scholarly journals A bipartite nuclear localization signal in the retinoblastoma gene product and its importance for biological activity

1993 ◽  
Vol 13 (8) ◽  
pp. 4588-4599
Author(s):  
E Zacksenhaus ◽  
R Bremner ◽  
R A Phillips ◽  
B L Gallie
Keyword(s):  
Biological Activity ◽  
Transcription Factors ◽  
Gene Product ◽  
Nuclear Localization ◽  
Simian Virus 40 ◽  
T Antigen ◽  
Retinoblastoma Gene ◽  
C Terminus ◽  
Retinoblastoma Gene Product ◽  
Cellular Transcription

The retinoblastoma gene product, p110RB1, appears to regulate cell growth by modulating the activities of nuclear transcription factors. The elements that specify the transport of p110RB1 into the nucleus have not yet been explored. We now report the identification of a basic region, KRSAEGGNPPKPLKKLR, in the C terminus of p110RB1, which has sequence similarity to known bipartite nuclear localization signals (NLSs). A two-amino-acid mutation introduced into this putative NLS [to give mutant NLS(NQ)] or deletion of the entire NLS (delta NLS) abrogated exclusive nuclear localization, yielding proteins which were distributed either equally throughout the cell or predominantly in the cytoplasm. A mutant protein [NLS(NQ)/delta 22] containing both the mutated NLS and a deletion of exon 22, previously shown to disrupt the interaction of p110RB1 with several cellular transcription factors and oncoproteins, accumulated only in the cytoplasm. When fused to the C terminus of Escherichia coli beta-galactosidase, the RB1 NLS directed this protein to the nucleus, indicating that the motif is not only necessary but also sufficient for nuclear transport. Neither NLS(NQ) nor delta NLS was hyperphosphorylated in vivo, but both retained their abilities to interact, in vitro, with simian virus 40 large T antigen, adenovirus E1a, and the cellular transcription factor E2F. When transfected at multiple copy number, the NLS mutant alleles displayed reduced biological activity, measured by inhibition of growth of the osteogenic sarcoma cell line Saos-2, which has no wild-type RB1. Naturally occurring mutations and deletions in exon 25 of RB1 which disrupt the NLS may lead to partial or complete inactivation of p110RB1 and may be responsible for some retinoblastoma and other tumors.

scholarly journals A bipartite nuclear localization signal in the retinoblastoma gene product and its importance for biological activity.

1993 ◽  
Vol 13 (8) ◽  
pp. 4588-4599 ◽  
Author(s):  
E Zacksenhaus ◽  
R Bremner ◽  
R A Phillips ◽  
B L Gallie
Keyword(s):  
Biological Activity ◽  
Transcription Factors ◽  
Gene Product ◽  
Nuclear Localization ◽  
Simian Virus 40 ◽  
T Antigen ◽  
Retinoblastoma Gene ◽  
C Terminus ◽  
Retinoblastoma Gene Product ◽  
Cellular Transcription

The retinoblastoma gene product, p110RB1, appears to regulate cell growth by modulating the activities of nuclear transcription factors. The elements that specify the transport of p110RB1 into the nucleus have not yet been explored. We now report the identification of a basic region, KRSAEGGNPPKPLKKLR, in the C terminus of p110RB1, which has sequence similarity to known bipartite nuclear localization signals (NLSs). A two-amino-acid mutation introduced into this putative NLS [to give mutant NLS(NQ)] or deletion of the entire NLS (delta NLS) abrogated exclusive nuclear localization, yielding proteins which were distributed either equally throughout the cell or predominantly in the cytoplasm. A mutant protein [NLS(NQ)/delta 22] containing both the mutated NLS and a deletion of exon 22, previously shown to disrupt the interaction of p110RB1 with several cellular transcription factors and oncoproteins, accumulated only in the cytoplasm. When fused to the C terminus of Escherichia coli beta-galactosidase, the RB1 NLS directed this protein to the nucleus, indicating that the motif is not only necessary but also sufficient for nuclear transport. Neither NLS(NQ) nor delta NLS was hyperphosphorylated in vivo, but both retained their abilities to interact, in vitro, with simian virus 40 large T antigen, adenovirus E1a, and the cellular transcription factor E2F. When transfected at multiple copy number, the NLS mutant alleles displayed reduced biological activity, measured by inhibition of growth of the osteogenic sarcoma cell line Saos-2, which has no wild-type RB1. Naturally occurring mutations and deletions in exon 25 of RB1 which disrupt the NLS may lead to partial or complete inactivation of p110RB1 and may be responsible for some retinoblastoma and other tumors.

A cellular protein that competes with SV40 T antigen for binding to the retinoblastoma gene product

Nature ◽  
1991 ◽  
Vol 350 (6314) ◽  
pp. 160-162 ◽  
Author(s):  
Shi Huang ◽  
Wen-Hwa Lee ◽  
Eva Y.-H. P. Lee
Keyword(s):  
Gene Product ◽  
Cellular Protein ◽  
T Antigen ◽  
Retinoblastoma Gene ◽  
Sv40 T Antigen ◽  
Retinoblastoma Gene Product

scholarly journals Nuclear localization conferred by the pocket domain of the retinoblastoma gene product

1999 ◽  
Vol 1451 (2-3) ◽  
pp. 288-296 ◽  
Author(s):  
Eldad Zacksenhaus ◽  
Zhe Jiang ◽  
Yong J. Hei ◽  
Robert A. Phillips ◽  
Brenda L. Gallie
Keyword(s):  
Gene Product ◽  
Nuclear Localization ◽  
Retinoblastoma Gene ◽  
Retinoblastoma Gene Product ◽  
Pocket Domain

scholarly journals Induction of S-phase entry by E2F transcription factors depends on their nuclear localization.

1997 ◽  
Vol 17 (9) ◽  
pp. 5508-5520 ◽  
Author(s):  
H Müller ◽  
M C Moroni ◽  
E Vigo ◽  
B O Petersen ◽  
J Bartek ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Nuclear Localization ◽  
Neutralizing Antibodies ◽  
Biological Activities ◽  
Simian Virus 40 ◽  
T Antigen ◽  
S Phase ◽  
Amino Terminus ◽  
Chimeric Proteins ◽  
E2f Transcription Factors

The E2F transcription factors are essential for regulating the correct timing of activation of several genes whose products are implicated in cell proliferation and DNA replication. The E2Fs are targets for negative regulation by the retinoblastoma protein family, which includes pRB, p107, and p130, and they are in a pathway that is frequently found altered in human cancers. There are five members of the E2F family, and they can be divided into two functional subgroups. Whereas, upon overexpression, E2F-1, -2, and -3 induce S phase in quiescent fibroblasts and override G1 arrests mediated by the p16INK4A tumor suppressor protein or neutralizing antibodies to cyclin D1, E2F-4 and -5 do not. Using E2F-1 and E2F-4 as representatives of the two subgroups, we showed here, by constructing a set of chimeric proteins, that the amino terminus of E2F-1 is sufficient to confer S-phase-inducing potential as well as the ability to efficiently transactivate an E2F-responsive promoter to E2F-4. We found that the E2F-1 amino terminus directs chimeric proteins to the nucleus. Surprisingly, a short nuclear localization signal derived from simian virus 40 large T antigen could perfectly substitute for the presence of the E2F-1 amino terminus in these assays. Thus, nuclearly localized E2F-4, when overexpressed, displayed biological activities similar to those of E2F-1. Furthermore, we showed that nuclear localization of endogenous E2F-4 is cell cycle regulated, with E2F-4 being nuclear in the G0 and early G1 phases and mainly cytoplasmic after the pRB family members have become phosphorylated. We propose a novel mechanism for the regulation of E2F-dependent transcription in which E2F-4 regulates transcription only from G0 until mid- to late G1 phase whereas E2F-1 is active in late G1 and S phases, until it is inactivated by cyclin A-dependent kinase in late S phase.

Retinoblastoma gene product activates expression of the human TGF-β2 gene through transcription factor ATF-2

Nature ◽  
1992 ◽  
Vol 358 (6384) ◽  
pp. 331-334 ◽  
Author(s):  
Seong-Jin Kim ◽  
Susanne Wagner ◽  
Fang Liu ◽  
Michael A. O'Reilly ◽  
Paul D. Robbins ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Gene Product ◽  
Retinoblastoma Gene ◽  
Retinoblastoma Gene Product

Specific transcription factors stimulate simian virus 40 and polyomavirus origins of DNA replication

1992 ◽  
Vol 12 (6) ◽  
pp. 2514-2524 ◽  
Author(s):  
Z S Guo ◽  
M L DePamphilis
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Dna Replication ◽  
Binding Sites ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Activation Domains ◽  
Auxiliary Component ◽  
Cell Extracts

The origins of DNA replication (ori) in simian virus 40 (SV40) and polyomavirus (Py) contain an auxiliary component (aux-2) composed of multiple transcription factor binding sites. To determine whether this component stimulated replication by binding specific transcription factors, aux-2 was replaced by synthetic oligonucleotides that bound a single transcription factor. Sp1 and T-antigen (T-ag) sites, which exist in the natural SV40 aux-2 sequence, provided approximately 75 and approximately 20%, respectively, of aux-2 activity when transfected into monkey cells. In cell extracts, only T-ag sites were active. AP1 binding sites could replace completely either SV40 or Py aux-2. Mutations that eliminated AP1 binding also eliminated AP1 stimulation of replication. Yeast GAL4 binding sites that strongly stimulated transcription in the presence of GAL4 proteins failed to stimulate SV40 DNA replication, although they did partially replace Py aux-2. Stimulation required the presence of proteins consisting of the GAL4 DNA binding domain fused to specific activation domains such as VP16 or c-Jun. These data demonstrate a clear role for transcription factors with specific activation domains in activating both SV40 and Py ori. However, no correlation was observed between the ability of specific proteins to stimulate promoter activity and their ability to stimulate origin activity. We propose that only transcription factors whose specific activation domains can interact with the T-ag initiation complex can stimulate SV40 and Py ori-core activity.

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