Aurora-A with G198N Substitution Rescues Aurora B Location and Mitotic Function
Our previous work (Scrittori et al., 2005
), using shRNA resistant AurB and Aurora A and B chimeras, suggested that specific sequences within the conserved catalytic domain must be critical for distinctive AurA and AurB localization and function (A). Consistent with this, overexpressed HA-tagged wild-type Aurora A did not localize to centromeres, but to centrosomes, in the absence of Aurora B, and other passenger proteins were distributed throughout the chromosomes rather than on centromeres. These results suggested that Aurora A cannot replace Aurora B in the passenger protein complex (Supplemental Figure 1).
Data from other groups have provided evidence that the patterns of localization of both kinases are strictly dependent on unique catalytic domain binding partners (Carmena and Earnshaw, 2003
; Fu et al., 2007
), indicating that unique catalytic domain sequences are crucial for selective interactions with binding partners that determine localization. With this in mind, we first sought to examine the structural differences between the catalytic domains of AurA and AurB kinases with the goal of determining how AurA might be made to bind INCENP. The crystal structures of the catalytic domains of AurA with bound TPX2 (Bayliss et al., 2003
), and of AurB with a bound IN-box INCENP fragment (Sessa et al., 2005
), have been solved. Using computer model docking to analyze the surface accessibility of amino acid residues of native and complexed forms of AurB, we searched for key residues in AurB that were critical for INCENP binding. Of 29 candidate residues (Sessa et al., 2005
), nine were different from AurA. Of these only two Xenopus
AurB residues, phenylalanine 88 (F88) (equivalent to human AurB F72) and N158 (equivalent to human AurB N142), displayed clear chemical differences from aligned residues in AurA, and the corresponding AurA residues created substantial clashes for INCENP docking.
A crystal structure model (B, left) shows human AurB (green) in complex with INCENP (blue). The two key INCENP binding residues on AurB identified by our analysis are indicated with red dots. A close view of Xenopus
AurB-N158 interacting with INCENP through multiple hydrogen bonds is also shown (B, right). We estimated that modification of the AurA-G198 residue to the corresponding Asparagine would generate a reduction of ~5–9 kcal/mol in the interaction energy between Aurora and INCENP, based on the binding energy of 3 kcal/mol for a hydrogen bond in a protein backbone (Shulz and Schirmer, 1979
). Interestingly, it was shown previously in vitro that substitution of G198 to Asparagine prevented AurA from binding to TPX2 (Bayliss et al., 2004
). Structural analysis of the AurA TPX2 complex argued for a conformation of the activation loop of the kinase that is stabilized in the presence of TPX2 and ready to accept the substrate (Bayliss et al., 2004
). Furthermore, a similar mutation in Xenopus
AurA resulted in a kinase with lower in vitro intrinsic kinase activity matching that of AurB activity in the absence of any binding partners (Eyers et al., 2005
For molecular analysis, we used an approach that we established previously (Scrittori et al., 2005
) that combines shRNA induced suppression of native AurB with expression of AurB mutants in the same cell. As we reported previously (Scrittori et al., 2005
), at 48 h after transfection with AurB shRNA, 46 ± 1% of all mitotic cells are negative for AurB and this suppression persists to 72 h. As a result, 28 ± 4% of the shRNA-treated total cell population is bi/multinucleate at 72 h versus 3% of the untreated cells.
Importantly, as demonstrated previously (Scrittori et al., 2005
), double transfection with AurB shRNA and wild-type AurB rescue plasmid (HA-AurB*, fully described below) completely rescues AurB function. Thus, 5 ± 2% of double-transfected cells are multinucleated versus 3 ± 3% of controls at 48 h. These results have validated the approach and permitted the conclusion that each cell that accepts the shRNA plasmid also incorporates the rescue plasmid, permitting genetic rescue analysis on a cell-by-cell basis.
We used the suppression and rescue approach to determine the effect of mutation of AurA residues to their AurB complements. First, we asked whether F72 or N142 were key residues for INCENP binding, centromere targeting, and function for AurB. We found that mutation of W128 of AurA to the aligned phenylalanine (F72) of AurB was without effect on AurA localization or function (data not shown). In contrast, mutation of G198 to the aligned asparagine caused a dramatic change in AurA. In the absence of AurB, ectopically expressed HA-tagged AurA-G198N localized correctly to the centromeres at metaphase and to the spindle midzone in anaphase/telophase (C). There were no apparent deficits in mitotic progression that are normally evident in the absence of AurB (A). Comparison of AurA-G198N to CREST anti-centromere serum showed that the mutant AurA localized to the inner centromere at metaphase, the normal position of a passenger protein (C, inset). In anaphase, AurA-G198N separated from the centromeres stained with CREST serum, and relocalized at the spindle midzone, mimicking the distribution of native AurB (C). Quantitation showed that when AurB suppression was combined with AurA-G198N mutant rescue, 69 ± 2% of mitotic cells that expressed AurA-G198N contained AurA on centromeres, versus 0% of cells rescued with wild-type AurA.
Figure 2. Effect of expression of AurA-G198N on passenger protein distribution in cells depleted of AurB. (A) Alignment of chromosomes in AurB suppressed mitotic cells. In the absence of AurB* rescue, there is no HA antigen signal (left), and other passenger proteins (more ...)
Suppression of AurB by shRNA (A) inhibits localization of the passenger proteins to centromeres, causing redistribution of survivin, telophase disc 60 (TD60), and INCENP to the whole chromosome (A), whereas rescue of AurB suppression with the shRNA resistant HA-AurB* leads to correct inner centromere targeting of AurB itself and AurB partners such as survivin, TD60, and INCENP (B; Scrittori et al., 2005
). HA-AurB* is HA-tagged AurB, mutated so it cannot be shRNA suppressed nor identified with an antibody to AurB N terminus but is otherwise wild-type in function, as described in Scrittori et al. (2005)
. Cells transfected both with shRNA plasmid targeting AurB and with plasmid expressing AurB* can thus be identified as both lacking native AurB and, using HA-antibody, as expressing HA-AurB*. We have demonstrated previously that HA-AurB* fully rescues AurB localization and function (Scrittori et al., 2005
). All rescue experiments with AurB are conducted with shRNA-resistant AurB*. Because AurB* behaves as native AurB (Scrittori et al., 2005
), further text does not explicitly mention its use.
Interestingly, rescue of Aurora B suppressed cells by AurA-G198N also rescued correct localization of survivin, TD60, and INCENP, which colocalized with AurA-G198N at the position normally occupied by AurB both on metaphase centromeres and at the spindle midzone during anaphase and telophase (C). These results demonstrate that construction of the entire passenger protein complex at the kinetochore critically depends on INCENP binding to Aurora, and that other than capacity to bind INCENP, there is no specific constraint on Aurora sequence that clearly distinguishes AurB from AurA as a passenger protein.
The localization of AurA-G198N to centromeres and to the spindle midzone, and rescue of passenger protein localization after AurB suppression, suggested that AurA-G198N could rescue AurB function in chromosome segregation and cell cleavage. If so, AurA-G198N rescue of AurB suppression should retain normal cell morphology after mitosis, reflected in the presence of mononucleate cells. We therefore quantitated the effect of rescue on the generation of binucleate and multinucleate cells that result from AurB suppression. The result (see D) shows that AurA-G198N rescued AurB suppression with efficiency similar to AurB* rescue, because it restored normal cell cleavage and maintained the mononucleate cell population.
Figure 7. Assay of histone H3 phosphorylation and functional rescue of AurB suppression by AurB-N142G, AurA-G198N, and AurA-Δ120 G198N constructs. (A) Histone H3 phosphorylation at serine 10 in AurB-depleted cells expressing the indicated Aurora kinase (more ...)
Correct localization of AurB to the inner centromere has been shown to result from interaction with INCENP (Klein et al., 2006
). Because the residue substitution was designed to enable AurA to interact with INCENP, we conducted an in vitro pull-down assay of the capacity of different 35
S-labeled AurA and B constructs to bind the GST-IN-box of INCENP after their in vitro transcription/translation. The results show full-length AurA-G198N binds the GST-IN-box of INCENP, as does the AurB control, whereas, as expected, wild-type AurA kinase does not bind (D). We conclude that the single residue substitution G198N in AurA transforms AurA into an INCENP binding protein that is competent to localize and function as AurB in mitosis.
A previous report showed that the substitution of G198 to asparagine (N) prevented AurA from binding to TPX2 in vitro (Bayliss et al., 2004
). Our in vitro results demonstrate that this substitution is also, remarkably, sufficient to create the appropriate protein interface required for INCENP binding. Structural analysis of the AurA TPX2 complex argued for a conformation of the activation loop of the kinase that is stabilized in the presence of TPX2 and ready to accept the substrate (Bayliss et al., 2004
). The apparent binding of INCENP to AurA-G198N in vitro and the observed rescue of AurB suppression by AurA-G198N in our cell-based assays provide evidence that the AurA-G198N kinase is active in cells. We confirmed that AurA-G198N is active, because it is competent to phosphorylate histone H3 (see ).
The result suggests that the binding of INCENP to AurA-G198N not only drives the kinase toward its proper localization but also may confer stability to the activation loop of the kinase in the extended conformation even in the absence of TPX2 by a mechanism similar to the allosteric mechanism of activation of AurB bound to the INCENP IN-box motif, as described previously (Sessa et al., 2005
Aurora B with N142G Substitution Affects Neither Aurora B Location Nor Mitotic Function
Given the unexpected capacity of AurA-G198N to rescue AurB suppression, we tested whether mutation of the critical N142 on AurB to glycine would yield a corresponding loss of AurB function in rescue experiments consequent to a loss of INCENP binding and, furthermore, if it would bind TPX2 and translocate to the centrosome.
Remarkably, despite the clear requirement for asparagine mutation to transform AurA into an INCENP-binding protein, HA-AurB-N142G rescued suppression of Aurora-B. Indeed, AurB-N142G remained fully competent to associate with centromeres in early mitosis and to migrate to the spindle midzone during anaphase (A). Furthermore, other passenger proteins, survivin and TD60, also retained correct cell localization in AurB-N142G rescue cells. In vitro pull-down assays confirmed that AurB-N142G binds to INCENP at levels comparable with AurB or AurA-G198N (B). We conclude that although N142 of AurB is important to INCENP binding, other IN-box binding residues of AurB must be sufficient to compensate for its absence, retaining INCENP binding capacity. Furthermore, we found no evidence that AurB-N142G even partially relocates to the centrosome, nor does it bind TPX2 (see A). As a consequence, although AurA can be transformed into functional AurB by a single residue substitution, the inverse mutant does not transform AurB into functional AurA. Indeed, quantitative analysis of capacity of AurB-N142G to substitute for AurB showed the mutation did not interfere with its ability to rescue AurB function (see D).
Figure 3. Effect of expression of AurB-N142G on passenger protein distribution in cells depleted of AurB. (A) Confocal microscopy visualization of AurB-suppressed cells transfected with AurB-N142G. Cells, stained for different passenger proteins, are shown in prometaphase/metaphase, (more ...)
Role of the Aurora N Terminus in Protein Localization
We had previously demonstrated that the absence of the N-terminal region of AurB did not negatively affect AurB rescue (Scrittori et al., 2005
). Indeed, neither N-terminal truncation of AurB nor substitution of the N terminus of AurB with N-terminal sequence of AurA affected AurB function. As seen previously (Scrittori et al., 2005
), the mitotic distribution of a chimera of N-terminal AurA coupled to C-terminal AurB is that of a passenger protein (A), although the N-terminal part of AurA alone localizes to the centrosome (Giet and Prigent, 2001
Figure 4. Mitotic distribution of different AurB chimeric, N142G and truncated constructs. (A) A chimera with N-terminal AurA and C-terminal AurB sequence (labeled with HA-tag) distributes as AurB, as described previously (Scrittori et al., 2005 ). (B) Images showing (more ...)
To reconcile these apparently contradictory data, we constructed and expressed this N-terminal AurA and C-terminal AurB chimera, also containing the AurB INCENP binding site N142G mutation (AurA1-133–AurB78-344 (N142G)). Expression of this construct showed a protein distribution intermediate between that of AurA and that of AurB. In different cells, either centrosome or centromere staining predominated, or both occurred simultaneously (B). In cleaving cells, midbodies were stained. This result permitted two conclusions. First, although the N terminus of AurA is important for Aurora localization to centrosomes, the kinetochore targeting capability of the catalytic domain is dominant for chimera localization (A). Second, although the N142G substitution in the catalytic domain is of no apparent consequence in full-length AurB, the combination of the N terminus of AurA with the N142G substitution results in centrosomal targeting in addition to kinetochore targeting.
Centrosome localization of the AurA1-133–AurB78-344 (N142G) chimera, however, is not associated with loss of INCENP binding because this chimera binds in vitro to the GST-IN-box (D). A possible contributing factor in the redistribution of the AurA1-133–AurB78-344 (N142G) mutant could be the lack of the AurB N terminus. To test for this possibility we constructed an AurB-Δ66 N-terminal truncation-N142G mutant and assayed its distribution in rescue of cells where endogenous AurB had been suppressed with shRNA. This mutant showed the same distribution in mitotic cells as wild-type AurB, and it colocalized with the passenger proteins TD60 and survivin (C). Most importantly, the AurB-Δ66 N-terminal truncation-N142G mutant behaved like wild-type AurB in rescue experiments, by the criterion of absence of bi/multinucleate cells (see D).
GST-IN-box pull-down (D) showed that the different AurA/AurB chimeras (AurA1-133–AurB78-344; AurA1-133–AurB78-344–N142G) and AurB mutants (AurB-Δ66 N-terminal truncation; AurB-Δ66 N-terminal truncation-N142G) all bound INCENP in vitro, in accord with microscopy results. Although the AurA1-133–AurB78-344–N142G chimera partially redistributed to centrosomes, it associated well with INCENP (D) but not with TPX2 (see ).
The results with the AurA1-133–AurB78-344–N142G mutant suggested that the N terminus of AurA is important to tethering AurA to the centrosome. To test this possibility, we rescued AurB shRNA suppressed cells with an AurA-Δ120 N-terminal truncation construct. The result (A) was striking. AurA-Δ120 shows no association with centrosomes but instead redistributes to centromeres (A). We assayed whether AurA-Δ120 would bind INCENP, and we found, remarkably, that redistribution to the centromere occurs in a mutant that does not bind INCENP (C). It was further surprising that the AurA-Δ120 mutant did not leave centromeres in anaphase and therefore lacked typical passenger protein mitotic behavior.
Figure 5. AurA-Δ120 and AurA-Δ120-G198N mitotic distribution and recruitment of INCENP, surviving, and TD60. (A) Localization of AurA-Δ120 (HA-tag) in AurB-depleted cells. Inset, AurA-Δ120 localization at the kinetochore, counterstained (more ...)
It is noteworthy that AurA-Δ120 binds to the kinetochore, rather than the inner centromere site of passenger proteins such as AurB (A, CREST, inset). Furthermore, other passenger proteins such as survivin associated with the whole chromosome rather than the centromere with AurA-Δ120 rescue (A). This strongly suggested that the catalytic domain of AurA has the “information” to bind to kinetochores and that INCENP is apparently necessary for the relocation from the kinetochore to the inner centromere. To test this, we created an AurA-Δ120-G198N mutant and studied its localization and capacity to rescue AurB suppression. This mutant, in contrast to AurA-Δ120, showed typical passenger protein localization (B), and was able to bind INCENP (C). Importantly, at metaphase, the AurA-Δ120-G198N mutant apparently localized to the inner centromere, thus lying between the two CREST kinetochore signals in B. In addition, all the passenger proteins in cells lacking endogenous AurB and expressing AurA-Δ120-G198N mutant exhibited proper protein passenger localization yielding signals that overlapped with AurA-Δ120-G198N (B). We conclude that a single G198N substitution in the catalytic domain of AurA creates the condition for localization to the inner centromere of both Aurora and the other passenger proteins.
We have assayed for the capacity of different Aurora constructs to bind to TPX2. Just as AurA-Δ120 did not bind INCENP, and yet localized to centromeres (), we found that although AurA1-133–AurB78-344–N142G localized to centrosomes (B), it did not bind TPX2 (). Indeed, no Aurora N142 or G198 mutant tested detectably bound TPX2 (), regardless of localization, under conditions where wild-type AurA binding was evident (). In addition, the intracellular mitotic spindle distribution of the TPX2 was not affected by the expression of the different Aurora mutants (data not shown).
Mutant Rescue of Mitotic H3 Phosphorylation and Mitotic Exit after AurB Suppression
If the single residue AurA substitution mutant is indeed transformed into an AurB kinase, then it should substitute for AurB catalytic activity. Because AurB is responsible for phosphorylation of residue S10 on histone H3 during mitosis (Goto et al., 2002
; Soncini et al., 2006
), we analyzed the phosphorylation status of S10 upon AurB depletion and rescue.
Depletion of AurB with shRNA, abolishes S10 phosphorylation on histone H3 as expected (A, no rescue). Rescue of AurB depletion with the AurB dead kinase K106A restores proper kinetochore localization of the mutant kinase but not histone H3S10 phosphorylation. In contrast, AurB wild-type and AurB-N142G mutants rescue H3S10 phosphorylation. In accord with in vitro Aurora kinase assays (, B and C), AurA-G198N and AurA-Δ120-G198N mutants rescue AurB suppression and restore histone H3S10 phosphorylation (A).
We also determined the ability of the different Aurora kinase constructs to rescue proper mitotic exit in cells where native AurB had been suppressed with shRNA (D). AurB-K106A dead kinase did not rescue the absence of AurB and serves as the control for inability to properly exit mitosis, as scored by accumulation of bi/multinucleate cells that result from mitotic and cell cleavage errors. The ~52% mitotic failure rate obtained with AurB-K106A dead kinase rescue is consistent with the overall 46% of cells in which AurB expression was observed previously to be suppressed by shRNA (Scrittori et al., 2005
). In contrast, AurA-G198N and AurA-Δ120-G198N, which bind INCENP (C), rescued mitotic exit in a manner statistically indistinguishable from wild-type AurB (D).