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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

Rapamycin-mediated heterodimerization of p53 and SUMO inhibits p53 transactivation function. (A) Schematic diagram of p53-2xFKBP and fusion of SUMO-1 or -3 with FRB and their heterodimerization mediated by rapamycin. GFP-SUMO-3 served as a control. (B) Tethering SUMO to p53 inhibited its transactivation function. H1299 cells were transfected with a firefly luciferase reporter under the control of the p21 promoter along with a control plasmid or various combinations of the indicated DNA constructs. The transfected cells were untreated (white bars) or treated with 0.1 μM of rapamycin at 6 h after transfection (black bars). Cells were lysed for dual luciferase assay 24 h after transfection. Statistical significance of pairwise comparisons was evaluated with Student's t test; down-regulation of reporter activity by heterodimerization of p53-2xFKBP and SUMO-1-FRB is significantly different from that by the control (p < 0.05). (C) p53-SUMO-3 fusion represses endogenous p21 expression. H1299 cells were stably transduced with lentiviral vectors for tetracycline-inducible expression of wt p53 or p53-SUMO-3 fusion. The vehicle (dimethyl sulfoxide) or tetracycline was added to the parental H1299 cells and wt p53- and p53-SUMO-3–expressing cells as indicated. The cells were then exposed to vehicle or 10 μM etoposide for 18 h. The cells were lysed for Western blotting analysis with antibodies against the indicated proteins. PCNA was used as a loading control.
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Figure 1: Rapamycin-mediated heterodimerization of p53 and SUMO inhibits p53 transactivation function. (A) Schematic diagram of p53-2xFKBP and fusion of SUMO-1 or -3 with FRB and their heterodimerization mediated by rapamycin. GFP-SUMO-3 served as a control. (B) Tethering SUMO to p53 inhibited its transactivation function. H1299 cells were transfected with a firefly luciferase reporter under the control of the p21 promoter along with a control plasmid or various combinations of the indicated DNA constructs. The transfected cells were untreated (white bars) or treated with 0.1 μM of rapamycin at 6 h after transfection (black bars). Cells were lysed for dual luciferase assay 24 h after transfection. Statistical significance of pairwise comparisons was evaluated with Student's t test; down-regulation of reporter activity by heterodimerization of p53-2xFKBP and SUMO-1-FRB is significantly different from that by the control (p < 0.05). (C) p53-SUMO-3 fusion represses endogenous p21 expression. H1299 cells were stably transduced with lentiviral vectors for tetracycline-inducible expression of wt p53 or p53-SUMO-3 fusion. The vehicle (dimethyl sulfoxide) or tetracycline was added to the parental H1299 cells and wt p53- and p53-SUMO-3–expressing cells as indicated. The cells were then exposed to vehicle or 10 μM etoposide for 18 h. The cells were lysed for Western blotting analysis with antibodies against the indicated proteins. PCNA was used as a loading control.

Mentions: Because the SUMO moiety is quickly removed from SUMO-conjugated substrates in cells, we used two approaches to model the effect of p53 SUMOylation on its function. We fused SUMO in-frame to the C-terminus of p53 to generate a p53-SUMO construct. We also used rapamycin-induced heterodimerization to chemically attach a SUMO moiety to the C-terminus of p53 based on a similar approach (Zhu et al., 2006), as shown in Figure 1A.


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)

Rapamycin-mediated heterodimerization of p53 and SUMO inhibits p53 transactivation function. (A) Schematic diagram of p53-2xFKBP and fusion of SUMO-1 or -3 with FRB and their heterodimerization mediated by rapamycin. GFP-SUMO-3 served as a control. (B) Tethering SUMO to p53 inhibited its transactivation function. H1299 cells were transfected with a firefly luciferase reporter under the control of the p21 promoter along with a control plasmid or various combinations of the indicated DNA constructs. The transfected cells were untreated (white bars) or treated with 0.1 μM of rapamycin at 6 h after transfection (black bars). Cells were lysed for dual luciferase assay 24 h after transfection. Statistical significance of pairwise comparisons was evaluated with Student's t test; down-regulation of reporter activity by heterodimerization of p53-2xFKBP and SUMO-1-FRB is significantly different from that by the control (p < 0.05). (C) p53-SUMO-3 fusion represses endogenous p21 expression. H1299 cells were stably transduced with lentiviral vectors for tetracycline-inducible expression of wt p53 or p53-SUMO-3 fusion. The vehicle (dimethyl sulfoxide) or tetracycline was added to the parental H1299 cells and wt p53- and p53-SUMO-3–expressing cells as indicated. The cells were then exposed to vehicle or 10 μM etoposide for 18 h. The cells were lysed for Western blotting analysis with antibodies against the indicated proteins. PCNA was used as a loading control.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 1: Rapamycin-mediated heterodimerization of p53 and SUMO inhibits p53 transactivation function. (A) Schematic diagram of p53-2xFKBP and fusion of SUMO-1 or -3 with FRB and their heterodimerization mediated by rapamycin. GFP-SUMO-3 served as a control. (B) Tethering SUMO to p53 inhibited its transactivation function. H1299 cells were transfected with a firefly luciferase reporter under the control of the p21 promoter along with a control plasmid or various combinations of the indicated DNA constructs. The transfected cells were untreated (white bars) or treated with 0.1 μM of rapamycin at 6 h after transfection (black bars). Cells were lysed for dual luciferase assay 24 h after transfection. Statistical significance of pairwise comparisons was evaluated with Student's t test; down-regulation of reporter activity by heterodimerization of p53-2xFKBP and SUMO-1-FRB is significantly different from that by the control (p < 0.05). (C) p53-SUMO-3 fusion represses endogenous p21 expression. H1299 cells were stably transduced with lentiviral vectors for tetracycline-inducible expression of wt p53 or p53-SUMO-3 fusion. The vehicle (dimethyl sulfoxide) or tetracycline was added to the parental H1299 cells and wt p53- and p53-SUMO-3–expressing cells as indicated. The cells were then exposed to vehicle or 10 μM etoposide for 18 h. The cells were lysed for Western blotting analysis with antibodies against the indicated proteins. PCNA was used as a loading control.
Mentions: Because the SUMO moiety is quickly removed from SUMO-conjugated substrates in cells, we used two approaches to model the effect of p53 SUMOylation on its function. We fused SUMO in-frame to the C-terminus of p53 to generate a p53-SUMO construct. We also used rapamycin-induced heterodimerization to chemically attach a SUMO moiety to the C-terminus of p53 based on a similar approach (Zhu et al., 2006), as shown in Figure 1A.

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