechanisms regulating E2F-3 accumulation in mouse fibroblasts. We have determined that E2F-3 DNA binding activity is restricted to the G1/S transition and S phase in both normal BALB/c-3T3 fibroblasts and in an SV40 virus-transformed BALB/c-3T3 derivative. Immunoblot analysis indicates that G0 and G1 cells have little or no E2F-3 polypeptide and that the increase in the DNA binding activity of E2F-3 at the G1/S boundary is reflected by an increase in total E2F-3 protein. In contrast to the E2F-3 polypeptide, RNAse protection assays demonstrate that the E2F-3 mRNA is clearly present in G0 and G1 cells. Finally, pulse/chase experiments indicate that the half-life of E2F-3 is approximately 40-fold greater in cells blocked in S phase relative to asynchronously growing cells. Together, these results indicate that the accumulation E2F-3 at S phase may be regulated, at least in part, at the level of protein stability.

3T3 Cells↗
PubMed1997

Subunit composition determines E2F DNA-binding site specificity.

The product of the retinoblastoma (Rb) susceptibility gene, Rb-1, regulates the activity of a wide variety of transcription factors, such as E2F, in a cell cycle-dependent fashion. E2F is a heterodimeric transcription factor composed of two subunits each encoded by one of two related gene families, denoted E2F and DP. Five E2F genes, E2F-1 through E2F-5, and two DP genes, DP-1 and DP-2, have been isolated from mammals, and heterodimeric complexes of these proteins are expressed in most, if not all, vertebrate cells. It is not yet clear whether E2F/DP complexes regulate overlapping and/or specific cellular genes. Moreover, little is known about whether Rb regulates all or a subset of E2F-dependent genes. Using recombinant E2F, DP, and Rb proteins prepared in baculovirus-infected cells and a repetitive immunoprecipitation-PCR procedure (CASTing), we have identified consensus DNA-binding sites for E2F-1/DP-1, E2F-1/DP-2, E2F-4/DP-1, and E2F-4/DP-2 complexes as well as an Rb/E2F-1/DP-1 trimeric complex. Our data indicate that (i) E2F, DP, and Rb proteins each influence the selection of E2F-binding sites; (ii) E2F sites differ with respect to their intrinsic DNA-bending properties; (iii) E2F/DP complexes induce distinct degrees of DNA bending; and (iv) complex-specific E2F sites selected in vitro function distinctly as regulators of cell cycle-dependent transcription in vivo. These data indicate that the specific sequence of an E2F site may determine its role in transcriptional regulation and suggest that Rb/E2F complexes may regulate subsets of E2F-dependent cellular genes.

Animals↗
PubMed1996

A role for a bent DNA structure in E2F-mediated transcription activation.

We examined the role of promoter architecture, as well as that of the DNA-bending capacity of the E2F transcription factor family, in the activation of transcription. DNA phasing analysis revealed that a consensus E2F site in the E2F1 promoter possesses an inherent bend with a net magnitude of 40 +/-2 degrees and with an orientation toward the major groove relative to the center of the E2F site. The inherent DNA bend is reversed upon binding of E2F, generating a net bend with a magnitude of 25 +/- 3 degrees oriented toward the minor groove relative to the center of the E2F site. We also found that three members of the E2F family, in conjunction with the DP1 protein, bend the DNA toward the minor groove, suggesting that DNA bending is a characteristic of the entire E2F family. The Rb-E2F complex, on the other hand, does not reverse the intrinsic DNA bend. Analysis of a series of E2F1 deletion mutants defined E2F1 sequences which are not required for DNA binding but are necessary for the DNA-bending capacity of E2F. An internal region of E2F1, previously termed the marked box, which is highly homologous among E2F family members, was particularly important in DNA bending. We also found that a bent DNA structure can be a contributory component in the activation of the E2F1 promoter but is not critical in the repression of that promoter in quiescent cells. This finding suggests that E2F exhibits characteristics typical of modular transcription factors, with independent DNA-binding and transcriptional activation functions, but also has features of architectural factors that alter DNA structure.

Adenovirus E2 Proteins↗
PubMed1994

Oncogenic capacity of the E2F1 gene.

Previous experiments have identified the E2F transcription factor as a potential downstream target for the action of cellular regulatory activities, such as the Rb tumor suppressor protein, that control cell growth and that, when altered, contribute to the development of human tumors. In light of these findings, we have assayed the ability of the E2F1 and DP1 genes, which encode heterodimeric partners that together create E2F activity, to act in an oncogenic fashion. We find that E2F1, particularly in combination with the DP1 product, cooperates with an activated ras oncogene to induce the formation of morphologically transformed foci in primary rat embryo fibroblast cultures. In addition, an E2F1 chimeric protein, in which sequences involved in Rb binding have been replaced with the herpesvirus VP16 activation domain, exhibits increased transformation activity. Cells transfected with E2F1 and DP1 or the E2F1-VP16 chimera form colonies in soft agar and induce tumor formation in nude mice. We conclude that deregulated E2F1 expression and function can have oncogenic consequences.

Animals↗
PubMed1994

Interacting domains of E2F1, DP1, and the adenovirus E4 protein.

Recent experiments demonstrate that a family of related proteins constitute the E2F transcription factor activity and that the interaction of two of these gene products, E2F1 and DP1, generates a heterodimer with DNA binding and transcriptional activating capacity. Previous experiments have shown that the adenovirus E4 19-kDa protein facilitates the formation of a stable E2F dimer on the adenovirus E2 promoter. We now show that coexpression of the E2F1 and DP1 products in transfected SAOS-2 cells, together with the E4 product, generates a multicomponent complex with specificity to the adenovirus E2 promoter. Using a yeast two-hybrid assay system, we find that the E2F1 hydrophobic heptad repeat (E2F1 amino acid residues 206 to 283) allows interaction with a corresponding domain of the DP1 protein (amino acids 196 to 245). We also find that the adenovirus E4 protein interacts with the DP1 hydrophobic heptad repeat domain, but we could not detect a direct interaction between E2F1 and E4. Additional assays demonstrate that the E4 protein can dimerize. Since our previous experiments have shown that mutations within the E2F1 hydrophobic heptad repeat element abolish the E4-mediated transcription enhancement in transfection assays, we conclude that the E4 protein likely interacts with the E2F1-DP1 heterodimer by directly binding to the DP1 product. As a consequence of the ability of E4 to dimerize, we propose that the stable complex formed on the two E2F sites within the E2 promoter is composed of two E2F1-DP1 heterodimers held together by an E4 dimer.

Adenovirus E4 Proteins↗
PubMed1993

Expression of transcription factor E2F1 induces quiescent cells to enter S phase.

Several lines of evidence implicate the E2F transcription factor as an important component of cell proliferation control. First, E2F binding sites are found in the promoters of genes responsive to proliferation signals and the level of E2F binding activity increases at a time when many of these genes are activated. Second, the tumour suppressor protein Rb, as well as the related p107 protein, complexes with E2F, resulting in an inhibition of E2F transcriptional activity. Third, oncogenic products of the DNA tumour viruses can dissociate these E2F complexes. We provide here direct evidence that E2F is involved in cellular proliferation control. Specifically, we demonstrate that overexpression of the E2F1 complementary DNA can activate DNA synthesis in cells that would otherwise growth-arrest, with an efficiency that is similar to that achieved by the expression of the adenovirus E1A gene. Moreover, microinjection of the E2F1 cDNA into quiescent cells can induce S-phase entry, whereas two E2F1 mutants, which are unable to transactivate the DHFR and TK promoters, are unable to induce S phase. We conclude that the E2F transcription factor plays an important role in progression into S phase and that this probably coincides with its capacity to stimulate transcription.

Adenoviridae↗
PubMed1993

A genetic analysis of the E2F1 gene distinguishes regulation by Rb, p107, and adenovirus E4.

The cellular transcription factor E2F appears to be a target for the regulatory action of the retinoblastoma tumor suppressor gene product. The recent isolation of the E2F1 cDNA clone, which encodes a polypeptide with properties characteristic of E2F, has now allowed a more detailed analysis of the regulation of E2F function by Rb as well as the Rb-related p107 protein and the adenovirus 19-kDa E4 gene product. Previous experiments have shown that each of these regulatory proteins can modulate the activity of cellular E2F. We find that each of these regulatory events can be mediated through the E2F1 product. Moreover, an examination of various E2F1 mutations reveals distinct specificities for these regulatory proteins. For instance, the ability of E4 to alter E2F1 function is dependent upon sequences within a putative leucine repeat of E2F1 as well as within the C-terminal acidic domain. In contrast, the leucine repeat element was not important for Rb- or p107-mediated inhibition of E2F1 activity. Although the C-terminal acidic domain of E2F1, previously shown to be important for Rb binding, appears to be a site for regulation of E2F1 by Rb and p107, point mutations within this region distinguish recognition by Rb and p107. These results underscore the complexity of E2F regulatory interactions and also demonstrate a qualitative distinction in the interactions of Rb and p107 with E2F1, perhaps reflective of functional differences.

Adenovirus E4 Proteins↗
PubMed1991

Reduced binding of TFIID to transcriptionally compromised mutants of VP16.

Activator proteins that control transcription initiation by RNA polymerase II usually have two domains: one binds to DNA, and the other activates transcription. A particularly potent acidic activation domain at the C terminus of the herpes simplex virus protein VP16 binds directly and selectively to the human and yeast TATA box-binding factor TFIID. We have now investigated the biological significance of this in vitro interaction by using mutant forms of VP16. For changes at the critical phenylalanine residue at position 442 of VP16 there was a good correlation between transactivation activity in vivo and the binding of VP16 to TFIID in vitro. In contrast, mutants with reduced negative charge were more defective for binding than for activation.

DNA Mutational Analysis↗
PubMed1991

Critical structural elements of the VP16 transcriptional activation domain.

Virion protein 16 (VP16) of herpes simplex virus type 1 contains an acidic transcriptional activation domain. Missense mutations within this domain have provided insights into the structural elements critical for its function. Net negative charge contributed to, but was not sufficient for, transcriptional activation by VP16. A putative amphipathic alpha helix did not appear to be an important structural component of the activation domain. A phenylalanine residue at position 442 was exquisitely sensitive to mutation. Transcriptional activators of several classes contain hydrophobic amino acids arranged in patterns resembling that of VP16. Therefore, the mechanism of transcriptional activation by VP16 and other proteins may involve both ionic and specific hydrophobic interactions with target molecules.

Amino Acid Sequence↗
PubMed1990

Selective inhibition of activated but not basal transcription by the acidic activation domain of VP16: evidence for transcriptional adaptors.

The interaction between the chimeric activator GAL4-VP16, consisting of the DNA binding domain of GAL4 and the acidic activation domain of VP16, and its target in the transcriptional machinery was studied in vitro. GAL4-VP16 stimulated transcription from a promoter bearing GAL4 sites, and greatly inhibited transcription from a promoter bearing binding sites for the dA:dT activator and from a basal promoter bearing only a TATA box. Mutations in the acidic domain that reduced activation from the GAL4 site promoter also reduced inhibition from the dA:dT promoter, indicating a similar interaction between VP16 and its target in both processes. Strikingly, if the DNA binding domain of GAL4-VP16 was occupied by a GAL4 site oligonucleotide, the protein inhibited activation by the dA:dT activator but did not inhibit basal transcription. We propose that, under these conditions, GAL4-VP16 acted to titrate an "adaptor" that bridges an interaction between the upstream activator and the basic transcriptional machinery at the TATA box.

Binding Sites↗