Genetic analysis of p53 nuclear importation

Li, Q; Falsey, Ryan R.; Gaitonde, Supriya; Sotello, V; Kislin, Kerri L.; Martinez, Jesse D.

doi:10.1038/sj.onc.1210597

Cited by 21 publications

(14 citation statements)

References 12 publications

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“…A search of the literature found only indirect evidence for the role of importin in the transport of most proteins and no direct evidence for a role in the transport of p53 (Kim et al ., 2000; Li et al ., 2007; Liang and Clarke, 1999). We conducted immunoprecipitation experiments to assess the extent of importin association with p53 and its putative role in the nuclear transport of p53.…”

Section: Resultsmentioning

confidence: 99%

Impaired p53 binding to importin: a novel mechanism of cytoplasmic sequestration identified in oxaliplatin-resistant cells

et al. 2009

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Previous studies have described one nuclear localization signal (NLSI) in p53 and speculated on two additional sites termed NLSII and NLSIII. Drug-resistant KB cells selected with cisplatin or oxaliplatin were found to have increased p53 levels and in oxaliplatin-selected cells, a larger p53 predominantly in the cytoplasm. In oxaliplatin-selected cells a single nucleotide deletion in the sequence-encoding amino acid 382, part of NLSIII, resulted in a frame shift and a 420 amino acid protein (p53 420 ). We investigated explanations for the cytoplasmic sequestration of p53 420 while assessing the role, if any, of NLSII and NLSIII in p53 nuclear import. We found that neither NLSII nor NLSIII are essential for p53 nuclear localization. Furthermore, we confirmed p53 420 is able to tetramerize, transactivate a p21 promoter, bind dynein and that the reduced nuclear accumulation is not a consequence of increased p53 nuclear export. However, the association of p53 420 with importin-b, essential for nuclear import, was significantly impaired. We conclude that despite sequence similarity to consensus NLSs neither NLSII nor NLSIII have roles in p53 nuclear transport. We also identified impaired association with importin as a novel mechanism of p53 cytoplasmic sequestration that impairs nuclear transport rendering cells functionally deficient in p53.

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Section: Resultsmentioning

confidence: 99%

Impaired p53 binding to importin: a novel mechanism of cytoplasmic sequestration identified in oxaliplatin-resistant cells

et al. 2009

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“…We examined three cell lines. A1-5 cells are the parental cells from which the ALTR cells were derived (9). ALTR12 cells lack expression of HSF1 and are defective for nuclear importation of p53 (10).…”

Section: Resultsmentioning

confidence: 99%

“…Previously we characterized the nuclear localization of p53 through derivation of a set of cell lines known as ALTR cell lines in which nuclear accumulation of p53 was defective (9). Subsequently we showed that in one of those lines, ALTR12, exclusion of p53 from the nucleus was due to loss of the heat-shock factor 1 (HSF1) (10).…”

Section: Introductionmentioning

confidence: 99%

Loss of HSF1 Results in Defective Radiation-Induced G₂Arrest and DNA Repair

Martinez

2011

Radiation Research

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HSF1 is a transcription factor that plays a key role in the response to heat stress and was previously shown by us to also be essential for importation of p53 into the nucleus. Here we extend these studies and show that loss of HSF1 renders cells more sensitive to killing by ionizing radiation. Cells that lack a functional HSF1 were unable to arrest in G2 after exposure to ionizing radiation, suggesting that HSF1 activity was essential for activation of this cell cycle checkpoint. In addition, cells with no HSF1 showed a reduced capacity to repair radiation-induced double-stranded DNA breaks. We found that in these cells 53BP1 did not accumulate at sites of DNA damage, suggesting that HSF1 was also essential for the functioning of this DNA damage mediator. Taken together our results indicate that HSF1 plays an important role in checkpoint activation and DNA repair and suggest that there is overlap between the heat stress response pathway and the pathway that responds to ionizing radiation.

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“…Like nuclear transport factors, many cargos shuttle between the nucleus and the cytoplasm, and their relative distributions can regulate their function (6,10,11). Prominent examples that are subject to this type of localization-mediated regulation are protein kinases and phosphatases, transcriptional regulators, and nuclear transporters for proteins or RNA (6,10,(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23).…”

Section: Introductionmentioning

confidence: 99%

Analysis of Signaling Events by Combining High-Throughput Screening Technology with Computer-Based Image Analysis

2008

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Intracellular signaling and cell-to-cell communication depend on the coordination of numerous signaling events, and this large flow of information has to be properly organized in space and time. Common and critical to all of these processes and the ultimate cellular response is the correct spatial distribution of signaling components and their targets. This fundamental concept applies to a large number of signaling processes. It is frequently important to quantify the localization of signaling molecules within different cellular compartments to detect subtle changes or to define threshold levels of signaling molecules in a certain location that are necessary to trigger subsequent events. Of particular importance is the separation of nuclear and cytoplasmic events, and sensitive methods are required to measure their contribution to signal transduction. Procedures described here allow the quantification of fluorescence signals located in the nucleus, cytoplasm, or at the nuclear envelope. The methods rely on high-throughput imaging equipment, confocal microscopy, and software modules that measure the fluorescence intensity in the compartment of interest. We discuss the rationale for selecting the appropriate equipment for image acquisition and the proper software modules to quantify fluorescence in distinct cellular compartments. Initially, high-throughput technology for high-speed image acquisition was developed for multiwell plates. We adapted high-throughput technology for image acquisition for cells grown on cover slips. Images of higher spatial resolution along the z axis were acquired by confocal microscopy. For subsequent analyses, the choice of appropriate software modules is critical for rapid and reliable quantification of fluorescence intensities.

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Genetic analysis of p53 nuclear importation

Cited by 21 publications

References 12 publications

Impaired p53 binding to importin: a novel mechanism of cytoplasmic sequestration identified in oxaliplatin-resistant cells

Impaired p53 binding to importin: a novel mechanism of cytoplasmic sequestration identified in oxaliplatin-resistant cells

Loss of HSF1 Results in Defective Radiation-Induced G₂Arrest and DNA Repair

Analysis of Signaling Events by Combining High-Throughput Screening Technology with Computer-Based Image Analysis

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Genetic analysis of p53 nuclear importation

Cited by 21 publications

References 12 publications

Impaired p53 binding to importin: a novel mechanism of cytoplasmic sequestration identified in oxaliplatin-resistant cells

Impaired p53 binding to importin: a novel mechanism of cytoplasmic sequestration identified in oxaliplatin-resistant cells

Loss of HSF1 Results in Defective Radiation-Induced G2Arrest and DNA Repair

Analysis of Signaling Events by Combining High-Throughput Screening Technology with Computer-Based Image Analysis

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Loss of HSF1 Results in Defective Radiation-Induced G₂Arrest and DNA Repair