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. 2007 Aug 29;2(8):e784.
doi: 10.1371/journal.pone.0000784.

Interphase nucleo-cytoplasmic shuttling and localization of SIRT2 during mitosis

Brian J North  1 , Eric Verdin
Affiliations

Interphase nucleo-cytoplasmic shuttling and localization of SIRT2 during mitosis

Brian J North et al. PLoS One. .

Abstract

The human NAD+-dependent protein deacetylase SIRT2 resides predominantly in the cytoplasm where it functions as a tubulin deacetylase. Here we report that SIRT2 maintains a largely cytoplasmic localization during interphase by active nuclear export in a Crm1-dependent manner. We identified a functional, leptomycin B-sensitive, nuclear export signal sequence within SIRT2. During the cell cycle, SIRT2 becomes enriched in the nucleus and is associated with mitotic structures, beginning with the centrosome during prophase, the mitotic spindle during metaphase, and the midbody during cytokinesis. Cells overexpressing wild-type or a catalytically inactive SIRT2 exhibit an increase in multinucleated cells. The findings suggest a novel mechanism of regulating SIRT2 function by nucleo-cytoplasmic shuttling, as well as a role for SIRT2 in the nucleus during interphase and throughout mitosis.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Cytoplasmic localization of SIRT2 is dependent on constitutive nuclear export.
(A) HeLa cells were transfected with GFP-SIRT2 and analyzed by immunofluorescence for SIRT2 localization. (B) Cells from (A) were scored for their distribution as cytoplasmic or pancellular. (C) HeLa cells were transfected with GFP-SIRT2 and incubated with or without leptomycin B (LMB). (D) Cells from (C) were scored for localization of GFP-SIRT2 in the nucleus. (E) HeLa cells were transfected with GFP-SIRT2 followed by treatment with LMB. At indicated times, cells were fixed and visualized for nuclear localization.
Figure 2
Figure 2. A Crm1-dependent NES is located in the amino-terminus of SIRT2.
(A) HeLa cells were transfected with GFP or full-length and deletion mutants of GFP-SIRT2 and visualized for subcellular distribution. (B) 293T cells were transfected with cDNAs used in (A) and lysates were separated by SDS-PAGE and visualized by western blotting with an antiserum specific for GFP. (C) Schematic diagram of deletion analysis GFP-SIRT2 subcellular distribution. The region required for cytoplasmic localization is indicated. (D) Schematic of consensus Rev-like NES and proposed SIRT2 NES. Nuclear export signal sequences from proteins exported from the nucleus in a Crm1-dependent manner.
Figure 3
Figure 3. Mutational analysis of SIRT2 NES sequence.
(A) HeLa cells were transfected with GFP-SIRT2 wild-type or single and double point mutants of the proposed NES sequence. (B) Subcellular distribution results of all single and double mutants of SIRT2 NES analyzed in (A). (C) 293T cells were transfected with GFP-SIRT2 single and double point mutants and lysates were separated by SDS–PAGE and visualized by western blotting with an antiserum specific for GFP. (D) HeLa cells were transfected with SIRT2-FLAG and fibrillarin-GFP, treated with or without LMB for 2 hrs and subsequently stained for FLAG and visualize by confocal microscopy.
Figure 4
Figure 4. Colocalization of SIRT2 with the centrosome.
(A) U2OS cells expressing SIRT2-HA were stained for HA (green) and for γ-tubulin (red) and analyzed by confocal microscopy. (B) HeLa cells were stained with antisera for endogenous SIRT2 (green) and Aurora A (red) and analyzed by confocal microscopy. (C) HeLa cells were stained with antisera for SIRT2 (green), acetylated tubulin (red), and Aurora A (blue) and analyzed by confocal microscopy. (D) 293T cells were transfected with Myc-Aurora A with or without SIRT2-FLAG. Cellular lysates were immunoprecipitated with anti-FLAG and probed by western blotting with antisera specific for FLAG and Myc. 10% of protein input was analyzed by western blotting with antisera for FLAG or Myc.
Figure 5
Figure 5. Enrichment of the three proteins on the midbody is shown in the merge images.
(A) HeLa cells were stained with antisera for SIRT2 (green) and Aurora B (blue) and acetylated tubulin (red), and analyzed by confocal microscopy. Enrichment of the three proteins on the midbody is shown in the merged images. (B) 293T cells were transfected with Myc-Aurora A with or without SIRT2-FLAG. Cellular lysates were immunoprecipitated with anti-FLAG and probed by western blotting with antisera specific for FLAG and Myc. 10% of protein input was analyzed by western blotting with antisera for FLAG or Myc.
Figure 6
Figure 6. Overexpression of SIRT2 leads increases mutlinucleation.
HeLa cells were transfected with GFP, and wild-type or H187Y GFP-SIRT2 and 48 hours after transfection, cells were scored for the presence of multiple nuclei. Percentages represent average of counting ∼200 cells in three independent transfections with error bars representing standard deviation.
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