Genetic analysis of polyomavirus large T nuclear localization: nuclear localization is required for productive association with pRb family members

Howes, Stacey; Bockus, B J; Schaffhausen, Brian

doi:10.1128/jvi.70.6.3581-3588.1996

Cited by 17 publications

(17 citation statements)

References 64 publications

(89 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…What is the role of the J region of LT? The one character- (21). To test the importance of LT sequences, two different reporters containing E2F sites adjoining the CAT gene were transfected, along with mutant or wild-type LTs.…”

Section: Resultsmentioning

confidence: 99%

“…CMV vectors expressing adenovirus E1A 12S and human papillomavirus (HPV) E7 have been described previously (15). The EC113 E2CAT (22), provided by Elliot Androphy, contains the adenovirus E2 promoter with its E2F sites; pA10-E2F-CAT plasmid had the Ϫ85-to-Ϫ30 adenovirus E2 promoter sequence and a minimal simian virus 40 (SV40) promoter fused to a chloramphenicol acetyltransferase (CAT) gene (21,52). Rous sarcoma virus (RSV) ␤-galactosidase (␤-Gal) (2,11) vector was provided by Amy Yee.…”

Section: Plasmids and Mutagenesis Pcmv Ltmentioning

confidence: 99%

See 1 more Smart Citation

The DnaJ domain of polyomavirus large T antigen is required to regulate Rb family tumor suppressor function

Sheng

Denis

Ratnofsky

et al. 1997

J Virol

View full text Add to dashboard Cite

Tumor suppressors of the retinoblastoma susceptibility gene family regulate cell growth and differentiation. Polyomavirus large T antigens (large T) bind Rb family members and block their function. Mutations of large T sequences conserved with the DnaJ family affect large T binding to a cellular DnaK, heat shock protein 70. The same mutations abolish large T activation of E2F-containing promoters and Rb binding-dependent large T activation of cell cycle progression. Cotransfection of a cellular DnaJ domain blocks wild-type large T action,showing that the connection between the chaperone system and tumor suppressors is direct. Although they are inactive in assays dependent on Rb family binding, mutants in the J region retain the ability to associate with pRb, p107, and p130. This suggests that binding of Rb family members by large T is not sufficient for their inactivation and that a functional J domain is required as well. This work connects the DnaJ and DnaK molecular chaperones to regulation of tumor suppressors by polyomavirus large T.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Plasmids and Mutagenesis Pcmv Ltmentioning

confidence: 99%

The DnaJ domain of polyomavirus large T antigen is required to regulate Rb family tumor suppressor function

Sheng

Denis

Ratnofsky

et al. 1997

J Virol

View full text Add to dashboard Cite

show abstract

“…Finally, the data also underscore additional Rb-independent mechanisms for LT transactivation. The notion that LT might have Rb-independent functions in transactivation is not novel (34,42); in SV40 LT, several different targets of transactivation have been demonstrated (26,87). In addition to targeting transcription factors, LT may also target the basal transcriptional machinery, since this appears to happen for SV40 LT (59).…”

Section: Discussionmentioning

confidence: 99%

“…Much of what LT does to cells depends upon its interactions with the Rb tumor suppressor family through an LXCXE motif starting at residue 141 (17,32). Immortalization (22,34,41), induction of cell DNA synthesis (70), overcoming p53-induced cell cycle arrest (16), and prevention of differentiation (52) can all depend on Rb family binding. Transactivation of the thymidine kinase, polymerase ␣, PCNA, dihydrofolate reductase, and thymidylate synthase genes (54) as well as regulation of interferon-inducible gene expression (80) also requires an intact pRb/p107 binding site on LT and is mediated via the cellular transcription factor E2F.…”

mentioning

confidence: 99%

J Domain-Independent Regulation of the Rb Family by Polyomavirus Large T Antigen

Sheng¹,

Love²,

Schaffhausen³

2000

J. Virol.

View full text Add to dashboard Cite

The ability of polyomavirus large T antigen (LT) to promote cell cycling, to immortalize primary cells, and to block differentiation has been linked to its effects on tumor suppressors of the retinoblastoma susceptibility (Rb) gene family. Our previous studies have shown that LT requires an intact N-terminal DnaJ domain, in addition to an Rb binding site, for activation of simple E2F-containing promoters and stimulation of cell cycle progression. Here we show that some LT effects dependent on interaction with the Rb family are largely DnaJ independent. In differentiating C2C12 myoblasts, overexpression of LT caused apoptosis. Although this activity of LT completely depended on Rb binding, LTs with mutations in the J domain remained able to kill. Comparisons of Rb ؊ and J ؊ LTs revealed additional differences. Wild-type but not Rb ؊ LT activated the cyclin A promoter under serum starvation conditions. Genetic analysis of the promoter linked the Rb requirement to an E2F site in the promoter. LTs with mutations in the J domain were still able to activate the promoter. Finally, J mutant LTs caused changes in phosphorylation of both pRb and p130. In the case of p130, Thr-986 was shown to be a site that is regulated by J mutant LT. Taken together, these observations reveal that LT regulation of Rb function can be separated into both DnaJ-dependent and DnaJ-independent pathways.

show abstract

“…Both SV40 (14, 31, 32; reviewed in reference 46) and Py (17,30,41,63) T Ags possess an LXCXE motif within their N termini which is necessary for their interaction with the retinoblastoma tumor suppressor protein (pRb) and related family members. It is well documented that the pRb protein plays a critical role in controlling the progression of cells from G 1 to S (reviewed in references 3, 28, 35, and 44).…”

mentioning

confidence: 99%

The Retinoblastoma Protein Alters the Phosphorylation State of Polyomavirus Large T Antigen in Murine Cell Extracts and Inhibits Polyomavirus Origin DNA Replication

Reynisdóttir

Bhattacharyya

Zhang

et al. 1999

J Virol

View full text Add to dashboard Cite

The retinoblastoma tumor suppressor protein (pRb) can associate with the transforming proteins of several DNA tumor viruses, including the large T antigen encoded by polyomavirus (Py T Ag). Although pRb function is critical for regulating progression from G1 to S phase, a role for pRb in S phase has not been demonstrated or excluded. To identify a potential effect of pRb on DNA replication, pRb protein was added to reaction mixtures containing Py T Ag, Py origin-containing DNA (Py ori-DNA), and murine FM3A cell extracts. We found that pRb strongly represses Py ori-DNA replication in vitro. Unexpectedly, however, this inhibition only partially depends on the interaction of pRb with Py T Ag, since a mutant Py T Ag (dl141) lacking the pRb interaction region was also significantly inhibited by pRb. This result suggests that pRb interferes with or alters one or more components of the murine cell replication extract. Furthermore, the ability of Py T Ag to be phosphorylated in such extracts is markedly reduced in the presence of pRb. Since cyclin-dependent kinase (CDK) phosphorylation of Py T Ag is required for its replication function, we hypothesize that pRb interferes with this phosphorylation event. Indeed, the S-phase CDK complex (cyclin A-CDK2), which phosphorylates both pRb and Py T Ag, alleviates inhibition caused by pRb. Moreover, hyperphosphorylated pRb is incapable of inhibiting replication of Py ori-DNA in vitro. We propose a new requirement for maintaining pRb phosphorylation in S phase, namely, to prevent deleterious effects on the cellular replication machinery.

show abstract

Genetic analysis of polyomavirus large T nuclear localization: nuclear localization is required for productive association with pRb family members

Cited by 17 publications

References 64 publications

The DnaJ domain of polyomavirus large T antigen is required to regulate Rb family tumor suppressor function

The DnaJ domain of polyomavirus large T antigen is required to regulate Rb family tumor suppressor function

J Domain-Independent Regulation of the Rb Family by Polyomavirus Large T Antigen

The Retinoblastoma Protein Alters the Phosphorylation State of Polyomavirus Large T Antigen in Murine Cell Extracts and Inhibits Polyomavirus Origin DNA Replication

Contact Info

Product

Resources

About