The levels of the Bora protein fluctuate in the cell cycle
Bora is induced transcriptionally in G2 (http://genome-www.stanford.edu/cgi-bin/Human-CellCycle/Hela/graph/mwhitSearch.pl?gene=IMAGE:824753
; Whitfield et al., 2002
). We examined the levels of the Bora protein across the cell cycle and compared it to key cell cycle regulators. HeLa S3 cells were arrested at the G1/S boundary by a double thymidine treatment (Thy–Thy) and then released into fresh media (Fang et al., 1998b
). The cell cycle profile of released cells was analyzed by FACS (). The mitotic time-points were also determined by levels of phosphorylated histone H3 (). The levels of Bora were low at the G1/S boundary but increased in late S, peaked in G2 (8–9 h after release), and then gradually decreased in mitosis (). The Bora protein accumulated earlier than other mitotic regulators, such as Aurora A and cyclin B, and its level decreased at a time when these mitotic regulators peaked (10 h after release; ). Consistent with this, the levels of Bora were low in unsynchronized cells (AS) and in late prometaphase cells shaken-off from the thymidine-nocodazole–arrested culture (TN0), even though a low level of Bora was still detectable in prometaphase cells (). Bora is phosphorylated in G2 and in mitosis, as treatment of G2/M lysates with λ-phosphatase reduced the mobility of Bora (). We concluded that Bora is a cell cycle–regulated protein.
Figure 1. Bora is a cell cycle protein interacting with Plk1. (A and B) The levels of Bora fluctuate in the cell cycle. HeLa S3 cells were synchronized at the G1/S boundary by a double thymidine arrest (Thy–Thy), released into fresh media, and harvested (more ...)
Bora interacts with Plk1
In an independent project, we investigated the function and regulation of Plk1 by purifying Plk1 complexes from mitotic cells stably expressing a GFP- and S-peptide–tagged Plk1 transgene. Proteomic analysis of associated proteins by mass spectrometry using liquid chromatography tandem mass spectrometry revealed that Bora is a Plk1-interacting protein (). This interaction was confirmed in a transient transfection experiment by coimmunoprecipitation of myc-Plk1 with GFP-Bora but not with GFP ().
As Plk1 interacts with and promotes the degradation of several cell cycle regulators (Barr et al., 2004
), we investigated the interaction of Bora and Plk1 during down-regulation of Bora. HeLa S3 cells were synchronized to late G2 and early mitosis by a release from a double thymidine arrest for 9–10 h (TT9 and 10). Plk1 and Bora mutually coimmunoprecipitated with each other in G2 and in mitosis (TT9 and 10; ). As cells exit from mitosis (TT12), the extent of Plk1-Bora association was substantially reduced, partly because of the decreased abundance of Bora. This interaction is specific, as control IgG precipitated neither Plk1 nor Bora (), and the anti-Bora antibody did not immunoprecipitate other mitotic kinases such as Aurora A (not depicted). We concluded that Bora interacts with Plk1 at late G2 and in mitosis.
Bora is degraded by proteasomes in a Plk1-dependent manner
Next, we investigated whether the down-regulation of Bora is mediated by proteasome-dependent proteolysis. HeLa S3 cells were synchronized by a double thymidine arrest and then released into fresh media (Fang et al., 1998b
). At 7, 8, and 9 h after release (corresponding to early to late G2), the proteasome inhibitor MG132 was added to the culture media and cells were harvested 2 h later. Inhibition of proteasomes stabilized Bora, especially at late G2 and mitosis (, TT10 and 11). This stabilization was not caused by a change in the cell cycle profile of proteasome-treated cells as assayed by FACS. We concluded that Bora is degraded by proteasomes.
Figure 2. Bora is degraded in a Plk1- and proteasome-dependent manner. (A) Proteasome-dependent degradation of Bora. HeLa S3 cells were synchronized with a double thymidine arrest and then released into fresh media. At 7, 8, and 9 h after release, the proteasome (more ...)
Degradation of Bora requires Plk1, as knockdown of Plk1 by a specific siRNA or inhibition of Plk1 by the small molecule inhibitor BI 2536 promoted accumulation of Bora in mitosis ( and not depicted). Knockdown of Plk1 also increased the half-life of Bora (), although this effect could be indirect, as depletion of Plk1 also delays the entry into mitosis (Hansen et al., 2004
), a cell cycle phase in which Bora is degraded. Furthermore, the kinase activity of Plk1 is required for the degradation of Bora, as cotransfection of the kinase-active myc-Plk1 with GFP-Bora destabilized GFP-Bora (, left, lanes 2 vs. 1), whereas cotransfection of the kinase-dead myc-Plk1-K82R mutant stabilized GFP-Bora (, left, compare lanes 1–3). In contrast, GFP-Bora coimmunoprecipitated with myc-Plk1 independent of its kinase activity (, right), indicating that binding of Bora to Plk1 is not sufficient for its degradation and that Plk1-mediated phosphorylation is required.
Next, we reconstituted the ubiquitination of Bora in extracts of synchronized HeLa S3 cells in vitro. When incubated, in the presence of recombinant ubiquitin, ATP, and MG132, with extracts (TN0) of prometaphase cells arrested by a thymidine-nocodazole treatment, 35S-labeled Bora migrated as a smear above the phosphorylated Bora (P-Bora; , left). This slow migrating smear corresponded to ubiquitinated Bora, as GST-ubiquitin conjugates purified from the TN0 extracts by glutathione beads contained the slow migrating 35S-Bora-GST-ub conjugates (, lane 2). Furthermore, ubiquitination of Bora in TN0 extracts requires Plk1, as immunodepletion of Plk1 from extracts (>90% depletion) substantially reduced the degree of Bora ubiquitination (). Ubiquitination of Bora is cell cycle regulated, as incubation of 35S-Bora with extracts (TT0) of G1 cells arrested by a double thymidine treatment generated neither phosphorylation nor ubiquitination of Bora (, right). Thus, Bora is ubiquitinated in mitotic extracts in a Plk1-dependent manner.
Figure 3. Ubiquitination of Bora requires Plk1. (A) In vitro ubiquitination assay. In vitro–synthesized 35S-Bora was incubated, in the presence of MG132, with extracts of HeLa S3 cells arrested by a thymidine-nocodazole treatment (TN0) or by a double thymidine (more ...)
Bora is ubiquitinated in a β-TrCP1–dependent manner
To investigate the mechanism of Bora degradation, we purified the Bora complex from HeLa S3 cells stably expressing a GFP and S-peptide–tagged Bora transgene. Proteomic analysis of associated proteins by mass spectrometry revealed three subunits of the SCF–β-TrCP ligase (Cul1, Skp1, and β-TrCP1/2) as Bora-interacting proteins (). Interaction between β-TrCP1 and Bora was confirmed in a transient transfection experiment by coimmunoprecipitation of HA-β-TrCP1 with GFP-Bora but not with GFP (). Furthermore, endogenous SCF–β-TrCP ligase coprecipitated with the anti-Bora antibody beads but not with the nonspecific IgG beads ( and not depicted).
Figure 4. Bora is ubiquitinated in a β-TrCP1–dependent manner. (A) Identification of the SCF–β-TrCP subunits as Bora-interacting proteins. Listed are peptides of Cul1, Skp1, and β-TrCP1/2 identified by mass spectrometry in (more ...)
The interaction between Bora and SCF–β-TrCP ligase requires the Plk1 activity. In the presence of TN0 extracts, in vitro–translated 35S-Bora efficiently coprecipitated with HA-β-TrCP1 (, compare lanes 5 and 6). This interaction depends on Plk1 in the TN0 extracts, as immunodepletion of Plk1 abolished the complex formation (, lane 2).
The stability of endogenous Bora is controlled by β-TrCP. Expression of a dominant-negative mutant (β-TrCP1-δF) of β-TrCP1 that lacks the F box (aa 30–179 deleted; Hansen et al., 2004
) stabilized the endogenous Bora in mitosis (), which is consistent with the ability of β-TrCP1-δF to stabilize other substrates of the SCF–β-TrCP ligase such as Emi1 (Hansen et al., 2004
). Furthermore, knockdown of β-TrCP using a previously characterized siRNA (Mailand et al., 2006
) increased the half-lives of both Bora and Emi1 (). Thus, Bora levels are under the control of the β-TrCP1 pathway in vivo.
A DSGxxT degron is required for Bora degradation
Next, we determined the molecular recognition of Bora by β-TrCP. β-TrCP recognizes in substrates a consensus motif, DSGxxS, in which both serine residues are phosphorylated (Cardozo and Pagano, 2004
; Ang and Wade Harper, 2005
; Petroski and Deshaies, 2005
). Although Bora does not contain such a motif, it does have the 496-DSGYNT-501 sequence that is conserved among human, rat, and Xenopus laevis
(). Thus, we investigated the role of this sequence in Bora degradation by mutating S497 and T501 to A, either singly or doubly. In the in vitro ubiquitination assay performed in TN0 extracts, mutations of S497, T501, or both blocked ubiquitination of Bora (), indicating that these two residues are critical for its degradation. Consistent with this, the 35
S-Bora-AA mutant failed to interact with HA-β-TrCP1 both in the in vitro binding assay and in the cotransfection experiment in vivo (). Thus, S497 and T501 are directly recognized by β-TrCP. In contrast, these two residues are dispensable for the interaction between Bora and Plk1, as GFP-Bora-AA efficiently coprecipitated the myc-Plk1 in cotransfection experiments (). This experiment further indicated that mutations of S497 and T501 do not globally alter the folding of the mutant proteins.
Figure 5. The DSGxxT degron is required for Bora degradation. (A) The conserved β-TrCP degron in Bora from different species. Residues important for recognition by β-TrCP are underlined. (B) The β-TrCP degron is required for ubiquitination (more ...)
The DSGYNT degron was directly phosphorylated by Plk1, as the Bora-AA mutant was phosphorylated much less efficiently by recombinant Plx1, the X. laevis homologue of Plk1 (). Furthermore, this degron controls the levels of the Bora protein in vivo. HeLa cells were transfected with either GFP-Bora or GFP-Bora-AA, and the levels of expressed proteins were determined in asynchronous cells and in prometaphase cells arrested by a thymidine-nocodazole treatment (). The level of GFP-Bora-AA was only marginally higher than that of GFP-Bora in asynchronous cells, the majority of which were in G1, whereas GFP-Bora-AA was significantly more abundant than GFP-Bora in prometaphase cells, a cell cycle stage with active Plk1. Interestingly, ectopic expression of the nondegradable Bora variant also increased the level of Plk1 in prometaphase cells, whereas expression and phosphorylation of Aurora A were not affected ().
Degradation of Bora is required for normal mitotic progression
To analyze the physiological requirement of Bora degradation, we constructed HeLa cell lines stably expressing GFP-Bora and GFP-Bora-AA resistant to knockdown (GFP-rBora and GFP-rBora-AA). Multiple clones were analyzed and data from representative clones are presented in this section. To avoid complication of mitotic defects arising from knockdown of Bora (see and ), we analyzed the effect of rBora and rBora-AA expression on mitosis without depletion of endogenous Bora.
Figure 7. Knockdown of Bora delays anaphase onset. (A and B) Analysis of Bora knockdown efficiency. HeLa cells were either control transfected or transfected with siRNAs targeting three regions of the Bora gene (siBora-A, -B, and -C and a mixture of all three) (more ...)
Figure 8. Bora controls spindle stability and microtubule growth in mitosis. (A–E) Shown in A, B, and D are maximum projections from deconvolved z stacks of representative HeLa cells stained for β-tubulin (red) and DNA (blue). Cells were transfected (more ...)
rBora and rBora-AA-c1 (clone 1) cells expressed transgenes at levels between one and twofold of the endogenous Bora, whereas rBora-AA-c2 (clone 2) cells expressed the transgene at a level ~16 times that of the endogenous protein (). Importantly, rBora was degraded in prometaphase cells (TN0) but rBora-AA was stable (), which is consistent with the requirement of the DSGYNT degron for Bora degradation.
Figure 6. Expression of nondegradable Bora accumulates cells in mitosis and delays anaphase onset. (A–C) Control, GFP-rBora, GFP-rBora c1, and GFP-rBora c2 cells were collected at G2 (TT8) or in mitosis (TN0), and the levels of GFP fusion proteins and endogenous (more ...)
Expression of rBora-AA increased mitotic index (), although the distribution of cells in G1, S, and G2 were not altered (not depicted). Time-lapse analysis of mitotic progression upon transfection of GFP-Histone H2B indicated that the duration of prometaphase (from nuclear envelope breakdown to the initial formation of the metaphase plate) was not altered, but the duration of metaphase (from initial metaphase to anaphase onset) was lengthened by >60% in rBora-AA–expressing cells (). The higher the levels of Bora-AA expression, the longer the metaphase (compare c1 and 2 clones). Furthermore, rBora-AA–expressing metaphase cells frequently had more than three chromosomes unaligned outside of the metaphase plate for extended time, although these chromosomes eventually incorporated into the metaphase plate before anaphase onset (). Similarly, rBora-expressing cells, but not the GFP-expressing control cells, also had a small percentage of metaphase cells with greater than three unaligned chromosomes (), probably because of the increased level of Bora protein in these cells. We conclude that degradation of Bora is required for timely mitotic progression through metaphase.
Bora functions in the Aurora A–Plk1 pathway (unpublished data). Thus, we analyzed the localization of Plk1 and Aurora A during mitosis. Expression of Bora, either the wild-type or the nondegradable variant, at its physiological level did not alter the localization of either kinase within the detection sensitivity of our assays (). However, expression of rBora-AA at a level 16 times that of the endogenous protein greatly diminished Plk1 signals on centrosomes and Aurora A signals on spindle pole microtubules in rBora-AA-c2 cells (). Quantitative analysis indicated a 50% reduction in the centrosomal Plk1 signals in rBora-AA-c2 metaphase cells compared with rBora metaphase cells. Changes in Plk1 and Aurora A function are likely to contribute to mitotic defects observed in cells expressing nondegradable Bora.
As the majority of endogenous Bora was degraded before metaphase, we expected an effect of nondegradable Bora on chromosome movement in anaphase. Expression of rBora did not affect the rate of anaphase chromosome segregation (), as rBora was degraded before anaphase onset (). However, expression of rBora-AA reduced the velocity of chromosome segregation by 25–35% at anaphase A (), although the fidelity of sister chromatid segregation was not affected in our assay (not depicted). Thus, timely degradation of Bora is required for efficient anaphase.
Bora controls interkinetochore tension and is required for timely anaphase onset
We investigated the function of Bora in the cell cycle. Bora did not associate with any specific cellular structure across the cell cycle, based on our anti-Bora antibody staining and our analysis of GFP-Bora stable cell lines (unpublished data). Thus, Bora likely acts as a soluble factor. Efficient knockdown of Bora by three independent siRNAs () increased the mitotic index, even though the distribution of cells in G1, S, and G2 phase was not altered (). Time-lapse analysis of HeLa cells stably expressing GFP-Histone H2B indicated that knockdown of Bora did not substantially affect the progression through prometaphase but significantly prolonged the length of metaphase (). Furthermore, metaphase cells depleted of Bora frequently had unaligned chromosomes outside of the metaphase plate for a prolonged time (). Even though these unaligned chromosomes eventually congressed to the metaphase plate, cells still stayed at metaphase without unaligned chromosomes for an extended time which was followed by sister chromatid segregation.
Anaphase onset requires the establishment of interkinetochore tension generated by the pulling force derived from dynamic turnover of attached microtubules. A prolonged metaphase suggested a lack of tension across sister kinetochores, which can be measured by interkinetochore distance (Maiato et al., 2004
). In control cells, the presence of the pulling force increased the interkinetochore distance in control cells from prometaphase (0.80 ± 0.02 μm) to metaphase (2.05 ± 0.07 μm; ). However, the interkinetochore distance of chromosomes aligned at the metaphase plate in Bora-depleted cells was substantially shorter than that in control metaphase cells (). Thus, depletion of Bora reduced the interkinetochore tension in metaphase cells.
Microtubule attachment to kinetochores and tension across sister kinetochores are monitored by the spindle checkpoint proteins Mad2 and BubR1, respectively (Chan et al., 1999
; Skoufias et al., 2001
). We observed a twofold increase in kinetochore BubR1 signals for chromosomes aligned at the metaphase plate in Bora knockdown cells compared with control metaphase cells (), confirming a defect in interkinetochore tension. In contrast, kinetochore Mad2 signals were comparable between Bora-depleted and control metaphase cells, indicating that kinetochores from chromosomes at the metaphase plate are attached to microtubules in Bora-depleted cells (unpublished data). Thus, knockdown of Bora activates the tension-sensitive spindle checkpoint and delays anaphase onset.
Bora controls spindle dynamics in mitosis
As interkinetochore tension is generated by dynamic polymerization and depolymerization of kinetochore microtubules in metaphase cells (Maiato et al., 2004
), we directly measured the microtubule growth in Bora knockdown cells. siRNA-transfected mitotic cells were treated with nocodazole to completely depolymerize spindle microtubules and then washed into fresh media to allow microtubules to repolymerize. Although knockdown of Bora did not affect the depolymerization of spindle microtubules (t = 0 min; ), mitotic cells depleted of Bora repolymerized microtubules with a substantially faster kinetics (). 6 min after release from nocodazole treatment, mitotic cells depleted of Bora had three times more microtubules polymerized compared with control mitotic cells (). Conversely, ectopic expression of Bora reduced the rate of microtubule repolymerization ().
An independent assay for spindle turnover is the level of acetylated α-tubulin, a marker for stabilized microtubules (Piperno et al., 1987
; de Pennart et al., 1988
). The mean immunofluorescence intensity of acetylated α-tubulin was increased by over 50% in the metaphase spindle of Bora knockdown cells compared with that of control cells (). This change in microtubule stability specifically resulted from Bora knockdown, as expression of a Bora transgene resistant to the Bora siRNA rescued the acetylated α-tubulin phenotype in Bora knockdown cells (). Changes in the spindle dynamics are expected to affect the rate of chromosome segregation at anaphase. Indeed, the velocity of anaphase chromosome movement was reduced by 40% in Bora knockdown cells (). Thus, Bora regulates the spindle stability and microtubule polymerization, which, in turn, controls the interkinetochore tension at metaphase and the rate of chromosome segregation at anaphase.