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Two nuclear localization signals in USP1 mediate nuclear import of the USP1/UAF1 complex.

Garcia-Santisteban I, Zorroza K, Rodriguez JA - PLoS ONE (2012)

Bottom Line: Using a cellular relocation assay based on these results, we map the UAF1-binding site to a highly conserved 100 amino acid motif in USP1.Importantly, our findings have practical implications for the development of USP1-directed therapies.First, the UAF1-interacting region of USP1 identified here might be targeted to disrupt the USP1/UAF1 interaction with therapeutic purposes.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country UPV/EHU, Leioa, Spain.

ABSTRACT
The human deubiquitinase USP1 plays important roles in cancer-related processes, such as the DNA damage response, and the maintenance of the undifferentiated state of osteosarcoma cells. USP1 deubiquitinase activity is critically regulated by its interaction with the WD40 repeat-containing protein UAF1. Inhibiting the function of the USP1/UAF1 complex sensitizes cancer cells to chemotherapy, suggesting that this complex is a relevant anticancer target. Intriguingly, whereas UAF1 has been reported to locate in the cytoplasm, USP1 is a nuclear protein, although the sequence motifs that mediate its nuclear import have not been functionally characterized. Here, we identify two nuclear localization signals (NLSs) in USP1 and show that these NLSs mediate the nuclear import of the USP1/UAF1 complex. Using a cellular relocation assay based on these results, we map the UAF1-binding site to a highly conserved 100 amino acid motif in USP1. Our data support a model in which USP1 and UAF1 form a complex in the cytoplasm that subsequently translocates to the nucleus through import mediated by USP1 NLSs. Importantly, our findings have practical implications for the development of USP1-directed therapies. First, the UAF1-interacting region of USP1 identified here might be targeted to disrupt the USP1/UAF1 interaction with therapeutic purposes. On the other hand, we describe a cellular relocation assay that can be easily implemented in a high throughput setting to search for drugs that may dissociate the USP1/UAF1 complex.

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USP1 NLSs mediate nuclear import of the USP1/UAF1 complex.A. Confocal images showing that Xpress-UAF1 localizes to the cytoplasm in transfected 293T cells, and it does not relocate to the nucleus in the presence of LMB. B. Confocal images of 293T cells co-expressing Xpress-UAF1 with GFP, GFP-USP1 wild type (WT) or GFP-USP1NLS1/2m. Xpress-UAF1 (red) relocated to the nucleus when co-expressed with GFP-USP1 wild type, but remained in the cytoplasm when co-expressed with the import deficient mutant GFP-USP1NLS1/2m. Cells were counterstained with Hoechst to show the nuclei (DNA panels). C Semiquantitative analysis of Xpress-UAF1 nucleocytoplasmic distribution when co-expressed with YFP, GFP-USP1 wild type (WT) or GFP-USP1NLS1/2m. Graphs show the percentage of co-transfected cells showing nuclear (N), nuclear and cytoplasmic (NC) or cytoplasmic (C) localization of Xpress-UAF1. The number of cells counted in each sample (n) is indicated within the graph. D. A model illustrating the ability of USP1 to bind importins (imp) and UAF1, and thus, mediate the nuclear import of the USP1/UAF1 complex.
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pone-0038570-g003: USP1 NLSs mediate nuclear import of the USP1/UAF1 complex.A. Confocal images showing that Xpress-UAF1 localizes to the cytoplasm in transfected 293T cells, and it does not relocate to the nucleus in the presence of LMB. B. Confocal images of 293T cells co-expressing Xpress-UAF1 with GFP, GFP-USP1 wild type (WT) or GFP-USP1NLS1/2m. Xpress-UAF1 (red) relocated to the nucleus when co-expressed with GFP-USP1 wild type, but remained in the cytoplasm when co-expressed with the import deficient mutant GFP-USP1NLS1/2m. Cells were counterstained with Hoechst to show the nuclei (DNA panels). C Semiquantitative analysis of Xpress-UAF1 nucleocytoplasmic distribution when co-expressed with YFP, GFP-USP1 wild type (WT) or GFP-USP1NLS1/2m. Graphs show the percentage of co-transfected cells showing nuclear (N), nuclear and cytoplasmic (NC) or cytoplasmic (C) localization of Xpress-UAF1. The number of cells counted in each sample (n) is indicated within the graph. D. A model illustrating the ability of USP1 to bind importins (imp) and UAF1, and thus, mediate the nuclear import of the USP1/UAF1 complex.

Mentions: As previously reported [16], [17], Xpress-tagged UAF1 localized to the cytoplasm of 293T cells (Figure 3A). LMB treatment did not induce its nuclear relocation, suggesting that CRM1-mediated nuclear export is not necessary to maintain the cytoplasmic localization of UAF1.


Two nuclear localization signals in USP1 mediate nuclear import of the USP1/UAF1 complex.

Garcia-Santisteban I, Zorroza K, Rodriguez JA - PLoS ONE (2012)

USP1 NLSs mediate nuclear import of the USP1/UAF1 complex.A. Confocal images showing that Xpress-UAF1 localizes to the cytoplasm in transfected 293T cells, and it does not relocate to the nucleus in the presence of LMB. B. Confocal images of 293T cells co-expressing Xpress-UAF1 with GFP, GFP-USP1 wild type (WT) or GFP-USP1NLS1/2m. Xpress-UAF1 (red) relocated to the nucleus when co-expressed with GFP-USP1 wild type, but remained in the cytoplasm when co-expressed with the import deficient mutant GFP-USP1NLS1/2m. Cells were counterstained with Hoechst to show the nuclei (DNA panels). C Semiquantitative analysis of Xpress-UAF1 nucleocytoplasmic distribution when co-expressed with YFP, GFP-USP1 wild type (WT) or GFP-USP1NLS1/2m. Graphs show the percentage of co-transfected cells showing nuclear (N), nuclear and cytoplasmic (NC) or cytoplasmic (C) localization of Xpress-UAF1. The number of cells counted in each sample (n) is indicated within the graph. D. A model illustrating the ability of USP1 to bind importins (imp) and UAF1, and thus, mediate the nuclear import of the USP1/UAF1 complex.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3368879&req=5

pone-0038570-g003: USP1 NLSs mediate nuclear import of the USP1/UAF1 complex.A. Confocal images showing that Xpress-UAF1 localizes to the cytoplasm in transfected 293T cells, and it does not relocate to the nucleus in the presence of LMB. B. Confocal images of 293T cells co-expressing Xpress-UAF1 with GFP, GFP-USP1 wild type (WT) or GFP-USP1NLS1/2m. Xpress-UAF1 (red) relocated to the nucleus when co-expressed with GFP-USP1 wild type, but remained in the cytoplasm when co-expressed with the import deficient mutant GFP-USP1NLS1/2m. Cells were counterstained with Hoechst to show the nuclei (DNA panels). C Semiquantitative analysis of Xpress-UAF1 nucleocytoplasmic distribution when co-expressed with YFP, GFP-USP1 wild type (WT) or GFP-USP1NLS1/2m. Graphs show the percentage of co-transfected cells showing nuclear (N), nuclear and cytoplasmic (NC) or cytoplasmic (C) localization of Xpress-UAF1. The number of cells counted in each sample (n) is indicated within the graph. D. A model illustrating the ability of USP1 to bind importins (imp) and UAF1, and thus, mediate the nuclear import of the USP1/UAF1 complex.
Mentions: As previously reported [16], [17], Xpress-tagged UAF1 localized to the cytoplasm of 293T cells (Figure 3A). LMB treatment did not induce its nuclear relocation, suggesting that CRM1-mediated nuclear export is not necessary to maintain the cytoplasmic localization of UAF1.

Bottom Line: Using a cellular relocation assay based on these results, we map the UAF1-binding site to a highly conserved 100 amino acid motif in USP1.Importantly, our findings have practical implications for the development of USP1-directed therapies.First, the UAF1-interacting region of USP1 identified here might be targeted to disrupt the USP1/UAF1 interaction with therapeutic purposes.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country UPV/EHU, Leioa, Spain.

ABSTRACT
The human deubiquitinase USP1 plays important roles in cancer-related processes, such as the DNA damage response, and the maintenance of the undifferentiated state of osteosarcoma cells. USP1 deubiquitinase activity is critically regulated by its interaction with the WD40 repeat-containing protein UAF1. Inhibiting the function of the USP1/UAF1 complex sensitizes cancer cells to chemotherapy, suggesting that this complex is a relevant anticancer target. Intriguingly, whereas UAF1 has been reported to locate in the cytoplasm, USP1 is a nuclear protein, although the sequence motifs that mediate its nuclear import have not been functionally characterized. Here, we identify two nuclear localization signals (NLSs) in USP1 and show that these NLSs mediate the nuclear import of the USP1/UAF1 complex. Using a cellular relocation assay based on these results, we map the UAF1-binding site to a highly conserved 100 amino acid motif in USP1. Our data support a model in which USP1 and UAF1 form a complex in the cytoplasm that subsequently translocates to the nucleus through import mediated by USP1 NLSs. Importantly, our findings have practical implications for the development of USP1-directed therapies. First, the UAF1-interacting region of USP1 identified here might be targeted to disrupt the USP1/UAF1 interaction with therapeutic purposes. On the other hand, we describe a cellular relocation assay that can be easily implemented in a high throughput setting to search for drugs that may dissociate the USP1/UAF1 complex.

Show MeSH
Related in: MedlinePlus