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p53 SUMOylation promotes its nuclear export by facilitating its release from the nuclear export receptor CRM1.

Santiago A, Li D, Zhao LY, Godsey A, Liao D - Mol. Biol. Cell (2013)

Bottom Line: The CRM1 Huntington, EF3, a subunit of PP2A, and TOR1 9 (HEAT9) loop, which regulates GTP-binding nuclear protein Ran binding and cargo release, contains a prototypical SIM.Remarkably, disruption of this SIM in conjunction with a mutated SIM-binding groove of SUMO-1 markedly enhances the binding of CRM1 to p53-SUMO-1 and their accumulation in the nuclear pore complexes (NPCs), as well as their persistent association in the cytoplasm.We propose that SUMOylation of a CRM1 cargo such as p53 at the NPCs unlocks the HEAT9 loop of CRM1 to facilitate the disassembly of the transporting complex and cargo release to the cytoplasm.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Cell Biology, UF Health Cancer Center, and UF Genetics Institute, University of Florida College of Medicine, Gainesville, FL 32610, USA.

ABSTRACT
Chromosomal region maintenance 1 (CRM1) mediates p53 nuclear export. Although p53 SUMOylation promotes its nuclear export, the underlying mechanism is unclear. Here we show that tethering of a small, ubiquitin-like modifier (SUMO) moiety to p53 markedly increases its cytoplasmic localization. SUMO attachment to p53 does not affect its oligomerization, suggesting that subunit dissociation required for exposing p53's nuclear export signal (NES) is unnecessary for p53 nuclear export. Surprisingly, SUMO-mediated p53 nuclear export depends on the SUMO-interacting motif (SIM)-binding pocket of SUMO-1. The CRM1 C-terminal domain lacking the NES-binding groove interacts with tetrameric p53, and the proper folding of the p53 core domain, rather than the presence of the N- or C-terminal tails, appears to be important for p53-CRM1 interaction. The CRM1 Huntington, EF3, a subunit of PP2A, and TOR1 9 (HEAT9) loop, which regulates GTP-binding nuclear protein Ran binding and cargo release, contains a prototypical SIM. Remarkably, disruption of this SIM in conjunction with a mutated SIM-binding groove of SUMO-1 markedly enhances the binding of CRM1 to p53-SUMO-1 and their accumulation in the nuclear pore complexes (NPCs), as well as their persistent association in the cytoplasm. We propose that SUMOylation of a CRM1 cargo such as p53 at the NPCs unlocks the HEAT9 loop of CRM1 to facilitate the disassembly of the transporting complex and cargo release to the cytoplasm.

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Related in: MedlinePlus

Effect of mutations in the SIM-binding groove of the p53-SUMO-1 fusion and the HEAT9 loop of CRM1 on their localization at NPCs. (A) The GFP-CRM1 (full-length) construct was cotransfected with an indicated p53-SUMO-1 fusion construct to Saos-2 cells. The transfected cells were fixed for immunofluorescence microscopy as in Figure 2. (B) The GFP-CRM1 (full-length) V430K mutant was cotransfected with an indicated p53-SUMO-1 fusion construct into Saos-2 cells. The transfected cells were fixed for immunofluorescence microscopy as described. (C) The p53-SUMO-1 fusion construct was coexpressed with the GFP-CRM1 V430K mutant in Saos-2 cells. The transfected cells were fixed 24 h after transfection and stained with antibodies to p53 and Nup153. p53, CRM1, and Nup153 were detected in the blue, green and red channels, respectively. Colocalization of CRM1, p53-SUMO-1, and Nup153 at the NPCs, as well as that of CRM1 and p53-SUMO-1 in the cytoplasm, is denoted with white arrows. Bottom, images of NPC colocalization of p53-SUMO-1, CRM1 V430K mutant, and Nup153 shown at a higher magnification, corresponding to the boxed area at the top. Lack of colocalization of CRM1 with the bright spots of p53-SUMO-1 in the nucleoplasm is indicated with a yellow arrow. (D) A model explaining a potential regulatory role for the cargo SUMOylation in cargo release and the disassembly of a CRM1 export complex. p53 (tetramer) is shown as the cargo (yellow oval). The HEAT9 loop is depicted as a hairpin in red. N, N-terminus; C, C-terminus; S1, SUMO-1. See the text for details.
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Figure 11: Effect of mutations in the SIM-binding groove of the p53-SUMO-1 fusion and the HEAT9 loop of CRM1 on their localization at NPCs. (A) The GFP-CRM1 (full-length) construct was cotransfected with an indicated p53-SUMO-1 fusion construct to Saos-2 cells. The transfected cells were fixed for immunofluorescence microscopy as in Figure 2. (B) The GFP-CRM1 (full-length) V430K mutant was cotransfected with an indicated p53-SUMO-1 fusion construct into Saos-2 cells. The transfected cells were fixed for immunofluorescence microscopy as described. (C) The p53-SUMO-1 fusion construct was coexpressed with the GFP-CRM1 V430K mutant in Saos-2 cells. The transfected cells were fixed 24 h after transfection and stained with antibodies to p53 and Nup153. p53, CRM1, and Nup153 were detected in the blue, green and red channels, respectively. Colocalization of CRM1, p53-SUMO-1, and Nup153 at the NPCs, as well as that of CRM1 and p53-SUMO-1 in the cytoplasm, is denoted with white arrows. Bottom, images of NPC colocalization of p53-SUMO-1, CRM1 V430K mutant, and Nup153 shown at a higher magnification, corresponding to the boxed area at the top. Lack of colocalization of CRM1 with the bright spots of p53-SUMO-1 in the nucleoplasm is indicated with a yellow arrow. (D) A model explaining a potential regulatory role for the cargo SUMOylation in cargo release and the disassembly of a CRM1 export complex. p53 (tetramer) is shown as the cargo (yellow oval). The HEAT9 loop is depicted as a hairpin in red. N, N-terminus; C, C-terminus; S1, SUMO-1. See the text for details.

Mentions: Because the CRM1 V430K mutant and the p53-SUMO-1 (F36A) construct forms a stable complex, one consequence could be an accumulation of the CRM1-p53 complex at the NPCs. Furthermore, p53-SUMO-1 may remain stably associated with CRM1 in the cytoplasm even after the complex is released from the NPCs. To test this possibility, we coexpressed GFP-CRM1 or the V430K mutant with p53-SUMO-1 or a corresponding construct with a mutation in the SIM-binding pocket of SUMO-1 (F36A or Y51A). We observed striking colocalizations of p53 and CRM1 in a mesh of regularly spaced dots around the nuclear rim and across the entire span of the nucleus (Figure 11, A and B). This pattern is more apparent in cells expressing the CRM1 V430K mutant and one of the p53-SUMO-1 fusion with a mutated SIM-binding groove (Figure 11B; e.g., 1–3). The nuclear pore localization of the p53-SUMO-1/CRM1 complex was confirmed, as both p53-SUMO-1 and CRM1 V430K colocalized with Nup153, a component of the NPC (Figure 11C, bottom, spots highlighted with white arrows). Colocalization of p53-SUMO-1, CRM1 V430K, and Nup153 was also obvious at a different focal plane in the same cell (Supplemental Figure S6). Remarkably, p53-SUMO-1/CRM1 V430K aggregates are clearly seen in the cytoplasm (Figure 11C, top; also see Figure 11, A and B, white arrows). The cytoplasmic p53-SUMO-1/CRM1 aggregates were observed in the vast majority (>80%) of the transfected cells that coexpressed GFP-CRM1 (V430K) mutant and any of the three p53-SUMO-1 fusions. Of interest, whereas several bright dots of p53-SUMO-1 appeared in the nucleus, CRM1 did not localize to those nuclear p53-SUMO-1 aggregates (Figure 11C, yellow arrow). Greater than 50% of the transfected cells contain nuclear aggregates of p53-SUMO-1, but none of these nuclear bodies colocalized with CRM1. Thus the stable p53-SUMO-1/CRM1 V430K mutant complex appears to reside only in the cytoplasm and not in the nucleoplasm.


p53 SUMOylation promotes its nuclear export by facilitating its release from the nuclear export receptor CRM1.

Santiago A, Li D, Zhao LY, Godsey A, Liao D - Mol. Biol. Cell (2013)

Effect of mutations in the SIM-binding groove of the p53-SUMO-1 fusion and the HEAT9 loop of CRM1 on their localization at NPCs. (A) The GFP-CRM1 (full-length) construct was cotransfected with an indicated p53-SUMO-1 fusion construct to Saos-2 cells. The transfected cells were fixed for immunofluorescence microscopy as in Figure 2. (B) The GFP-CRM1 (full-length) V430K mutant was cotransfected with an indicated p53-SUMO-1 fusion construct into Saos-2 cells. The transfected cells were fixed for immunofluorescence microscopy as described. (C) The p53-SUMO-1 fusion construct was coexpressed with the GFP-CRM1 V430K mutant in Saos-2 cells. The transfected cells were fixed 24 h after transfection and stained with antibodies to p53 and Nup153. p53, CRM1, and Nup153 were detected in the blue, green and red channels, respectively. Colocalization of CRM1, p53-SUMO-1, and Nup153 at the NPCs, as well as that of CRM1 and p53-SUMO-1 in the cytoplasm, is denoted with white arrows. Bottom, images of NPC colocalization of p53-SUMO-1, CRM1 V430K mutant, and Nup153 shown at a higher magnification, corresponding to the boxed area at the top. Lack of colocalization of CRM1 with the bright spots of p53-SUMO-1 in the nucleoplasm is indicated with a yellow arrow. (D) A model explaining a potential regulatory role for the cargo SUMOylation in cargo release and the disassembly of a CRM1 export complex. p53 (tetramer) is shown as the cargo (yellow oval). The HEAT9 loop is depicted as a hairpin in red. N, N-terminus; C, C-terminus; S1, SUMO-1. See the text for details.
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Figure 11: Effect of mutations in the SIM-binding groove of the p53-SUMO-1 fusion and the HEAT9 loop of CRM1 on their localization at NPCs. (A) The GFP-CRM1 (full-length) construct was cotransfected with an indicated p53-SUMO-1 fusion construct to Saos-2 cells. The transfected cells were fixed for immunofluorescence microscopy as in Figure 2. (B) The GFP-CRM1 (full-length) V430K mutant was cotransfected with an indicated p53-SUMO-1 fusion construct into Saos-2 cells. The transfected cells were fixed for immunofluorescence microscopy as described. (C) The p53-SUMO-1 fusion construct was coexpressed with the GFP-CRM1 V430K mutant in Saos-2 cells. The transfected cells were fixed 24 h after transfection and stained with antibodies to p53 and Nup153. p53, CRM1, and Nup153 were detected in the blue, green and red channels, respectively. Colocalization of CRM1, p53-SUMO-1, and Nup153 at the NPCs, as well as that of CRM1 and p53-SUMO-1 in the cytoplasm, is denoted with white arrows. Bottom, images of NPC colocalization of p53-SUMO-1, CRM1 V430K mutant, and Nup153 shown at a higher magnification, corresponding to the boxed area at the top. Lack of colocalization of CRM1 with the bright spots of p53-SUMO-1 in the nucleoplasm is indicated with a yellow arrow. (D) A model explaining a potential regulatory role for the cargo SUMOylation in cargo release and the disassembly of a CRM1 export complex. p53 (tetramer) is shown as the cargo (yellow oval). The HEAT9 loop is depicted as a hairpin in red. N, N-terminus; C, C-terminus; S1, SUMO-1. See the text for details.
Mentions: Because the CRM1 V430K mutant and the p53-SUMO-1 (F36A) construct forms a stable complex, one consequence could be an accumulation of the CRM1-p53 complex at the NPCs. Furthermore, p53-SUMO-1 may remain stably associated with CRM1 in the cytoplasm even after the complex is released from the NPCs. To test this possibility, we coexpressed GFP-CRM1 or the V430K mutant with p53-SUMO-1 or a corresponding construct with a mutation in the SIM-binding pocket of SUMO-1 (F36A or Y51A). We observed striking colocalizations of p53 and CRM1 in a mesh of regularly spaced dots around the nuclear rim and across the entire span of the nucleus (Figure 11, A and B). This pattern is more apparent in cells expressing the CRM1 V430K mutant and one of the p53-SUMO-1 fusion with a mutated SIM-binding groove (Figure 11B; e.g., 1–3). The nuclear pore localization of the p53-SUMO-1/CRM1 complex was confirmed, as both p53-SUMO-1 and CRM1 V430K colocalized with Nup153, a component of the NPC (Figure 11C, bottom, spots highlighted with white arrows). Colocalization of p53-SUMO-1, CRM1 V430K, and Nup153 was also obvious at a different focal plane in the same cell (Supplemental Figure S6). Remarkably, p53-SUMO-1/CRM1 V430K aggregates are clearly seen in the cytoplasm (Figure 11C, top; also see Figure 11, A and B, white arrows). The cytoplasmic p53-SUMO-1/CRM1 aggregates were observed in the vast majority (>80%) of the transfected cells that coexpressed GFP-CRM1 (V430K) mutant and any of the three p53-SUMO-1 fusions. Of interest, whereas several bright dots of p53-SUMO-1 appeared in the nucleus, CRM1 did not localize to those nuclear p53-SUMO-1 aggregates (Figure 11C, yellow arrow). Greater than 50% of the transfected cells contain nuclear aggregates of p53-SUMO-1, but none of these nuclear bodies colocalized with CRM1. Thus the stable p53-SUMO-1/CRM1 V430K mutant complex appears to reside only in the cytoplasm and not in the nucleoplasm.

Bottom Line: The CRM1 Huntington, EF3, a subunit of PP2A, and TOR1 9 (HEAT9) loop, which regulates GTP-binding nuclear protein Ran binding and cargo release, contains a prototypical SIM.Remarkably, disruption of this SIM in conjunction with a mutated SIM-binding groove of SUMO-1 markedly enhances the binding of CRM1 to p53-SUMO-1 and their accumulation in the nuclear pore complexes (NPCs), as well as their persistent association in the cytoplasm.We propose that SUMOylation of a CRM1 cargo such as p53 at the NPCs unlocks the HEAT9 loop of CRM1 to facilitate the disassembly of the transporting complex and cargo release to the cytoplasm.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Cell Biology, UF Health Cancer Center, and UF Genetics Institute, University of Florida College of Medicine, Gainesville, FL 32610, USA.

ABSTRACT
Chromosomal region maintenance 1 (CRM1) mediates p53 nuclear export. Although p53 SUMOylation promotes its nuclear export, the underlying mechanism is unclear. Here we show that tethering of a small, ubiquitin-like modifier (SUMO) moiety to p53 markedly increases its cytoplasmic localization. SUMO attachment to p53 does not affect its oligomerization, suggesting that subunit dissociation required for exposing p53's nuclear export signal (NES) is unnecessary for p53 nuclear export. Surprisingly, SUMO-mediated p53 nuclear export depends on the SUMO-interacting motif (SIM)-binding pocket of SUMO-1. The CRM1 C-terminal domain lacking the NES-binding groove interacts with tetrameric p53, and the proper folding of the p53 core domain, rather than the presence of the N- or C-terminal tails, appears to be important for p53-CRM1 interaction. The CRM1 Huntington, EF3, a subunit of PP2A, and TOR1 9 (HEAT9) loop, which regulates GTP-binding nuclear protein Ran binding and cargo release, contains a prototypical SIM. Remarkably, disruption of this SIM in conjunction with a mutated SIM-binding groove of SUMO-1 markedly enhances the binding of CRM1 to p53-SUMO-1 and their accumulation in the nuclear pore complexes (NPCs), as well as their persistent association in the cytoplasm. We propose that SUMOylation of a CRM1 cargo such as p53 at the NPCs unlocks the HEAT9 loop of CRM1 to facilitate the disassembly of the transporting complex and cargo release to the cytoplasm.

Show MeSH
Related in: MedlinePlus