SIRT2 regulates nuclear envelope reassembly through ANKLE2 deacetylation

Abstract

Sirtuin 2 (SIRT2) is an NAD-dependent deacetylase known to regulate microtubule dynamics and cell cycle progression. SIRT2 has also been implicated in the pathology of cancer, neurodegenerative diseases and progeria. Here, we show that SIRT2 depletion or overexpression causes nuclear envelope reassembly defects. We link this phenotype to the recently identified regulator of nuclear envelope reassembly ANKLE2. ANKLE2 acetylation at K302 and phosphorylation at S662 are dynamically regulated throughout the cell cycle by SIRT2 and are essential for normal nuclear envelope reassembly. The function of SIRT2 therefore extends beyond the regulation of microtubules to include the regulation of nuclear envelope dynamics.

Keywords: ANKLE2; Cell cycle; Nuclear envelope; SIRT2.

Fig. 1.

Depletion or overexpression of SIRT2 causes nuclear envelope defects. (A) U2OS cells stably expressing LAP2β–GFP were transfected with siRNA pools targeting SIRT2 (siSIRT2 #1, ON-TARGETplus; #2, siGENOME) or a non-silencing control (si Control), or with HA empty vector, or vectors expressing HA–SIRT2 or HA–SIRT2 H150Y. Arrows indicate cells exhibiting a lobulated nuclear envelope. Scale bars: 10 μm. (B) Western blots showing SIRT2 silencing efficiency and overexpression levels. Tubulin was used as a loading control. (C) Quantification of cells exhibiting a lobulated nuclear envelope 72 h after silencing SIRT2 or 48 h after overexpressing SIRT2. Mean±s.e.m. based on four independent experiments. **P<0.01; ***P<0.001 (two-tailed Student's t-test).

Fig. 2.

Proteomics analysis of new SIRT2-binding partners identified by STREP-HA TAP or BioID. (A,B) Immunofluorescence images of stable HEK293 cell lines expressing (A) HA-STREP–SIRT2 and (B) an empty vector control, HA-STREP–mycBirA–SIRT2 or SIRT2–BirAmyc under the control of a doxycycline-inducible promoter. Doxycycline (dox) was added for 24 h prior to processing for immunofluorescence microscopy using antibodies against the Myc tag or the HA tag (red), a fluorescently labelled streptavidin probe (green) and DAPI to counterstain DNA (blue). Negative controls were left uninduced; all media in (B) were supplemented with 50 µM biotin for 16 h to facilitate biotinylation. Scale bars: 10 µm. (C) A schematic presentation of STREP-HA TAP and western blot analysis of affinity purification of HA-STREP-tagged SIRT2. SIRT2 cytoplasmic fractions are first applied to a streptactin affinity column, bound proteins are eluted with biotin, and are then incubated with anti-HA magnetic beads and eluted with acidic glycine. 0.4% of the input, 0.4% of the supernatant (Sup), 2% of the streptactin eluate (Elu) and 50% of the anti-HA eluate were loaded on the gel. (D) A schematic presentation of BioID and streptavidin–HRP western blotting of BioID streptavidin pulldowns from whole-cell lysates (WCL) or cytoplasmic fractions (cyto) of N- and C-terminal SIRT2 BirA fusions. (E) A heat map of the top 50 SIRT2 interactors sorted by the descending global score. The hue indicates SAINT interaction probability. The height of a rectangle shows the proportion of replicates above the cut-offs (SAINT probability >0.98 and fold change >10). The global score is calculated based on the number of rectangles that go over the two dashed lines corresponding to a specific method. If the rectangle is over the two dashed lines, the corresponding method contributes one point to the global score.

Fig. 3.

SIRT2 deacetylates the regulator of nuclear envelope reassembly ANKLE2. (A) Western blots of anti-HA co-immunoprecipitation (IP) from asynchronous and mitotic HEK293T cells overexpressing HA–SIRT2 and ANKLE2–GFP. HA–PARP3 was used as a negative control. (B) In vitro acetylation of recombinant ANKLE2 with 0.15 μg of p300, PCAF and CBP acetyltransferases. hMOF did not acetylate ANKLE2. 0.015, 0.15 or 1.5 μg of ANKLE2 was present in the loaded sample. (C) In vitro deacetylation of ANKLE2 by SIRT2. 1.5 μg of ANKLE2, 0.15 μg of acetyltransferase and 0.15/1.5 μg of SIRT2 or SIRT2 H150Y were present in the loaded samples.

Fig. 4.

SIRT2 regulates ANKLE2 acetylation and phosphorylation dynamics in vivo. (A,B) ANKLE2–GFP (A) acetylation and (B) phosphorylation sites identified in asynchronous (circles) and mitotic (square) cells in three independent experiments. Acetylated lysine and phosphorylated serine residues are coloured according to their average abundance relative to unmodified peptides. (C–F) Changes in the relative abundance of (C,E) ANKLE2 acetylation sites and (D,F) a phosphorylation site upon SIRT2 overexpression (C,D) or silencing (E,F). Mean±s.e.m. values from two to four independent experiments are shown. The acetylation sites shown are characterized by a >0.1 relative abundance and a reproducible reduction in acetylation upon SIRT2 overexpression or increase in acetylation upon SIRT2 silencing. The phosphorylation site fulfils the criteria of a >1 relative abundance and a reproducible increase in mitotic phosphorylation. Relative abundance ratios show considerable variability due to inherent limitations of the shotgun mass spectrometry analysis of substoichiometric modifications.

Fig. 5.

ANKLE2 depletion results in defective nuclear envelope reassembly. (A) U2OS cells stably expressing LAP2β–GFP were transfected with siANKLE2 (#1, ON-TARGETplus; #2, siGENOME) or a non-silencing control (si Control). (B) Quantification (mean±.s.e.m.) of cells exhibiting a lobulated nuclear envelope 72 h after silencing ANKLE2, based on four independent experiments. **P<0.01; ***P<0.001 (two-tailed Student's t-test). (C) Western blot showing siANKLE2 efficiency; tubulin served as a loading control. (D) Still images of live-cell imaging movies of U2OS LAP2β–GFP-expressing cells 48–72 h after transfection of siANKLE2 or siSIRT2. Arrows indicate cells with lobulated nuclear envelopes. Scale bars: 10 μm.

Fig. 6.

SIRT2 directly regulates nuclear envelope reassembly by deacetylating ANKLE2. (A) Immunofluorescence images of U2OS cells stably expressing ANKLE2–GFP or siANKLE2-resistant wild-type (WT) or acetylation or phosphorylation site mutant ANKLE2–GFP. The cells were transfected with siRNA targeting ANKLE2 or a non-silencing control (si Control) for 48 h, and co-stained for lamin A and C. ANKLE2 K302R and K302Q and ANKLE2 S662A, S662C and S662D mutants cannot rescue the polylobed phenotype caused by ANKLE2 depletion. Scale bars: 10 µm. (B,C) Quantification (mean±.s.e.m.) of the polylobed phenotype for ANKLE2 (B) acetylation and (C) phosphorylation mutants, based on two to four independent experiments. *P<0.05; ***P<0.001 (two-tailed Student's t-test).

Fig. 7.

A model of SIRT2-mediated cell cycle regulation of ANKLE2 acetylation and phosphorylation. (A) Under normal conditions ANKLE2 K302 acetylation is low in interphase and high in mitosis, whereas S662 phosphorylation is low throughout the cell cycle. (B) SIRT2 silencing increases interphase but reduces mitotic K302 acetylation, and increases mitotic S662 phosphorylation. (C) SIRT2 overexpression reduces mitotic K302 acetylation and increases mitotic S662 phosphorylation. (B,C) Perturbation of ANKLE2 K302 acetylation levels and S662 phosphorylation upon SIRT2 depletion or overexpression result in a polylobed nuclear envelope phenotype.