Analysis of Ipl1-mediated phosphorylation of the
Ndc80 kinetochore protein in Saccharomyces cerevisiae
Bungo Akiyoshi*, §, Christian R. Nelson*, Jeffrey A. Ranish† and Sue Biggins*
*Division of Basic Sciences, Fred Hutchinson Cancer Research Center
Seattle, WA 98109, USA
§Molecular and Cellular Biology Program, University of Washington
Seattle, WA 98195, USA
†Institute for Systems Biology, Seattle, WA 98103, USA
Genetics: Published Articles Ahead of Print, published on October 12, 2009 as 10.1534/genetics.109.109041
Running Head: Ndc80 phosphorylation by Ipl1
Keywords: Ndc80/Hec1, Ipl1/Aurora B protein kinase, chromosome segregation, spindle
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Phosphorylation of the Ndc80 kinetochore protein by the Ipl1/Aurora B kinase
reduces its microtubule binding activity in vitro. We found that kinetochore-bound
Ndc80 is phosphorylated on Ipl1 sites in vivo, but this phosphorylation is not essential.
Instead, we show that additional Ipl1 targets contribute to segregation and the spindle
The faithful transmission of genetic material requires chromosomes to biorient
such that sister kinetochores attach to spindle microtubules from opposite poles. In the
absence of biorientation, cells activate the spindle checkpoint to halt the cell cycle and
give cells time to fix errors. The conserved Ipl1 protein kinase is essential for both error
correction and checkpoint activation because it destabilizes aberrant interactions between
kinetochores and microtubules, creating unattached kinetochores that trigger the
checkpoint (BIGGINS et al. 1999; BIGGINS and MURRAY 2001; TANAKA et al. 2002;
PINSKY et al. 2006b). The Ndc80 outer kinetochore protein that directly binds to
microtubules has been proposed to be a key target of Ipl1 for biorientation because
phosphorylation of its unstructured N-terminus reduces its affinity toward microtubules
in vitro (CHEESEMAN et al. 2006; DELUCA et al. 2006; WEI et al. 2007; CIFERRI et al.
2008; GUIMARAES et al. 2008; MILLER et al. 2008). However, it is not known whether
Ndc80 is phosphorylated on kinetochores in vivo. In addition, the relative contributions
of Ipl1-mediated phosphorylation of Ndc80 to segregation and the spindle checkpoint
have not been analyzed.
We recently developed a method to detect phosphorylation on centromere-bound
kinetochore proteins in vivo by isolating minichromosomes and then performing mass
spectrometry (MS) (B. Akiyoshi, C. R. Nelson, J. A. Ranish, S. Biggins, unpublished
data). We applied this method to identify Ipl1 phosphorylation on kinetochores by
purifying minichromosomes from conditions where the activity of the Glc7 protein
phosphatase 1 (PP1) that opposes Ipl1 was decreased (FRANCISCO et al. 1994). We
overexpressed the Gip4 regulatory subunit that titrates Glc7 from the nucleus to enrich
for cells arrested in mitosis with increased phosphorylation on Ipl1 targets (PINSKY et al.
2006a). When we analyzed the minichromosomes purified from this condition by MS,
we detected three phosphorylated peptides corresponding to Ipl1 consensus sites in the N-
terminus of Ndc80 (S37, T54 and T74, Figure 1A and data not shown), providing the first
evidence that kinetochore-bound Ndc80 is phosphorylated in vivo on its N-terminal
domain. In addition, it was previously reported that S100 on Ndc80 is phosphorylated in
vivo (CHEESEMAN et al. 2002). All of these residues map to the Ndc80 unstructured N-
terminal domain that is phosphorylated by Ipl1/Aurora in vitro and implicated in
regulating microtubule attachments to kinetochores in multicellular eukaryotes
(CHEESEMAN et al. 2006; DELUCA et al. 2006; CIFERRI et al. 2008; GUIMARAES et al.
2008; MILLER et al. 2008). We therefore examined the potential relevance of
phosphorylation of the budding yeast Ndc80 N-terminal region (1-112 residues) on the
Ipl1 consensus sites in this domain by mutating all of them to create the Ndc80-7A
mutant (Figure 1A). We confirmed that phosphorylation of the Ndc80-7A protein by Ipl1
is decreased in vitro (Figure 1B). The residual phosphorylation of Ndc80-7A is likely
due to the presence of other putative Ipl1 sites outside of the N-terminus. However, these
sites are not conserved in multicellular eukaryotes and do not map to the microtubule
binding region of Ndc80, and phosphorylation on these residues in vivo has not been
We tested whether Ndc80-7A is functional in vivo by testing whether it could
complement an ndc80? mutant. Strikingly, ndc80-7A cells were viable and did not
exhibit any obvious growth defects at various temperatures (Figure 1C and data not
shown), or defects in bipolar spindle assembly (data not shown). These data are
consistent with the report that cells are viable when this domain is completely deleted
(KEMMLER et al. 2009). In addition, there was no delay in cell cycle progression when
the levels of the anaphase inhibitor Pds1 were monitored as cells were released from G1
(Figure 1D). However, although Ndc80-7A protein levels are similar to Ndc80 (data not
shown), we found that ndc80-7A mutant cells exhibited hyper-sensitivity to the
microtubule depolymerizing agent, benomyl (Figure 1C). Spindle checkpoint mutants
are benomyl sensitive because the cell cycle continues when microtubules are
depolymerized (HOYT et al. 1991; LI and MURRAY 1991). However, ndc80-7A cells
activate the checkpoint and arrest in metaphase with high levels of Pds1 when treated
with the microtubule destabilizing drug, nocodazole (Figure 1E). Therefore, the ndc80-
7A mutant cells likely have altered microtubule dynamics that result in a benomyl
We next examined the importance of individual phosphorylation sites by
reversing each site in ndc80-7A back to WT, creating seven different ndc80-6A mutants.
These mutants exhibited various degrees of sensitivity to benomyl and revealed that S37,
S95 and S100 are more important than other residues (Figure 2A). To test whether
specific sites are sufficient to lead to benomyl sensitivity, we constructed ndc80-2A
(S95A S100A), ndc80-3A (S37A S95A S100A) and ndc80-4A (S37A T54A T71A T74A)
mutants. However, none of these mutants were hyper-sensitive to benomyl (Figure 2B).
Taken together, these results demonstrate that the presence of multiple sites is important,
but that no single specific site is critical for Ndc80 function. The interaction between
Ndc80 and microtubules is primarily electrostatic (the former being positively charged
and the latter negatively charged), and the N-terminal unstructured domain is highly
positively charged to strengthen the interaction (CHEESEMAN et al. 2006; DELUCA et al.
2006; WEI et al. 2007; CIFERRI et al. 2008; GUIMARAES et al. 2008; MILLER et al. 2008).
Therefore, it is most likely that the overall charge state of the N-terminal domain, but not
any specific site, is important for the function.
We previously found that Ipl1 is essential for checkpoint activation when
kinetochores are not under tension, possibly due to its role in generating unattached
kinetochores that signal the checkpoint (BIGGINS and MURRAY 2001; PINSKY et al.
2006b). We therefore tested whether the ndc80-7A mutant is defective in the tension
checkpoint by analyzing Pds1 levels in two conditions that create tension defects (a
cohesion defect and a bipolar spindle assembly defect). Cells containing ndc80-7A
activated the checkpoint in both conditions (Figure 3, A and B), suggesting that Ipl1
phosphorylates one or more additional targets or sites in Ndc80 to activate the spindle
If Ndc80 contributes to Ipl1 functions in vivo, we expected that the ndc80-7A
mutant would exhibit genetic interactions with ipl1 mutants. Consistent with this, we
found that ndc80-7A is synthetically lethal with the ipl1-321 temperature sensitive mutant
(data not shown). It is noteworthy that an ndc80-1 temperature sensitive allele does not
exhibit lethal interactions with ipl1-321 (PINSKY et al. 2006b), suggesting that ndc80-7A
is more specifically defective in an Ipl1-related function than the ndc80-1 mutant. This
result also suggested that ipl1-321 cells retain enough activity for survival when Ndc80
sites can be phosphorylated by Ipl1, but becomes lethal when Ndc80 has reduced
capacity to be phosphorylated by Ipl1. Taken together, these data strongly suggest that
there are unidentified targets of Ipl1. Interestingly, ndc80-7A is also synthetically lethal
with the mps1-1 temperature sensitive mutant (data not shown). Because Mps1 targets
some of the Ipl1 sites of Ndc80 (KEMMLER et al. 2009), this result may suggest that Ipl1
and Mps1 have overlapping substrate specificity in vivo. Alternatively, Mps1 may be
required for activating Ipl1 or vice versa (JELLUMA et al. 2008).
To directly analyze whether Ndc80 phosphorylation contributes to the
biorientation function of Ipl1, we created a conditional ndc80-7A deg-ipl1 double mutant
to analyze chromosome segregation. We previously found that although deg-ipl1 has
reduced function, it retains viability even when transcription is repressed by the addition
of doxycycline (Dox) (NG et al. 2009). Consistent with this, deg-ipl1 ndc80-7A double
mutant cells grew in the absence of Dox, but deg-ipl1 function was sufficiently reduced
to be inviable when combined with ndc80-7A in the presence of Dox (Figure 3C). This
allowed us to analyze segregation in the double mutant cells by releasing them from G1
into media containing Dox and monitoring the fate of a fluorescently marked
chromosome (STRAIGHT et al. 1996). Strikingly, 18 % of cells mis-segregated ChrIV to
the same pole instead of opposite poles (Figure 3D), strongly suggesting that the lethality
of the double mutant is due to a biorientation defect. If every chromosome exhibited a
similar biorientation defect, few cells would remain viable. The chromosome segregation
defect in these cells should lead to activation of the spindle checkpoint, so we analyzed
Pds1 levels in cells released from G1. However, similar to ipl1 mutants (BIGGINS and
MURRAY 2001; PINSKY et al. 2006b), the ndc80-7A deg-ipl1 cells did not activate the
checkpoint in response to the segregation defect (Figure 3E). Taken together, these data
suggest that N-terminal Ndc80 phosphorylation contributes to both the segregation and
checkpoint functions of Ipl1.
In summary, our studies show that multiple Ipl1 sites on the N-terminal domain of
Ndc80 are phosphorylated when it is associated with kinetochores in vivo. Strikingly,
although this phosphorylation contributes to the role of Ipl1 in both chromosome
segregation and spindle checkpoint activation, our data strongly support the idea that
additional key targets of Ipl1 must be phosphorylated in combination with Ndc80 to carry
out the precise functions of the kinase. In the future, it will be critical to identify the
complete complement of these phosphorylation events to fully understand the mechanism
by which Ipl1 mediates segregation and checkpoint activation to ensure genomic
We thank Min Yuan and the ISB facility for proteomic analyses. This work was
supported by NIH grant GM064386 to S. B. and NIGMS grant (PM50
GMO76547/Center for Systems Biology) to J. A. R. S. B. is a Scholar of the Leukemia
and Lymphoma Society.
FIGURE 1. An N-terminal Ndc80 mutant lacking Ipl1 phosphorylation sites is benomyl
sensitive but spindle checkpoint proficient.
(A) A schematic of Ndc80 shows that it contains seven putative Ipl1 phosphorylation
Ser/Thr sites (shown in bold) in the unstructured N-terminus. Phosphorylation sites
detected by MS are underlined. Minichromosomes were purified from cells expressing
Gip4 from the galactose promoter (SBY6247) for three hours and subjected to MS
analysis ((KIM et al. 2007) and unpublished data). Yeast strains used in this study are
listed in Supplemental Table S1. (B) Phosphorylation of the Ndc80-7A phospho-
deficient mutant by Ipl1 is reduced in vitro. Ndc80-WT-Myc (WT, SBY7258) and
Ndc80-7A-Myc (7A, SBY7259) proteins were immunoprecipitated with anti-Myc
antibodies and used as substrates in vitro for an Ipl1 kinase assay as previously described
(BUVELOT et al. 2003). M are the molecular weight markers. (C) Ndc80-7A mutant cells
are viable, but show hyper-sensitivity to benomyl that is rescued by endogenous NDC80.
Serial dilutions (5-fold) of NDC80-WT and ndc80-7A cells in the presence or absence of
an endogenous NDC80 were plated on YPD plates with or without 7.5 µg/ml benomyl
(SBY7258, SBY7259, SBY6859 and SBY6860). (D) Ndc80-7A cells do not exhibit
defects in cell-cycle progression. Cells containing Pds1-Myc and either NDC80-WT
(SBY7120) or ndc80-7A (SBY7123) were released from G1 and lysates were prepared at
the indicated time points and monitored for Pds1-Myc by immunoblot as previously
described (BIGGINS et al. 1999). (E) Ndc80-7A cells activate the spindle checkpoint in
response to microtubule depolymerization. The experiment in (D) was repeated by
releasing cells into 10 µg/ml nocodazole.
FIGURE 2. Multiple phosphorylations on Ndc80 are important for proper microtubule
(A) Ndc80-6A mutants exhibit varying degrees of sensitivity to benomyl. Cells
containing six mutations were assayed for benomyl sensitivity as in Figure 1A
(SBY7257, SBY7255, SBY7252, SBY7328, SBY7329, SBY7326, SBY7327, SBY7258
and SBY7259). (B) Ndc80-4A, ndc80-2A and ndc80-3A mutants do not exhibit benomyl
hyper-sensitivity, indicating that there is no specific site required for the phenotype
(SBY7258, SBY7259, SBY7296, SBY7325 and SBY8597).
FIGURE 3. The Ndc80 N-terminal domain is not the sole Ipl1 downstream target for
segregation and spindle checkpoint activation.
(A, B) Ndc80-7A mutant cells do not bypass the checkpoint in response to a defect in
cohesion (A; mcd1-1) or bipolar spindle assembly (B; mps2-1). Cells containing either
NDC80-WT (SBY7131, SBY7118) or ndc80-7A (SBY7133, SBY7121) were arrested in
G1, released to 37˚, and analyzed as in Figure 1D. (C) Ndc80-7A is synthetically lethal
with deg-ipl1. Serial dilutions (5-fold) of deg-ipl1 NDC80-WT (SBY8325), deg-ipl1
ndc80-7A (SBY8241), and WT (SBY818) cells were plated onto YPD with or without 25
?g/ml Dox. (D, E) The ndc80-7A deg-ipl1 mutant exhibits chromosome mis-segregation
but does not activate the spindle checkpoint. Cells containing deg-ipl1 NDC80-WT or
deg-ipl1 ndc80-7A were arrested in G1 for 2 hrs and then 25 ?g/ml Dox was added for an
additional 1.5 hrs. Cells were released into media containing Dox and either fixed at 140
min for microscopy (D) as previously described (BIGGINS et al. 1999) (examples of
proper segregation (left), mis-segregation to the mother cell (middle) or daughter cell
(right) are shown) or monitored for Pds1 levels at the indicated time points (E).
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NDC80-WT + NDC80
ndc80-7A + NDC80
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NDC80-WT + NDC80
ndc80-7A + NDC80
WT 7A WT 7AM WT 7AWT 7AM
Ipl1 consensus: [R/K]-X-[S/T]-[V/I/L/X]
ndc80-4A (S37A T54A T71A T74A)
ndc80-2A (S95A S100A)
ndc80-3A (S37A S95A S100A)
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Proper segregation Mis-segregation
NDC80-WT deg-ipl1ndc80-7A deg-ipl1