ATP-Dependent Simian Virus 40 T-Antigen–Hsc70 Complex Formation

ABSTRACT Simian virus 40 large T antigen is a multifunctional oncoprotein that is required for numerous viral functions and the induction of cellular transformation. T antigen contains a J domain that is required for many of its activities including viral DNA replication, transformation, and virion assembly. J-domain-containing proteins interact with Hsc70 (a cellular chaperone) to perform multiple biological activities, usually involving a change in the conformation of target substrates. It is thought that Hsc70 associates with T antigen to assist in performing its numerous activities. However, it is not clear if T antigen binds to Hsc70 directly or induces the binding of Hsc70 to other T-antigen binding proteins such as pRb or p53. In this report, we show that T antigen binds Hsc70 directly with a stoichiometry of 1:1 (dissociation constant = 310 nM Hsc70). Furthermore, the T-antigen–Hsc70 complex formation is dependent upon ATP hydrolysis at the active site of Hsc70 (ATP dissociation constant = 0.16 μM), but T-antigen–Hsc70 complex formation does not require nucleotide hydrolysis at the T-antigen ATP binding site. N136, a J domain-containing fragment of T antigen, does not stably associate with Hsc70 but can form a transient complex as assayed by centrifugation analysis. Finally, T antigen does not associate stably with either of two yeast Hsc70 homologues or an amino-terminal fragment of Hsc70 containing the ATPase domain. These results provide direct evidence that the T-antigen–Hsc70 interaction is specific and that this association requires multiple domains of both T antigen and Hsc70. This is the first demonstration of a nucleotide requirement for the association of T antigen and Hsc70 and lays the foundation for future reconstitution studies of chaperone-dependent tumorigenesis induced by T antigen.

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The Molecular Chaperone Activity of Simian Virus 40 Large T Antigen Is Required To Disrupt Rb-E2F Family Complexes by an ATP-Dependent Mechanism

Molecular and Cellular Biology ◽

10.1128/mcb.20.17.6233-6243.2000 ◽

2000 ◽

Vol 20 (17) ◽

pp. 6233-6243 ◽

Cited By ~ 75

Author(s):

Christopher S. Sullivan ◽

Paul Cantalupo ◽

James M. Pipas

Keyword(s):

Atp Hydrolysis ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Cellular Growth ◽

Binding Motif ◽

Large T Antigen ◽

Consensus Binding Site ◽

J Domain ◽

Immortalized Cell

ABSTRACT The simian virus 40 large T antigen (T antigen) inactivates tumor suppressor proteins and therefore has been used in numerous studies to probe the mechanisms that control cellular growth and to generate immortalized cell lines. Binding of T antigen to the Rb family of growth-regulatory proteins is necessary but not sufficient to cause transformation. The molecular mechanism underlying T-antigen inactivation of Rb function is poorly understood. In this study we show that T antigen associates with pRb and p130-E2F complexes in a stable manner. T antigen dissociates from a p130–E2F-4–DP-1 complex, coincident with the release of p130 from E2F-4–DP-1. The dissociation of this complex requires Hsc70, ATP, and a functional T-antigen J domain. We also report that the “released” E2F–DP-1 complex is competent to bind DNA containing an E2F consensus binding site. We propose that T antigen disrupts Rb-E2F family complexes through the action of its J domain and Hsc70. These findings indicate how Hsc70 supports T-antigen action and help to explain the cisrequirement for a J domain and Rb binding motif in T-antigen-induced transformation. Furthermore, this is the first demonstration linking Hsc70 ATP hydrolysis to the release of E2F bound by Rb family members.

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The amino-terminal transforming region of simian virus 40 large T and small t antigens functions as a J domain.

Molecular and Cellular Biology ◽

10.1128/mcb.17.8.4761 ◽

1997 ◽

Vol 17 (8) ◽

pp. 4761-4773 ◽

Cited By ~ 165

Author(s):

A Srinivasan ◽

A J McClellan ◽

J Vartikar ◽

I Marks ◽

P Cantalupo ◽

...

Keyword(s):

Amino Acid ◽

Atpase Activity ◽

Mammalian Cells ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Large T Antigen ◽

Amino Terminal ◽

J Domain ◽

Small T Antigen

Simian virus 40 (SV40) encodes two proteins, large T antigen and small t antigen that contribute to virus-induced tumorigenesis. Both proteins act by targeting key cellular regulatory proteins and altering their function. Known targets of the 708-amino-acid large T antigen include the three members of the retinoblastoma protein family (pRb, p107, and p130), members of the CBP family of transcriptional adapter proteins (cap-binding protein [CBP], p300, and p400), and the tumor suppressor p53. Small t antigen alters the activity of phosphatase pp2A and transactivates the cyclin A promoter. The first 82 amino acids of large T antigen and small t antigen are identical, and genetic experiments suggest that an additional target(s) important for transformation interacts with these sequences. This region contains a motif similar to the J domain, a conserved sequence found in the DnaJ family of molecular chaperones. We show here that mutations within the J domain abrogate the ability of large T antigen to transform mammalian cells. To examine whether a purified 136-amino-acid fragment from the T antigen amino terminus acts as a DnaJ-like chaperone, we investigated whether this fragment stimulates the ATPase activity of two hsc70s and discovered that ATP hydrolysis is stimulated four- to ninefold. In addition, ATPase-defective mutants of full-length T antigen, as well as wild-type small t antigen, stimulated the ATPase activity of hsc70. T antigen derivatives were also able to release an unfolded polypeptide substrate from an hsc70, an activity common to DnaJ chaperones. Because the J domain of T antigen plays essential roles in viral DNA replication, transcriptional control, virion assembly, and tumorigenesis, we conclude that this region may chaperone the rearrangement of multiprotein complexes.

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Species-Specific Elements in the Large T-Antigen J Domain Are Required for Cellular Transformation and DNA Replication by Simian Virus 40

Molecular and Cellular Biology ◽

10.1128/mcb.20.15.5749-5757.2000 ◽

2000 ◽

Vol 20 (15) ◽

pp. 5749-5757 ◽

Cited By ~ 55

Author(s):

Christopher S. Sullivan ◽

James D. Tremblay ◽

Sheara W. Fewell ◽

John A. Lewis ◽

Jeffrey L. Brodsky ◽

...

Keyword(s):

Dna Replication ◽

Jc Virus ◽

Simian Virus 40 ◽

Cellular Transformation ◽

Simian Virus ◽

T Antigen ◽

Large T Antigen ◽

Sv40 Large T Antigen ◽

J Domain ◽

Species Specific

ABSTRACT The J domain of simian virus 40 (SV40) large T antigen is required for efficient DNA replication and transformation. Despite previous reports demonstrating the promiscuity of J domains in heterologous systems, results presented here show the requirement for specific J-domain sequences in SV40 large-T-antigen-mediated activities. In particular, chimeric-T-antigen constructs in which the SV40 T-antigen J domain was replaced with that from the yeast Ydj1p or Escherichia coli DnaJ proteins failed to replicate in BSC40 cells and did not transform REF52 cells. However, T antigen containing the JC virus J domain was functional in these assays, although it was less efficient than the wild type. The inability of some large-T-antigen chimeras to promote DNA replication and elicit cellular transformation was not due to a failure to interact with hsc70, since a nonfunctional chimera, containing the DnaJ J domain, bound hsc70. However, this nonfunctional chimeric T antigen was reduced in its ability to stimulate hsc70 ATPase activity and unable to liberate E2F from p130, indicating that transcriptional activation of factors required for cell growth and DNA replication may be compromised. Our data suggest that the T-antigen J domain harbors species-specific elements required for viral activities in vivo.

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Loss of p19ARF Eliminates the Requirement for the pRB-Binding Motif in Simian Virus 40 Large T Antigen-Mediated Transformation

Molecular and Cellular Biology ◽

10.1128/mcb.20.20.7624-7633.2000 ◽

2000 ◽

Vol 20 (20) ◽

pp. 7624-7633 ◽

Cited By ~ 20

Author(s):

Herta H. A. Chao ◽

Ann M. Buchmann ◽

James A. DeCaprio

Keyword(s):

Simian Virus 40 ◽

Cellular Transformation ◽

Simian Virus ◽

T Antigen ◽

Binding Domain ◽

Large T Antigen ◽

Wild Type ◽

J Domain ◽

Lxcxe Motif ◽

Inactivating Mutations

ABSTRACT At least three domains of simian virus 40 large T antigen (TAg) participate in cellular transformation. The LXCXE motif of TAg binds to all members of the retinoblastoma protein (pRB) family of tumor suppressors. The N-terminal 70 residues of TAg have significant homology to the J domain of Hsp40/DnaJ and cooperate with the LXCXE motif to inactivate the pRB family. A bipartite C-terminal domain of TAg binds to p53 and thereby disrupts the ability of p53 to act as a sequence-specific transcription factor. The contribution of these three domains of TAg to cellular transformation was evaluated in cells that contained inactivating mutations in the pRB and p53 pathways. Cells that stably expressed wild-type or selected mutant forms of TAg were generated in mouse embryo fibroblasts (MEFs) containing homozygous deletions in the RB, INK4a, and ARFloci. It was determined that the J domain, the LXCXE motif, and the p53-binding domain of TAg were required for full transformation of wild-type and RB −/− MEFs. In contrast,INK4a −/− MEFs that lacked expression of p16 INK4a and p19 ARF andARF −/− MEFs that lacked p19 ARF but expressed p16 INK4a acquired anchorage-independent growth when expressing wild-type TAg or mutant derivatives that disrupted either the pRB-binding or p53-binding domain. The expression and function of the pRB family members were not overly disrupted inARF −/− MEFs expressing LXCXE mutants of TAg. These results suggest that inactivating mutations of p19 ARF can relieve the requirement for the LXCXE motif in TAg-mediated transformation and that TAg may have additional functions in transformation.

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Hyperphosphorylation of the retinoblastoma gene product is determined by domains outside the simian virus 40 large-T-antigen-binding regions.

Molecular and Cellular Biology ◽

10.1128/mcb.10.12.6586 ◽

1990 ◽

Vol 10 (12) ◽

pp. 6586-6595 ◽

Cited By ~ 24

Author(s):

P A Hamel ◽

B L Cohen ◽

L M Sorce ◽

B L Gallie ◽

R A Phillips

Keyword(s):

Cell Cycle ◽

Complex Formation ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Phosphorylation Sites ◽

Dependent Manner ◽

Large T Antigen ◽

T Complex ◽

Cycle Dependent

With the murine retinoblastoma (RB) cDNA, a series of RB mutants were expressed in COS-1 cells and the pRB products were assessed for their ability (i) to bind to large T antigen (large T), (ii) to become modified by phosphorylation, and (iii) to localize in the nucleus. All point mutations and deletions introduced into regions previously defined as contributing to binding to large T abolished pRB-large T complex formation and prevented hyperphosphorylation of the RB protein. In contrast, a series of deletions 5' to these sites did not interfere with binding to large T. While some of the 5' deletion mutants were clearly phosphorylated in a cell cycle-dependent manner, one, delta Pvu, failed to be phosphorylated depsite binding to large T. pRB with mutations created at three putative p34cdc2 phosphorylation sites in the N-terminal region behaved similarly to wild-type pRB, whereas the construct delta P5-6-7-8, mutated at four serine residues C terminal to the large T-binding site, failed to become hyperphosphorylated despite retaining the ability to bind large T. All of the mutants described were also found to localize in the nucleus. These results demonstrate that the domains in pRB responsible for binding to large T are distinct from those recognized by the relevant pRB-specific kinase(s) and/or those which contain cell cycle-dependent phosphorylation sites. Furthermore, these data are consistent with a model in which cell cycle-dependent phosphorylation of pRB requires complex formation with other cellular proteins.

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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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Enterocyte Proliferation and Intestinal Hyperplasia Induced by Simian Virus 40 T Antigen Require a Functional J Domain

Journal of Virology ◽

10.1128/jvi.00922-07 ◽

2007 ◽

Vol 81 (17) ◽

pp. 9481-9489 ◽

Cited By ~ 12

Author(s):

Abhilasha V. Rathi ◽

M. Teresa Sáenz Robles ◽

James M. Pipas

Keyword(s):

Transgenic Mice ◽

Family Members ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Wild Type ◽

Morphological Examination ◽

Enterocyte Proliferation ◽

J Domain ◽

Intestinal Hyperplasia

ABSTRACT Transgenic mice expressing the simian virus 40 large T antigen (TAg) in enterocytes develop intestinal hyperplasia that progresses to dysplasia with age. This induction requires TAg action on the retinoblastoma (Rb) family of tumor suppressors and is independent of the p53 pathway. In cell culture systems, the inactivation of Rb proteins requires both a J domain in TAg that interacts with hsc70 and an LXCXE motif that directs association with Rb proteins. Together these elements are sufficient to release E2Fs from their association with Rb family members. We have generated transgenic mice that express a J domain mutant (D44N) in villus enterocytes. In contrast to wild-type TAg, the D44N mutant is unable to induce enterocyte proliferation. Histological and morphological examination revealed that mice expressing the J domain mutant have normal intestines without loss of growth control. Unlike mice expressing wild-type TAg, mice expressing D44N do not reduce the protein levels of p130 and are also unable to dissociate p130-E2F DNA binding complexes. Furthermore, mice expressing D44N in a null p130 background are still unable to develop hyperplasia. These studies demonstrate that the ectopic proliferation of enterocytes by TAg requires a functional J domain and suggest that the J domain is necessary to inactivate all three pRb family members.

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Induction versus progression of brain tumor development: differential functions for the pRB- and p53-targeting domains of simian virus 40 T antigen.

Molecular and Cellular Biology ◽

10.1128/mcb.14.4.2686 ◽

1994 ◽

Vol 14 (4) ◽

pp. 2686-2698 ◽

Cited By ~ 73

Author(s):

M T Sáenz Robles ◽

H Symonds ◽

J Chen ◽

T Van Dyke

Keyword(s):

Tumor Progression ◽

Choroid Plexus ◽

Simian Virus 40 ◽

Cell Types ◽

Simian Virus ◽

T Antigen ◽

Cellular Proteins ◽

Wild Type ◽

Binding Region ◽

Amino Terminal

The ability of simian virus 40-encoded large T antigen to disrupt the growth control of a variety of cell types is related to its ability to interfere with certain cellular proteins, such as p53 and the retinoblastoma susceptibility gene product (pRB). We have used wild-type and mutant forms of T antigen in transgenic mice to dissect the roles of pRB, p53, and other cellular proteins in tumorigenesis of different cell types. In this study, using a cell-specific promoter to target expression specifically to brain epithelium (the choroid plexus) and to B and T lymphoid cells, we characterize the tumorigenic capacity of a T-antigen fragment that comprises only the amino-terminal 121 residues. This fragment (dl1137) retains the ability to interact with pRB and p107 but lacks the p53-binding domain. While loss of the p53-binding region results in loss of the capacity to induce lymphoid abnormalities, dl1137 retains the ability to induce choroid plexus tumors that are histologically indistinguishable from those induced by wild-type T antigen. Tumors induced by dl1137 develop much more slowly, however, reaching an end point at around 8 months of age rather than at 1 to 2 months. Analysis of tumor progression indicates that tumor induction by dl1137 does not require secondary genetic or epigenetic events. Rather, the tumor growth rate is significantly slowed, indicating that the T-antigen C-terminal region contributes to tumor progression in this cell type. In contrast, the pRB-binding region appears essential for tumorigenesis as mutation of residue 107, known to disrupt pRB and p107 binding to wild-type T antigen, abolishes the ability of the dl1137 protein to induce growth abnormalities in the brain.

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