Borealin interacts with components of the SUMO system and is modified by SUMO in vitro. (A and B) Interaction of Borealin with Ubc9 (A) and the conjugatable forms of SUMO1 and SUMO2 (B, top two rows), but not the unconjugatable forms (SUMO^GA) (B, bottom two rows) in directed yeast two-hybrid assays. −LW indicates plates lacking leucine and tryptophane, whereas QDO indicates plates lacking leucine, tryptophane, histidine, and adenine. (C and D) Borealin is modified by SUMO in vitro. ³⁵S-Labeled CPC subunits, generated by in vitro transcription/translation, were incubated with recombinant E1, E2, and either SUMO1 (C) or SUMO2 (D) in the presence of ATP. In control reactions, the E1 enzyme was omitted. SUMO–Borealin conjugates are indicated by arrows.

Borealin is modified by SUMO in vivo and is preferentially targeted by SUMO2/3. (A) Myc-tagged Borealin and HA- or His-tagged versions of SUMO1 or SUMO2, respectively, were coexpressed in COS-7 cells. His-SUMO conjugates were recovered on Ni-NTA beads and subjected to Western blotting by using anti-myc antibody. (B) Myc-Survivin was tested for sumoylation as described in A. (C) Myc-tagged Borealin and either wild-type (SUMO1^wt, SUMO2^wt) or mutant versions of His-tagged SUMO forms (SUMO1^K16R, SUMO2^K11R) were coexpressed in COS-7 cells and analyzed as described in A. (D) Borealin is modified by endogenous SUMO2/3, but not SUMO1. HeLa cells expressing His-tagged Borealin were arrested in prometaphase by taxol treatment for 16 h. His-Borealin was recovered on Ni-NTA beads, and immunoblotting was performed with anti-Borealin, anti-SUMO1, anti-SUMO2/3, or anti-ubiquitin antibodies. The Borealin-reactive bands at ∼45 kDa (asterisk) are interpreted as a Borealin–SUMO2/3 degradation product. (E) Endogenous Borealin is conjugated to SUMO2/3. Immunoprecipitations were performed with anti-SUMO2/3 or control IgGs from taxol-arrested HeLa cells and probed by Western blotting with the indicated antibodies.

Sumoylation of Borealin is cell cycle regulated and requires RanBP2. (A) Sumoylation of Borealin is cell cycle regulated. HeLa cells expressing His-Borealin were arrested in G1 (lane 1), metaphase (lane 2), or allowed to enter anaphase (lanes 3 and 4). His-Borealin and His-Borealin-conjugates were detected by Western blotting with anti-Borealin and anti-SUMO2/3 antibodies (lane 5–8). Bands marked by asterisk are interpreted as Borealin–SUMO2/3 degradation products. The mitotic status was assayed by monitoring Cyclin B1 levels. (B) RanBP2 binds to the CPC. Immunoprecipitations were performed with rabbit anti-Borealin antibodies or preimmune serum from taxol-arrested HeLa cells and probed by Western blotting with the indicated antibodies. The anti-PIAS2 antibody is directed against the α and β isoforms. Asterisk denotes an anti-PIAS2 cross-reactive band. (C) RanBP2 stimulates sumoylation of Borealin in vitro. ³⁵S-labeled Borealin or p53, generated by in vitro transcription/translation, were subjected to an in vitro sumoylation assay under limiting E2 concentrations. RanBP2^ΔFG was added at a concentration of 500 ng (lane 3), 50 ng (lane 4), and 5 ng (lane 5). SUMO conjugates are indicated by arrows. (D) RanBP2 is required for sumoylation of Borealin in vivo. Myc-tagged Borealin and His-SUMO2 were coexpressed in HeLa cells treated with siRNA oligonucleotides directed against indicated proteins. Depletion was verified by Western blotting. The siRNA directed against PIAS2 targets the α and β isoform (Yang et al., 2005). The asterisk in the anti-PIAS2 Western blot denotes a cross-reactive band. His–SUMO2 conjugates were recovered on Ni-NTA beads, and Western blotting was performed with anti-myc antibodies.

RanBP2^ΔFG restores sumoylation of Borealin in RanBP2 depleted cells. (A) FLAG-tagged Borealin and His-SUMO2 were coexpressed in HeLa cells treated with siRNA oligonucleotides directed against RanBP2 or GL2 as control. In parallel, cells were transfected with either empty myc-vector (−), myc-tagged wild-type RanBP2^ΔFG (wt) or catalytically inactive RanBP2^ΔFG (AA) bearing the mutations L2651A and L2653A. Depletion of RanBP2 and expression of myc-tagged RanBP2^ΔFG constructs was verified by Western blotting. His-SUMO2 conjugates were recovered on Ni-NTA beads, and Western blotting was performed with anti-FLAG antibodies to detect Borealin-SUMO conjugates. (B–E) Overexpression of RanBP2^ΔFG causes chromosome missegregation during mitosis. (B) Untransfected mitotic control cells stained with Borealin antibodies. (C) HeLa cells were transfected with myc-tagged RanBP2^ΔFG and stained with myc- and Borealin antibodies. Note the massive chromosome missegregation after anaphase onset. (D) Experiment as in C. Bottom, costaining of myc- and tubulin antibodies. (E) Experiment as in C, but the catalytically inactive RanBP2^ΔFG construct (RanBP2^{ΔFG AA}) was transfected. DNA was stained with 4,6-diamidino-2-phenylindole (DAPI). Bar, 10 μm. (F) RanBP2 protein level at distinct cell cycle stages. HeLa cells were released from a double thymidine block into nocodazole, and lysates were prepared at different time points after nocodazole release and detected by anti-RanBP2 antibodies. The mitotic status was assayed by monitoring Cyclin B1 levels. Tubulin protein level serves as a loading control. as. = asynchronous growing cells. (G) RanBP2 and Borealin mRNA levels upon cell cycle progression were determined in synchronized HeLa cells by using quantitative RT-PCR.

CPC assembly and localization to the centromere and central spindle are independent of Borealin sumoylation. (A) RanBP2 does not influence CPC localization to the kinetochore/centromere region and vice versa. HeLa cells were treated with indicated siRNA duplexes for 48 h and stained for RanBP2 and Borealin. DNA was stained with DAPI. Bar, 10 μm. (B) HeLa cells were treated with siRNA duplexes specific for the 3′ untranslated region of Borealin and simultaneously transfected with HA-Borealin or HA-Borealin^25KR. Immunofluorescence was performed with antibodies directed against HA, phospho(S7)-CENP-A, and PRC1. DNA was stained with DAPI. Bar, 10 μm. (C) HeLa cells were transfected with the indicated constructs, arrested in S phase by thymidine treatment, and released for 10 h to enter mitosis. Mitotic lysates were prepared and immunoprecipitations were performed with anti-HA antibodies and probed by Western blotting with the indicated antibodies. Asterisk denotes immunoglobulin heavy chain.

SENP3 colocalizes and interacts with Borealin. (A) FLAG-tagged SENP3 and myc-tagged constructs of Borealin were coexpressed in HeLa cells. Immunoprecipitations were performed using anti-myc antibodies. The two fragments of Borealin show a different electrophoretic mobility due to different isoelectric points. Asterisks denote immunoglobulins. (B) SENP3 and Borealin colocalize in interphase nucleoli. HeLa cells were transfected with FLAG-SENP3 and HA-Borealin. Localization was determined by immunostaining with anti-HA and anti-FLAG antibodies. (C) Localization of SENP3 during mitosis. HeLa cells were transfected with FLAG-tagged SENP3 and incubated for 48 h. Immunostaining was performed with anti-FLAG and anti-Borealin antibodies. SENP3 localizes to the cytoplasm during mitosis and showed partial overlap with Borealin from prophase until anaphase. (C′) During telophase SENP3 accumulates at the reforming nuclear envelope and reenters the nucleolus during cytokinesis. DNA was stained with DAPI. Bar, 10 μm.

SENP3 catalyzes desumoylation of Borealin. (A) FLAG-tagged versions of wild-type SENP3 (SENP3^wt, lanes 3 and 8) and SENP5 (SENP5^wt, lanes 5 and 10), or the catalytically inactive mutants (SENP3^C532S, lanes 4 and 9, and SENP5^C713S, lanes 6 and 11), generated by in vitro translation/transcription, were added to in vitro sumoylated Borealin. The anti-FLAG Western blot serves as a loading control for the proteases. Note deconjugation of SUMO2/3, but not SUMO1, of Borealin when incubated with SENP3^wt but not SENP3^C532S. (B) Myc-tagged Borealin and His-SUMO constructs were coexpressed with FLAG-tagged SENP3^wt (lanes 2, 5, 8, and 11) or SENP3^C532S (lanes 3, 6, 9, and 12) in HeLa cells. His-SUMO conjugates were recovered on Ni-NTA beads (lanes 7–12), and Western blotting was performed with anti-myc antibodies. Expression of SENP3 constructs was verified by anti-FLAG Western blotting. (C and C′) SENP3 depletion leads to accumulation of SUMO2/3 modified Borealin. HeLa cells were transfected with His-tagged Borealin and indicated siRNA duplexes, arrested in S phase by thymidine treatment, and released for 10 h to enter mitosis. Mitotic lysates were prepared and Ni-NTA precipitation was performed as described above. Immunoblotting was performed with indicated antibodies to demonstrate depletion of corresponding proteins (C′) and monitor the sumoylation status of Borealin (C). The Borealin reactive bands at ∼45 kDa (asterisk in C) is interpreted as a Borealin–SUMO2/3 degradation product. Note that knock-down of SENP3, but not SENP5, enhances Borealin sumoylation compared with control-depleted cells. In contrast, RanBP2 knockdown results in a loss of Borealin modification.