Expression of a form of the mitotic Clb2 cyclin that was resistant to ubiquitin-mediated protein degradation arrests cells in late anaphase, after the completion Keywords anaphase; Cdc14
Trang 1A decade of Cdc14 – a personal perspective
Delivered on 9 July 2007 at the 32nd FEBS Congress in Vienna,
Austria
Angelika Amon
David H Koch Institute for Integrative Cancer Research, Cambridge, MA, USA
Discovery of Cdc14 as the key
regulator of exit from mitosis in
budding yeast
In 1995 my scientific life changed I was given the
unique opportunity to become a Whitehead Fellow
The Whitehead Fellow’s Program allows young
scien-tists, mostly straight out of graduate school, to pursue
their independent research program At the time I was
a postdoctoral fellow in Ruth Lehman’s laboratory
and Ruth had decided to move to New York I could
not go with her (and also realized that flies were not
my thing) and decided to give this a try But what
should I work on?
As a graduate student in Kim Nasmyth’s labora-tory I had studied the regulation of cyclin-dependent kinases (CDKs), which are composed of a catalytic kinase subunit and a regulatory cyclin subunit A specific form of these CDKs, the mitotic CDKs (Clb1-, Clb2-, Clb3- and Clb4–CDKs in budding yeast), promote entry into mitosis [1] Previous work
by Andrew Murray and Marc Kirschner had shown that degradation of the regulatory mitotic cyclin sub-unit was required for exit from mitosis in Xenopus [2] I could show that this was also the case in yeast Expression of a form of the mitotic Clb2 cyclin that was resistant to ubiquitin-mediated protein degradation arrests cells in late anaphase, after the completion
Keywords
anaphase; Cdc14; cell cycle; chromosome
segregation; FEAR network; mitosis; mitotic
exit; mitotic exit network; nucleolus;
phosphatase
Correspondence
A Amon, David H Koch Institute for
Integrative Cancer Research, Howard
Hughes Medical Institute, Massachusetts
Institute of Technology, E17-233, 40 Ames
Street, Cambridge, MA 02139, USA
Fax: +1 617 258 6558
Tel: +1 617 258 8964
E-mail: angelika@mit.edu
(Received 29 July 2008, revised 15
September 2008, accepted 18 September
2008)
doi:10.1111/j.1742-4658.2008.06693.x
In budding yeast, the protein phosphatase Cdc14 is a key regulator of late mitotic events Research over the last decade has revealed many of its func-tions and today we know that this protein phosphatase orchestrates several aspects of chromosome segregation and is the key trigger of exit from mitosis Elucidation of the mechanisms controlling Cdc14 activity through nucleolar sequestration now serves as a paradigm for how regulation of the subcellular localization of proteins regulates protein function Here I review these findings focusing on how discoveries in my laboratory helped eluci-date the function and regulation of Cdc14
Abbreviations
APC ⁄ C, anaphase promoting complex or cyclosome; cdc, cell division cycle; CDK, cyclin-dependent kinase; GAP, GTPase activating protein; GEF, guanine nucleotide exchange factor; MEN, mitotic exit network; SIN, septation initiation network; SPB, spindle pole bodies.
Trang 2of chromosome segregation but prior to spindle
disassembly [3] Subsequent work by the Kirschner,
Nasmyth, Ruderman and Hershko laboratories
showed that a ubiquitin ligase known as the
anaphase-promoting complex or cyclosome (APC⁄ C)
degraded mitotic cyclins at the end of mitosis [4–6]
How the ubiquitin ligase was activated at the end of
mitosis was, however, not clear
As a Whitehead fellow I decided to return to this
question In particular, I was interested in determining
the role of CDKs in the regulation of mitotic Clb cyclin
degradation, by examining the consequences of CDK
inactivation on mitotic Clb cyclin degradation I
arrested cells in a stage of the cell cycle when Clb
cyc-lins are normally stable because the APC⁄ C is inactive
(in metaphase) and then examined the consequences of
CDK inactivation on Clb protein levels To inhibit
Clb-CDKs, I overexpressed Sic1, a Clb-CDK inhibitor
that inhibits Clb kinases by binding to them [1] To
inhibit Cln-CDKs I treated cells with pheromone This
leads to the production of Far1, an inhibitor of
Cln-CDKs [1] In this experiment, which we ended up
calling the ‘sic-trick’, Clb cyclins were efficiently
degraded [7] Inactivation of the APC⁄ C prevented Clb
cyclin degradation in the ‘sic-trick’ experiment,
indicat-ing that degradation of Clb cyclins was brought about
by the same mechanisms responsible for degrading Clb
cyclins at the end of mitosis [7] This result was not very
well received by the field because it was rather
unex-pected Mitotic cyclin degradation is initiated in the
presence of high CDK activity, and phosphorylation of
the APC⁄ C-promoted cyclin degradation in vitro Thus,
everyone assumed that mitotic CDKs promoted rather
than inhibited mitotic cyclin degradation This is
cer-tainly true, though only in part APC⁄ C bound to its
activating subunit Cdc20 degrades mitotic cyclins in the
presence of high mitotic CDK activity However, today
we know that a second APC⁄ C activator, Cdh1, is
inhibited by CDK-dependent phosphorylation [8] In
the ‘sic-trick’ experiment it presumably is this form of
APC⁄ C that becomes active when CDKs are inhibited
How do APC⁄ C–Cdc20 and APC ⁄ C–Cdh1 regulate
cyclin degradation? In most eukaryotes the bulk of
mitotic cyclin degradation occurs at the metaphase–
anaphase transition and is mediated by APC⁄ C–Cdc20
APC⁄ C–Cdh1 is needed during G1 to keep mitotic
CDK activity low In budding yeast, however, a
signifi-cant amount of mitotic CDK activity persists until late
anaphase [9] This pool of mitotic CDK activity is
targeted for degradation by APC⁄ C–Cdh1, which is
inhibited by CDK-dependent phosphorylation (Fig 1)
[8,10–12] Thus, for APC⁄ C–Cdh1 to degrade Clb
cyclins at the end of mitosis a phosphatase needs to
dephosphorylate Cdh1 At the time, we all assumed that it must be CDK downregulation that initiates Cdh1 dephosphorylation allowing constitutively active phosphatases to win the upper hand This idea was of course wrong Today we know that a phosphatase – namely Cdc14 – is activated to bring about Cdh1 dephopshorylation It is also clear now that regulation
of phosphatases is important for many cell-cycle events
So how did we realize that Cdc14 was the key regu-lator of exit from mitosis? A number of screens, most notable among them the cell division cycle (cdc) screen
by Lee Hartwell and colleagues [13], had identified sev-eral temperature-sensitive mutants that, when shifted
to restrictive temperatures, arrest with high levels of Clb cyclins in late anaphase, prior to exit from mitosis [9] However, we did not know whether this inability
to degrade Clb cyclins was due to these mutants being defective in Clb cyclin degradation or due to the mutants arresting in a cell-cycle stage when Clb degra-dation had not yet commenced The ability to induce Clb cyclin degradation at will and in stages of the cell cycle when Clb cyclins are normally stable enabled me
to distinguish between these possibilities The cdc mutants, which arrested in anaphase with high levels
of Clb-CDK activity, were also defective in cyclin degradation in metaphase-arrested cells in which Clb degradation was induced using the ‘sic-trick’ (Fig 2)
Cln-CDKs
APC/C-Cdh1 Sic1
Clb-CDKs
Cdc14
Fig 1 Cdc14 promotes Clb-CDK inactivation Clb-CDKs and the factors that inactivate it mutually inhibit each other During the S phase and early mitosis, Clb-CDKs inhibit APC ⁄ C–Cdh1 by prevent-ing the association of Cdh1 with APC ⁄ C Clb-CDKs also prevent the accumulation of Sic1 They prevent entry of the transcription factor Swi5 into the nucleus thereby inhibiting SIC1 transcription They also phopshorylate Sic1, which targets it for degradation Acti-vation of Cdc14 during anaphase dephosphorylates Cdh1, which causes activation of the APC–Cdh1 and hence mitotic cyclin degra-dation Cdc14 also dephosphorylates Swi5 and Sic1, causing SIC1 transcription and Sic1 stabilization, respectively This leads to the Clb-CDK inhibitors to gain the upper hand, keeping Clb-CDKs in the inactive state Cln-CDKs break this inhibition once they are active at the G1–S phase transition They target Sic1 for degradation and phosphorylate Cdh1, allowing Clb-CDKs to accumulate again.
Trang 3Thus, the inability of these mutants to degrade Clb
cyclins was not a mere consequence of the cell-cycle
arrest they caused
The ‘sic-trick’ not only identified a number of genes
as being important for cyclin degradation but also
pointed towards the phosphatase Cdc14 as being
criti-cally important for Clb cyclin degradation In cdc14-3
mutants inactivation of CDKs did not induce Clb2
degradation Inactivation of other genes required for
exit from mitosis such as CDC15 also delayed Clb2
cyclin degradation upon CDK inactivation, but the
effects were not as dramatic (Fig 2) Among all the cdc
mutants that were defective in exit from mitosis, cdc14
mutants exhibited the most severe defect in Clb2
degra-dation in the ‘sic-trick’ experiment We now understand
why this is the case Cdc14 is the phosphatase that
dephosphorylates Cdh1 Its inactivation has the most
dramatic effect on Clb cyclin degradation CDC15 is a
component of one of the two pathways that activate
Cdc14 In cdc15 mutants, Cdc14 is thus not completely
inactive The observation that inactivation of CDC14
led to the most severe defect in Clb cyclin degradation
led me to focus on understanding how this phosphatase
regulates Clb-CDK inactivation and hence exit from
mitosis, and how it itself is controlled
Cdc14 triggers CDK inactivation by multiple mechanisms
I was very lucky for Rosella Visintin to be the first person to have joined my laboratory She came in March 1997 and decided to first investigate how Cdc14 controls exit from mitosis She found that cells lacking Cdc14 function arrest in late anaphase with high mitotic CDK activity [14] Conversely, overexpression
of CDC14 results in inappropriate mitotic CDK inacti-vation Rosella could further show that Cdc14 promotes mitotic CDK inactivation by reversing CDK phosphorylation events Cdc14 dephosphorylates Cdh1, which promotes its association with the APC⁄ C, thereby activating it [14,15] Cdc14 also triggers Sic1 accumulation by dephosphorylating Sic1 and its tran-scription factor Swi5, which leads to the stabilization
of Sic1 and the upregulation of SIC1 transcription, respectively [14] It is now clear that Cdc14 has many substrates in the cell and it is likely that Cdc14 dep-hosphorylates many, if not all, Clb-CDK substrates and perhaps substrates of other mitotic kinases such as the Polo kinase Cdc5 and the Aurora B kinase Ipl1 This general reversal of mitotic phosphorylation events rapidly triggers exit from mitosis
- Kar2
- Clb2
- Histone H1
Time (h) 4 3 2 1 0
0 1 2 3 4
0 1 2 3 4
Wild-type
A
B
C
Wild-type
Fig 2 CDC14 is required for Clb2 degradation brought about by ectopic CDK inactivation GAL–SIC1 (A810), cdc14-3 GAL–SIC1 (A831) and cdc15-2 GAL–SIC1 (A844) cells were arrested with nocodazole (15 lgÆmL)1) for 165 min in YEP medium containing 2% raffinose at 23 C The 0 time point was taken and cells were shifted to 37 C for 30 min Then 2% galactose and a)factor (5 lgÆmL )1) were added to induce
Sic1 production and to inhibit Cln-CDKs, respectively Nocodazole (5 lgÆmL)1) was re-added at the same time to ensure that microtubules remain depolymerized Samples were taken at the indicated times after temperature shift to determine the amount of Clb2 protein (A), Clb2-associated histone H1 kinase activity (B) and DNA content (C) Kar2 was used as a loading control in western blots Methods were
as described in Amon [7].
Trang 4Regulation of Cdc14 – the nucleolus
moves into the limelight
The finding that CDC14 was unique among the genes
required for mitotic exit in that it was necessary and
sufficient to bring about mitotic CDK inactivation
begged the question whether the activity of Cdc14 was
regulated during the cell cycle Insight into this
ques-tion came from localizaques-tion studies on Cdc14 Rosella
found that Cdc14 was localized in the nucleolus from
G1 until the onset of anaphase During anaphase,
Cdc14 was present throughout the nucleus and
cyto-plasm This result was very exciting All our previous
studies on Cdc14 predicted that the phosphatase ought
to become active during anaphase to bring about
mito-tic CDK inactivation The observation that the
phos-phatase changed its localization during the stage of the
cell cycle when CDK inactivation commences raised
the very interesting possibility that this change in
sub-cellular localization reflects a change in Cdc14 activity
We now know that this is the case When Cdc14 is
located in the nucleolus it is bound to a competitive
inhibitor Cfi1⁄ Net1 [16,17] During anaphase, the
phos-phatase is released from its inhibitor and spreads into
the nucleus and cytoplasm, allowing it to
dephosphory-late its substrates [16,17] We identified Cfi1 as a
Cdc14-interacting protein in a yeast two-hybrid screen [17] At
the same time, Wenying Shou in Ray Deshaies’s
labora-tory isolated yeast mutants that were able to proliferate
in the absence of CDC15, a gene required for exit from
mitosis The screen revealed mutations in the same
gene, which Ray called NET1 [16] This elegant screen
not only identified loss-of-function alleles in NET1 but
also gain-of-function alleles in CDC14 as suppressors of
cdc15D mutants The simplest interpretation of these
findings was that NET1 functioned, directly or
indi-rectly as an inhibitor of CDC14 and that CDC15 was
needed to alleviate this inhibition This idea turned out
to be correct Using biochemical approaches, the
Des-haies and Charbonneau laboratories showed that Cfi1⁄
Net1 functions as an inhibitor of Cdc14 in vitro [16,18]
Consistent with this idea, overexpression of CFI1⁄
NET1prevented exit from mitosis [17]
I would like to note here that at first I was not very
excited about the fact that Cdc14 resided in the
nucleolus A cool cell-cycle protein like Cdc14 could
not possibly be in a place as mundane as the nucleolus
Today, it is clear that nucleolar sequestration is a
mechanism employed by cells to regulate many
pro-teins involved in numerous different cellular processes,
from transcription factors such as Hand1 [19],
check-point proteins such as Pch2 [20] to tumor suppressors
such as p19–Arf [21]
MEN
If release of Cdc14 from its inhibitor was a key step in the regulation of exit from mitosis, mutants that fail to
do so should also not be able to exit from mitosis Indeed, Rosella in my laboratory and Wenying in Ray Deshaies’ laboratory both found that mutants that failed to exit from mitosis arrested in late anaphase with Cdc14 sequestered in the nucleolus Furthermore, inac-tivation of the Cdc14 inhibitor Cfi1⁄ Net1 suppressed the temperature-sensitive lethality of these mutants, indicating that releasing Cdc14 from its inhibitor during exit from mitosis was the essential function of these genes [16,17] Earlier, David Morgan had studied the genes required for exit from mitosis and identified many genetic interactions between them, therefore, collec-tively calling them the mitotic exit network (MEN) [22] The MEN is essential for exit from mitosis and was the first signal transduction pathway shown to regulate Cdc14 localization The pathway resembles a Ras-like GTPase signaling cascade (Fig 3) [9,23] Research aimed at understanding how the individual compo-nents within MEN functioned to promote Cdc14 release from the nucleolus was guided by work on the homologous pathway in Schizosaccharomyces pombe Kathy Gould, Dan McCollum and Viesturs Simanis studied cytokinesis in fission yeast and identified a Ras-like signaling cascade essential for this process [24,25] Using genetic, cell biological and biochemical techniques they quickly ordered the genes into a path-way that they named the septation initiation network (SIN) [26] Similar analyses revealed that the MEN components function in an analogous order as their SIN counterparts [26,27] (Fig 3) The GTPase Tem1 is positively regulated by the GTPase-activating protein (GAP) complex Bub2–Bfa1 Lte1 resembles a guanine nucleotide exchange factor (GEF) that positively regu-lates Tem1, although whether it actually functions as a GEF for Tem1 is not known [28,29] Activated Tem1
is thought to propagate a signal to the protein kinase Cdc15 [4,30–33], which in turn activates the protein kinase complex Dbf2–Mob1 [34–37] Spindle pole bodies (SPBs), the yeast equivalent of centrosomes, appear to be the signaling center of the MEN, with Nud1 functioning as a scaffold [38–43] How locali-zation of MEN components to SPBs is utilized to contribute to the integration of exit from mitosis with spindle position will be discussed below How activa-tion of the MEN ultimately leads to the release of Cdc14 from the nucleolus is still not understood Pre-sumably phosphorylation of Cdc14 and⁄ or Cfi1 ⁄ Net1
or as yet unknown factors by the MEN protein kinase Dbf2 brings about this event
Trang 5… and FEAR
Discovery of the second signaling pathway that
regu-lates Cdc14 was a great piece of detective work When
Rosella examined the localization of Cdc14 in MEN
mutants progressing through the cell cycle in a
syn-chronous manner she noticed that Cdc14 was briefly
released from the nucleolus during early anaphase but
was sequestered again when cells entered the late
ana-phase arrest Having convinced ourselves that this
transient release was not simply due to incomplete
inactivation of the MEN in temperature-sensitive
MEN mutants we concluded that there must be a
sec-ond pathway that controls Cdc14 localization during
early anaphase [44] Elmar Schiebel’s and Akio Toh-E’
groups came to the same conclusion [29,45]
But what controlled Cdc14 localization during early
anaphase? Insight into this question came from a
pub-lication by Orna Cohen-Fix in Doug Koshland’s
labo-ratory and Rachel Tinker-Kulberg in David Morgan’s
laboratory They had previously shown that the key
regulator of the metaphase–anaphase transition,
Separ-ase, a protease that triggers the separation of sister
chromatids at the metaphase–anaphase transition [46]
also regulates cyclin degradation and hence exit from
mitosis [47,48] Frank Stegmeier, a graduate student in
the laboratory, took Rosella’s observation and the
result from the Koshland and Morgan laboratories and put them together Could Separase (known as Esp1 in yeast) be required for releasing Cdc14 from the nucleolus during early anaphase in a MEN-inde-pendent manner? The answer was yes Frank had dis-covered another pathway controlling Cdc14 activity
He subsequently identified several other factors impor-tant for Cdc14 localization during early anaphase Based on the observation that all these proteins regulated Cdc14 localization during early anaphase, we collectively called them the FEAR network, short for Cdc14 early anaphase release network
How does the FEAR network bring about Cdc14 release from the nucleolus? Work by Ray Deshaies and Frank Uhlmann provided insight into this ques-tion CDK-dependent phosphorylation of Cfi1⁄ Net1
on six CDK consensus sites brings about the transient release of Cdc14 from the nucleolus during early ana-phase [49] How are Clb-CDKs induced to phosphory-late the inhibitor? Interestingly, the FEAR network components Esp1 and Slk19 do not promote activation
of the kinase but rather inactivate the protein phos-phatase PP2A associated with the targeting subunit Cdc55 [50] (Fig 3) Esp1 and Slk19 also promote Spo12 phosphorylation by Clb-CDKs, which is essen-tial for Cdc14 release during early anaphase (B Tom-son, personal communication) [9] (Fig 3)
MEN
Lte1 Bub2-Bfa1
Pds1
Tem1 Esp1/Slk19
Clb-CDKs
Pds1
Nud1 Cdc15
Dbf2-Mob1 Spo12
Cdc55-PP2A
Cdc14
Fob1
Metaphase
Cfi1/Net1
Early anaphase Late anaphase
Fig 3 The FEAR network and the MEN control Cdc14 localization The degradation of Pds1 and hence activation of Esp1 marks the onset of anaphase Esp1 then not only cleaves cohesins to bring about sister chromatid segregation, but together with Slk19 also promotes the down-regulation of the protein phosphatase PP2A This allows Clb-CDKs to phosphorylate Cfi1 ⁄ Net1 and Spo12, which brings about the dissociation
of Cdc14 from its inhibitor Phosphorylation of Cfi1 ⁄ Net1 directly disrupts the Cdc14–Cfi1 ⁄ Net1 complex Phosphorylation of Spo12 promotes the protein to inhibit Fob1, which inhibits Cdc14–Cfi1 ⁄ Net1 dissociation Movement of the MEN-bearing SPB into the bud, where Lte1 is located, downregulation of Bub2–Bfa1 activity by Cdc5, inactivation of Kin4 and other unknown signals promote MEN activation during anaphase Tem1, presumably in its GTP bound form, then activates Cdc15, which activates Dbf2 in a Mob1-dependent manner Dbf2–Mob1 promotes Cdc14 release from the nucleolus by an unknown mechanism Nud1 functions as a scaffold for MEN components at the SPB.
Trang 6The polo kinase Cdc5 is also required for the release
of Cdc14 from the nucleolus during early anaphase
How Cdc5 functions in the FEAR network is not yet
clear Epistasis analyses place Cdc5 either downstream
of Esp1–Slk19 or in parallel with the complex [51]
Furthermore, Cdc5 promotes phosphorylation of
Cdc14 in vivo [51–53] and can directly bind to and
phosphorylate the phosphatase in vitro [52–54]
(R Rahal and R Visintin, personal communication)
suggesting that it functions in parallel with the Esp1–
Slk19 branch to dissociate Cdc14 from its inhibitor
Identifying the Cdc5 phosphorylation sites in the
Cdc14–Cfi1⁄ Net1 complex and examining the
conse-quences of mutating them to residues resistant to
phosphorylation will be essential to elucidate Cdc5’s
role in the MEN and the FEAR network
Cellular events controlling Cdc14
activation
The discovery that two pathways control Cdc14
activ-ity immediately raised the question as to which cellular
events control Cdc14 and why cells employ two
path-ways to control the phosphatase Two components of
the FEAR network, the Separase Esp1 and the polo
kinase Cdc5, are also key regulators of sister
chroma-tid separation This dual employment certainly
pro-vides a means for the cell to ensure that exit from
mitosis does not occur prior to the onset of sister
chro-matid separation [44,55] but it does not provide a
mechanism for ensuring that sister chromatid
separa-tion occurs prior to exit from mitosis Other regulatory
signals such as the high-osmolarity MAP kinase
path-way have also being implicated in regulation of FEAR
network activity [56], but how these pathways help
regulate Cdc14 activity through the FEAR network is
not really understood and is an important question
that we need to address in the future
The function of the MEN in coordinating Cdc14
activation with other cellular events is better defined
Today we know that the MEN is employed to ensure
that exit from mitosis only occurs when the nucleus is
positioned correctly along the mother–bud axis This
connection was first made by my first graduate student
Allison Bardin and at the same time by Gislene Pereira
in Elmar Schiebel’s laboratory Allison joined the
labo-ratory in 1999 when it became clear that the MEN
was an important regulator of Cdc14 activity Given
that components of the MEN resembled constituents
of the RAS pathway, Allison wanted to identify
poten-tial signals controlling the MEN Her approach was
simple She asked: can we learn something about
puta-tive MEN signals by determining where in the cell
MEN components that function at the top of the path-way are located? She found that Tem1 (and we now know all other MEN components) localized to the SPB that migrates into the bud during anaphase The MEN activator Lte1 localizes to the bud cortex con-comitant with bud formation [38,40] This localization pattern led us to hypothesize that MEN activation does not occur until the Tem1-bearing SPB migrates into the bud during anaphase [38,40]
This hypothesis appeared to be correct Allison as well as Schiebel and co-workers showed that restrain-ing MEN activity was essential to prevent exit from mitosis in cells that failed to pull the nucleus into the bud during anaphase In 1995, Kerry Bloom’s labora-tory had shown that cells that fail to align the spindle correctly along the mother–bud axis and hence elon-gate the spindle in the mother cell, arrest prior to cyto-kinesis until the spindle position defect was corrected [57] He called the mechanisms responsible for this cell-cycle delay the spindle position checkpoint Allison and Schiebel’s laboratory showed that spindle mis-position restrained MEN activity, in part through sequestration of Lte1 in the bud and in part through regulating the activity of the Tem1 GAP, Bub2–Bfa1 (Fig 3) Several recent studies provided insight into how Bub2–Bfa1 activity is regulated by the spindle position checkpoint The protein kinase Kin4 phospho-rylates Bfa1 This precludes phosphorylation and hence inactivation of the GAP by Cdc5 [58–62] (Fig 3) Although the spindle position checkpoint restrains MEN activity when the spindle is not correctly aligned along the mother–bud axis, it is clear that during an unperturbed cell cycle this pathway alone is not solely responsible for controlling MEN activity Identifying the pathways that regulate MEN activity during an unper-turbed cell cycle will be an important task in the future
Turning off MEN and FEAR Inactivation of Cdc14 following mitotic exit is as important for successful cell division as its activation during anaphase Cells with unrestrained Cdc14 activ-ity exhibit severe growth defects [14,16,17] FEAR net-work activity appears to be restricted to a very brief period during early anaphase, as Cdc14 becomes re-sequestered into the nucleolus during late anaphase
in cells lacking a functional MEN [29,44,45] How FEAR network activity is restricted to early anaphase
is unknown However, how both the MEN and FEAR network are silenced once mitotic exit has been com-pleted is understood Cdc14 plants the seeds of its own inactivation The protein phosphatase activates APC– Cdh1, which targets Cdc5 for degradation [54] Other
Trang 7factors also contribute to the silencing of the MEN,
such as dephopshorylation of Bfa1 and hence
reactiva-tion of the GAP [45,58] Lte1 activity may also be
controlled by Cdc14 [28,63], but Cdc5 degradation
appears to be mainly responsible for silencing the
machinery that promotes Cdc14 activation [54]
Cdc14 – more than just promoting CDK
inactivation
A question that arises from the observation that at
least two pathways control Cdc14 localization is why
budding yeast utilizes two pathways rather than one to
regulate Cdc14? One possibility is that Cdc14 released
by the FEAR network performs functions during
mito-sis that are different from that of Cdc14 released by
the MEN This appears to be the case Cdc14 released
by the FEAR network has important roles in
promot-ing MEN activation and in regulatpromot-ing chromosome
segregation, mitotic spindle dynamics and
chromo-somal passenger proteins localization [64–70] (Fig 4)
Cdc14 released by the MEN triggers CDK inactivation
and hence exit from mitosis [16,17] How Cdc14 brings
about these many different events is understood, at
least in some instances in detail, and summarized in a
review by D’Amours and Amon [71]
Here, rather than focusing on the details of Cdc14’s
functions in chromosome segregation, I want to ask
the more fundamental question: Why are events
neces-sary for the faithful segregation of chromosomes regu-lated by a transient FEAR network-dependent burst
of Cdc14 activity, whereas exit from mitosis by a sustained level of MEN-dependent Cdc14 activity? I suspect that the gradual activation of Cdc14 is not unlike that of Clb-CDK activation earlier during mito-sis A recent study by Rami Rahal in my laboratory showed that entry into mitosis requires less Clb-CDK activity than progression through the metaphase–ana-phase transition This dependency of early mitotic events on increasing amounts of Clb-CDK activity may help establish the order of events during early mitosis [72] Perhaps a gradual activation of Cdc14 and hence gradual decrease of Clb-CDK activity after entry into anaphase accomplishes a similar task A low-level transient activation of Cdc14 by the FEAR network helps orchestrate anaphase events by either locally inhibiting Clb-CDKs or dampening the effects
of the kinases throughout the cell Full activation of Cdc14 by the MEN eliminates Clb-CDKs and hence promotes exit from mitosis (Fig 5) Perhaps mitosis is all about fine-tuning mitotic CDK activity
How do FEAR and MEN bring about the release of Cdc14 from the nucleolus – a model
So how does it all work? Once chromosomes have correctly aligned on the metaphase spindle, Securin is
Fig 4 Cdc14 orchestrates anaphase events At the onset of anaphase, Cdc14 is activated by the FEAR network and controls many aspects
of anaphase chromosome movement The protein phosphatase promotes rDNA segregation by targeting condensins to the rDNA It stabi-lizes the anaphase spindle by dephosphorylating kinetochore and spindle proteins such as Ask1, Ndc10, Fin1 and the chromosomal passen-ger complex Cdc14 promotes the localization of the chromosomal passenpassen-ger protein complex to the spindle midzone and controls nuclear position Cdc14 also promotes MEN activity, which is necessary to maintain Cdc14 in the released active state during late stages of ana-phase Once activated the MEN further promotes Cdc14 activity This sustained activation of Cdc14 then brings about exit from mitosis.
Trang 8degraded leading to the activation of Separase (Fig 3).
The protease then not only initiates anaphase
chromo-some movement but also, as a component of the
FEAR network promotes the release of Cdc14 from
the nucleolus By downregulating PP2A, Esp1 bound
to Slk19 leads to high levels of Clb-CDK activity
(per-haps especially in the nucleolus) Clb-CDKs in turn
phosphorylate Cfi1⁄ Net1 and Spo12 These Clb-CDK
phosphorylation sites could then function as binding
sites for Cdc5 Its phosphorylation of Cdc14 (and
Cfi1⁄ Net1) then promotes the release of Cdc14 from
the nucleolus The reverse could of course also be true
Cdc5 phosphorylation could be a prerequisite for
Clb-CDKs to promote the dissociation of Cdc14 from
its inhibitor Spo12 phosphorylation could either aid in
this process or through its interaction with Fob1
further destabilize the interaction between Cdc14 and
its inhibitor Once Cdc14 is released, the protein
phosphatase orchestrates anaphase chromosome
segre-gation and stimulates MEN activity thereby setting in
motion exit from mitosis (Fig 4)
Exit from mitosis is triggered when the MEN is fully
activated FEAR network function, spindle position
and perhaps other signals all converge on Tem1 to
bring about activation of the MEN In the face of
declining Clb-CDK activity, brought about by
APC⁄ C–Cdc20 activity and a transient activation of
Cdc14 that dephosphorylates Clb-CDK substrates,
Dbf2–Mob1 could take over Clb-CDK function Cdc14 then remains spread throughout the cell giving the phosphatase time to set in motion the events that lead to Clb-CDK inactivation
What is left to do?
During the last decade we have made great strides towards understanding how Cdc14 controls anaphase progression and exit from mitosis and how the phos-phatase is itself regulated However, several important questions remain to be answered How is the release of Cdc14 from the nucleolus regulated at the molecular level and how do the protein kinases implicated in the phosphatase’s regulation function together to bring about this event? In in vitro reconstitution experiments several kinases are able to disrupt the Cdc14–Cfi1⁄ Net1 complex indicating that such reconstitution approaches are not likely to yield answers to this ques-tion It will therefore be necessary to obtain a complete phosphorylation landscape of Cdc14, Cfi1⁄ Net1 and perhaps other binding partners to fully understand how Cdc14 activity is controlled
We are still lacking a thorough understanding of the signaling events in the FEAR network and the MEN The MEN resembles a Ras-like signaling cascade, which
is in many respects unusual A thorough biochemical characterization, particularly of the GTPase and its reg-ulators will be necessary to not only obtain a detailed understanding of the pathway, but also to generate new tools to understand how the pathway is regulated
in vivo Is one essential pathway activating the MEN during anaphase or are many nonessential signals func-tioning as inputs to bring about activation of the path-way as the spindle pole moves into the bud during anaphase? Similarly, we are still lacking a thorough understanding of the FEAR network How does Separ-ase inhibit PP2A? How does the Spo12–Fob1 branch of the pathway contribute to the disassembly of the inhibi-tory complex during anaphase and so forth
How Cdc14 itself brings about the various ana-phase events is understood in quite some detail, but several questions remain Most pressing among them are the role of Cdc14 in rDNA segregation and the relationship between Cdc14 and the MEN GAP com-plex Bub2–Bfa1 Furthermore, identifying additional functions of Cdc14 during anaphase will be an impor-tant task The use of substrate trap alleles of Cdc14
in mass spectrometry or yeast two-hybrid approaches will yield new putative substrates and roles for Cdc14 Finally, the most important question that remains to be addressed is whether or not Cdc14 is active in the nucleolus Biochemical analyses indicate
Clb-CDK threshold 2:
Metaphase-anaphase, Anaphase chromosome
Clb-CDK threshold 3:
APC/C-Cdc20 activation
Spindle elongation
movement
Clb-CDK threshold 4:
Mitotic exit
CDK activity Clb-CDK threshold 1:
Mitotic entry,
Bipolar spindle assembly
Chromosome
condensation
Spindle disassembly
Anaphase Metaphase
Fig 5 Different Clb-CDK and Cdc14 thresholds establish order in
the progression through mitosis A certain amount of Clb-CDK
activ-ity is required for cells to enter mitosis (Threshold 1) The amount of
Clb-CDK activity needed for entry into mitosis is not as high as that
for anaphase initiation (Threshold 2) This is illustrated by the fact that
inactivation of Clb1–4 leads to a G2 arrest Inactivation of Clb2 and
Clb1 or Clb2 and Clb3 results in a metaphase delay The need for
increasing amounts of Clb-CDK activity could help establish an order
of events as cells progress from G 2 into metaphase Increasing levels
of Cdc14 activity and hence decreasing levels of Clb-CDK activity
could also help establish order to the progression from metaphase to
G 1 Once cells enter anaphase Clb-CDK activity needs to decline
some for accurate anaphase chromosome movement (Threshold 3).
Cdc14 released by the FEAR network brings about this event For
cells to exit from mitosis, Clb-CDK activity needs to be lowered even
further (Threshold 4) Eventually all Clb-CDKs are inactivated
reset-ting the cell cycle to a GAP phase state.
Trang 9that Cfi1⁄ Net1 is a competitive inhibitor with a high
specific activity (Ki= 3 nm), which would argue that
this is not the case [16,18] However, recent evidence
indicates that Cfi⁄ Net1 is not the only factor that
binds Cdc14 in the nucleolus Tof2 also binds Cdc14
and appears to be an activator rather than an
inhibi-tor of Cdc14 [73] It is thus possible that Cdc14 is
also active in the nucleolus and there could regulate
important aspects of rRNA biogenesis
Finally, we must determine the broader significance
of the findings in budding and fission yeast Homologs
of Cdc14 exist in mammals, with one isoform
(Cdc14B) residing in the nucleolus during interphase
but not during mitosis and one isoform (Cdc14A)
located at centrosomes [74–76] The mechanisms
whereby Cdc14B is anchored in the nucleolus and
Cdc14A at centrosomes are not understood However,
it appears that as in yeast, mammalian Cdc14
func-tions as antagonists of CDKs [74] and has been
impli-cated in a broad range of cellular processes ranging
from centrosome function to cytokinesis [75,76] It is,
however, important to note that several of the
described phenotypes of Cdc14-loss of function may
be artifacts of the knock-down procedure A recent
analysis of human cells deleted for Cdc14B revealed
no significant mitotic defects [77] Thus, the two
Cdc14 isoforms are either truly redundant (at least in
a tissue culture setting) or Cdc14 is dispensable for
cell proliferation in mammals Clearly, a Cdc14A
Cdc14B double knock-out will be needed to answer
this question Whether the signaling pathways that
control Cdc14 in yeast exist in higher eukaryotes is also
not clear Some components of the FEAR network
and the MEN exist but whether the same pathways
control Cdc14 localization in higher eukaryotes is not
known Figuring this out will probably take another
decade
Acknowledgements
I thank Brett Tomson, Rami Rahal and Rosella Visintin
for communicating unpublished results I am grateful to
the members of the Amon lab, past and present for their
hard work and dedication I thank Jenny Cimino for
help with the preparation of this manuscript and Leon
Chan, Fernando Monje-Casas, Jeremy Rock, Frank
Stegmeier and Rosella Visintin for comments on the
manuscript I would also like to apologize to the many
colleagues whose work I could not discuss because of
space constrains Work in my laboratory was supported
by a grant from the National Institutes of Health
(GM056800) I am also an investigator of the Howard
Hughes Medical Institute
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