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

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.13.8.4588-4599.1993 ◽

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.

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

Nature ◽

10.1038/358331a0 ◽

1992 ◽

Vol 358 (6384) ◽

pp. 331-334 ◽

Cited By ~ 172

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

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Control of Cell Cycle Progression in Human Natural Killer Cells Through Redox Regulation of Expression and Phosphorylation of Retinoblastoma Gene Product Protein

Blood ◽

10.1182/blood.v89.11.4092 ◽

1997 ◽

Vol 89 (11) ◽

pp. 4092-4099 ◽

Cited By ~ 31

Author(s):

Akira Yamauchi ◽

Eda T. Bloom

Keyword(s):

Cell Cycle ◽

Nk Cells ◽

Natural Killer ◽

Gene Product ◽

Redox Regulation ◽

Interleukin 2 ◽

Retinoblastoma Gene ◽

Regulation Of Expression ◽

Retinoblastoma Gene Product

Abstract Using thiol deprivation, we have previously shown that the response of natural killer (NK) cells to interleukin-2 (IL-2) is subject to redox regulation downstream of IL-2 binding and internalization. We have now used the IL-2–dependent cell line, NK3.3 to study redox regulation of NK cells further, and found that NK3.3 cells neither incorporated [3H]-thymidine nor completed the G1-S phase transition in medium lacking the thiol-related compounds, L-cystine, and glutathione, despite the presence of sufficient IL-2. Thiol deprivation did not alter the induction of DNA interferon-γ activated sequence (GAS)-binding activity in response to IL-2. However, the retinoblastoma gene product (RB), a cyclin-dependent kinase (CDK) substrate, was phosphorylated within 24 hours after IL-2 stimulation in standard medium, but its expression and phosphorylation were reduced in thiol-depleted medium in both NK3.3 cells and freshly isolated NK cells. These reductions were not associated with an increased level of p27Kip1, an inhibitor of CDKs CDK6/2 in association with G1 cyclins. Reducing agents, N-acetylcysteine, reduced glutathione or 2-ME restored both RB phosphorylation and DNA synthesis in thiol-deprived NK3.3 cells. The in vitro kinase activities of CDK6 and CDK2 were prematurely increased by thiol deprivation. This enhancement was associated with CDK hyperphosphorylation and prolonged phosphorylation, and could be observed before and beyond IL-2 stimulation. The data suggest the possibility that the premature and prolonged enhancement of CDK activity in thiol-deprived NK cells is associated with, and therefore may contribute to, the reduced expression and phosphorylation of RB, and the associated cell cycle arrest.

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