Download Protein phosphatases and the regulation of mitosis

2323
Commentary
Protein phosphatases and the regulation of mitosis
Francis A. Barr1,2,*, Paul R. Elliott3 and Ulrike Gruneberg1,2
1
University of Liverpool, Cancer Research Centre, 200 London Road, Liverpool L3 9TA, UK
University of Oxford, Department of Biochemistry, South Parks Road, Oxford OX1 3QU, UK
3
University of Liverpool, Institute of Integrative Biology, Crown Street, Liverpool L69 7ZB, UK
2
*Author for correspondence ([email protected])
Journal of Cell Science 124, 2323-2334
© 2011. Published by The Company of Biologists Ltd
doi:10.1242/jcs.087106
Summary
Dynamic control of protein phosphorylation is necessary for the regulation of many cellular processes, including mitosis and
cytokinesis. Indeed, although the central role of protein kinases is widely appreciated and intensely studied, the importance of protein
phosphatases is often overlooked. Recent studies, however, have highlighted the considerable role of protein phosphatases in both the
spatial and temporal control of protein kinase activity, and the modulation of substrate phosphorylation. Here, we will focus on recent
advances in our understanding of phosphatase structure, and the importance of phosphatase function in the control of mitotic spindle
formation, chromosome architecture and cohesion, and cell division.
Journal of Cell Science
Key words: Cdk1, Kinase, Mitosis, PP1, PP2A, Phosphatase
Introduction
Post-translational modifications are crucial for the control of the
remarkable changes in cellular architecture that are observed as
cells enter and exit mitosis. Of central importance among these
modifications is protein phosphorylation, which is carried out by a
series of conserved serine/threonine protein kinases of the cyclindependent kinase (Cdk), Polo, Aurora and Nek families. These
protein kinases have well-established roles in phosphorylating key
mitotic substrates and have been reviewed extensively elsewhere
(Barr et al., 2004; Lindqvist et al., 2009; Nigg, 2001; O’Farrell,
2001; Ruchaud et al., 2007). Protein phosphorylation is typically a
short-lived highly dynamic modification and, accordingly, in recent
years, equally important roles in mitosis have begun to emerge for
specific protein phosphatases (Chen et al., 2007; Gharbi-Ayachi
et al., 2010; Kitajima et al., 2006; Mochida et al., 2009; Mochida
et al., 2010; Riedel et al., 2006; Zeng et al., 2010). It is this balance
of kinase and phosphatase activity that orchestrates the changes
seen during cell division. Interference with either type of activity
will alter the amount and half-life of substrate phosphorylation
(Heinrich et al., 2002), and thus disturb orderly mitotic progression.
This Commentary will focus on a number of recent advances
that show the central importance of phosphatase function in the
dynamic control of protein phosphorylation during mitosis. The
salient structural features of specific phosphatase holoenzymes,
which are thought to be important for substrate recognition, will
also be described. Particular attention will be paid to recent reports
that discuss the regulation of mitotic spindle formation,
chromosome architecture and cohesion, and cell division by specific
phosphatase holoenzyme complexes.
Introducing the protein phosphatase
superfamily
The phosphatases encoded by the human genome can be split into
two main groups according to the specific amino acids that they
dephosphorylate; these groups can then be further subdivided
according to the sequence similarity of their catalytic subunits,
sensitivity to inhibitors, and regulatory subunit structure (Lu et al.,
2004; Tonks, 2006; Trinkle-Mulcahy and Lamond, 2006). The first
major group is the protein tyrosine phosphatase (PTP) family,
which includes the dual-specificity tyrosine and serine/threonine
phosphatases (DUSPs); these have been reviewed expertly
elsewhere (Tonks, 2006). Tyrosine phosphorylation and
dephosphorylation have a central role in signal transduction;
however, with the notable exception of the phosphorylation and
dephosphorylation of tyrosine 15 of Cdk1 by Wee1 and celldivision cycle 25 (Cdc25), respectively, they are of less importance
for the regulation of mitosis (Kumagai and Dunphy, 1991; Mueller
et al., 1995). Instead, serine/threonine phosphorylation and
dephosphorylation are the key regulatory events during cell
division. We will therefore focus on this second main group of
phosphatases, the serine/threonine-specific phosphatases (PSTPs),
and their many roles in mitosis and cytokinesis. PSTPs can be
further subdivided into the PPM family of metallo-dependent
phosphatases (including PPM1) and the phospho-protein
phosphatases (PPP) family, which are both dependent on metal
ions for catalysis. PPM family members function in signal
transduction and DNA damage pathways (Lu et al., 2004), and it
is therefore the PPP family that is most commonly associated with
mitotic regulation (Axton et al., 1990; Felix et al., 1990; Picard
et al., 1989). Remarkably, brief treatment of cells with the algal
toxin okadaic acid, which is specific for PPP enzymes, triggers
many of the changes that are associated with mitosis, including
chromatin condensation and structural rearrangement of organelles
such as the Golgi apparatus (Lucocq et al., 1991; Yamashita et al.,
1990). This finding indicates that mitotic entry is normally opposed
by PPP enzymes and suggests that inhibition of PPP activity – and
not simply protein kinase activation – is required to allow cells to
progress into mitosis.
Catalytic mechanisms and inhibitor sensitivity
PSTPs and PTPs use completely different catalytic mechanisms to
dephosphorylate their substrates (Barford et al., 1998). Briefly, the
PTPs share the conserved active site motif HCX5R, in which the
essential catalytic cysteine acts as a nucleophile and forms an
intermediate phospho-cysteine during hydrolysis (Barford et al.,
1998; Fauman and Saper, 1996). By contrast, the phosphatases of
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Journal of Cell Science 124 (14)
the PSTP family use two metal ions for catalysis, typically
manganese (Mn2+) and iron (Fe2+), which are coordinated by a set
of conserved amino acid residues (Shi, 2009). These bound metal
ions coordinate the phosphate group of the substrate and stabilize
the negative charge, thus facilitating nucleophilic attack on the
phosphorus by a water molecule and hydrolysis of the phosphate
ester bond (Goldberg et al., 1995). The catalytic subunits of the
PPP family are so similar (Fig. 1A) that all family members are
inhibited by the potent algal toxins microcystin-LR and okadaic
acid (Fig. 1B), and share three substrate-peptide-binding grooves
that lead to the catalytic site (Fig. 1C).
A
PPP5C
PP5
PPP2CA
PPP2CB
PP2A subfamily
PPP6C
PPP4C
PPP1Cβ
PPP1Cγ
0.05
PP1 subfamily
PPP1Cα
Despite their similarity, the catalytic subunits of PPP family
members PP1 and PP2A can be distinguished, to some extent, by
their sensitivity to inhibition by okadaic acid. PP2A is inhibited by
okadaic acid in the subnanomolar range [half-maximal inhibitory
concentration (IC50)0.1 nM], whereas PP1 requires 100-fold
higher concentrations of okadaic acid for inhibition (Xing et al.,
2006). At a structural level, this can be explained by the absence
of specific hydrophobic residues in the catalytic cleft of PP1 that
are present in PP2A and are required for tight binding of the
hydrophobic end of okadaic acid (Xing et al., 2006). However,
although the catalytic subunits of PP1 and PP2A are highly active
in isolation, and can be discriminated in terms of toxin sensitivity,
they lack appreciable substrate specificity (Agostinis et al., 1992;
Imaoka et al., 1983; Mumby et al., 1987). This close similarity and
activity of their catalytic subunits are difficult to reconcile with
their biological functions and specific substrates observed in cells.
It is therefore unsurprising that these catalytic subunits associate
with specific regulatory subunits that direct the biological activity
of the PPP family phosphatases in living cells. As we will discuss
later, this is necessary to explain how PP1 and PP2A regulate
unique sets of substrates and cellular events during mitosis.
Journal of Cell Science
B
C
Hydrophobic
groove
Fig. 1. Conservation of the PPP family catalytic subunit. (A)The eight
human PPP family catalytic subunits were aligned using ClustalX and a
neighbour-joining tree, plotted using NJplot (Larkin et al., 2007). This
approach reveals that the PPP catalytic subunits fall into three subgroups
containing PP1,  and ; PP2A, PP4 and PP6; and PP5. (B)The catalytic
subunit of PP2A (PDB code 2IE4) is shown with conserved catalytic residues
of the PPP family – GDxHG, GDxVDRG, GNHE, HGG, RG, AHQ (D57,
H59, D85, N117, H167, R206 and H241 in PPA2) – in stick representation.
(C)Surface of the catalytic subunit (in an analogous orientation to B) coloured
according to residue conservation within the PPP superfamily, on the basis of
analysis by ConSurf (Landau et al., 2005). The active site of the catalytic
subunit is highly conserved (red) with the two active-site metal centres (orange
spheres). The active site forms an extended Y-shaped groove and the tumourinducing toxin okadaic acid (yellow) is shown binding across the hydrophobic
and C-terminal grooves. A 90° rotation of the catalytic subunit on the righthand side reveals a highly variable region (blue), which permits binding of
specific regulatory subunits to the catalytic subunit. Figure prepared using
Pymol (The PyMOL Molecular Graphics System, version 1.2r3pre,
Schrödinger, LLC.).
Subunit structure of protein phosphatase
holoenzymes
Phosphatases of the PPP family act as multimeric holoenzyme
complexes formed from a specific catalytic subunit, which defines
the phosphatase (e.g. PP1 or PP2A), and one or more of a number
of associated regulatory and scaffolding subunits (Fig. 2A).
Phosphatase holoenzyme complexes that contain a PP1 catalytic
subunit (C-subunit) associate with a single regulatory or R-subunit,
whereas PP2A subfamily enzymes typically possess a scaffolding
or A-subunit, in addition to one of the four regulatory B-subunits
– B, B⬘, B⬙ or B⬘⬙ (Janssens and Goris, 2001). Other enzymes
within the PPP family have a similar subunit composition. PP6 has
a similar architecture to PP2A, with an ankyrin repeat domain
subunit and a SIT4 phosphatase-associated protein (SAPS)
domain scaffolding subunit (Stefansson and Brautigan, 2006;
Stefansson et al., 2008), whereas PP4 is thought to possess a single
regulatory subunit (Chen et al., 2008). This makes it possible for
a relatively small number of catalytic subunits to form a multitude
of functionally distinct holoenzymes with unique substrate
specificity, regulatory properties and localization within the cell.
Binding of these regulatory subunits has the dual role of directing
phosphatase activity towards a specific substrate, and also reducing
its activity towards other phosphorylated proteins (Agostinis et al.,
1992; Imaoka et al., 1983; Mumby et al., 1987). This results in an
inhibitory effect towards phosphorylated non-substrate proteins,
hence the apparent misnaming of many of these proteins as
regulatory inhibitor subunits.
Given the large number of protein kinases and substrate proteins,
one would therefore expect equivalent numbers of phosphatase
regulatory subunits. Indeed, PP1 is claimed to have in the region
of 150–200 ‘validated’ interaction partners (Bollen et al., 2010),
which are probably regulatory subunits important for targeting PP1
to diverse subcellular structures and for restricting its specificity
(Cohen, 2002). Although many PP1 interaction partners have
been described, particularly in the field of mitosis, few have been
rigorously validated. It is important to note that PP1 has three
potential catalytic subunits, PPP1C,  and , and that these might
form complexes with specific regulatory subunits. The best
examples in the context of mitosis are the PP1–Repo-man
Protein phosphatases regulating mitosis
A
PP1
PP2A
B, B⬘,
B⬙,B⵮
R
PP4
C
PP6
SAPS
1–3
R
C
ANKRD28/44/52
B PP1 regulatory subunit binding
C PP1–substrate binding
K or R
V
PP1–MYPT
Acidic
groove
Spinophilin
K
R or T
F
PP1–spinophilin
C
C
A
>150
2325
Extended
Ct groove
Hydrophobic
groove
RVXF docking motifs
D PP2A regulatory subunit binding
E PP2A–substrate recognition
B⬘γ-subunit
Bα-subunit
Hydrophobic
groove
MYPT
B⬘γ-subunit Substrate binding
Substrate binding
Journal of Cell Science
Bα-subunit
Regulatory
phosphorylation
C-subunit
C-subunit
A-subunit
PP2A–Bα
A-subunit
PP2A–B⬘γ
HEAT
repeats
C-subunit
PP2A–Bα
C-subunit
PP2A–B⬘γ
HEAT
repeats
Fig. 2. PPP holoenzymes and substrate selectivity. (A)Schematic showing PPP subunit composition. PP1 catalytic subunits (C) associate with a single regulatory
subunit (R) drawn from a pool of over 150 potential partners. PP2A has a trimeric structure, with one each of the two possible catalytic and A-subunit variants, and
a B, B⬘, B⬙ or B⵮ subunit. Multiple isoforms of the four B, B⬘, B⬙ and B⵮ subunits exist. PP4 has a single catalytic and regulatory subunit. PP6 is similar to PP2A,
and comprises a single catalytic subunit in conjunction with one each of the ankyrin repeat domain or SAPS domain subunits. For all PPP enzymes, there is a
standard gene-naming convention of the form PPP#C for catalytic subunits and PPP#R## for regulatory subunits, where # is a number that indicates the PPP
enzyme (1, 2A, 4, 5 and 6) and ## is a unique number. Where there are multiple catalytic or regulatory subunit isoforms, these numbers are followed by letters , ,
 or A, B, C. For example, MYPT is composed of PPP1C and the MYPT regulatory subunit PPP1R12A. See text for more details. (B,C)Electrostatic potential of
the catalytic face of PP1 shown in complex with either spinophilin (yellow) or MYPT (green). A single regulatory subunit binding to the PP1 catalytic subunit is
enough to determine substrate specificity. In addition to the typical RVXF motif, extensive contacts with the catalytic subunit occur through other regions of the
regulatory subunits. Residues of the RVXF motifs of spinophilin (RKIKF, yellow) and MYPT (TKVKF, green) are shown in the enlarged region (dotted outline,
upper part) and bind to the same surface of PP1 (shown with spinophilin in the lower part). To date, only two complexes between PP1 and its associated regulatory
subunits have been solved and both display differential levels of substrate recognition. In C, the catalytic face of PP1 is shown with the catalytic Y-shaped groove in
red. Binding of spinophilin (yellow surface) masks the C-terminal (Ct) groove, permitting one orientation of substrate across the catalytic surface (dotted line). By
contrast, binding of MYPT (green surface) to the catalytic face of PP1 creates an extended binding interface, which forms two alternative paths for substrate
binding (blue and brown lines). (D) The HEAT-repeat-containing A-subunit of PP2A (green surface) adopts an L-shaped conformation, which positions the
B-type subunits towards the catalytic subunits (electrostatic surfaces). The B-subunit binds to the first seven HEAT repeats of the scaffold subunit, whereas the
B⬘ subunit binds to HEAT repeats 2–8 and the catalytic subunit. (E)Substrate recognition is provided through the B-subunits. Structures of the PP2A–B and
PP2A–B⬘ holoenzymes have demonstrated that the B-subunits have different folds, which provides different substrate-recognition surfaces. The B-subunit is
composed of HEAT repeats (shown as cartoon), which is analogous to the scaffold subunit, whereas the B⬘ subunit has a WD40 fold (shown as cartoon) and
generates a different substrate-recognition interface. Figure prepared using Pymol and CCP4mg (Potterton et al., 2004).
(CDCA2) holoenzyme and the PP1–PPP1R7 (SDS22) holoenzyme
(Posch et al., 2010; Trinkle-Mulcahy et al., 2006; Vagnarelli et al.,
2006).
Substrate recognition by protein phosphatase
holoenzymes
To date, the crucial question of how substrate selection is achieved
has only been addressed by structural studies in a small number of
cases (Ragusa et al., 2010; Xu et al., 2008). Two well-studied
examples of PP1–regulatory subunit complexes are the
PP1–spinophilin complex found in neurons (Ragusa et al., 2010),
and the PP1–MYPT (myosin phosphatase; PPP1R12A) complex
that acts as a myosin phosphatase in muscle and is also important
in mitosis (Terrak et al., 2004; Yamashiro et al., 2008). Spinophilin,
a neuronal PP1-targeting protein, blocks one of the three potential
substrate-binding grooves on the PP1 catalytic subunit (Fig. 2B,C)
Journal of Cell Science
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Journal of Cell Science 124 (14)
and thus allows dephosphorylation of the specific substrate,
glutamate-receptor 1 subunit, but not phosphorylase-, the
substrate of a different PP1 holoenzyme (Ragusa et al., 2010).
Structural studies have shown two different mechanisms for
substrate recognition by PP1, through the binding of regulatory
subunits. In the case of spinophilin, the binding site for PP1 is
unstructured in solution, but on binding to PP1 it adopts an ordered
conformation, which coordinates the catalytic subunit through four
distinct regions (Ragusa et al., 2010). Extensive contacts are made
between spinophilin and the C-terminal groove of PP1, thus
restricting substrate access to the catalytic site, as mentioned above
(Fig. 2B). Binding of MYPT again occurs through several extensive
sites on PP1 (Terrak et al., 2004), in addition to the RVXF motif
of MYPT (Egloff et al., 1997). However, MYPT provides substrate
selectivity through the extension of the C-terminal and acidic
grooves (Fig. 2B,C).
Similarly, in the case of PP2A, PP2A–B holoenzyme
complexes exhibit high phosphatase activity towards the
phosphorylated form of the microtubule-stabilising protein tau,
whereas PP2A–B⬘ holoenzyme complexes have little activity
towards the same substrate (Xu et al., 2008). In fact, PP2A–B⬘
holoenzymes dephosphorylate tau less well than the PP2A core
enzyme devoid of a B-subunit (Xu et al., 2008). In this case,
specific substrate recognition is achieved by the interaction of two
lysine-rich sequences in the phosphorylated tau substrate protein
with an acidic groove in the -propeller region of the B-subunit
(Xu et al., 2008). In PP2A, the position of the substrate-binding Bsubunit relative to the catalytic C-subunit is determined by the Aor scaffolding subunit (Fig. 2D). This A-subunit has 15 tandem
HEAT (Huntingtin, elongation factor 3, A-subunit of PP2A, TOR1
yeast PI3-kinase) 39-amino-acid -helical repeats that form an
L-shaped molecule. The PP2A C-subunits bind to HEAT repeats
11–15, whereas the different B-subunits bind within the first eight
HEAT repeats (Fig. 2E). The B⬘-subunit binds to HEAT repeats
2–8 (Xing et al., 2006; Xu et al., 2006) and also makes contacts
with the catalytic subunit (Cho and Xu, 2007), whereas the Bsubunit binds to HEAT repeats 1–7 (Xu et al., 2008). This
arrangement positions the substrate so that it faces the catalytic
cleft of the PP2A holoenzyme. Although no structural data are
available for PP6, it is notable that the PP6 scaffolding subunits
contain multiple ankyrin repeats (Stefansson et al., 2008). Ankyrin
33-amino-acid -helical repeats form structures that are comparable
to HEAT repeats (Michaely et al., 2002), suggesting that PP6 has
a holoenzyme structure that is similar to that of PP2A. These
findings provide compelling evidence for the idea that regulatory
subunits are the key determinants of phosphatase selectivity, both
increasing activity towards specific subunits and reducing activity
towards non-substrate phospho-proteins.
Phosphatase holoenzyme regulation
The existence of large holoenzyme complexes raises the question
of how they are assembled and regulated. In the case of PP2A,
there is evidence that post-translational modifications, including
phosphorylation of regulatory subunits and phosphorylation or
carboxymethylation of catalytic subunits, might determine
holoenzyme composition (Cho and Xu, 2007; Stanevich et al.,
2011). The protein kinase Erk prevents assembly of active PP2A–
B⬘ holoenzymes by phosphorylating the B⬘ regulatory subunit at
serine 337 (Cho and Xu, 2007; Letourneux et al., 2006). As this
region of the B⬘-subunit makes direct contact with the PP2A
catalytic subunit, any conformational change is therefore likely to
affect this interaction (Fig. 2D). Phosphorylation of PP2A and PP1
catalytic subunits by the tyrosine kinase Src at tyrosine 307 and the
mitotic kinase Cdk1 at threonine 320, respectively, inhibits catalytic
activity (Chen et al., 1992; Dohadwala et al., 1994; Kwon et al.,
1997). In both cases, the phosphorylated residue is close to the
C-terminus and phosphorylation in this region is predicted to
disrupt the interaction between the catalytic and B-subunits in
PP2A (Cho and Xu, 2007).
Other factors implicated in the assembly of functional PP2A
holoenzymes are the PP2A phosphatase activator (PTPA) (Fellner
et al., 2003) and the ubiquitin-binding 4 protein (Kong et al.,
2009; Lenoue-Newton et al., 2011; McConnell et al., 2010; Prickett
and Brautigan, 2006). PTPA does not seem to be required for the
assembly of holoenzymes, but in its absence PP2A shows reduced
activity and stability in cells, suggesting that it has a chaperonelike function and maintains PP2A activity (Fellner et al., 2003).
Similar findings have also been reported for 4, although this is
not restricted to PP2A, as 4 has been found to interact with the
form of PP6 active in mitosis (Prickett and Brautigan, 2006; Zeng
et al., 2010). In the case of PP2A, the 4 protein inhibits catalytic
subunit ubiquitylation and proteasomal degradation (LenoueNewton et al., 2011; McConnell et al., 2010), possibly to ensure
that there is a pool of PP2A catalytic subunits available for assembly
into functional holoenzymes. Post-translational modifications of
both catalytic and regulatory subunits are therefore important
considerations, both for the assembly of specific phosphatase
holoenzyme complexes and for their subsequent activity during
mitosis.
Protein phosphatases and the regulation of
mitosis
As outlined above, the requirement for specific protein kinases for
the control of entry into and passage through mitosis implies the
existence of specific protein phosphatases (Barr et al., 2004;
Lindqvist et al., 2009; Nigg, 2001; O’Farrell, 2001; Ruchaud et al.,
2007). Here, we will discuss a number of recent examples that
highlight the importance of protein phosphatase holoenzyme
complexes in controlling both temporal and spatial aspects of
protein phosphorylation during mitosis.
Entering mitosis: inhibition of PP2A–B by the
Greatwall kinase
Entry into mitosis is driven by the Cdk1 kinase and its associated
activator protein cyclin B (Lindqvist et al., 2009; Nigg, 2001;
O’Farrell, 2001). Cdk1 is subject to dual regulation. First, the
synthesis and stability of cyclin B are controlled such that its levels
increase in G2-phase cells, where it can therefore bind to and
activate Cdk1. Second, Cdk1 is inhibited by phosphorylation by
Wee1 family kinases at threonine 14 and tyrosine 15 (Mueller
et al., 1995); these phosphorylations must be removed by the Cdc25
phosphatase for full activity to be achieved (Dunphy and Kumagai,
1991; Kumagai and Dunphy, 1991). This activation of Cdk1 is
generally assumed to be sufficient to drive cells into mitosis, and
degradation of cyclin B and Cdk1 inactivation sufficient to allow
exit from mitosis (Deibler and Kirschner, 2010; Nigg, 2001;
O’Farrell, 2001; Pomerening et al., 2003; Potapova et al., 2006).
A number of lines of evidence show, however, that this is only part
of the picture, as a specific okadaic-acid-sensitive phosphatase,
which dephosphorylates Cdk1 substrates, needs to be inhibited for
mitosis to proceed normally (Burgess et al., 2010; Castilho et al.,
2009; Vandre and Wills, 1992; Vigneron et al., 2009; Zhao et al.,
Protein phosphatases regulating mitosis
2008). Moreover, this phosphatase inhibitory activity needs to be
relieved before cells can exit mitosis (Skoufias et al., 2007).
Greatwall is a protein kinase that was originally identified in the
fruit fly Drosophila melanogaster as a mutant displaying defective
chromosome condensation and progression through mitosis
(Archambault et al., 2007; Yu et al., 2004); similar findings were
also reported for the human homologue microtubule-associated
serine/threonine kinase-like enzyme (MASTL) (Burgess et al.,
2010; Voets and Wolthuis, 2010). A series of studies has revealed
that Greatwall inhibition of PP2A sensitizes cells to Cdk1 activation
and can thus promote entry into mitosis (Burgess et al., 2010;
Castilho et al., 2009; Lorca et al., 2010; Vigneron et al., 2009;
Metaphase
Cdc25
APC/C
Cyclin B
Interphase
Metaphase
Anaphase
ARPP-19
P
ARPP-19
ENSA
Greatwall
PP2A
C
P
PP2A
C
P
P
ARPP-19
B⬘
ENSA
Plk1
P
Bδ
Cohesin
cleavage
Cohesin
dissociation
(Inactive)
P
ENSA
Separase
T- P
(Active)
Substrate
Plk1
Cdk1
T- P
T-OH
(Inactive)
Bδ
Cyclin B
Cdk1
Cdk1
?
Bδ
A-subunit
A-subunit
A-subunit
(Inactive)
(Active)
PP2A
C
Sister
chromatids
A-subunit
PP2A
C
(Active)
Sgo1
Arm
Centromere
Arm
D Organelle reassembly: Golgi and PP2A–B
C Spindle pole formation: PP6 and Aurora A
Interphase
Mitosis
Anaphase
SAPS
2/3 PPP6C
ANKRD22/44
TPX2
Aurora A
Aurora A
T- P
Journal of Cell Science
Anaphase
P
4T1 5
Y1
P
Zhao et al., 2008). Intriguingly, this inhibitory effect is not mediated
by direct phosphorylation of PP2A catalytic or regulatory subunits,
but, instead, involves two closely related small heat-stable proteins
called
endosulfine-
(ENSA)
and
c-AMP-regulated
phosphoprotein-19 (ARPP-19) (Gharbi-Ayachi et al., 2010;
Mochida et al., 2010). ENSA and ARPP-19 are phosphorylated by
Greatwall at serine 67 within a very highly conserved sequence,
and can then bind specifically to PP2A holoenzyme complexes
that contain B-subunits encoded by the PPP2R2D gene (GharbiAyachi et al., 2010; Mochida et al., 2010). This interaction results
in a near-complete inhibition of PP2A–B activity. PP2A–B
activity is therefore the inverse of that of Cdk1–cyclin B, and is
B Cohesin protection: Shugoshin–PP2A–B⬘
A Mitotic entry: Greatwall PP2A–B pathway
Interphase
2327
Cdk1–
cyclin B
T-OH
TPX2
+++
GM130
P
+++
Spindle
(Active)
Cytosol
(Inactive)
PP2A–
Bα
---
p115
---
+++
---
Fig. 3. Spatial control of phosphatase function in mitosis. (A)In interphase cells, Cdk1 activity is inhibited, owing to regulatory phosphorylation of threonine 14
and tyrosine 15, and PP2A–B is active. At the G2-M transition, Cdc25 dephosphorylates Cdk1 at these sites, and Cdk1 associates with cyclin B. Cdk1 activates the
Greatwall kinase, which phosphorylates the ENSA and ARPP-19 family inhibitors and thereby blocks PP2A–B activity. Cdk1 substrates then accumulate in
phosphorylated form. Once the spindle checkpoint is satisfied, the APC/C ubiquitylates cyclin B and it is degraded by the proteasome. ENSA and ARPP-19
inhibition of PP2A–B is relieved and Cdk1 substrates become dephosphorylated. (B)In prophase, Plk1 phosphorylates cohesin complexes and they dissociate from
the chromosomes. This is prevented in the centromeric region by a local pool of PP2A–B⬘ bound to Sgo1 that maintains the cohesin complexes in a
dephosphorylated state and thereby opposes Plk1. (C)Assembly of spindle poles and microtubule organisation requires a set of proteins collectively called spindle
assembly factors (SAFs) (Clarke and Zhang, 2008). The activity of these proteins is under the control of a system involving the Aurora A kinase, which is reviewed
in detail elsewhere (Barr and Gergely, 2007; Clarke and Zhang, 2008). Aurora A is activated and concentrated at spindle poles by interaction with TPX2, and
Aurora A activity is shown by red arrows. This is reversed by the PP6 phosphatase present in the cytoplasm, creating a gradient of Aurora A activity that is high at
the spindle and low in the cytoplasm. Aurora A phosphorylation promotes the activity of SAFs involved in pole separation, pole integrity and kinetochore
microtubule dynamics. (D)During mitosis, the Golgi apparatus is disassembled through the activity of the Cdk1–cyclin B mitotic kinase, reviewed in detail
elsewhere (Lowe and Barr, 2007). In interphase, the Golgi matrix protein GM130 interacts with the p115 vesicle-tethering factor and helps link Golgi membranes
and vesicles together. In mitosis, Cdk1–cyclin B phosphorylates GM130 within a basic region that interacts with an acidic patch in p115, thereby disrupting this
interaction and linkages between Golgi cisternae. At the onset of anaphase, PP2A–B complexes dephosphorylate GM130, allowing reassembly of linkages between
Golgi vesicles and the recreation of ordered stacked Golgi cisternae. The conserved threonine residue in the kinase activation loop is depicted in nonphosphorylated (inactive) and phosphorylated (active) forms by T-OH and T-P, respectively.
Journal of Cell Science
2328
Journal of Cell Science 124 (14)
high in interphase and low in mitosis (Fig. 3A). Forms of PP2A
containing B⬘-, B⬙- and B⵮-subunits are not targets for
phosphorylated ENSA and ARPP-19 (Gharbi-Ayachi et al., 2010;
Mochida et al., 2010), suggesting that this mode of regulation is
highly specific for B-subunits. This is in contrast to inhibitors such
as okadaic acid and microcystins, which bind directly to the
catalytic subunit of the enzyme and show little specificity for
specific PPP family members, as discussed above.
The existing studies differ in their findings on the importance of
endogenous ENSA and ARPP-19 in the control of mitotic entry
(Gharbi-Ayachi et al., 2010; Mochida et al., 2010). One study
reported a role for endogenous ENSA (Mochida et al., 2010),
whereas the other shows an exclusive role for ARPP-19 in
controlling entry into mitosis (Gharbi-Ayachi et al., 2010). Why
two such closely related proteins with similar biochemical function
exist is unclear, but it might relate to other regulatory inputs or
indicate that redundancy in a crucial cellular pathway controlling
mitosis is advantageous. Further studies are therefore needed to
clarify the roles of ENSA and ARPP-19. An important consequence
of this mechanism is that both the extent and half-life of the pool
of Cdk1 phosphorylations normally recognised by PP2A–B in
mitosis is increased (Mochida et al., 2010). This suggests that,
during mitosis, these Cdk1 phosphorylation sites are essentially
stable, and their rapid or dynamic turnover is only associated with
mitotic entry at the G2–M transition and the metaphase–anaphase
transition, which follows spindle checkpoint inactivation.
ENSA and ARPP-19: the first of many mitotic
phosphatase inhibitors?
ENSA and ARPP-19 are not the only phosphorylated phosphatase
inhibitory proteins found in cells that might play a role in mitosis.
PP1 also has heat-stable inhibitors, including inhibitor-1, a protein
of the dopamine- and c-AMP-regulated phosphoprotein family
(DARPP-32; PPP1R1A-C) (Hemmings et al., 1984; Wang, X. et
al., 2008), nuclear inhibitor of protein phosphatase 1 (NIPP1;
PPP1R8), protein kinase C potentiated inhibitor of PP1 (CPI-17;
PPP1R14A) and phosphatase holoenzyme inhibitor 1 (PHI-1;
PPP1R14B) (Beullens et al., 2000; Elbrecht et al., 1990; Eto, 2009;
Huang and Glinsmann, 1975), and inhibitor-2 (PPP1R2), which
have high potency when phosphorylated (Li et al., 2007). Like the
PP2A inhibitors, there is evidence for holoenzyme specificity and
PPP1R14A has selectivity towards PP1–MYPT (PPP1R12A) (Eto
et al., 2004), suggesting that it has a function in mitosis. Intriguingly,
the PP1 inhibitors also bind to certain protein kinases, and might
thereby link kinase activity and phosphatase inhibition. Inhibitor-1
has been shown to regulate Cdk5 activity in neurons (Bibb et al.,
1999), whereas inhibitor-2 can directly activate the Aurora A
mitotic kinase and is required for normal mitotic progression
(Satinover et al., 2004; Wang, W. et al., 2008). Thus, like the
Greatwall–ENSA–ARPP-19 pathway, other PP1 inhibitors might
function in mitosis to promote a switch-like state change, in which
substrate phosphorylation is promoted by simultaneous kinase
activation and phosphatase inhibition.
Cohesion control in mitosis and meiosis: the
PP2A–shugoshin pathway
When cells enter mitosis, DNA condenses to form distinct bodies,
the chromosomes. These comprise two sister chromatids that
correspond to two discrete copies of the genome. The sister
chromatids are linked by proteinaceous multisubunit cohesin
complexes that form rings around the two sister chromatids
(Nasmyth and Haering, 2009). In mammalian cells, most cohesin
is removed from the chromosome arms during prophase by
phosphorylation mediated by Polo-like kinase 1 (Plk1) and Aurora
B (Hauf et al., 2005). However, cohesin-mediated linkages at the
centromeres are protected at this time and thus give mitotic
chromosomes their characteristic X-shaped morphology. This
arrangement is needed for the attachment of chromosomes to the
mitotic spindle, such that one sister chromatid is attached to
microtubules that emanate from one pole of the spindle and the
paired sister chromatid is linked to the opposite pole. The spindle
checkpoint signalling pathway, reviewed extensively elsewhere
(Kops, 2008), prevents further progression through mitosis until
this geometry is achieved at all chromosomes. How, then, is
centromeric cohesion protected? Shugoshin 1 (Sgo1) was originally
discovered as a fission yeast factor required to protect the meiotic
cohesin protein Rec8 at the centromere from cleavage by separase
during anaphase I of meiosis (Kitajima et al., 2004). The fission
yeast paralogue Sgo2 has a similar role in fission yeast mitosis
(Kitajima et al., 2004). Mammals have two shugoshins, Sgo1 and
Sgo2, which have discrete functions in mitosis and meiosis,
respectively. Sgo2-knockout mice are viable but infertile, indicating
that mammalian Sgo2 is important for meiosis, but not for mitosis
(Llano et al., 2008), whereas mammalian Sgo1 is required for
mitosis (Salic et al., 2004; Tang et al., 2004). In human cells, Sgo1
localises to centromeres and its depletion results in the premature
loss of all sister chromatid cohesion in early mitosis (Kitajima
et al., 2005; McGuinness et al., 2005; Salic et al., 2004), which
supports the idea that it protects centromeric cohesion during
M-phase. The loss of sister chromatid cohesion in human Sgo1depleted cells can be suppressed by expressing a nonphosphorylatable cohesin SA2 subunit (McGuinness et al., 2005).
The explanation for these properties came from the analysis of
purified Sgo1 complexes, which were found to contain specific
PP2A holoenzymes with B⬘-subunits encoded by the PPP2R5A-E
genes (Kitajima et al., 2006; Riedel et al., 2006; Tang et al., 2006).
A similar mechanism operates for Sgo2 (Kitajima et al., 2006;
Tanno et al., 2010); however, although Sgo1 binds only to
assembled PP2A–B⬘ holoenzymes, Sgo2 can bind to PP2A catalytic
subunits alone, as well as to PP2A holoenzymes (Xu et al., 2009).
It is important to note that the interaction of Sgo1 and Sgo2 with
PP2A–B⬘ complexes does not obviously influence their catalytic
activity (Xu et al., 2009), suggesting that they are specific PP2A–
B⬘ targeting or localisation factors.
Together, these findings suggest that Sgo1 recruits a pool of
PP2A–B⬘ holoenzymes to the centromeres, where they oppose
phosphorylation by Plk1 and Aurora B by dephosphorylating specific
substrates (Fig. 3B). This maintains centromeric cohesin in a
dephosphorylated state, and thereby prevents the prophase pathway
from removing cohesin at centromeres, but not at chromosome arms.
A further important point that is highlighted by these findings is the
need to simultaneously promote phosphorylation by one pathway,
while opposing phosphorylation by another. In mitosis, this problem
is solved in the case of the Greatwall–ENSA–ARPP-19 pathway
and the shugoshin pathways by their interaction with specific PP2A
holoenzymes and either inhibiting them or locally activating them,
respectively. As discussed above, it is the specific class of regulatory
subunit found in each of these PP2A holoenzymes that imparts these
specific regulatory and substrate-binding properties. In this way,
phosphorylation of Cdk1 substrates is promoted, whereas, at the
same time, phosphorylation of centromeric cohesion complexes is
prevented.
Protein phosphatases regulating mitosis
Journal of Cell Science
Spindle formation: PP1 regulation of Nek2
Formation of a stable bipolar spindle structure is a key step in
mitosis and is required for accurate segregation of the
chromosomes (Wittmann et al., 2001). The first step in spindle
formation, as cells enter mitosis, involves the separation of the
duplicated centrosomes to create two independent microtubuleorganising centres, which ultimately give rise to the two spindle
poles. This centrosome-splitting event is controlled by the Nek2
protein kinase and an opposing okadaic-sensitive phosphatase of
the PP1 family (Ghosh et al., 1998; Helps et al., 2000; Meraldi
and Nigg, 2001; Mi et al., 2007). PP1 docks with an RVXF-type
motif in Nek2, and has a role in controlling both kinase activity
and dephosphorylation of Nek2 substrates, such as the centriolarlinker protein C-Nap1 (Helps et al., 2000). More recent evidence
suggests that both PP1 and PP1 can bind Nek2, but it is PP1
that is more crucial for the regulation of Nek2 activity in vivo
(Mi et al., 2007).
Spindle formation: PP6 regulation of Aurora A
Once centrosome splitting has occurred, the centrosomes move
apart and their microtubule-organising properties are remodelled
to promote bipolar spindle formation. These events are controlled
by two main kinases, Aurora A and Plk1, and inhibition or depletion
of either kinase leads to monopolar spindles that are unable to
mediate chromosome segregation (Girdler et al., 2006; Glover
et al., 1995; Lane and Nigg, 1996; Lenart et al., 2007; Sumara et al.,
2004; Sunkel and Glover, 1988). Like many protein kinases,
Aurora A and Plk1 are regulated by phosphorylation of the
conserved so-called activation or T loop at a conserved threonine
residue (Bayliss et al., 2003; Huse and Kuriyan, 2002; Sessa et al.,
2005). This phosphorylation event is carried out either in an
autocatalytic fashion or by an upstream activating kinase, and
results in the correct positioning of key catalytic residues for the
phosphotransfer reaction (Huse and Kuriyan, 2002). Targeting and
activation of Aurora A and the related kinase Aurora B are
accompanied by binding to the activator proteins TPX2 (originally
identified as a targeting protein for Xenopus kinesin-like motor
protein 2) and inner centromere protein (INCENP), respectively
(Bayliss et al., 2003; Kufer et al., 2002; Ruchaud et al., 2007;
Sessa et al., 2005). This interaction protects the phosphorylated
T loop from dephosphorylation and stabilises the active form of
the kinase (Bayliss et al., 2003). A prevailing view in the field of
mitosis is that mitotic kinases, such as Aurora A, Plk1 and Aurora
B, are ‘switched on’ by T-loop phosphorylation upon entry into
mitosis and are ‘switched off’, generally by degradation, at the end
of mitosis (Barr et al., 2004; Nigg, 2001; Ruchaud et al., 2007).
However, such a static model does not take into account that the
activity of these mitotic kinases is both spatially and temporally
regulated at multiple sites and different times during mitosis,
therefore suggesting that there is a highly dynamic equilibrium
between kinase T-loop phosphorylation and dephosphorylation.
Surprisingly, until recently, there was little information concerning
the identity of the T-loop phosphatases that regulate the major
mitotic kinases.
Recent efforts from our own groups have resulted in the
identification of the PP2A family phosphatase PP6 as the mitotic
T-loop phosphatase for Aurora A–TPX2 complexes in vivo (Zeng
et al., 2010). Both PP1 and PP2A catalytic subunits had been
previously implicated as regulators of free Aurora A T-loop
phosphorylation in vitro (Bayliss et al., 2003; Eyers and Maller,
2004; Tsai et al., 2003), but our study demonstrates that the PP6
2329
holoenzyme is the major Aurora A–TPX2 T-loop phosphatase in
both mitotic extracts and intact mitotic cells (Zeng et al., 2010).
Depletion of PP6 catalytic or regulatory subunits, but not PP1 or
PP2A, leads to the stabilization of Aurora A T-loop phosphorylation
(Zeng et al., 2010). As a consequence, Aurora A is hyperactive, and
Aurora-A-regulated spindle assembly factors are misregulated and
fail to target to the mitotic spindle (Fig. 3C). This then results in
impaired bipolar spindle assembly and defective chromosome
segregation (Chen et al., 2007; Zeng et al., 2010). Aurora A
overexpression and amplification is considered to be a driving
force for tumour formation; therefore, regulation of Aurora A
activity by PP6 has relevance beyond the cell biology of mitosis
(Bischoff et al., 1998). Depletion of PP6 might have a similar
effect to amplification of Aurora A, and, indeed, at least one tumour
cell line is known in which both PP6 alleles are mutated (Zeng
et al., 2010). Furthermore, loss of PP6 activity, which limits
Aurora A kinase activity, might be detrimental when Aurora A is
already amplified and might lead to elevated cell death due to
mitotic catastrophe.
Spindle formation: PP1 regulation of Aurora B
and Plk1
Another specific PPP holoenzyme has been implicated in the
regulation of the T loop of the related kinase Aurora B, which
acts in regulating bipolar attachment of chromosomes to the
forming mitotic spindle. Similar to Aurora A, Aurora B binds to
the activator protein INCENP, which mediates both its activation
and targeting to chromosomes (Ruchaud et al., 2007). On the
basis of a series of cell biological studies, the PP1–PPP1R7
(Sds22) holoenzyme complex has been suggested to act as the Tloop phosphatase for Aurora B (Posch et al., 2010; Sugiyama et
al., 2002). Cells depleted of PP1 or PPP1R7 (Sds22) have defects
in the regulation of chromosome attachment to the mitotic spindle.
Biochemical analysis showing that PP1–PPP1R7 has T-loop
phosphatase activity towards activated Aurora B–INCENP
complexes is currently lacking, but, consistent with this idea,
cells that are depleted of PPP1R7 have increased Aurora B Tloop phosphorylation (Posch et al., 2010). Similarly, there is
evidence that a MYPT-targeted pool of PP1 can influence the
activity and level of T-loop phosphorylation of Plk1 (Yamashiro
et al., 2008). Plk1 activation and localisation involves docking to
phosphorylated binding partners (Barr et al., 2004), and MYPT
phosphorylation by Cdk1 in M phase creates such a Plk1-docking
site (Yamashiro et al., 2008). It seems counter-intuitive that Plk1
would dock with a protein that would then simply reduce its
kinase activity, and it is possible that this interaction might
therefore reflect the need to coordinate phosphorylation and
dephosphorylation of downstream substrates, rather than of Plk1
itself.
One remaining puzzle relates to how PP6 or PP1–PPP1R7
access the phosphorylated T loop in activated Aurora A–TPX2 or
Aurora B–INCENP complexes. In contrast to most substrates, the
T loop is buried in the kinase active site, rather than being exposed
on the surface of the protein. PP6 and PP1–PPP1R7 therefore need
to interact with the activated kinase to trigger local unfolding or
restructuring of the T-loop peptide, so that it becomes accessible to
the phosphatase catalytic site. Once dephosphorylated, the kinase–
activator complex would disassemble and dissociate from the
phosphatase. Although this is an intriguing speculation, it highlights
the need for relevant structures of phosphatase–substrate complexes
to be resolved.
Journal of Cell Science
2330
Journal of Cell Science 124 (14)
Tension between kinase and phosphatase:
Aurora B and PP1
In addition to direct regulation of Aurora B, PP1 is also involved
in the dephosphorylation of Aurora B substrates at kinetochores,
thereby stabilizing microtubule attachments at kinetochores under
tension (Liu et al., 2010) and helping to promote spindle checkpoint
silencing (Meadows et al., 2011; Pinsky et al., 2009; Vanoosthuyse
and Hardwick, 2009). This is mediated by a pool of PP1 that
directly associates with conserved RVXF and SILK docking motifs
in the KNL1 subunit of the microtubule attachment site formed by
the KNL1–Mis12–Ndc80 (KMN) network (Liu et al., 2010; Wan
et al., 2009; Welburn et al., 2010). In fission yeast, the KNL1
homologue, Spc7, and two additional factors, Klp-5 and Klp-6,
which form the kinesin-8 motor complex, bind directly to PP1 and
have a similar role (Meadows et al., 2011). In the absence of
tension, the centromere-associated pool of Aurora B is in close
spatial proximity to the kinetochores and can phosphorylate KNL1
near to the PP1-docking site. This modification prevents docking
of PP1 to KNL1 and therefore allows Aurora B substrates to
become phosphorylated, which destabilizes microtubule attachments
(Liu et al., 2010; Meadows et al., 2011). If microtubule attachment
leads to the generation of tension, which stretches the kinetochore
away from the centromere, Aurora B can no longer phosphorylate
KNL1 (Liu et al., 2010). PP1 can then bind to KNL1 and
dephosphorylate Aurora B substrates at kinetochores, thus
stabilizing microtubule attachments at kinetochores under tension
(Liu et al., 2010).
Leaving mitosis: PP1 and PP2A clean up after
the party
Once all of the chromosomes have been aligned to form a
metaphase plate and the spindle checkpoint is satisfied, the
anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase
modifies its two key substrate proteins – cyclin B and the separase
inhibitor securin. Cdk1 inactivation by cyclin B degradation allows
exit from mitosis and entry into anaphase, whereas separase cleaves
cohesin linkages at centromeres and allows sister chromatid
separation in anaphase (Musacchio and Salmon, 2007). This is
insufficient to allow exit from mitosis, as the mitotic
phosphorylation carried out by Cdk1 and other kinases still needs
to be reversed. In budding yeast, this is carried out by the Cdc14
phosphatase, whose activity is under the control of a complex
signalling pathway, the mitotic exit network (Stegmeier and Amon,
2004). Although there is some evidence that Cdc14 has a role in
anaphase regulation and cytokinesis in multicellular eukaryotes, it
is unlikely to be the major phosphatase controlling Cdk1 substrate
dephosphorylation. Recent evidence indicates that vertebrate Cdc14
has a more important function in DNA repair than in mitotic
regulation (Mocciaro and Schiebel, 2010; Mocciaro et al., 2010).
Studies on a Drosophila abnormal anaphase resolution (aar)
mutant suggest that the phosphatase pathways that promote
anaphase onset are different in yeast and higher eukaryotes. The
aar mutation maps to the gene for the fruit fly PP2A–B complex
and, as the name suggests, results in abnormal separation of
chromosomes in anaphase (Mayer-Jaekel et al., 1993). Additionally,
mutations in Drosophila PP1 cause defective sister chromatid
segregation and abnormal chromatin condensation (Axton et al.,
1990). More recent publications show that both PP1 and PP2A are
required for mitotic exit (Mochida et al., 2009; Schmitz et al.,
2010; Wu et al., 2009). This reflects the need for additional
phosphatases that act on substrates required for processes other
than sister chromatid separation and for local control of phosphatase
activity. The PP1–Repo-man (CDCA2) pathway, which regulates
chromatin architecture in anaphase, provides an example of the
need for local control (Trinkle-Mulcahy et al., 2006; Vagnarelli
et al., 2006). Cdk1–cyclin B negatively regulates Repo-man by
direct phosphorylation until the onset of anaphase, when Repoman becomes dephosphorylated and then targets PP1 to chromatin
(Trinkle-Mulcahy et al., 2006; Vagnarelli et al., 2006). In chicken
B-cell lines that lack condensin complexes, chromosomes lose
their structure during anaphase, which results in abnormal mitosis
(Vagnarelli et al., 2006). This defect can be suppressed by depletion
of Repo-man or overexpression of a B-type cyclin (Vagnarelli
et al., 2006). The authors of this paper suggest that an as yet
unidentified activity, which they term regulator of chromatin
architecture (RCA), is inactivated, and presumably
dephosphorylated, by PP1–Repo-man. More recent evidence
indicates that one substrate of the Repo-man pathway is the form
of histone H3 that is phosphorylated by the protein kinase haspin
at threonine 3 (Qian et al., 2011). Threonine-3-phosphorylated
histone H3 is found predominantly at centromeres, where it forms
the chromosome binding site for the survivin subunit of the Aurora
B chromosomal passenger complex (Dai et al., 2006; Kelly et al.,
2010; Wang et al., 2010). A Repo-man-bound pool of PP1 actively
dephosphorylates histone H3 at threonine 3 during metaphase and
as cells exit mitosis, and prevents the spread of Aurora B away
from the centromeres and onto the chromosome arms (Qian et al.,
2011).
Although globally increased phosphatase activity is important
for the removal of the phosphorylations carried out by Cdk1–
cyclin kinase, if anaphase is to occur, other protein kinases must
remain active and have essential functions in the later events of
cell division. Both Plk1 and Aurora B have to phosphorylate
substrates that are required for cell contractility and the proper
timing of cytokinesis (Bastos and Barr, 2010; Douglas et al., 2010;
Neef et al., 2007; Neef et al., 2006; Petronczki et al., 2007; Wolfe
et al., 2009). The activity of a number of these substrates is
inhibited by Cdk1 and activated by Aurora B and Plk1
phosphorylation, respectively (Mishima et al., 2004; Neef et al.,
2007; Toure et al., 2008). It is therefore essential that the
phosphatase holoenzymes that remove the phosphorylation carried
out by Cdk1, Plk1 and Aurora B during mitosis do not act on Plk1
or Aurora B phosphorylation sites that are required for anaphase
and cytokinesis. Although a clearer picture has begun to emerge of
the importance of phosphatase holoenzyme complexes that act on
different classes of phosphorylation site for the proper regulation
of mitosis and cytokinesis, many details remain to be resolved.
Membrane organelles: rebuilt behind the
Greatwall
Cells exiting mitosis also need to reassemble organelles of
the secretory and endocytic pathways, as well as segregate the
chromosomes and rebuild an interphase nucleus (Lowe and Barr,
2007). The role of phosphorylation in the assembly and disassembly
of the Golgi apparatus is well understood, and a number of key
substrates for mitotic kinases and phosphatases have been identified,
as reviewed elsewhere (Lowe and Barr, 2007). In the case of one
of these substrates, the Golgi matrix protein and vesicle-tethering
factor GM130, dephosphorylation during anaphase at a conserved
Cdk1 phosphorylation site has been shown to involve the PP2A–B
(PPPR2A) holoenzymes (Fig. 3D) (Lowe et al., 2000; Lowe
et al., 1998). Intriguingly, this is the same PP2A holoenzyme class
Protein phosphatases regulating mitosis
that is inhibited by Greatwall (Gharbi-Ayachi et al., 2010; Mochida
et al., 2010; Schmitz et al., 2010), suggesting that this pathway
also contributes to Golgi disassembly and reassembly in mitosis.
Importantly, neither PP2A–B⬘ (PPP2R5A) holoenzymes nor PP1
complexes can dephosphorylate GM130 (Lowe et al., 2000). Once
GM130 is dephosphorylated, Golgi membrane vesicles become
tethered together and self-organise to form a stacked structure that
is characteristic of interphase cells (Lowe et al., 2000; Lowe et al.,
1998; Shorter and Warren, 1999).
Journal of Cell Science
A dynamic future for protein phosphatases in
mitosis
Emerging evidence has started to provide a clearer picture of how
substrate phosphorylation is controlled during mitosis. Multiple
inhibitory mechanisms exist to reduce the activity of some PP1
and PP2A holoenzymes towards their substrates. Importantly, this
inhibition is coordinated with the activation of mitotic kinases such
as Cdk1–cyclin B. As a consequence, phosphorylation of these
substrates will increase during prophase and metaphase.
Furthermore, these phosphorylations will be essentially stable
during this time (long half-life), owing to the absence of specific
phosphatase activity (summarised in Fig. 4A). Dynamic turnover
A
of these sites will occur only during the mitotic entry and exit
transitions. Simultaneously, pools of other phosphatase
holoenzymes remain active during mitosis and might even be
concentrated at specific cellular structures. A pool of PP2A–B⬘ is
concentrated at centromeres, where it reverses Plk1 phosphorylation
of cohesin complexes and thereby prevents their dissociation from
chromatin. Cohesin at chromatin arms will thus not be protected
and is subsequently phosphorylated and displaced (Fig. 4B).
Although there has been a large amount of progress in our
understanding of phospho-regulation and the role of specific protein
phosphatases in mitosis, many questions remain. Phosphatase
regulators have been identified for a number of crucial pathways
in mitosis and, because of the known importance of dynamic
phosphorylation in other pathways, many more must remain to be
identified. Examples include spindle checkpoint control,
centrosome and microtubule function, cell shape and adhesion,
and central spindle control during cytokinesis. In this Commentary,
we have stressed the importance of the identification and
verification of phosphatase holoenzyme complexes that are linked
to discrete biological functions. Phosphatase catalytic subunits do
not act alone, and it is unlikely that useful information can be
obtained using them in isolation in vivo or in vitro. As discussed
Mitotic entry
S
G2
Prophase-Metaphase
G2/M
2331
Mitotic exit
Anaphase-Telophase
APC/C activation
G1
Cyclin B destruction
Cyclin B levels
Cdk1 activity
PP2A–Bδ activity
Greatwall activity and ENSA phosphorylation
PP1 activity
Cdk1 inhibition of PP1 catalytic
and regulatory subunits
Rapid turnover
Rapid turnover
Cdk1 substrate phosphorylation
Half-life of phosphorylation
Stable phosphorylation of
PP2A–Bδ and PP1 substrates
B
Plk1 activity
PP2A–B⬘ activity at centromeres (Shugoshin)
Arm cohesin phosphorylation
Centromeric cohesin phosphorylation
Cohesin cleavage/destruction
Fig. 4. Temporal control of phosphorylation site dynamics by coordinated kinase and phosphatase activity. (A)The levels and activity of key kinase and
phosphatase regulators of mitosis. Cdk1, cyclin B, PP1, PP2A–B and the Greatwall pathway are depicted, together with the extent and half-life of protein
phosphorylation. (B)PP2A–B⬘ remains active during mitosis and is concentrated at centromeres, where it protects cohesins from Plk1 activity.
Journal of Cell Science
2332
Journal of Cell Science 124 (14)
here, there are also spatial and temporal considerations – simply
put, is the phosphatase active at the right time and in the right place
to dephosphorylate the substrate in question (Figs 3 and 4)? Caution
is especially necessary when interpreting in vitro data, because,
although a particular enzyme might dephosphorylate a given
substrate, it might not actually be active at the appropriate time and
place within cells.
The discovery of the algal toxins okadaic acid and microcystin
created much excitement (Bialojan and Takai, 1988; Cohen et al.,
1990; MacKintosh et al., 1990), but, as discussed in this
Commentary, this was tempered by the realisation that they show
little specificity for individual PPP family members. For this and
other reasons, PPP enzymes have not been viewed as promising
targets for drugs or small inhibitory molecules. This view has
started to change, owing to a number of discoveries. First, there is
the identification of highly specific cellular inhibitors of PP2A
holoenzymes, which have added to the known group of specific
PP1 inhibitors. Second, a small compound called guanabenz can
be used to disrupt specific PP1–PPP1R15A holoenzyme complexes
(Tsaytler et al., 2011). Structures of PP2A holoenzymes bound to
ENSA and ARPP-19 inhibitors, or PPP1R15A bound to guanabenz,
will therefore provide valuable insight into how holoenzymespecific inhibition can be achieved. These principles should prove
useful to aid the development of specific phosphatase inhibitory
compounds and drugs with a multitude of potential uses, both in
the laboratory and in the clinic.
We thank Anja Dunsch for comments on the manuscript and Kang
Zeng for many discussions. Research in our groups is supported by
a Cancer Research UK programme grant to F.A.B. (C20079/A9473)
and a Cancer Research UK career development fellowship to U.G.
(C24085/A8296).
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