Cdc15 integrates Tem1 GTPase-mediated
spatial signals with Polo kinase-mediated
temporal cues to activate mitotic exit
Jeremy M. Rock and Angelika Amon1
David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts
02139, USA; Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA
In budding yeast, a Ras-like GTPase signaling cascade known as the mitotic exit network (MEN) promotes exit
from mitosis. To ensure the accurate execution of mitosis, MEN activity is coordinated with other cellular
events and restricted to anaphase. The MEN GTPase Tem1 has been assumed to be the central switch in MEN
regulation. We show here that during an unperturbed cell cycle, restricting MEN activity to anaphase can occur
in a Tem1 GTPase-independent manner. We found that the anaphase-specific activation of the MEN in the
absence of Tem1 is controlled by the Polo kinase Cdc5. We further show that both Tem1 and Cdc5 are required to
recruit the MEN kinase Cdc15 to spindle pole bodies, which is both necessary and sufficient to induce MEN
signaling. Thus, Cdc15 functions as a coincidence detector of two essential cell cycle oscillators: the Polo kinase
Cdc5 synthesis/degradation cycle and the Tem1 G-protein cycle. The Cdc15-dependent integration of these
temporal (Cdc5 and Tem1 activity) and spatial (Tem1 activity) signals ensures that exit from mitosis occurs only
after proper genome partitioning.
[Keywords: Cdc14; Cdc15; Cdc5; mitotic exit network; NDR kinase; Polo kinase; Tem1]
Supplemental material is available for this article.
Received June 17, 2011; revised version accepted August 12, 2011.
The creation of a daughter cell requires the faithful du-
plication and segregation of the genome. The success of
this process necessitates the temporal and spatial co-
ordination of genome segregation with the final cell cycle
transition, exit from mitosis, when the mitotic spindle is
disassembled, nuclei are reformed, and cytokinesis splits
the cell into two. In the absence of such coordination,
significant genetic and epigenetic changes occur. Thus, as
might be expected, the inability to coordinate genome
segregation with exit from mitosis is strongly associated
with cancer (Kops et al. 2005; Gonzalez 2007).
In budding yeast, exit from mitosis is controlled by the
essential protein phosphatase Cdc14. Cdc14 antagonizes
mitotic cyclin-dependent kinases (Clb-CDKs), the inacti-
et al. 1998; Visintin et al. 1998; Zachariae et al. 1998).
Cdc14 activity is tightly regulated. In cell cycle stages
prior to anaphase, Cdc14 is sequestered within the nu-
cleolus as a result of its association with its nucleolar-
localized inhibitor, Cfi1/Net1 (Shou et al. 1999; Visintin
et al. 1999). Upon anaphase entry, Cdc14 is released from
the nucleolus and spreads throughout the nucleus and, to
a significantly lesser extent, the cytoplasm. This early
anaphase release of Cdc14 is mediated by the FEAR
network and results in a pulse of Cdc14 activity (Pereira
et al. 2002; Stegmeier et al. 2002; Yoshida et al. 2002).
While not essential, FEAR network-mediated release of
Cdc14 from the nucleolus is crucial for the accurate
execution of anaphase (Rock and Amon 2009). Cdc14
release from the nucleolus during late anaphase is pro-
moted by the mitotic exit network (MEN), which drives
the sustained release of Cdc14 in both the nucleus and
the cytoplasm and results in exit from mitosis (Stegmeier
and Amon 2004).
The MEN is a Ras-like GTPase signal transduction
cascade (see Figure 7B, below, for a pathway diagram;
for review, see Stegmeier and Amon 2004). As in other
G-protein signaling pathways, the GTPase Tem1 is
thought to be the central switch regulating MEN activity
(Cooper and Nelson 2006; Wang and Ng 2006; Geymonat
et al. 2009; Chan and Amon 2010). Tem1 is negatively
regulated by its two-component GTPase-activating pro-
tein (GAP), Bub2–Bfa1. The Bub2–Bfa1 complex is regu-
lated by two protein kinases. The Polo kinase Cdc5
phosphorylates Bfa1, which reduces Bub2–Bfa1 GAP
activity. The protein kinase Kin4 functions in opposition
to Cdc5, phosphorylating Bfa1 and thus rendering the
GAP insensitive to Cdc5-dependent inhibitory phosphor-
Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.17257711.
GENES & DEVELOPMENT 25:1943–1954 ? 2011 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/11; www.genesdev.org1943
ylation (Maekawa et al. 2007). Tem1 is positively regu-
lated by Lte1, which inhibits Kin4 in the bud (Bertazzi
et al. 2011; Falk et al. 2011).
During late anaphase, Tem1-GTP is thought to bind to
and activate the protein kinase Cdc15, which then
activates the downstream kinase Dbf2 associated with
its activating subunit, Mob1. Based on binding data and
homology with known scaffolds, Nud1 is thought to
function as a scaffold for the core MEN components
Tem1, Bub2–Bfa1, Cdc15, and Dbf2-Mob1 at spindle pole
bodies (SPBs) (Gruneberg et al. 2000; Valerio-Santiago and
Monje-Casas 2011). Tem1 SPB localization is essential for
MEN activation, and it is thought that SPB localization of
Cdc15, Dbf2, and Mob1 is also essential (Valerio-Santiago
and Monje-Casas 2011). Activation of Dbf2-Mob1 results,
at least in part, in the phosphorylation of Cdc14’s nuclear
localization sequence and causes the retention of Cdc14
in the cytoplasm, where it can act on its substrates
(Mohl et al. 2009). Activation of the MEN in late ana-
phase is essential for the sustained release of Cdc14
from the nucleolus, which ultimately promotes exit from
The mechanisms by which MEN activity and exit from
mitosis are temporally and spatially coordinated with
genome segregation are beginning to be understood. MEN
activity is controlled by spindle position. When the ana-
phase spindle is not correctly aligned along the mother–
daughter cell axis, MEN signaling is inhibited (Yeh et al.
1995). This spindle position control of MEN signaling is
accomplished by a system composed of a MEN inhibitory
and a MEN-activating zone and a sensor that moves
between them. The MEN inhibitor Kin4 is located in
the mother cell, the MEN activator Lte1 is located in the
bud, and the MEN GTPase Tem1 is localized to the SPB
(Bardin et al. 2000; Pereira et al. 2000; D’Aquino et al.
2005; Maekawa et al. 2007). Only when the MEN-bearing
SPB escapes the MEN inhibitor Kin4 in the mother cell
and moves into the bud where the MEN activator Lte1
resides can exit from mitosis occur. In this manner,
spatial information is sensed and translated to regulate
Spindle position cannot be the only event controlling
MEN activity, as exit from mitosis occurs at the appro-
priate time in lte1D or kin4D cells with correctly posi-
tioned spindles. Here, we describe the identification of
a novel role for Cdc5 in regulating the timing of MEN
activation. Interestingly, this essential Cdc5-dependent
MEN-activating signal does not regulate the GTPase
Tem1, but rather the Tem1 effector Cdc15. We found
that Cdc5 is essential for the anaphase-specific recruit-
ment of Cdc15 to SPBs. Furthermore, the artificial
targeting of Cdc15 to the SPB bypasses the requirement
for both Tem1 and Cdc5 in MEN activation. Our results
indicate that multiple signals converge on the MEN
effector kinase Cdc15 to integrate spatial (spindle po-
sition) and temporal (Cdc5 activation) cues with mi-
totic exit. Thus, Cdc15 functions as a coincidence de-
tector, integrating spatial and temporal signals to ensure
that exit from mitosis only occurs after proper genome
LTE1 and KIN4-independent activation of the MEN
LTE1 and KIN4 are the central mediators of MEN
regulation by spindle position (Bardin et al. 2000; Pereira
et al. 2000; Castillon et al. 2003; D’Aquino et al. 2005;
Pereira and Schiebel 2005; Geymonat et al. 2009; Bertazzi
et al. 2011; Falk et al. 2011). The subcellular partitioning
of these two proteins ensures that cells that have a mis-
positioned anaphase spindle do not prematurely activate
the MEN. It is unclear, however, whether LTE1 and KIN4
are also important for regulating the proper timing of
MEN activation in cells where spindles are correctly
aligned along the mother–bud axis. To address this ques-
tion, we examined the consequences of deleting KIN4
and LTE1 on MEN activity. Cells were arrested in G1
using pheromone and then released to allow them to
progress through the cell cycle in a synchronous manner.
MEN activity was monitored by measuring the kinase
activity of the most downstream MEN kinase, Dbf2-
Mob1. Dbf2 kinase activity was restricted to anaphase
in wild-type cells (Fig. 1A,B; Toyn and Johnston 1994).
Similar results were obtained in lte1D kin4D cells (Fig.
1A,B). Thus, there must exist Kin4- and Lte1-independent
mechanisms that restrict MEN activity to anaphase in
cells with correctly positioned spindles.
Anaphase-specific activation of the MEN
in the absence of TEM1
Our data indicate that regulatory mechanisms other than
spindle position control MEN activity. To identify these
signals, we first asked whether, as in other GTPase
signaling cascades, all critical MEN regulation is medi-
ated by the GTPase Tem1. To this end, we measured Dbf2
kinase activity in cells lacking the essential MEN GTPase
Tem1 but kept alive by overexpression of CDC15 (hence-
forth tem1D CDC15-UP) (Pereira et al. 2000). Surprisingly,
growth of tem1D CDC15-UP cells was indistinguishable
from that of wild-type cells (Fig. 1C), and cell cycle pro-
gression occurred with near wild-type kinetics (Fig. 1D).
activity remained restricted to anaphase in tem1D CDC15-
Control of MEN activity by spindle position was,
however, lost in the tem1D CDC15-UP strain. Cells
lacking cytoplasmic dynein (dyn1D cells) frequently mis-
position their spindles at low temperature and arrest in
anaphase because the MEN GTPase Tem1 is inhibited by
Bub2–Bfa1 (for review, see Fraschini et al. 2008). When
the GAP is inactivated by deleting BUB2 or BFA1, cells
with mispositioned spindles will not arrest in anaphase,
but rather exit mitosis to produce anucleate and multi-
nucleate cells (Supplemental Fig. 1; Bardin et al. 2000;
Bloecher et al. 2000; Pereira et al. 2000). As in bub2D
cells, tem1D CDC15-UP cells did not arrest in anaphase
in response to spindle misposition (Supplemental Fig. 1).
Our data confirm that spindle position control of the
MEN is mediated by Tem1. Our data also indicate that
Rock and Amon
1944 GENES & DEVELOPMENT
the Tem1 GTPase is not the sole switch controlling MEN
activity and that there must exist GTPase-independent
mechanisms of MEN regulation that restrict Dbf2-Mob1
kinase activity to anaphase in an unperturbed cell cycle.
The FEAR network is not required for MEN activity
in tem1D CDC15-UP cells
The phosphatase Cdc14 is an activator of the MEN
(Jaspersen and Morgan 2000; Stegmeier et al. 2002; Konig
et al. 2010). Cdc14 activated by the FEAR network
dephosphorylates Cdc15 and Mob1 and thereby promotes
their activity (see Figure 7B, below). Although not essen-
tial for MEN activation, inactivation of the FEAR net-
work leads to a delay in MEN activation, as judged by
Dbf2 kinase activity (Stegmeier et al. 2002). To determine
whether the FEAR network was also required for MEN
activity in tem1D CDC15-UP cells, we examined the
consequences of deleting FEAR network components in
this strain. SPO12; its close homolog, BNS1; and SLK19
are components of the FEAR network; loss-of-function
mutations in these genes inactivate the FEAR network
and greatly reduce the release of Cdc14 from the nucle-
olus in early anaphase (Stegmeier et al. 2002; Visintin
et al. 2003). Deletion of these FEAR network components
did not affect the growth of tem1D CDC15-UP cells (Fig.
2A). More importantly, inactivation of the essential
FEAR network component Separase (Esp1) or the ulti-
mate FEAR network effector Cdc14 had a similar effect
on the kinetics of Dbf2 activation in tem1D CDC15-UP
cells as in wild-type cells. Dbf2 kinase activation was
delayed by ;10 min (Fig. 2B–D; Supplemental Fig. 2). Our
results indicate that the FEAR network regulates MEN
activity in tem1D CDC15-UP and wild-type cells in
a similar manner. Thus, the FEAR network promotes
but is not essential for the anaphase-specific activation of
the MEN in tem1D CDC15-UP cells.
Anaphase entry is not required for MEN activity
in the absence of TEM1
We next sought to determine the mechanism underlying
the GTPase-independent activation of the MEN in ana-
phase. We first asked whether entry into anaphase was
a prerequisite for MEN activation in tem1D CDC15-UP
cells. The fact that MEN activation occurred with similar
kinetics in tem1D CDC15-UP and tem1D CDC15-UP
esp1-1 cells (Supplemental Fig. 2), which cannot undergo
anaphase spindle elongation due to an inability to elim-
inate sister chromatid cohesion, already suggested that
absence of TEM1. (A,B) Wild-type (A2747) and lte1D kin4D
(A26379) cells containing 3HA-Cdc14 and 3MYC-Dbf2 fusion
proteins were arrested in G1 with a-factor pheromone (5 mg/mL)
in YEP medium containing glucose (YEPD). When the arrest was
complete (after 150 min), cells were released into pheromone-
free YEPD medium. After 80 min, a-factor pheromone (10 mg/
mL) was re-added to prevent entry into the subsequent cell
cycle. The percentage of cells with metaphase spindles (closed
squares; A), anaphase spindles (closed circles; A), and 3HA-
Cdc14 released from the nucleolus (open circles; A), and the
amount of Dbf2-associated kinase activity (Dbf2 kinase; B)
and immunoprecipitated 3MYC-Dbf2 (Dbf2 IP; B) were deter-
mined at the indicated times. (C) Wild-type (A2747) and tem1D
CDC15-UP (A22670) cells containing 3HA-Cdc14 and 3MYC-
Dbf2 fusion proteins were spotted on YEP plates containing
raffinose and galactose (YEPRG) at 30°C. Approximately 3 3 104
cells were deposited in the first spot, and each subsequent
spot is a 10-fold serial dilution. The picture shown depicts 3 d
of growth. (D,E) Wild-type (A2747) and tem1D CDC15-UP
(A22670) cells containing 3HA-Cdc14 and 3MYC-Dbf2 fusion
proteins were arrested in G1 with a-factor pheromone (5 mg/mL)
in YEPRG medium. When the arrest was complete (after 2 h 50
min), cells were released into pheromone-free YEPRG medium.
After 60 min, a-factor pheromone (10 mg/mL) was added to
prevent entry into the subsequent cell cycle. The percentage of
cells with metaphase spindles (closed squares; D), anaphase
spindles (closed circles; D), and 3HA-Cdc14 released from the
nucleolus (open circles; D), and the amount of Dbf2-associated
kinase activity (Dbf2 kinase; E) and immunoprecipitated 3MYC-
Dbf2 (Dbf2 IP; E) were determined at the indicated times. (F)
The amount of Dbf2-associated kinase activity and immuno-
precipitated 3MYC-Dbf2 from E was determined by quantitative
autoradiography and quantitative Western blot, respectively.
Shown is the specific Dbf2-associated kinase activity.
Anaphase-specific activation of the MEN in the
Cdc15 integrates Tem1 and Cdc5 signals
GENES & DEVELOPMENT1945
spindle elongation was not essential for MEN activation
in tem1D CDC15-UP cells.
To determine whether other aspects of anaphase entry
were necessary for MEN activation, we arrested tem1D
CDC15-UP cells in metaphase. Entry into anaphase is
triggered by the activation of a ubiquitin ligase known as
APC/CCdc20. Activation of the spindle assembly check-
point by microtubule depolymerization results in the
inhibition of APC/CCdc20and arrests cells in metaphase
(Musacchio and Salmon 2007). We found that entry into
anaphase was not required for Dbf2-Mob1 activation in
tem1D CDC15-UP cells. tem1D CDC15-UP cells acti-
vated Dbf2-Mob1 with nearly identical kinetics in the
presence or absence of the microtubule depolymerizing
drug nocodazole (Supplemental Fig. 3). Similar results
were obtained when anaphase entry was blocked by
depletion of the APC/C coactivator CDC20 (JM Rock,
unpubl.). Thus, although MEN activity is restricted to
anaphase in an unperturbed cell cycle, anaphase entry is
not a prerequisite for MEN activation in tem1D CDC15-
UP cells. In contrast, anaphase entry is required to ac-
tivate the MEN in cells with a wild-type MEN. Dbf2 ac-
tivation is greatly delayed in cdc23-1 mutants, which are
defective in APC/C activity (Visintin and Amon 2001).
We conclude that the dependence of MEN activation on
anaphase entry is mediated by the MEN GTPase Tem1.
However, the observation that MEN activation occurs 70
min after pheromone release irrespective of whether
tem1D CDC15-UP cells enter anaphase (Supplemental
Fig. 3) indicates that a Tem1 GTPase-independent MEN
regulatory timing mechanism must exist. Furthermore,
this timing mechanism must be independent of Separase
Polo kinase Cdc5 controls MEN activity in the absence
The Polo kinase Cdc5 is a key regulator of exit from
mitosis (Lee et al. 2005). As a component of the FEAR
network, Cdc5 promotes the release of Cdc14 from the
nucleolus during early anaphase, which then promotes
MEN activity (Stegmeier et al. 2002; Visintin et al. 2003).
Cdc5 also regulates the MEN GAP Bub2–Bfa1. Cdc5
phosphorylates Bfa1, which reduces Bub2–Bfa1 GAP
activity (Hu et al. 2001; Geymonat et al. 2003). Could
Cdc5 have additional roles in regulating the MEN and
conferMENactivationintem1D CDC15-UPcells? If Cdc5
was required for MEN activity in tem1D CDC15-UP cells,
then inactivating CDC5 should abrogate MEN activation.
Consistent with this hypothesis, we found that the tem1D
CDC15-UP allele combinationexhibitssyntheticlethality
with the temperature-sensitive cdc5-1 and cdc5-2 alleles
at the permissive temperature (data not shown). However,
we were able to construct a tem1D CDC15-UP cdc5-7
strain. We found that inactivation of CDC5 abolishes the
ability of the tem1D CDC15-UP strain to activate Dbf2-
essential to activate the MEN in the absence of TEM1.
Is Cdc5 also sufficient for MEN activation in a tem1D
CDC15-UP strain? Cdc5 protein levels are tightly regu-
lated during the cell cycle. Cdc5 is absent during G1,
begins to accumulate late in S phase, and peaks at the
metaphase-to-anaphase transition. During exit from mi-
tosis, Cdc5 is rapidly degraded by the APC/CCdh1(Charles
et al. 1998; Cheng et al. 1998; Shirayama et al. 1998). If
Cdc5 was limitingforMEN activationina tem1D CDC15-
UP strain, then the premature expression of Cdc5 might
result in the premature activation of the MEN. To test this
hypothesis, we expressed a stable form of Cdc5 (Cdc5Ddb)
from the conditional MET25 promoter in tem1D CDC15-
UP cells. We found that the premature accumulation of
Cdc5 results in the premature activation of Dbf2-Mob1 in
a tem1D CDC15-UP strain (Fig. 3C,D). It should be noted
in tem1D CDC15-UP cells. (A) Wild-type (A2747), tem1D CDC15-
UP (A22670), tem1D spo12D bns1D CDC15-UP (A23392), and
tem1D slk19D CDC15-UP (A23387) cells containing 3HA-Cdc14
and 3MYC-Dbf2 fusion proteins were spotted on YEPRG plates,
as in Figure 1C. (B,C) tem1D CDC15-UP (A23782) and tem1D
cdc14-3 CDC15-UP (A23712) cells containing a 3MYC-Dbf2
fusion protein were arrested in G1 with a-factor pheromone (5
mg/mL) in YEPRG medium at room temperature. Thirty min-
utes prior to release, the cells were shifted to 35°C. When the
arrest was complete (after 3 h 30 min), cells were released into
pheromone-free YEPRG medium at 35°C. After 65 min, a-factor
pheromone (10 mg/mL) was re-added to prevent entry into the
subsequent cell cycle. The percentage of cells with metaphase
spindles (closed squares, B) and anaphase spindles (closed circles,
B) and the amount of Dbf2-associated kinase activity (Dbf2
kinase, C) and immunoprecipitated 3MYC-Dbf2 (Dbf2 IP, C) were
determined at the indicated times. (D) The amount of Dbf2-
associated kinase activity and immunoprecipitated 3MYC-Dbf2
from C was determined as in Figure 1F. Shown is the specific
Dbf2-associated kinase activity. Note that the specific Dbf2-
associated kinase activity continues to rise in the tem1D cdc14-
3 CDC15-UP strain as a result of a prolonged anaphase arrest.
The FEAR network is not required for Dbf2 activity
Rock and Amon
1946GENES & DEVELOPMENT
that the premature activation of Dbf2-Mob1 upon
Cdc5Ddb expression is likely due to the premature ac-
tivation of both the FEAR network and the MEN. Our
results demonstrate that Cdc5 is essential for MEN ac-
tivation in the absence of Tem1 GTPase function. More-
over, Cdc5 is sufficient to stimulate MEN signaling in
other stages of the cell cycle.
Cdc5 promotes localization of Cdc15 to SPBs
To determine how Cdc5 controls MEN activity in the
absence of Tem1, we examined the consequences of
modulating Cdc5 activity on Cdc15 localization. Cdc15
localization in wild-type cells is dynamic. During G1, S,
G2, and metaphase, Cdc15 is localized diffusely through-
out the cytoplasm. Shortly after the metaphase-to-ana-
phase transition, Cdc15 localizes to the SPB that is pulled
into the daughter and, in late anaphase, is found on both
SPBs (Hu et al. 2001; Visintin and Amon 2001; Molk et al.
2004; Konig et al. 2010). Because Cdc15 recruitment to
SPBs coincides with MEN activation and depends on
TEM1, it is thought that localization of Cdc15 to SPB(s) is
essential for MEN function (Visintin and Amon 2001).
Although Cdc15 is highly overexpressed in the tem1D
CDC15-UP strain (these cells harbor two overexpression
constructs: GAL-CDC15 and GPD-CDC15), Cdc15 did
not localize to SPBs prematurely, and association with
this organelle remained largely restricted to anaphase
(Fig. 4A). The anaphase-restricted Cdc15 SPB localization
in tem1D CDC15-UP cells suggests a simple possible
mechanism by which Cdc5 activates the MEN in parallel
to Tem1: Cdc5 functions to promote Cdc15 SPB locali-
zation. To test this prediction, we followed Cdc15 local-
ization in tem1D CDC15-eGFP-UP cells containing an
inhibitor-sensitive allele of CDC5 (cdc5-as1). In the
presence of the inhibitor, Cdc15 is no longer able to
localize to SPBs in the tem1D CDC15-eGFP-UP cdc5-as1
cells (Fig. 4B). As CDC5 is sufficient to activate the MEN
in theabsence of Tem1(Fig. 3D), it might be expected that
the premature expression of Cdc5 results in the premature
loading of Cdc15 onto SPBs. Indeed, we found that the
premature activation of Cdc5 with the CDC5Ddb allele
led to the premature recruitment of Cdc15 to SPBs in
metaphase (Fig. 4C). Taken together, these data indicate
that CDC5 functions in parallel to TEM1 to promote the
association of Cdc15 with SPBs.
Cdc15 functions as a coincidence detector of Tem1
and Cdc5 activity
Our data suggest that both CDC5 and TEM1 function to
promote Cdc15 SPB localization. If true, Cdc15 could
function as a coincidence detector of Cdc5 and Tem1
activity. By this model, wild-type levels of Cdc15 might
integrate essential inputs from Tem1 and Cdc5, both of
which are required for MEN activation. A prediction of
this hypothesis is that both Tem1 and Cdc5 should be
essential for Cdc15 SPB localization and Dbf2-Mob1
activity in a wild-type cell. We first monitored Cdc15
localization in a strain depleted of Tem1 but wild-type for
CDC5. Consistent with previously published data, de-
pletion of Tem1 abolishes the localization of Cdc15 to
SPBs (Fig. 5A; Johnson et al. 1992; Visintin and Amon
2001). CDC5 was also essential for Cdc15 association
with SPBs. Cdc15 did not localize to SPBs in anaphase
cells depleted of Cdc5 (Fig. 5B). Similar results were
obtained in bub2D cells depleted of Cdc5 (Fig. 5B).
Importantly, depletion of Cdc5 did not affect Tem1
localization to the SPB (Supplemental Fig. 4). These
findings exclude the possibility that Cdc5 affects Cdc15
SPB localization indirectly by inactivating the Bub2–Bfa1
GAP complex or perturbing Tem1 SPB localization.
absence of TEM1. (A,B) tem1D CDC15-UP (A22670) and tem1D
cdc5-7 CDC15-UP (A24305) cells containing 3HA-Cdc14 and
3MYC-Dbf2 fusion proteins were arrested in G1 with a-factor
pheromone (5 mg/mL) in YEPRG medium at 30°C. Thirty
minutes prior to release, the cells were shifted to 37°C. When
the arrest was complete (after 3 h), cells were released into
pheromone-free YEPRG medium at 37°C. After 65 min, a-factor
pheromone (10 mg/mL) was added to prevent entry into the
subsequent cell cycle. The percentage of cells with metaphase
spindles (closed squares; A) and anaphase spindles (closed
circles; B) and the amount of Dbf2-associated kinase activity
(Dbf2 kinase; B) and immunoprecipitated 3MYC-Dbf2 (Dbf2 IP;
B) were determined at the indicated times. (C,D) tem1D CDC15-
UP (A22670) and tem1D MET25-CDC5Ddb CDC15-UP (A25175)
cells containing 3HA-Cdc14 and 3MYC-Dbf2 fusion proteins
were arrested in G1 with a-factor pheromone (5 mg/mL) in
YEPRG medium. Ninety minutes prior to release, the cells were
transferred to ?Met medium containing raffinose and galactose
(?MetRG; to induce the expression of Cdc5Ddb) supplemented
with a-factor pheromone (5 mg/mL). When the arrest was com-
plete (after 3 h), cells were released into pheromone-free ?MetRG
medium. After 70 min, a-factor pheromone (10 mg/mL) was re-
added to prevent entry into the subsequent cell cycle. The per-
centage of cells with metaphase spindles (closed squares; C) and
anaphase spindles (closed circles; C) and the amount of Dbf2-
associated kinase activity (Dbf2 kinase; D) and immunoprecipitated
3MYC-Dbf2 (Dbf2 IP; D) were determined at the indicated times.
Polo-like kinase Cdc5 controls MEN activity in the
Cdc15 integrates Tem1 and Cdc5 signals
GENES & DEVELOPMENT 1947
To further validate an essential role for Cdc5 in
activating the MEN in wild-type cells, we monitored
depleted for Cdc5. To control for Cdc5’s role in activating
the FEAR network and in inactivating Bub2–Bfa1, these
experiments were performed in a cdc14-3 bub2D back-
ground. The BUB2 deletion eliminates the role of CDC5
in MEN GAP down-regulation, and the cdc14-3 mutation
eliminates Cdc5-dependent FEAR network activation. As
expected, Dbf2 kinase activity peaked in anaphase in the
tem1D CDC15-eGFP-UP (A25630) cells containing a mCherry-
Tub1 fusion protein were arrested in G1 with a-factor phero-
mone (5 mg/mL) in YEPRG medium. When the arrest was
complete (after 2 h 50 min), cells were released into phero-
mone-free YEPRG medium and imaged after a brief paraformal-
dehyde fixation. Cell cycle stage was determined based on
spindle morphology and correlated with Cdc15 localization at
SPBs (n $ 100 cells for each cell cycle stage). Representative
images of G1/S, metaphase, and anaphase cells are shown.
Cdc15 is shown in green, microtubules are shown in red, and
DNA is shown in blue. (B) tem1D CDC15-eGFP-UP (A25630)
and tem1D CDC15-eGFP-UP cdc5-as1 (A25633) cells contain-
ing a mCherry-Tub1 fusion protein were arrested in G1 as in A.
Cells were released into pheromone-free YEPRG medium sup-
plemented with 5 mM CMK (cdc5-as1 inhibitor). Cells were
scored as in A. Representative images of anaphase cells are shown.
(C) tem1D CDC15-eGFP-UP (A25744) and tem1D CDC15-eGFP-
UP MET25-CDC5DN70 (tem1D CDC15-eGFP-UP CDC5-UP;
A25983) cells containing a Spc42-mCherry fusion protein were
arrested in G1 with a-factor pheromone (5 mg/mL) in YEPRG
medium supplemented with 8 mM methionine. Ninety minutes
prior to release, the cells were transferred to ?MetRG medium (to
induce the expression of Cdc5DN70) supplemented with a-factor
pheromone. When the arrest was complete (after 3 h), cells were
released into pheromone-free ?MetRG medium. Cells were
imaged and scored as in A. Representative images of metaphase
cells are shown. Cdc15 is shown in green, Spc42 is shown in red,
and DNA is shown in blue.
Cdc5 promotes localization of Cdc15 to SPBs. (A)
and Cdc5 activity. (A) CDC15-eGFP (A26481) and CDC15-eGFP
GAL-UPL-TEM1 (A27055) cells containing a mCherry-Tub1
fusion protein were arrested in G1 with a-factor pheromone (5
mg/mL) in YEPRG medium. Ubiquitin-proline-LacZ (UPL) acts
as a destabilizing module that permits rapid degradation of
appended proteins. One hour prior to release, glucose was added
to a final concentration of 2% (to repress expression of GAL-
UPL-TEM1). When the arrest was complete (after 2 h 40 min),
cells were released into pheromone-free YEPD medium. Cells
were imaged and scored as in Figure 4A. (B) CDC15-eGFP
(A26481), CDC15-eGFP bub2D (A26480), CDC15-eGFP GAL-
URL-3HA-CDC5 (A26556), and CDC15-eGFP bub2D GAL-URL-
3HA-CDC5 (A26558) cells containing a mCherry-Tub1 fusion
protein were arrested in G1 with a-factor pheromone (5 mg/mL)
in YEPRG medium. Ubiquitin-arginine-LacZ (URL) acts as a desta-
bilizing module that permits rapid degradation of appended pro-
teins. Two hours prior to release, glucose was added to a final
concentration of 2% (to repress expression of GAL-URL-3HA-
CDC5). When the arrest was complete (after 2 h 45 min), cells
were released into pheromone-free YEPD medium. Cells were
imaged and scored as in Figure 4A. (C,D) bub2D cdc14-3 (A26844)
and bub2D cdc14-3 GAL-URL-3HA-CDC5 (A26842) cells contain-
ing a 3MYC-Dbf2 fusion protein were arrested in G1 with a-factor
pheromone (5 mg/mL) in YEPRG medium. Two hours prior to
release, glucose was added to repress expression of GAL-URL-
3HA-CDC5. When the arrest was complete (after 2 h 45 min), cells
were released into pheromone-free YEPD medium. The percentage
of cells with metaphase spindles (closed squares; C) and anaphase
spindles (closed circles; C) and the amount of Dbf2-associated
kinase activity (Dbf2 kinase; D) and immunoprecipitated 3MYC-
Dbf2 (Dbf2 IP; D) were determined at the indicated times.
Cdc15 functions as a coincidence detector of Tem1
Rock and Amon
1948 GENES & DEVELOPMENT
cdc14-3 bub2D strain (Fig. 5C,D). Consistent with the
Cdc15 localization observations, Dbf2-Mob1 was not
activated in the cdc14-3 bub2D strain depleted of Cdc5
(Fig. 5C,D). We conclude that Cdc5 is essential for MEN
activation and regulates this pathway at multiple steps.
Cdc5 stimulates MEN activity through its role in the
FEAR network, partially inhibits the Tem1 GAP Bub2–
Bfa1, and promotes the localization of Cdc15 to SPBs. Our
data further indicate that Cdc15 behaves like a coinci-
dence detector, requiring inputs from both Tem1 and
Cdc5 to localize to the SPB and thus activate the MEN.
Targeting Cdc15 to the SPB bypasses the need for both
Tem1 and Cdc5 in MEN activation
Localization of Cdc15 to the SPB is thought to be
essential for MEN activation (Stegmeier and Amon
2004). Our observations suggest that the essential
MEN-activating function of both Tem1 and Cdc5 is to
promote Cdc15 SPB localization. To test this possibility,
we asked whether artificially targeting Cdc15 to SPBs
bypasses the need for Tem1 and Cdc5 in MEN activation.
We fused the CDC15-eGFP ORF to the ORF of the SPB
outer plaque component CNM67 to generate a Cdc15-
eGFP-Cnm67 fusion protein (hereafter referred to as
Cdc15-SPB). Expression of the fusion protein from the
CDC15 promoter is lethal (data not shown). We therefore
placed Cdc15-SPB under the transcriptional control of the
low-strength conditional MET3 promoter. Induction of
the Cdc15-SPB fusion was toxic (data not shown), but the
fusion protein was well tolerated when the MET3 pro-
moter was repressed. Under these conditions, the Cdc15-
SPB fusion protein was detectable by fluorescence mi-
croscopy (Fig. 6C) but was not detectable by Western blot
for TEM1 and CDC5 in MEN activation. (A) CDC15-
eGFP (CDC15; A20935), pMET3-CDC15-eGFP-
CNM67 (CDC15-SPB; A26417), and CDC15-eGFP-
UP (CDC15-UP; A25515) cells were grown to log
phase in either YEPRG + methionine (+MET) or ?Met
medium to determine the amount of Cdc15-eGFP
(a-GFP) in cells. Kar2 was used as a loading control
in Western blots. (B) Wild-type (A2587), cdc15-2
(A2597), pMET3-CDC15-eGFP-CNM67 (CDC15-SPB;
A26419), and pMET3-CDC15-eGFP-CNM67 cdc15-2
(CDC15-SPB cdc15-2; A26413) cells were spotted on
YEPRG plates supplemented with 8 mM methionine
as in Figure 1C. The picture shown depicts 2 d of
growth at 37°C and 3 d of growth at 23°C. (C) pMET3-
CDC15-eGFP-CNM67 (CDC15-SPB; A26486) cells
containing a mCherry-Tub1 fusion protein were grown
to log phase in YEPRG medium supplemented with
8 mM methionine and imaged after a brief parafor-
maldehyde fixation. Representative images of G1/S,
metaphase, and anaphase cells are shown. (D) Wild-
type (A2747), tem1D CDC15-UP (A22670), and tem1D
A26396) cells containing 3HA-Cdc14 and 3MYC-
Dbf2 fusion proteins were spotted on YEPRG plates
supplemented with 8 mM methionine as in Figure
1C. The picture shown depicts 3 d of growth. (E,F)
Wild-type (A2747) and tem1D GAL-URL-3HA-CDC5
3HA-CDC5 CDC15-SPB; A27051) cells containing
3HA-Cdc14 and 3MYC-Dbf2 fusion proteins were
arrested in G1 with a-factor pheromone (5 mg/mL) in
YEPRG medium supplemented with 8 mM methio-
nine. Two hours prior to release, glucose was added (to
repress expression of GAL-URL-3HA-CDC5). When
the arrest was complete (after 2 h 50 min), cells were
released into pheromone-free YEPD medium supple-
mented with 8 mM methionine. After 65 min, a-factor
pheromone (10 mg/mL) was added to prevent entry into
the subsequent cell cycle. The percentage of cells with
metaphase spindles (closed squares; E) and anaphase
Targeting Cdc15 to SPBs bypasses the need
spindles (closed circles; E) and the amount of Dbf2-associated kinase activity (Dbf2 kinase; F) and immunoprecipitated 3MYC-Dbf2 (Dbf2
IP; F) were determined at the indicated times. (G) The amount of Dbf2-associated kinase activity and immunoprecipitated 3MYC-Dbf2
from F was determined as in Figure 1F. Shown is the specific Dbf2-associated kinase activity.
Cdc15 integrates Tem1 and Cdc5 signals
GENES & DEVELOPMENT 1949
analysis (Fig. 6A, lane marked with asterisk). The fusion
protein was nevertheless present at high enough levels
under MET3-repressive conditions to allow the necessary
experimental manipulations to follow. We therefore per-
formed all experimentsinvolvingthis fusion protein under
conditions where the MET3 promoter was repressed.
First, we confirmed the functionality of the fusion.
While we were not able to measure kinase activity associ-
ated with the Cdc15-SPB fusion protein (presumably be-
cause the Cdc15-SPB protein is tightly bound to the SPB
and thus is not amenable to standard immunoprecipita-
tion kinase techniques), the CDC15-SPB fusion sup-
pressed the temperature-sensitive lethality of cells har-
boring the cdc15-2 allele as the sole source of CDC15 (Fig.
6B). Thus, the Cdc15-SPB protein is active as a kinase and
is capable of performing the essential function of Cdc15.
The fusion protein also exhibited the expected localiza-
tion pattern. Cdc15-SPB localizes to the SPB constitu-
tively throughout the cell cycle (Fig. 6C; Supplemental
Fig. 5). To determine whether the Cdc15-SPB fusion can
support the essential functions of TEM1 and CDC5 in
MEN activation, we constructed a tem1D GAL-URL-
3HA-CDC5 CDC15-SPB strain in which TEM1 was de-
leted and Cdc5 could be efficiently depleted (Bachmair
et al. 1986). We found that tem1D cells are viable when
they harbor the CDC15-SPB fusion (Fig. 6D); thus, the
essential function of TEM1 can be bypassed by the
CDC15-SPB allele. To determine whether CDC5 func-
tion in MEN activation was also bypassed by the Cdc15-
SPB fusion protein, we examined Dbf2 kinase activity in
tem1D cells that were also depleted for Cdc5. Strikingly,
provision of the CDC15-SPB allele in the tem1D GAL-
URL-3HA-CDC5 strain suppressed the defect in Dbf2-
Mob1 activation observed in cells that lack TEM1 or
CDC5 (cf. Figs. 5D and 6E–G; Visintin and Amon 2001).
Moreover, Dbf2 kinase activity was both premature and
hyperactive in this strain (Fig. 6F,G). Similar results were
obtained in wild-type cells expressing the Cdc15-SPB
fusion (Supplemental Fig. 6).
Our analysis of a C-terminally truncated CDC15 allele
[GAL-GFP-CDC15(1–750)] is consistent with the idea
that targeting Cdc15 to SPBs bypasses the requirement
for both Tem1 and Cdc5 in MEN activation (Bardin et al.
2003). Like the Cdc15-SPB fusion, Cdc15(1–750) localized
to the SPB throughout the cell cycle in a manner in-
dependent of Tem1 and Cdc5 (Supplemental Fig. 7A).
Consistent with these observations, we found that Dbf2
kinase was both premature and hyperactive upon over-
expression of Cdc15(1–750). Moreover, the overexpression
of Cdc15(1–750) was sufficient to activate Dbf2-Mob1 in
the absence of Cdc5 kinase activity (Supplemental Fig.
Interestingly, Dbf2 kinase activity still fluctuates dur-
ing the cell cycle in cells in which Cdc15 localizes to SPBs
constitutively (Supplemental Figs. 6–8). Thus, Dbf2-
Mob1 kinase activity must be regulated by mechanisms
in addition to Cdc15 SPB recruitment (see the Discus-
sion). It should also be noted that, despite premature and
hyperactive Dbf2 kinase activity in Cdc15-SPB-express-
ing cells, Cdc14 release from the nucleolus remained
restricted to anaphase (Supplemental Figs. 6A, 8). This
indicates that yet additional mechanisms control Cdc14
localization downstream from and/or in parallel to Dbf2-
Mob1 (see the Discussion). We conclude that the sole
essential MEN-activating function of both TEM1 and
CDC5 is to target Cdc15 to SPBs.
Multiple signals converge on Cdc15 to integrate MEN
activity with other cellular events
The MEN is essential for exit from mitosis. The MEN
GTPase Tem1 has been assumed to be the central switch
in MEN regulation. We show here that robust MEN
regulation occurs in a GTPase-independent manner and
identify the Tem1 effector Cdc15 as an integrator of cell
cycle signals. Cdc15 behaves like a coincidence detector
(Fig. 7A), integrating inputs from two essential cell cycle
oscillators: the Tem1 GTPase cycle and the Polo kinase
Cdc5 synthesis/degradation cycle. The Cdc15-dependent
integration of these temporal (Cdc5 and Tem1 activity)
and spatial (Tem1 activity) signals ensures that exit from
mitosis occurs only after proper genome partitioning.
Indeed, reliance on the timing signal alone (tem1D
CDC15-UP) results in the inability to coordinate MEN
activity with spindle position and the inappropriate exit
from mitosis in the presence of a mispositioned anaphase
spindle (Supplemental Fig. 1). Tem1 and Cdc5 activity are
read by the ability of Cdc15 to associate with the SPB.
from mitosis with spatial and temporal cues. (A)
Cdc15 functions as a coincidence detector of Tem1
and Cdc5 activity, both of which are required for
the association of Cdc15 with SPBs. See the text
for details. (B) Multiple signals control MEN activ-
ity. The core MEN components are shown in blue,
activators of the MEN are shown in green, and
inhibitors of the MEN are shown in red. Experi-
mentally validated interactions are shown with
solid lines, and more speculative interactions are
shown with dashed lines. See the text for details.
A model for the coordination of exit
Rock and Amon
1950GENES & DEVELOPMENT
Artificially targeting Cdc15 to SPBs by fusing Cdc15 to an
integral SPB component (Cdc15-SPB) bypasses the re-
quirement for both proteins in MEN activation. Thus, it
appears that recruitment of Cdc15 to SPBs is the essential
function of Cdc5 and Tem1 in MEN activation.
It is unclear why Cdc15 recruitment to SPBs is essen-
tial for MEN activity. Cdc15 kinase activity, at least as
measured by in vitro immunoprecipitation kinase assays,
does not change during the cell cycle (Jaspersen et al.
1998). It is possible that Cdc15 could be activated by
a SPB-associated protein, but such activation may not be
detectable using standard immunoprecipitation kinase
assay conditions. An alternative but not mutually exclu-
sive possibility is that a SPB scaffold, such as Nud1, may
be required to increase the efficiency of interaction
between MEN components to promote Cdc15-dependent
Dbf2-Mob1 activation. Although we do not yet know
why Cdc15 must associate with SPBs, we have some
understanding of how this association occurs. Tem1
recruits Cdc15 to SPBs via a region in Cdc15 immediately
adjacent to its kinase domain (Asakawa et al. 2001). How
Cdc5 promotes Cdc15 SPB localization is unknown.
Preliminary data suggest that Cdc15 is not a Cdc5 sub-
affected by modulating CDC5 activity (JM Rock,
unpubl.). In addition, mutation of Cdc5 consensus bind-
ing sites (SSP to AAP) in Cdc15 did not abrogate Cdc15-
dependent MEN activation (JM Rock, unpubl.). These
results and several other observations raise the possibility
that the putative SPB anchor for Cdc15, Nud1, might
be Cdc5’s essential MEN-activating target: (1) Nud1 is
thoughtto bind to and recruit Cdc15 to the SPB (Stegmeier
and Amon 2004), (2) nud1 temperature-sensitive mu-
tants arrest in late anaphase with an inactive MEN
(Gruneberg et al. 2000; Visintin and Amon 2001), (3)
Nud1 is a substrate of Cdc5 in vivo and in vitro (Maekawa
et al. 2007; Park et al. 2008), and (4) Nud1 hyperphos-
phorylation coincides with Cdc15 recruitment to SPBs
(Visintin et al. 2003; Maekawa et al. 2007; Park et al.
2008). Cdc5 could phosphorylate Nud1 in mitosis,
thereby creating a phospho-dependent SPB-binding site
for Cdc15. As Nud1 is the most extensively phosphory-
lated SPB component (>50 phosphosites) (Keck et al.
2011), testing this hypothesis will be extremely challeng-
ing. It is important to note, however, that the CDC15-
SPB fusion does not suppress the temperature-sensitive
lethality of cells harboring the nud1-44 allele as the sole
source of NUD1 (JM Rock, unpubl.). Thus, unlike TEM1
and CDC5, NUD1 has essential roles in MEN signaling in
addition to recruiting Cdc15 to SPBs.
32P incorporation into Cdc15 in vivo was not
Novel temporal regulators of the MEN
Our data indicate that MEN activity is regulated by
multiple inputs (Fig. 7B). The dependence of MEN activ-
ity on CDC5 ensures that the MEN can only be activated
during G2 and mitosis, when Cdc5 is active. Our data also
indicate that restricting MEN activity to anaphase is
mediated by the GTPase Tem1. In wild-type cells arrested
in metaphase, Dbf2-Mob1 activity remains low. In tem1D
CDC15-UP cells arrested in metaphase, however, Dbf2-
Mob1 is activated. Thus, an unknown anaphase event,
likely under the control of the APC/CCdc20, must be
responsible for activating Tem1 at anaphase onset or
keeping Tem1 inactive in earlier cell cycle stages. While
the FEAR network contributes to activating the MEN in
anaphase, the subtle effects of inactivating the FEAR
network on mitotic exit kinetics argues that alternative
pathways must regulate Tem1 activity.
As elaborated in this study, Cdc5 regulates the cell
cycle-dependent localization of Cdc15 to SPBs. Despite
the importance of regulating Cdc15 recruitment to SPBs,
it is clear that additional mechanisms function down-
stream from and/or in parallel to Cdc15 to regulate exit
from mitosis. Our data suggest that Dbf2 kinase activity
is controlled by mechanisms in addition to Cdc15 re-
cruitment to SPBs. Even though Dbf2 is hyperactive and
active well before metaphase in CDC15-SPB cells, Dbf2
kinase activity nevertheless fluctuates during the cell
cycle, being low in G1 and peaking in early anaphase
(Supplemental Figs. 6–8). Thus, there must exist a signal
that promotes Dbf2 kinase activity as cells progress
through S phase and mitosis or inhibits Dbf2 kinase
activity in G1. Given that Dbf2-Mob1 kinase activity
mirrors Clb-CDK activity in CDC15-SPB and GAL-GFP-
CDC15(1–750) cells, it is tempting to speculate that Clb-
CDKs directly or indirectly control Dbf2 kinase activity
in these cells.
Our data also indicate that Dbf2 kinase activation is
necessary but not sufficient to promote Cdc14 release
from the nucleolus. In CDC15-SPB cells, Dbf2-specific
activity is more than five times that seen in wild-type
cells, and substantial Dbf2-Mob1 kinase activity (equal to
the peak seen in a wild-type cell cycle) is achieved well
before metaphase in the CDC15-SPB strain. In GAL-GFP-
CDC15(1–750) cells, the difference is even more striking,
with Dbf2-specific activity levels >43 times that seen in
wild-type cells. The difference in Dbf2-specific activity in
these strains is likely due, at least in part, to the much
higher expression levels of the GAL-GFP-CDC15(1–750)
construct as compared with the MET3-CDC15-SPB con-
struct. Despite premature and hyperactive Dbf2 kinase
activity, Cdc14 is not released prematurely in these
strains (Supplemental Figs. 6–8). The mechanisms that
restrict Dbf2-Mob1-dependent Cdc14 release to anaphase
are unknown. Given that the overexpression of Cdc5 in
combination with the premature activation of the MEN
is sufficient to drive Cdc14 out of the nucleolus in any
cell cycle stage (Manzoni et al. 2010), we propose that
Cdc5 plays yet an additional key role in regulating Cdc14
release downstream from and/or in parallel to Dbf2-
Logic of MEN activation
Our results and those of previous studies suggest the
following model for how MEN activity is restricted to
anaphase and coupled to accurate spindle position by the
integration of multiple spatial and temporal cues (Fig. 7B).
As cells approach the metaphase-to-anaphase transition
Cdc15 integrates Tem1 and Cdc5 signals
GENES & DEVELOPMENT 1951
and Cdc5 kinase reaches high levels of activity, Cdc5
phosphorylates an as-yet-unidentified target, which
primes the MEN for activation by creating conditions
that promote theassociation of Cdc15 with the SPB. Cdc5
also phosphorylates Bub2–Bfa1, thereby lowering its GAP
activity. At the metaphase-to-anaphase transition, Cdc14
activated by the FEAR network dephosphorylates Cdc15
and Mob1, thereby stimulating MEN activity. This cou-
ples full MEN activation with the onset of chromosome
segregation, as components of the FEAR network are not
only MEN activators but are also essential for inducing
chromosome segregation. Additional unknown signals
regulate Tem1 and Dbf2-Mob1 to restrict their activity
to anaphase. Finally, spindle position is integrated with
MEN regulation via Tem1. As the spindle elongates along
the mother–daughter axis, the Tem1-bearing SPB leaves
the MEN inhibitory zone in the mother cell (defined by
Kin4) and enters the MEN-activating zone in the bud
(defined by Lte1). This allows for the activation of Tem1
and recruitment of Cdc15 to SPBs. Additional signals
functioning downstream from and/or in parallel to Dbf2-
Cdc14 from the nucleolus in anaphase in a sustained
manner. While much remains to be learned about MEN
regulation, it is clear that Cdc15 integrates both temporal
(Cdc5 and Tem1) and spatial (Tem1) signals to mediate the
robust and timely activation of the MEN in late anaphase.
MEN-like signaling pathways in other eukaryotes
The MEN is conserved in fission yeast, where it is called
the septation initiation network (SIN) and regulates
cytokinesis. Does Plo1 (Cdc5 homolog) regulate the SIN
in a manner similar to the way Cdc5 regulates the MEN?
plo1+has been shown genetically to act as an activator of
the SIN and placed to function upstream of spg1+(Tem1
homolog) (Tanaka et al. 2001). That said, the strong
similarities between the MEN and SIN, and particularly
between Saccharomyces cerevisiae Cdc15 and its homo-
log in Schizosaccharomyces pombe, Cdc7, suggest that
Plo1 may also regulate the association of Cdc7 with SPBs.
Cdc7 localizes to SPBs in mitosis, and this localization is
regulated by both Spg1 and Plo1 (Sohrmann et al. 1998;
Mulvihill et al. 1999). Both Cdc15 and Cdc7 can associate
with SPBs in at least two ways: One is mediated by
a GTPase interaction domain, and the other is mediated
by an independent SPB localization domain (Asakawa
et al. 2001; Bardin et al. 2003; Mehta and Gould 2006).
Consistent with both modes of SPB localization being cell
cycle-regulated, localization of Cdc7 to SPBs is restricted
to mitosis even when Cdc7 is overexpressed. Finally,
while the Cdc7–Spg1 interaction is essential for SIN
activation in wild-type cells, overexpression of cdc7+
can suppress the lethality of a strain deleted for spg1+
(Schmidt et al. 1997). Thus, just as is the case for the
MEN, there must exist GTPase-independent mecha-
nisms of SIN activation, and these mechanisms might
be mediated by Polo kinase.
The core MEN signaling module—consisting of Cdc15,
Dbf2, Mob1, and Nud1—also exists in higher eukaryotes.
In higher eukaryotes, these proteins are known as mam-
malian sterile-20-related kinases (MSTs; Cdc15 homo-
log), nuclear Dbf2-related kinases (NDRs; Dbf2 homolog),
Mob1 coactivators, and scaffolding (Nud1 homolog) fam-
ilies. While there are few known roles for these proteins
in regulating mitotic exit (Bothos et al. 2005), they are
essential components of signaling pathways that regulate
a multitude of other cellular processes. As part of the
Hippo pathway, this signaling module is essential for the
proper regulation of organ growth in Drosophila and
vertebrates (Halder and Johnson 2011). Like their fungal
counterparts, human NDR kinases and their Mob1 coac-
tivators localize to centrosomes, the mammalian equiv-
alent of SPBs (Hergovich et al. 2007; Wilmeth et al. 2010).
Intriguingly, as is the case in S. cerevisiae (Luca et al.
2001; JM Rock, unpubl.), the localization of Mob1 iso-
forms to the centrosome is dependent on Polo-like kinase
1 activity (Wilmeth et al. 2010). Finally, we note that
overexpression of human NDR1 results in centrosome
overduplication as does overexpression of Polo-like ki-
nase 4 (Plk4) (Habedanck et al. 2005; Hergovich et al.
2007). This raises the possibility that Plk4 plays a role in
activating the MST/NDR1 signaling cascade. It will be
interesting to explore whether or not Polo kinase acti-
vates NDR kinase signaling in higher eukaryotes.
Materials and methods
Yeast strains and growth conditions
All strains are derivatives of W303 (A2587) and are listed in
Supplemental Table S1. Growth conditions are described in the
All plasmids used in this study are listed in Supplemental Table
S2. Specifics of plasmid construction are detailed in the Supple-
For immunoblot analysis of Cdc15-eGFP, Cdc15-eGFP-Cnm67,
GFP-Cdc15, GFP-Cdc15(1-750), Pgk1, and Kar2, cells were in-
cubatedfor a minimum of 10 minin 5% trichloroacetic acid. The
acid was washed away with acetone, and cells were pulverized
with glass beads in 166 mL of lysis buffer (50 mM Tris-Cl at pH
7.5, 1 mM EDTA, 2.75 mM DTT, complete protease inhibitor
cocktail [Roche]) using a bead mill. Sample buffer was added, and
the cell homogenates were boiled. Cdc15-eGFP, Cdc15-eGFP-
Cnm67, GFP-Cdc15, and GFP-Cdc15(1–750) were detected using
an anti-GFP antibody (Clontech, JL-8) at a 1:1000 dilution. Pgk1
was detected using an anti-Pgk1 antibody (Invitrogen) at a 1:5000
dilution. Kar2 was detected using a rabbit anti-Kar2 antiserum
(Rose et al. 1989) at a 1:200,000 dilution.
Dbf2 kinase assays
Dbf2 kinase assays were performed as described previously
(Visintin and Amon 2001) with the following modifications:
Approximately 1.5 mg of total protein was used per immuno-
precipitation, and kinase reactions were incubated for 45 min
with gentle mixing. Histone H1 phosphorylation was quantified
Rock and Amon
1952GENES & DEVELOPMENT
using the PhosphorImaging system. Western blots were quanti-
fied using ECL Plus (GE Healthcare) and fluorescence imaging.
Indirect in situ immunofluorescence methods to detect Tub1
were performed as previously described (Kilmartin and Adams
1984). For imaging of Cdc15-eGFP and Cdc15-eGFP-Cnm67,
cells were fixed for 2 min in 4% paraformaldehyde (in 3.4%
sucrose solution). Cells were washed once in KPO4/sorbitol
(1.2 M sorbitol, 0.1 M KPO4 at pH 7.5) and resuspended in
KPO4/sorbitol supplemented with 1% Triton. Prior to imaging,
cells were stained with Prolong Gold anti-fade reagent (Invitro-
gen, P36935). Cells were imaged within 24 h on a Zeiss Axioplan
2 microscope and a Hamamatsu OCRA-ER digital camera.
Flow cytometric DNA quantitation was performed as described
by Haase and Reed (2002).
We thank Frank Solomon, Iain Cheeseman, Rosella Visintin,
and members of the Amon laboratory for comments on the
manuscript. This work was supported by the National Institutes
of Health (GM056800 to A.A.)and an NSF Predoctoral Fellowship
(to J.M.R.). A.A. is an investigator of the Howard Hughes Medical
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