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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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Effect of rapamycin-induced attachment of ubiquitin to p53 on its intracellular localization. Saos-2 cells were transfected with the indicated DNA constructs (p53-2xFKBP along with K-less-Ub(G76A)-FRB-HA or Ub(G76A)-FRB-HA). Cells were untreated or treated with rapamycin (0.1 μM) 6 h after transfection and fixed 24 h after transfection. Cells were stained with rabbit anti-HA and mouse anti-p53 (DO-1) antibodies. Goat anti-rabbit immunoglobulin G (IgG)–fluorescein and goat anti-mouse IgG-rhodamine conjugates were used as the secondary antibodies. The nuclei were stained with 4′,6-diamidino-2-phenylindole.
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Figure 5: Effect of rapamycin-induced attachment of ubiquitin to p53 on its intracellular localization. Saos-2 cells were transfected with the indicated DNA constructs (p53-2xFKBP along with K-less-Ub(G76A)-FRB-HA or Ub(G76A)-FRB-HA). Cells were untreated or treated with rapamycin (0.1 μM) 6 h after transfection and fixed 24 h after transfection. Cells were stained with rabbit anti-HA and mouse anti-p53 (DO-1) antibodies. Goat anti-rabbit immunoglobulin G (IgG)–fluorescein and goat anti-mouse IgG-rhodamine conjugates were used as the secondary antibodies. The nuclei were stained with 4′,6-diamidino-2-phenylindole.

Mentions: SUMOylation exerts diverse effects, ranging from regulation of transcription to intracellular trafficking (Gareau and Lima, 2010). Previous studies suggest that p53 SUMOylation enhances nuclear export of p53 (Carter et al., 2007; Carter and Vousden, 2008; Heo et al., 2011). To assess the potential effect of p53 SUMOylation on its intracellular localization in our experimental system, we immuno­stained p53 when it was fused to a SUMO moiety or when p53 was tethered to SUMO via rapamycin-induced heterodimerization in p53- Saos-2 cells. As shown in Figures 2 and 3, wt p53 or its fusion with 2xFKBP was predominantly found in the nucleus, and rapamycin did not influence p53 nuclear localization (Figure 2A). By contrast, the p53-SUMO-1 fusion construct showed dramatic cytoplasmic presence (Figure 3A). In rapamycin-mediated heterodimerization experiments, coexpression of p53-2xFKBP with either SUMO-1-FRB or FRB did not influence p53 nuclear localization in the absence of rapamycin (Figure 2A). Remarkably, tethering SUMO-1-FRB but not the FRB control to p53-2xFKBP dramatically shifted p53 from the nucleus to the cytoplasm (Figure 2, A and B). Similar results were observed when SUMO-3 was fused to p53 or tethered to it via rapamycin-mediated heterodimerization (Figure 4). We further assessed whether rapamycin-mediated attachment of ubiquitin could also promote p53 nuclear export. We found that tethering ubiquitin to p53 did not obviously induce its nuclear export, although the heterodimerization of a ubiquitin moiety lacking lysine residues (K-less-Ub(G76A)-FRB–hemagglutinin [HA]) with p53-2xFKBP did trigger notable p53 nuclear export (Figure 5). Thus, in agreement with previous findings, monoubiquitination seems to enable nuclear export of p53 (Li et al., 2003; Carter et al., 2007). The signal intensity of nuclear p53, however, was still far greater than that of p53 in the cytoplasm in cells expressing p53-2xFKBP and K-less-Ub (G76A)-FRB-HA in the presence of rapamycin (Figure 5). Collectively these results support the notion that SUMOylation of p53 facilitates its nuclear export.


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 rapamycin-induced attachment of ubiquitin to p53 on its intracellular localization. Saos-2 cells were transfected with the indicated DNA constructs (p53-2xFKBP along with K-less-Ub(G76A)-FRB-HA or Ub(G76A)-FRB-HA). Cells were untreated or treated with rapamycin (0.1 μM) 6 h after transfection and fixed 24 h after transfection. Cells were stained with rabbit anti-HA and mouse anti-p53 (DO-1) antibodies. Goat anti-rabbit immunoglobulin G (IgG)–fluorescein and goat anti-mouse IgG-rhodamine conjugates were used as the secondary antibodies. The nuclei were stained with 4′,6-diamidino-2-phenylindole.
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Related In: Results  -  Collection

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Figure 5: Effect of rapamycin-induced attachment of ubiquitin to p53 on its intracellular localization. Saos-2 cells were transfected with the indicated DNA constructs (p53-2xFKBP along with K-less-Ub(G76A)-FRB-HA or Ub(G76A)-FRB-HA). Cells were untreated or treated with rapamycin (0.1 μM) 6 h after transfection and fixed 24 h after transfection. Cells were stained with rabbit anti-HA and mouse anti-p53 (DO-1) antibodies. Goat anti-rabbit immunoglobulin G (IgG)–fluorescein and goat anti-mouse IgG-rhodamine conjugates were used as the secondary antibodies. The nuclei were stained with 4′,6-diamidino-2-phenylindole.
Mentions: SUMOylation exerts diverse effects, ranging from regulation of transcription to intracellular trafficking (Gareau and Lima, 2010). Previous studies suggest that p53 SUMOylation enhances nuclear export of p53 (Carter et al., 2007; Carter and Vousden, 2008; Heo et al., 2011). To assess the potential effect of p53 SUMOylation on its intracellular localization in our experimental system, we immuno­stained p53 when it was fused to a SUMO moiety or when p53 was tethered to SUMO via rapamycin-induced heterodimerization in p53- Saos-2 cells. As shown in Figures 2 and 3, wt p53 or its fusion with 2xFKBP was predominantly found in the nucleus, and rapamycin did not influence p53 nuclear localization (Figure 2A). By contrast, the p53-SUMO-1 fusion construct showed dramatic cytoplasmic presence (Figure 3A). In rapamycin-mediated heterodimerization experiments, coexpression of p53-2xFKBP with either SUMO-1-FRB or FRB did not influence p53 nuclear localization in the absence of rapamycin (Figure 2A). Remarkably, tethering SUMO-1-FRB but not the FRB control to p53-2xFKBP dramatically shifted p53 from the nucleus to the cytoplasm (Figure 2, A and B). Similar results were observed when SUMO-3 was fused to p53 or tethered to it via rapamycin-mediated heterodimerization (Figure 4). We further assessed whether rapamycin-mediated attachment of ubiquitin could also promote p53 nuclear export. We found that tethering ubiquitin to p53 did not obviously induce its nuclear export, although the heterodimerization of a ubiquitin moiety lacking lysine residues (K-less-Ub(G76A)-FRB–hemagglutinin [HA]) with p53-2xFKBP did trigger notable p53 nuclear export (Figure 5). Thus, in agreement with previous findings, monoubiquitination seems to enable nuclear export of p53 (Li et al., 2003; Carter et al., 2007). The signal intensity of nuclear p53, however, was still far greater than that of p53 in the cytoplasm in cells expressing p53-2xFKBP and K-less-Ub (G76A)-FRB-HA in the presence of rapamycin (Figure 5). Collectively these results support the notion that SUMOylation of p53 facilitates its nuclear export.

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