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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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Identification of two NLSs that mediate USP1 nuclear import.A. Schematic representation of USP1 deletion mutants. The positions of the cNLS and the NES are indicated. B. Immunoblot analysis demonstrating the expression and the correct size of the different mutant proteins. C. Confocal images of 293T cells showing the nucleocytoplasmic localization of each USP1 deletion mutant. All the fragments tested were nuclear, except for the (1–269) fragment, which was exclusively located to the cytoplasm. D. Images show that inhibition of the nuclear export receptor CRM1 using leptomycin B (LMB) induces a partial relocation of YFP-USP1(1–269) to the nucleus. E. Site directed mutagenesis of USP1 NLSs in the context of the full-length protein. On the left, drawings show a schematic representation of USP1 mutants bearing alanine substitutions (red) of several basic residues (green) in NLS1, NLS2 or both. Confocal microscopy images on the right show representative examples of the nucleocytoplasmic localization of each USP1 mutant in 293T cells. Mutation of both NLSs was necessary to abrogate USP1 nuclear import. Cells were counterstained with Hoechst to show the nuclei (DNA panels).
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pone-0038570-g002: Identification of two NLSs that mediate USP1 nuclear import.A. Schematic representation of USP1 deletion mutants. The positions of the cNLS and the NES are indicated. B. Immunoblot analysis demonstrating the expression and the correct size of the different mutant proteins. C. Confocal images of 293T cells showing the nucleocytoplasmic localization of each USP1 deletion mutant. All the fragments tested were nuclear, except for the (1–269) fragment, which was exclusively located to the cytoplasm. D. Images show that inhibition of the nuclear export receptor CRM1 using leptomycin B (LMB) induces a partial relocation of YFP-USP1(1–269) to the nucleus. E. Site directed mutagenesis of USP1 NLSs in the context of the full-length protein. On the left, drawings show a schematic representation of USP1 mutants bearing alanine substitutions (red) of several basic residues (green) in NLS1, NLS2 or both. Confocal microscopy images on the right show representative examples of the nucleocytoplasmic localization of each USP1 mutant in 293T cells. Mutation of both NLSs was necessary to abrogate USP1 nuclear import. Cells were counterstained with Hoechst to show the nuclei (DNA panels).

Mentions: In order to assess the contribution of each cNLS to USP1 nuclear localization, we generated a series of USP1 deletion mutants fused to YFP (Figure 2A). The expression and size of each USP1 fragment was confirmed by immunoblot (Figure 2B), and their nucleocytoplasmic localization was evaluated in transfected 293T cells (Figure 2C). Like the full-length protein, the fragment (1–350), lacking the carboxy-terminal half of USP1, but bearing the three cNLSs, was nuclear. The fragment (1–285), lacking cNLS[298–321], also localized to the nucleus. In contrast, the fragment (1–269), which lacks both cNLS[266–287] and cNLS[298–321], localized exclusively to the cytoplasm. These results indicate that cNLS[14–37] is not sufficient to maintain nuclear localization of USP1.


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

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

Identification of two NLSs that mediate USP1 nuclear import.A. Schematic representation of USP1 deletion mutants. The positions of the cNLS and the NES are indicated. B. Immunoblot analysis demonstrating the expression and the correct size of the different mutant proteins. C. Confocal images of 293T cells showing the nucleocytoplasmic localization of each USP1 deletion mutant. All the fragments tested were nuclear, except for the (1–269) fragment, which was exclusively located to the cytoplasm. D. Images show that inhibition of the nuclear export receptor CRM1 using leptomycin B (LMB) induces a partial relocation of YFP-USP1(1–269) to the nucleus. E. Site directed mutagenesis of USP1 NLSs in the context of the full-length protein. On the left, drawings show a schematic representation of USP1 mutants bearing alanine substitutions (red) of several basic residues (green) in NLS1, NLS2 or both. Confocal microscopy images on the right show representative examples of the nucleocytoplasmic localization of each USP1 mutant in 293T cells. Mutation of both NLSs was necessary to abrogate USP1 nuclear import. Cells were counterstained with Hoechst to show the nuclei (DNA panels).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038570-g002: Identification of two NLSs that mediate USP1 nuclear import.A. Schematic representation of USP1 deletion mutants. The positions of the cNLS and the NES are indicated. B. Immunoblot analysis demonstrating the expression and the correct size of the different mutant proteins. C. Confocal images of 293T cells showing the nucleocytoplasmic localization of each USP1 deletion mutant. All the fragments tested were nuclear, except for the (1–269) fragment, which was exclusively located to the cytoplasm. D. Images show that inhibition of the nuclear export receptor CRM1 using leptomycin B (LMB) induces a partial relocation of YFP-USP1(1–269) to the nucleus. E. Site directed mutagenesis of USP1 NLSs in the context of the full-length protein. On the left, drawings show a schematic representation of USP1 mutants bearing alanine substitutions (red) of several basic residues (green) in NLS1, NLS2 or both. Confocal microscopy images on the right show representative examples of the nucleocytoplasmic localization of each USP1 mutant in 293T cells. Mutation of both NLSs was necessary to abrogate USP1 nuclear import. Cells were counterstained with Hoechst to show the nuclei (DNA panels).
Mentions: In order to assess the contribution of each cNLS to USP1 nuclear localization, we generated a series of USP1 deletion mutants fused to YFP (Figure 2A). The expression and size of each USP1 fragment was confirmed by immunoblot (Figure 2B), and their nucleocytoplasmic localization was evaluated in transfected 293T cells (Figure 2C). Like the full-length protein, the fragment (1–350), lacking the carboxy-terminal half of USP1, but bearing the three cNLSs, was nuclear. The fragment (1–285), lacking cNLS[298–321], also localized to the nucleus. In contrast, the fragment (1–269), which lacks both cNLS[266–287] and cNLS[298–321], localized exclusively to the cytoplasm. These results indicate that cNLS[14–37] is not sufficient to maintain nuclear localization of USP1.

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