Antibodies specific for the human retinoblastoma protein identify a family of related polypeptides

Even though the retinoblastoma gene is one of the best-studied tumor suppressor genes, little is known about its functional role. Like all tumor suppressor gene products, the retinoblastoma protein (pRB) is thought to inhibit some aspect of cell proliferation. It also appears to be a cellular target of several DNA tumor virus-transforming proteins, such as adenovirus E1A, human papillomavirus E7, or simian virus 40 large T antigen. To help in the analysis of pRB, we have prepared a new set of anti-human pRB monoclonal antibodies. In addition to being useful reagents for the study of human pRB, these antibodies display several unexpected properties. They can be used to distinguish different subsets of the pRBs on the basis of their phosphorylation states. Some are able to recognize pRB homologs in other species, including mice, chickens, and members of the genus Xenopus. In addition, some of these antibodies can bind directly to other cellular proteins that, like pRB, were originally identified through their association with adenovirus E1A. These immunologically cross-reactive proteins include the p107 and p300 proteins, and their recognition by antibodies raised against pRB suggests that several members of the E1A-targeted cellular proteins form a structurally and functionally related family.

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Role of pRb-related proteins in simian virus 40 large-T-antigen-mediated transformation.

Molecular and Cellular Biology ◽

10.1128/mcb.15.10.5800 ◽

1995 ◽

Vol 15 (10) ◽

pp. 5800-5810 ◽

Cited By ~ 94

Author(s):

J Zalvide ◽

J A DeCaprio

Keyword(s):

Simian Virus 40 ◽

Cell Types ◽

Suppressor Gene ◽

Simian Virus ◽

T Antigen ◽

Cellular Proteins ◽

Large T Antigen ◽

Wild Type ◽

Relative Contribution ◽

Lxcxe Motif

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.

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The retinoblastoma protein-binding region of simian virus 40 large T antigen alters cell cycle regulation in lenses of transgenic mice.

Molecular and Cellular Biology ◽

10.1128/mcb.14.10.6743 ◽

1994 ◽

Vol 14 (10) ◽

pp. 6743-6754 ◽

Cited By ~ 62

Author(s):

L Fromm ◽

W Shawlot ◽

K Gunning ◽

J S Butel ◽

P A Overbeek

Keyword(s):

Cell Cycle ◽

Transgenic Mice ◽

Retinoblastoma Protein ◽

Simian Virus 40 ◽

Cell Types ◽

Simian Virus ◽

T Antigen ◽

Lens Fiber ◽

Large T Antigen ◽

Fiber Cells

Regulation of the cell cycle is a critical aspect of cellular proliferation, differentiation, and transformation. In many cell types, the differentiation process is accompanied by a loss of proliferative capability, so that terminally differentiated cells become postmitotic and no longer progress through the cell cycle. In the experiments described here, the ocular lens has been used as a system to examine the role of the retinoblastoma protein (pRb) family in regulation of the cell cycle during differentiation. The ocular lens is an ideal system for such studies, since it is composed of just two cell types: epithelial cells, which are capable of proliferation, and fiber cells, which are postmitotic. In order to inactivate pRb in viable mice, genes encoding either a truncated version of simian virus 40 large T antigen or the E7 protein of human papillomavirus were expressed in a lens-specific fashion in transgenic mice. Lens fiber cells in the transgenic mice were found to incorporate bromodeoxyuridine, implying inappropriate entry into the cell cycle. Surprisingly, the lens fiber cells did not proliferate as tumor cells but instead underwent programmed cell death, resulting in lens ablation and microphthalmia. Analogous lens alterations did not occur in mice expressing a modified version of the truncated T antigen that was mutated in the binding domain for the pRb family. These experimental results indicate that the retinoblastoma protein family plays a crucial role in blocking cell cycle progression and maintaining terminal differentiation in lens fiber cells. Apoptotic cell death ensues when fiber cells are induced to remain in or reenter the cell cycle.

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Association of the human papillomavirus type 16 E7 protein with the S-phase-specific E2F-cyclin A complex

Molecular and Cellular Biology ◽

10.1128/mcb.13.10.6537-6546.1993 ◽

1993 ◽

Vol 13 (10) ◽

pp. 6537-6546 ◽

Cited By ~ 1

Author(s):

M Arroyo ◽

S Bagchi ◽

P Raychaudhuri

Keyword(s):

Cell Cycle ◽

Human Papillomavirus ◽

Simian Virus 40 ◽

Cyclin A ◽

T Antigen ◽

S Phase ◽

Cellular Proteins ◽

Hpv 16 ◽

Adenovirus E1a ◽

Cell Extracts

The transcription factor E2F has been shown to be involved in the expression of several cell cycle-regulated genes, and the activity of this factor is controlled by cellular proteins such as pRB and p107. E2F is also a target of the DNA virus oncoproteins (adenovirus E1A, simian virus 40 T antigen, and human papillomavirus [HPV] E7) (see the review by J. R. Nevins [Science 258: 424-429, 1992]). These viral oncoproteins dissociate an inactive complex between E2F and the retinoblastoma tumor suppressor protein (pRB), and this dissociation of the E2F-pRB complex correlates with a stimulation of the E2F-dependent transcription. In the S phase of the cell cycle, E2F forms a complex with p107, cyclin A, and the cdk2 kinase (E2F-cyclin A complex). The cellular function of this S-phase-specific complex is unclear. The adenovirus E1A protein dissociates the E2F-cyclin A complex. The HPV type 16 (HPV-16) E7 protein, which possesses significant sequence homology with E1A, does not dissociate the E2F-cyclin A complex. We find that the HPV-16 E7 protein associates very efficiently with the E2F-cyclin A complex. This association is dependent on the sequences that are also necessary for the transforming activity of E7. Moreover, the E7 protein of a low-risk HPV (type 6b) is much less efficient in binding to the E2F-cyclin A complex compared with that of the high-risk type. We also find that the E2F-cyclin A complex remains endogenously associated with the E7 protein in extracts of Caski cells, which express high levels of HPV-16 E7 protein. Finally, we have extensively purified the E2F-cyclin A complex from mouse L-cell extracts and show that, in cell extracts, the E2F-cyclin A complex remains associated with other cellular proteins.

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Stimulation of the adenovirus E2 promoter by simian virus 40 T antigen or E1A occurs by different mechanisms

Molecular and Cellular Biology ◽

10.1128/mcb.6.6.2020-2026.1986 ◽

1986 ◽

Vol 6 (6) ◽

pp. 2020-2026

Author(s):

M R Loeken ◽

G Khoury ◽

J Brady

Keyword(s):

Simian Virus 40 ◽

Promoter Sequence ◽

Simian Virus ◽

Chloramphenicol Acetyltransferase ◽

T Antigen ◽

Base Pairs ◽

Promoter Sequences ◽

Adenovirus E1a ◽

E1a Gene ◽

Stimulation Of

We have examined the ability of simian virus 40 T antigen to stimulate transcription from the adenovirus E2 promoter. T antigen, produced from a cotransfected plasmid, stimulated chloramphenicol acetyltransferase enzyme and mRNA production from an E2 promoter-chloramphenicol acetyltransferase fusion plasmid (pEC113) in monkey kidney CV-1 cells. The level of stimulation of E2 transcription by simian virus 40 T antigen was equal to that observed in cotransfections of pEC113 and the adenovirus E1A gene product. Deletion mutations from the 5' end of the E2 promoter were examined for their ability to express basal, T-antigen, or E1A trans-activated promoter activity. In each case, deletion of upstream promoter sequences to -70 base pairs reduced chloramphenicol acetyltransferase expression to approximately 30% of the level observed with the intact E2 promoter. Deletion to -59 base pairs resulted in chloramphenicol acetyltransferase expression that was 3 to 5% of that observed with the intact E2 promoter. At saturating levels of the stimulatory proteins, the chloramphenicol acetyltransferase levels obtained in response to T antigen and adenovirus E1A were additive. COS-1 cells, which are derived from CV-1 cells and constitutively express simian virus 40 T antigen, do not support E2 promoter trans activation by T antigen. E1A trans activation of the E2 promoter is efficient in COS-1 cells. These results suggest that although promoter sequence requirements are similar, T antigen and E1A trans activate the E2 promoter by different mechanisms.

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Maintenance of cellular proliferation by adenovirus early region 1A in fibroblasts conditionally immortalized by using simian virus 40 large T antigen requires conserved region 1

Molecular and Cellular Biology ◽

10.1128/mcb.10.12.6664-6673.1990 ◽

1990 ◽

Vol 10 (12) ◽

pp. 6664-6673

Author(s):

T E Riley ◽

A Follin ◽

N C Jones ◽

P S Jat

Keyword(s):

Cell Line ◽

Growth Arrest ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Growth Defect ◽

Primary Cells ◽

Nonpermissive Temperature ◽

Large T Antigen ◽

Adenovirus E1a

Various mutants of adenovirus E1A were assayed for their ability to complement the growth defect at the nonpermissive temperature for the cell line tsa14 which was isolated by immortalizing rat embryo fibroblasts with the thermolabile large T antigen of tsA58. This cell line grows indefinitely at the permissive temperature but undergoes rapid growth arrest upon shift up to the nonpermissive temperature. Since this growth arrest can be overcome by introduction of wild-type simian virus 40 large T antigen, human papillomavirus 16 E7, and adenovirus E1A, the tsa14 cells provided an excellent system for defining regions of E1A necessary for complementation of the growth defect. We demonstrate that conserved region 1 (CR1) is the region of E1A required for complementation. While CR2 of E1A has been shown to be required for the immortalization of primary cells and is also necessary for the binding of the 105-kDa retinoblastoma protein, mutations within this region did not abrogate complementation of the growth defect. However, since both CR1 and CR2 have previously been shown to be absolutely required for immortalization of primary cells by adenovirus E1A, this evidence suggests that the tsa14 system assays for the maintenance of proliferation and that this requires CR1.

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Simian virus 40 large-T antigen expresses a biological activity complementary to the p300-associated transforming function of the adenovirus E1A gene products

Molecular and Cellular Biology ◽

10.1128/mcb.11.4.2116-2124.1991 ◽

1991 ◽

Vol 11 (4) ◽

pp. 2116-2124

Author(s):

P Yaciuk ◽

M C Carter ◽

J M Pipas ◽

E Moran

Keyword(s):

Biological Activity ◽

Rat Kidney ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Binding Region ◽

Adenovirus E1a ◽

P300 Binding ◽

Transforming Activity ◽

E1a Gene

In this report we present evidence that simian virus 40 T antigen encodes a biological activity that is functionally equivalent to the transforming activity lost by deletion of the E1A p300-binding region. T-antigen constructs from which the pRb-binding region has been deleted are virtually unable to induce foci of transformed cells in a ras cooperation assay in primary baby rat kidney cells. Nevertheless, such a construct can cooperate with an E1A N-terminal deletion mutant, itself devoid of transforming activity, to induce foci in this assay. The heterologous trans-cooperating activity observed between E1A and T-antigen deletion products is as efficient as trans cooperation between mutants expressing individual E1A domains. The cooperating function can be impaired by a deletion near the N terminus of T antigen. Such a deletion impairs neither the p53-binding function nor the activity of the pRb-binding region.

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Maintenance of cellular proliferation by adenovirus early region 1A in fibroblasts conditionally immortalized by using simian virus 40 large T antigen requires conserved region 1.

Molecular and Cellular Biology ◽

10.1128/mcb.10.12.6664 ◽

1990 ◽

Vol 10 (12) ◽

pp. 6664-6673 ◽

Cited By ~ 5

Author(s):

T E Riley ◽

A Follin ◽

N C Jones ◽

P S Jat

Keyword(s):

Cell Line ◽

Growth Arrest ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Growth Defect ◽

Primary Cells ◽

Nonpermissive Temperature ◽

Large T Antigen ◽

Adenovirus E1a

Various mutants of adenovirus E1A were assayed for their ability to complement the growth defect at the nonpermissive temperature for the cell line tsa14 which was isolated by immortalizing rat embryo fibroblasts with the thermolabile large T antigen of tsA58. This cell line grows indefinitely at the permissive temperature but undergoes rapid growth arrest upon shift up to the nonpermissive temperature. Since this growth arrest can be overcome by introduction of wild-type simian virus 40 large T antigen, human papillomavirus 16 E7, and adenovirus E1A, the tsa14 cells provided an excellent system for defining regions of E1A necessary for complementation of the growth defect. We demonstrate that conserved region 1 (CR1) is the region of E1A required for complementation. While CR2 of E1A has been shown to be required for the immortalization of primary cells and is also necessary for the binding of the 105-kDa retinoblastoma protein, mutations within this region did not abrogate complementation of the growth defect. However, since both CR1 and CR2 have previously been shown to be absolutely required for immortalization of primary cells by adenovirus E1A, this evidence suggests that the tsa14 system assays for the maintenance of proliferation and that this requires CR1.

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Multiple functional domains of human U2 small nuclear RNA: strengthening conserved stem I can block splicing.

Molecular and Cellular Biology ◽

10.1128/mcb.12.12.5464 ◽

1992 ◽

Vol 12 (12) ◽

pp. 5464-5473 ◽

Cited By ~ 10

Author(s):

J Wu ◽

J L Manley

Keyword(s):

Simian Virus 40 ◽

Suppressor Gene ◽

Simian Virus ◽

T Antigen ◽

Compensatory Mutation ◽

Base Pairing ◽

Recognition Sequence ◽

Site Mutation ◽

U6 Snrna ◽

Branch Site

We showed previously that a branch site mutation in simian virus 40 early pre-mRNA that prevented small t antigen mRNA splicing could be efficiently suppressed by a compensatory mutation in a coexpressed U2 small nuclear (sn) RNA gene. We have now generated second-site mutations in this suppressor gene to investigate regions of U2 RNA required for function. A number of mutations in a putative stem at the 5' end of the molecule inhibited splicing, indicating that bases in this region are important for activity. However, several lines of evidence suggested that formation of the entire stem is not essential for splicing. Indeed, mutations that strengthen the stem actually inhibited splicing, and evidence that this prevents a required base-pairing interaction with U6 snRNA is presented. These results suggest that the relative stabilities of competing intra- and intermolecular base-pairing interactions play an important role in the splicing reaction. Mutations in a conserved single-stranded region immediately 3' to the branch site recognition sequence all inhibited splicing, indicating that this region is required for U2 function, although its exact role remains unknown. Finally, two mutations in the loop of stem IV at the 3' end of the molecule, which destroy the binding site of U2 sn ribonucleoprotein B", prevented small t splicing; this finding contrasts with previous studies which utilized different assay systems. Analysis of the accumulation and subcellular localization of all of the mutant RNAs showed that they were similar to those of the parental suppressor U2 RNA, indicating that the effects observed indeed reflect defects in splicing.

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Simian Virus 40 Large T Antigen's Association with the CUL7 SCF Complex Contributes to Cellular Transformation

Journal of Virology ◽

10.1128/jvi.79.18.11685-11692.2005 ◽

2005 ◽

Vol 79 (18) ◽

pp. 11685-11692 ◽

Cited By ~ 38

Author(s):

Jocelyn S. Kasper ◽

Hiroshi Kuwabara ◽

Takehiro Arai ◽

Syed Hamid Ali ◽

James A. DeCaprio

Keyword(s):

Tumor Suppressor ◽

Mouse Embryo ◽

Family Members ◽

Simian Virus 40 ◽

Cellular Transformation ◽

Simian Virus ◽

T Antigen ◽

Mouse Embryo Fibroblasts ◽

Embryo Fibroblasts ◽

F Box Protein

ABSTRACT Simian virus 40 large T antigen (T Ag) is capable of immortalizing and transforming rodent cells. The transforming activity of T Ag is due in large part to perturbation of the tumor suppressor proteins p53 and the retinoblastoma (pRB) family members. Inactivation of these tumor suppressors may not be sufficient for T Ag-mediated cellular transformation. It has been shown that T Ag associates with an SCF-like complex that contains a member of the cullin family of E3 ubiquitin ligases, CUL7, as well as SKP1, RBX1, and an F-box protein, FBXW8. We identified T Ag residues 69 to 83 as required for T Ag binding to the CUL7 complex. We demonstrate that Δ69-83 T Ag, while it lost its ability to associate with CUL7, retained binding to p53 and pRB family members. In the presence of CUL7, wild-type (WT) T Ag but not Δ69-83 T Ag was able to induce proliferation of mouse embryo fibroblasts, an indication of cellular transformation. In contrast, WT and Δ69-83 T Ag enabled mouse embryo fibroblasts to proliferate to similarly high densities in the absence of CUL7. Our data suggest that, in addition to p53 and the pRB family members, T Ag serves to bind to and inactivate the growth-suppressing properties of CUL7. In addition, these results imply that, at least in the presence of T Ag, CUL7 may function as a tumor suppressor.

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