The Wellcome Trust Centre for Cell Biology, Institute of Cell and
Molecular Biology, The University of Edinburgh, Edinburgh EH9 3JR, UK
* Present address: MRC Human Genetics Unit, Western General Hospital, Edinburgh
EH4 2XU, UK
Author for correspondence (e-mail:
h.ohkura{at}ed.ac.uk)
Accepted 11 December 2002
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Polo, Kinase, Cell cycle, Fission yeast
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
One of the most intriguing aspects of polo kinase function is the variety
of tasks they execute throughout the cell cycle. Polo kinases are required at
several key points through mitosis, starting from control of the G2/M
transition through phosphorylation of Cdc25C and mitotic cyclins
(Abrieu et al., 1998;
Karaiskou et al., 1999
;
Kumagai and Dunphy, 1996
;
Ouyang et al., 1997
;
Qian et al., 1998
;
Toyoshima-Morimoto et al.,
2001
) and a role in the DNA damage checkpoint to prevent entry
into mitosis (Sanchez et al.,
1999
; Smits et al.,
2000
; Toczyski et al.,
1997
). At the beginning of mitosis, various proteins are recruited
to the centrosomes, a maturation process which requires polo kinases
(Sunkel and Glover, 1988
;
Lane and Nigg, 1996
). Polo
kinases are also required for the establishment of a bipolar spindle
(Ohkura et al., 1995
;
Lane and Nigg, 1996
;
Qian et al., 1998
), a
conserved function which is evident from the phenotype of the original
Drosophila polo1 mutant
(Llamazares et al., 1991
;
Sunkel and Glover, 1988
).
Equally, polo kinases are important for exit from mitosis. A role in the
metaphase to anaphase transition via an interaction with the anaphase
promoting complex/cyclosome (APC/C) has been demonstrated
(May et al., 2002;
Descombes and Nigg, 1998
;
Charles et al., 1998
;
Shirayama et al., 1998
). In
addition, budding yeast polo kinase phosphorylates cohesin to allow
proteolysis by separase in order to initiate anaphase
(Alexandru et al., 2001
).
Fission yeast polo kinase, Plo1, is required for formation and correct
positioning of the septum and overexpression induces septation even in
interphase cells (Ohkura et al.,
1995; Bahler et al.,
1998
). Overproduction of murine or budding yeast polo kinases in
budding yeast cells also induces septation in a non-catalytic domain dependent
manner (Lee and Erikson, 1997
;
Song et al., 2000
), and a
physical interaction has been demonstrated between the budding yeast polo
kinase, Cdc5p, and septins (Song and Lee,
2001
). This function in cytokinesis has also been shown to be
conserved in Drosophila (Carmena
et al., 1998
; Herrmann et al.,
1998
).
Dynamic, cell cycle regulated localisation and kinase activation during
mitosis is a feature common to all members of the family. Localisation to the
SPB/centrosome occurs early in mitosis and persists until late anaphase, when
the protein has been seen to relocalise to the midbody or to the future site
of cell cleavage (Bahler et al.,
1998; Golsteyn et al.,
1995
; Logarinho and Sunkel,
1998
; Moutinho-Santos et al.,
1999
; Mulvihill et al.,
1999
; Shirayama et al.,
1998
; Song et al.,
2000
). Detailed study in fission yeast has indicated that
localisation to the SPB takes place very early in mitosis, prior to full
activation of catalytic activity (Tanaka
et al., 2001
). This cell cycle regulated catalytic activation and
localisation is likely to play an important role in the execution of polo
kinase function.
Polo-like kinases are characterised by an amino terminal catalytic domain,
and a carboxy terminal non-catalytic domain consisting of three blocks of
conserved sequences known as polo boxes
(Glover et al., 1996)
(Fig. 1). Studies have been
carried out to identify the role of this non-catalytic domain in budding yeast
and mammalian cultured cells. In mammalian cells, the C-terminus of mammalian
Plk1 alone directs localisation to centrosomes
(Jang et al., 2002
;
Seong et al., 2002
). A
mutation in polo box 1 abolished the ability of mammalian or budding yeast
polo kinase to localise to the mitotic apparatus in budding yeast
(Lee et al., 1998
;
Song et al., 2000
) or to
complement a budding yeast mutant (Jang et
al., 2002
; Lee et al.,
1998
; Song et al.,
2000
). Also, the effects of overexpression of the kinase in
budding yeast or mammalian cells were shown to be dependent on polo boxes
(Lee et al., 1998
;
Seong et al., 2002
). However,
whether the non-catalytic region consists of functionally separable
sub-domains, in particular in the light of multi-functional nature of polo
kinase, has not been addressed.
|
Fission yeast is an excellent model organism for the dissection of the
multi-functional nature of polo kinase due to the ease with which it is
genetically manipulated and the clarity with which multiple functions are
observed. In fission yeast, deletion of the polo kinase gene,
plo1+, results in three major defects failure to
establish spindle bipolarity, failure to form septa or misplacement of septa
(Ohkura et al., 1995;
Bahler et al., 1998
). In
addition, overexpression of the polo kinase results in two clear phenotypes,
cells displaying multiple septa and mitotically arrested cells with monopolar
spindles (Ohkura et al.,
1995
). Involvement in entry into mitosis has been suggested
through the interaction with the SPB protein Cut12 and the NimA-like kinase
Fin1 (Mulvihill et al., 1999
;
Grallert and Hagan, 2002
) and
a role in metaphase to anaphase transition via an interaction with the APC/C
subunit Cut23 has also been demonstrated for fission yeast Plo1
(May et al., 2002
).
Furthermore, it has been shown that Plo1 is at the top of the septum
initiating network (SIN) (Tanaka et al.,
2001
) demonstrating that many of the conserved functions of polo
kinases may be studied using fission yeast as a model organism.
In this study we examined the roles of conserved amino acid sequences in the fission yeast polo-like kinase (Plo1) and in particular their relationship with multiple functions. Functions were explored by overexpression experiments and by stable expression of mutant genes at wild-type levels in a plo1 deletion background. We have shown, using a combination of site directed and a novel random mutagenesis method, that the polo boxes form a single functional unit required for in vivo function, SPB localisation and multiple protein-protein interactions.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Fission yeast techniques
Fission yeast analyses were carried out as described
(Moreno et al., 1991;
Ohkura et al., 1995
). Strains
used in this study are shown in Table
1.
|
In overexpression experiments, plo1 mutants were expressed from
the nmt1 promoter in pREP1
(Maundrell, 1993). Cells were
grown in the presence of thiamine and then washed in sterile water before
growth in two parallel cultures, either in the presence or absence of 4 µM
thiamine, for 15-16 hours.
For plasmid shuffling experiments, spores were prepared from a diploid
strain heterozygous for deletion of plo1 (Sp269) carrying wild-type
plo1 on a ura4+ marked plasmid
[pURplo1+ (May et al.,
2002)] and mutant plo1 in pREP1. Germination was carried
out in the presence of thiamine and plo1
haploids selected.
The presence of pURplo1+ maintains viability of the
deletion while thiamine represses expression of the plo1 mutants. The
strains were replica plated three times to media containing uracil (both in
the presence and absence of thiamine) to allow loss of the
pURplo1+ plasmid. Cells that had lost the plasmid were
selected by using 5-FOA, which kills Ura+ cells. Cells
were spotted on selective media in the presence or absence of both 5-FOA and
thiamine. Abilities of each plo1 mutant to complement a plo1
deletion were tested either at approximately wild-type levels of expression (+
thiamine, nmt1 repressed) or when overexpressed (- thiamine,
100
times that of wild-type expression levels, nmt1 de-repressed).
Spore germination analysis was carried out to determine whether expression
of the mutant genes at wild-type level from an integrated copy were able to
support growth in the absence of endogenous plo1+.
HA-tagged plo1 mutants under the control of the attenuated
nmt1 promoter (derived from pREP41) were integrated at the
leu1 locus of a
plo1::his3+/plo1+ diploid strain
(Sp269). Sp269 was created by replacing the entire coding region of one
plo1 gene with his3+ using a PCR-mediated method
(Bahler et al., 1998).
Integration was confirmed by PCR using primers 5' to leu1 and
within the polo box domain of plo1. Comparison of the number of
His+ (plo1
) and His-
(plo1+) haploids obtained following spore germination
revealed whether a particular mutant complemented the plo1
disruptant. Full rescue of the deletion phenotype resulted in the ratio of
His- to His+ haploids of 1:1. Where no rescue occurred.
no His+ haploids were found.
Fluorescence microscopy
Samples were prepared and fixed as described previously
(May et al., 2002;
Ohkura et al., 1995
).
Localisation of the Plo1 mutant proteins in vivo was observed in strains
expressing GFP-Plo1 under the control of the attenuated nmt1 promoter
by autofluorescence while Tat1 antibody
(Woods et al., 1989
) and Sad1
antibody (Hagan and Yanagida,
1995
) were used for visualising microtubules and SPB,
respectively. Cells were observed with an Axioplan 2 microscope (Zeiss).
Images were captured using a CCD camera (Hamamatsu) and processed using
Openlab2 (Improvision) and Photoshop (Adobe).
Two-hybrid analyses
Two-hybrid screening using plo1+ as a bait was carried
out as described (May et al.,
2002) and positive interactors confirmed by isolation and
reintroduction of the prey plasmids along with either
pBTM116plo1+ or pBTM116.
Isolation of plo1 mutants, which retain only a subset of
interactions was carried out as follows. For random mutagenesis of
plo1, pBTM116plo1+ was used as template in a PCR
reaction using Taq polymerase without proofreading activity (Roche). Primers
used in the reaction were complementary to the 5' end of
plo1+ and to the ADH1 terminator sequence, which
is 3' to the plo1+ gene in
pBTM116plo1+. Strain L40 was transformed with gapped
pBTM116 plasmid and PCR product. Gap repair in vivo resulted in the recreation
of pBTM116plo1 with mutations in plo1
(Muhlrad et al., 1992). Each
L40 stain carrying mutagenised plo1 bait constructs was mated with
Y187 strains carrying prey plasmids. Mating efficiency was determined by
growth on selective media and the two-hybrid interaction assessed by
expression of the HIS3 reporter gene (by growth on selective media
lacking histidine and containing 10 mM 3-AT after 2 days at 32°C).
Plasmids were isolated from strains with pBTM116plo1 constructs of
interest, re-transformed to confirm the interaction pattern, and sequenced to
determine the mutation site.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
First the mutant genes were overexpressed in wild-type cells under the
control of the nmt1 promoter on a multicopy plasmid, pREP1.
Overexpression of the wild-type plo1 gene resulted in two major cell
defects (Ohkura et al., 1995)
(Fig. 2B). One is the induction
of untimely septation, which can be seen in cells with multiple septa or
mononucleated cells with septa. The other is a failure to establish the
bipolarity of the mitotic spindle resulting in mitotic arrest, which can be
seen as cells with overcondensed chromosomes and monopolar spindles.
|
To see whether the non-catalytic domain consists of functionally separable units, we tested various mutants in the polo boxes. These include various point mutations at highly conserved amino acids in polo box 1 (W497F, G505A, YQL508AAA and FN519AA), polo box 2 (L577A) or polo box 3 (DHK625AAA), and series of C-terminal deletions (50 amino acids each; 1-633, 1-583, 1-533, and 1-483). We found that all of the mutations created in the non-catalytic domain show identical effects upon overexpression. They all abolished the mitotic arrest phenotype but had little effect on induction of untimely septation (Fig. 2C,D,K as examples, and data not shown for other mutations). This demonstrates that the overexpression effects of plo1+ on spindle formation and septation are separable and that the former is dependent on the integrity of all of the polo boxes but the latter is not. Mutations in any one of the three polo boxes led to an identical effect suggesting that the polo boxes may act as a single functional unit.
On the other hand, mutations in the kinase domain of Plo1 result in the
opposite effect. We tested various mutations (K69R, K69Q, D181R, D181N, E193V,
and T197V) in conserved residues that are known or thought to be important for
the activity of polo kinases. All of the mutants in the catalytic domain
resulted in mitotic arrest and, in some cases, lack of septum upon
overexpression (Fig. 2E,L).
Immunostaining indicated that the mitotic arrest was associated with monopolar
spindles in all kinase domain mutants (Fig.
2G-I). As one of the mutants (Plo1K69R) has been shown to have no
or little kinase activity (Tanaka et al.,
2001), this is likely to be due to dominant negative effects
caused by overexpression of inactive (or less active) kinase. To test whether
the mitotic arrest was due to activation of the spindle checkpoint, Plo1K69R
was overproduced in the checkpoint-defective mad2 deletion mutant.
Inactivation of the spindle checkpoint completely abolished the mitotic arrest
upon overexpression of kinase domain mutants, and instead gave rise to cells
with large nuclei probably resulting from a continuation of the cell cycle
without nuclear division (Fig.
2L).
We also examined whether this dominant negative effect is dependent on intact polo boxes. Mutations in either polo box 1 (YQL508AAA) or 3 (DHK625AAA) completely abolish the effects of overexpression of an inactive kinase Plo1K69R (Fig. 2F), confirming the importance of polo boxes for the plo1 function.
Polo boxes are required for cellular function
To determine whether the plo1 mutants we created retain function
in vivo, we first tested complementation of a plo1 disruptant using
plasmid shuffling. In these experiments, we first constructed a plo1
disruptant (plo1::his3+) which contains a wild-type copy
of plo1+ (pURAplo1+) on a
ura4+ marked plasmid and mutant plo1
(pREP1plo1*) on a LEU2 marked plasmid. We then assayed the
ability of pREP1plo1* to support the growth of the plo1
disruptant when pURAplo1+ is lost (selectable by 5-FOA
which kills Ura+ cells).
The wild-type plo1 gene was able to fully support the growth of a plo1 disruptant. Various mutants in the kinase domain which we tested (Plo1K69R, K69Q, D181R, T197V, and E193V) supported little or no growth of the disruptant. Mutants in the non-catalytic domain also failed to support the growth of the disruptant (W497F, G505A, YQL508AAA, FN519AA, YM572AA, DHK625AAA, 1-633 and 1-583).
In the above experiments it was not possible to control expression levels
accurately or to observe cytological phenotypes. Therefore we examined whether
expression of these mutants at wild-type levels from an integrated copy can
support growth of a plo1 disruptant. The viability of plo1
disruptants carrying integrated HA-tagged mutant plo1 under the
control of nmt41, which results in expression at a level comparable
to the native promotor (Mulvihill et al.,
1999) (Fig. 4E),
were assayed through spore germination (see Materials and Methods). Wild-type
plo1 expressed in this way was able to fully complement the lethality
of the disruptant. On the other hand, complementation was abolished by
mutations in the kinase domain (K69R, D181R, D181N, T197V and E193V) or the
non-catalytic domain (YQL508AAA, DHK625AAA, 1-633, 1-583 and 1-533).
Cytological examination of germinating spores indicated that 50%-90% of
disruptant cells expressing these mutant genes exhibited similar defects to
those seen in a plo1 disruptant
(Fig. 3A,B). These include
septation defects (either a lack of septation or the formation of defective
septa) and mitotic arrest. Multiple septation or untimely septation, which is
typical upon overexpression of plo1+, was not observed. As
a control, less than 5% of disruptant cells expressing the wild-type gene show
abnormalities upon spore germination. It is clear then that all three polo
boxes of Plo1, in addition to the catalytic domain, are required for cellular
function of the protein. The fact that mutations in different polo boxes
resulted in the same consequences is consistent with the idea that polo boxes
together form one functional unit.
|
|
The non-catalytic domain is sufficient for cell-cycle-regulated
localisation of Plo1 to the SPBs
The effect of the mutations on the cellular localisation of Plo1 protein
was tested in vivo by GFP tagging of the mutant proteins at the N-terminus.
Mutated plo1 genes fused in frame with the GFP gene were expressed
under the control of an attenuated nmt1 promoter from a copy
integrated at the leu1 locus of wild-type cells. Wild-type Plo1
tagged in this way is expressed at a level comparable to the endogenous
protein and displays cell cycle localisation identical to the endogenous
protein (Mulvihill et al.,
1999). In the case of catalytically inactive Plo1 (Plo1K69R),
localisation has been shown to be unaffected by the mutation
(Tanaka et al., 2001
).
There were no differences observed between localisation of kinase domain mutants (GFP-Plo1K69R and T197V) and wild-type protein. Like wild-type (Fig. 4A), GFP-Plo1K69R and GFP-Plo1T197V localised initially to unseparated SPBs and remained there before becoming weaker during anaphase (Fig. 4B). This prompted us to examine whether the entire catalytic domain is dispensable for cell-cycle regulated localisation. We constructed a strain expressing GFP fused to the non-catalytic domain of Plo1 protein (GFP-Plo1.313-683). GFP-Plo1.313-683 localises to the SPBs in a similar manner to GFP-Plo1. To follow this localisation pattern as cells progress through the cell cycle, newly-divided short cells (early G2 cells) were collected by centrifugal elutriation. These synchronised cells were cultured and samples taken at regular time intervals. Like wild-type Plo1, GFP-Plo1.313-683 accumulates at SPBs in late G2 or very early mitosis before separation of SPBs (where only one SPB signal is visible) and remains on SPBs as they separate (two SPB signals) until late anaphase (Fig. 4D). Unfortunately, as a protein consisting of just the polo box domain of Plo1 (GFP-Plo1.472-683) was not detected by immunoblotting (Fig. 4E), it could not be determined if this region of the protein alone is sufficient for cell-cycle localisation to the SPBs. However, these results indicate that the non-catalytic domain of Plo1 is sufficient for cell-cycle-regulated localisation to the SPBs.
In contrast, localisation to the SPBs was abolished in all of the non-catalytic domain mutants tested (W497F, YQL508AAA, DHK625AAA, 1-633, 1-583, 1-533). This was not due to differences in the amount of GFP-Plo1 proteins, as all except Plo1.1-483 and Plo1.472-683 (not included in our cytological analysis) were detected at comparable levels to that of untagged Plo1 by immunoblotting using an antibody against the non-catalytic domain of Plo1 (Fig. 4E).
In summary, SPB localisation is dependent on the polo boxes and the non-catalytic domain of Plo1 is sufficient for its cell-cycle-regulated localisation.
Plo1 interacts with multiple proteins in a polo-box-dependent
manner
Protein protein interactions may play an important role in Plo1 function.
To identify proteins potentially interacting with Plo1, we carried out a
two-hybrid screen of a mitoitc S. pombe cDNA library using full
length Plo1 as a bait. Positive two-hybrid interactors included known
interactors (cut23 and dmf1/mid1), genes previously
described in another context [sum2, sck1 and abp2
(Forbes et al., 1998;
Jin et al., 1995
;
Sanchez et al., 1998
)] and
previously uncharacterised genes (Table
2). cut23 and dmf1/mid1 encode a subunit of the
anaphase promoting complex, and a protein which localises to the pre-division
site and is required for correct septum positioning, respectively. Both gene
products have been shown to functionally and physically interact with Plo1
(Bahler et al., 1998
;
May et al., 2002
). sum2,
sck1 and abp2 encode a protein which may have a role in G2/M
transition, a non-essential protein kinase and a putative ARS-binding protein,
respectively (Forbes et al.,
1998
; Jin et al.,
1995
; Sanchez et al.,
1998
). The following three genes are previously uncharacterised
but encode proteins which have structural motifs or limited homologies to
other proteins SPAC1006.03c (containing predicted coiled-coil
regions), SPAC6B12.08 (with a Dna-J domain) and SPAC26H5.05
(containing ankyrin repeats).
|
Cut23 and Dmf1/Mid1 have been shown to interact with Plo1 through the
non-catalytic domain (Bahler et al.,
1998; May et al.,
2002
). To identify the region of Plo1 that mediates the
interaction with each interactor we have isolated, we tested each of them
against various Plo1 mutants in a directed two-hybrid assay. Without
exception, these interactors were all able to interact with the dead kinase
mutant (Plo1K69R) and Plo1 lacking the entire kinase domain (Plo1.313-683). On
the other hand, any of the mutations in the non-catalytic domain that we
tested (YQL508AAA, DHK625AAA, 1-633, 1-583, 1-533) abolished the interaction
with all of the two-hybrid interactors
(Table 2). This indicates that
the non-catalytic domain is sufficient for the two-hybrid interactions, and
that the integrity of the polo boxes is essential for interaction with all of
the proteins identified in our screen. This suggests that the polo boxes
together form a domain which interacts with multiple proteins.
The polo boxes are crucial for determining the specificity of protein
interactions
Our site-directed mutagenesis of conserved amino acids indicated that the
polo boxes are essential for the interaction with all of the proteins we
examined. It is not clear, however, whether the polo boxes determine which
proteins interact with Plo1. To gain an insight into this issue, we attempted
to isolate mutations in plo1 which specifically disrupt interactions
with only a subset of proteins by random mutagenesis.
The entire plo1 gene was randomly mutagenised by PCR and cloned into a bait plasmid in yeast by gap repair. In this experiment, the mutation rate assayed by sequencing was roughly 1 point mutation in every 1 kb. These mutant genes were simultaneously tested for interaction against four of the two-hybrid interactors (cut23, dmf1/mid1, SPAC1006.03c, and SPAC6B12.08) by mating individual yeast strains containing mutant plo1 with other yeast strains containing different prey plasmids (see details in Materials and Methods).
Of 1035 potential mutants tested, 60% were positive for interaction with all (except the empty activation domain vector control), 29% did not interact with any of the prey constructs and 11% displayed differential interactions (i.e., interact with some but not others). Among mutants which displayed differential interactions, the protein interaction profiles did not show a tendency for any two of the interactors behave similarly.
To determine which residues are responsible for the specificity of the protein interactions, we sequenced some of the mutants which display differential interactions. All of these mutations mapped within or close to the polo boxes, except those disrupting the interaction with SPAC6B12.08 (Fig. 5B), indicating that the polo boxes play a crucial role in determining protein interactions. At least three mutations which disrupt interaction with Cut23 mapped in three different polo boxes, confirming the view that the polo boxes together form one domain. Those mutations which disrupt the interaction with SPAC6B12.08 mapped in a cluster of residues in subdomain X of the catalytic domain (K251E, I252T and S256P). As the entire catalytic domain including subdomain X is dispensable for the interaction with SPAC6B12.08, this may be due to stereo-hindrance caused by a structural change.
|
We hoped that mutations which differentially affect protein interactions might disrupt a subset of plo1 functions in vivo. We tested complementation of a plo1 disruptant by expression of these mutant genes from an integrated copy in the genome. None of these mutants were able to fully support the growth of a plo1 disruptant. Further cytological analysis did not reveal defects specific to each of the mutants, perhaps reflecting the fact that Plo1 is likely to interact with a number of other proteins in vivo and that in no case merely a single interaction was compromised by the mutations. Nevertheless, our screen for differential two-hybrid interactions highlights the importance of the polo boxes for determining protein interactions.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our functional study is consistent with a very recent structural study of
the Sak polo-box domain (Leung et al.,
2002). It suggests that this part of the polo-box domain
(equivalent to polo box 1 and 2) autonomously folds and can interact with the
second polo box domain (equivalent to polo box 3 and the C-terminal tail) to
form a putative ligand-binding domain.
Polo boxes are essential for cellular function
Upon overexpression of plo1, mutations in the polo boxes do not
affect induction of septation but abolish the ability to interfere with
spindle formation. This is the first example that polo box mutations exhibit
distinct effects on two functions of polo-like kinase. The opposite effects of
mutations in either the kinase domain or in the polo boxes upon overexpression
of plo1 indicate that the polo boxes are not simply required for
kinase activity. In mammalian cells, carboxy terminal truncations of polo-like
kinase resulted in an increase in overall kinase activity
(Jang et al., 2002;
Mundt et al., 1997
). Our
preliminary results confirmed that polo box mutants retain cell cycle
regulated kinase activity in fission yeast (N.R. and H.O., unpublished).
In contrast to the overexpression assay, replacement of wild-type plo1 gene by mutants indicated that the polo boxes are essential for at least three detectable functions of Plo1 kinase in vivo. These functional studies suggest that the catalytic and non-catalytic domains work in concert but that the requirement for the polo boxes is not simply a requirement for catalytic activity.
A role for polo boxes in localisation to centrosomes/SPBs
So what is the role of the polo boxes? It has been shown in S.
cerevisiae, S. pombe, X. laevis, D. melanogaster and mammalian cultured
cells that polo kinases localise to the centrosomes/SPBs in a cell cycle
regulated manner and that mutations in the polo boxes abolish this
localisation (Bahler et al.,
1998; Golsteyn et al.,
1995
; Lee et al.,
1998
; Logarinho and Sunkel,
1998
; Moutinho-Santos et al.,
1999
; Mulvihill et al.,
1999
; Shirayama et al.,
1998
; Song et al.,
2000
). In mammalian cultured cells, the non-catalytic domain alone
has been shown to be sufficient for the localisation
(Jang et al., 2002
).
Consistently, in fission yeast it has been shown that a kinase inactive
mutant can localise to the SPBs (Tanaka et
al., 2001). Here we show that the polo boxes are essential, and
the non-catalytic domain is sufficient for cell-cycle regulated localisation
of Plo1 to the SPBs. Therefore at least one molecular role of the polo boxes
is to form an autonomous domain which directs cell cycle regulation of SPB
localisation.
Role of polo boxes in protein interactions
Although this and previous studies have identified a role for the polo box
domain in localisation to the SPB/centrosome, our study suggests that the polo
box domain is likely to play a more general role, which is to mediate
interaction with multiple proteins. It has been suggested that localisation is
required for polo kinase function based on the observation that mutations in
the polo boxes disrupt both localisation and in vivo function
(Lee et al., 1998;
Song et al., 2000
). In the
light of our findings, more caution is necessary to interpret these results as
polo box mutations simultaneously disrupt interaction with many proteins.
Site-directed mutagenesis in any individual polo box disrupts all of the functions and protein interactions of Plo1 kinase that we have examined. Therefore it is unlikely that each polo box forms a distinct subdomain which interacts with a different set of proteins. Most likely, the polo boxes together form one protein interaction domain.
Then how do the polo boxes participate in protein-protein interactions? It is possible that the polo boxes directly recognise interacting proteins and thereby determine specificity. Alternatively they may simply facilitate folding of the domain to allow intervening sequences to recognise target proteins. We have isolated mutations which disrupt only a subset of protein interactions. These mutations mapped mostly within or close to the polo boxes, despite the fact that the polo boxes occupy less than 20% of the non-catalytic domain. Therefore it is possible that the polo boxes play a crucial role in determining the specificity of protein interaction.
Plo1-interacting proteins
The two-hybrid interactors that we isolated include two previously
identified interactors, Dmf1/Mid1 and Cut23. Dmf1/Mid1 is a medial ring
protein required for positioning of the division site and Plo1 has been shown
to interact with it and to be required for its localisation
(Bahler et al., 1998). Fission
yeast Cut23 is a subunit of the APC/C which interacts with Plo1, and a
mutation in Plo1 which compromises that interaction fails to activate APC
mediated proteolysis (May et al.,
2002
). Therefore at least some of the two-hybrid interactors we
isolated have strong functional connections with Plo1 kinase.
Although the other two-hybrid interactors we have isolated have not yet
been shown to have a clear functional relationship with Plo1 kinase, our
preliminary results indicate that at least one of them (SPAC1006.3c) are
indeed co-immunoprecipitated with Plo1 (N.R. and H.O., unpublished). Moreover,
some studies, although limited, may suggest possible connections between some
of the interactors and Plo1 function. For example, sum2+
(suppressor of uncontrolled mitosis) is implicated in the G2/M transition, as
it was originally isolated as a suppressor of cdc25+
overproduction (Forbes et al.,
1998). Abp2 was originally identified as a putative ars binding
protein, but the deletion mutant shows aberrant chromatin and septal
structures and fails to arrest cell cycle when replication is inhibited
(Sanchez et al., 1998
).
Further detailed study of these two-hybrid interactors will reveal the
significance of these interactions.
In other organisms, several mitotic proteins, such as human TCTP,
Drosophila Asp, Xenopus Cdc25C, budding yeast septins and
tubulins, have been shown to physically interact with polo kinase. TCTP is a
microtubule associated protein which is phosphorylated by Plk1 kinase.
Overexpression of a non-phosphorylatable form disrupts nuclear division
(Yarm, 2002). Asp is another
microtubule associated protein which is implicated in microtubule assembly
from centrosomes (Gonzalez et al.,
1990
; Wakefield et al.,
2001
). Asp interacts physically with polo kinase and
phosphorylation by polo kinase is required for its activity
(do Carmo Avides et al., 2001
;
Gonzalez et al., 1998
).
Septins are required for cytokinesis in budding yeast and have been shown to
interact with the budding yeast polo kinase Cdc5p both physically and
functionally (Song and Lee,
2001
). In most cases, these interactions were mediated entirely
through the non-catalytic domain with the exceptions of the tubulins
(Feng et al., 1999
) and GRASP65
(Lin et al., 2000
). Therefore
protein interactions through the non-catalytic domain are likely to play a
crucial role for polo kinase function in general.
Interactions with multiple cell cycle regulators
Then what are the roles of these protein-protein interactions? It is
possible that some of the interactors are substrates of polo kinase. As the
region of the protein required for protein-protein interactions is separate
from the catalytic domain, this is unlikely to be a simple substrate/kinase
interaction. Rather, it is likely that physical interactions via the polo box
domain act as a `docking' mechanism to enhance the efficiency of substrate
recognition. If the role of an interaction is in docking, the interactor does
not have to be a direct substrate of polo kinase. The interactors can act as
`adaptors' which bring substrate and kinase together by interacting with both
polo kinase and particular substrates.
It is also possible that these interactors may act as regulators to influence kinase activity directly, either positively or negatively. Indeed polo kinase is catalytically activated in a cell cycle regulated manner. However, our preliminary results suggest that Plo1 kinase which has mutated polo boxes still exhibits cell cycle regulation, suggesting its catalytic activity is regulated in other ways.
Polo kinases exhibit multiple functions at different stages of mitosis. Cell cycle regulation of kinase activity alone may not be sufficient to achieve this complex task. Interaction with multiple mitotic regulators may provide means for complex temporal and spatial regulation of polo kinase, perhaps via independent control of interaction with individual proteins. Therefore the characterisation of these interactors and an analysis of their mode of interaction will be crucial to understanding the function and regulation of polo kinase in vivo.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abrieu, A., Brassac, T., Galas, S., Fisher, D., Labbe, J. C. and
Doree, M. (1998). The Polo-like kinase Plx1 is a component of
the MPF amplification loop at the G2/M-phase transition of the cell cycle in
Xenopus eggs. J. Cell Sci.
111,1751
-1757.
Alexandru, G., Uhlmann, F., Mechtler, K., Poupart, M. and Nasmyth, K. (2001). Phosphorylation of the cohesin subunit scc1 by polo/cdc5 kinase regulates sister chromatid separation in yeast. Cell 105,459 -472.[CrossRef][Medline]
Bahler, J., Steever, A. B., Wheatley, S., Wang, Y., Pringle, J.
R., Gould, K. L. and McCollum, D. (1998). Role of polo kinase
and Mid1p in determining the site of cell division in fission yeast.
J. Cell Biol. 143,1603
-1616.
Carmena, M., Riparbelli, M. G., Minestrini, G., Tavares, A. M.,
Adams, R., Callaini, G. and Glover, D. M. (1998). Drosophila
polo kinase is required for cytokinesis. J. Cell Biol.
143,659
-671.
Charles, J. F., Jaspersen, S. L., Tinker-Kulberg, R. L., Hwang, L., Szidon, A. and Morgan, D. O. (1998). The Polo-related kinase Cdc5 activates and is destroyed by the mitotic cyclin destruction machinery in S. cerevisiae. Curr. Biol. 8, 497-507.[Medline]
Descombes, P. and Nigg, E. A. (1998). The
polo-like kinase Plx1 is required for M phase exit and destruction of mitotic
regulators in Xenopus egg extracts. EMBO J.
17,1328
-1335.
do Carmo Avides, M., Tavares, A. and Glover, D. M. (2001). Polo kinase and Asp are needed to promote the mitotic organizing activity of centrosomes. Nat Cell Biol 3, 421-424.[CrossRef][Medline]
Donaldson, M. M., Tavares, A. A., Hagan, I. M., Nigg, E. A. and
Glover, D. M. (2001). The mitotic roles of Polo-like kinase.
J. Cell Sci. 114,2357
-2358.
Feng, Y., Hodge, D. R., Palmieri, G., Chase, D. L., Longo, D. L. and Ferris, D. K. (1999). Association of polo-like kinase with alpha-, beta- and gamma-tubulins in a stable complex. Biochem. J. 339,435 -442.[CrossRef][Medline]
Forbes, K. C., Humphrey, T. and Enoch, T.
(1998). Suppressors of cdc25p overexpression identify two
pathways that influence the G2/M checkpoint in fission yeast.
Genetics 150,1361
-1375.
Glover, D. M., Ohkura, H. and Tavares, A. (1996). Polo kinase: the choreographer of the mitotic stage? J. Cell Biol. 135,1681 -1684.[Medline]
Glover, D. M., Hagan, I. M. and Tavares, A. A.
(1998). Polo-like kinases: a team that plays throughout mitosis.
Genes Dev. 12,3777
-3787.
Golsteyn, R. M., Mundt, K. E., Fry, A. M. and Nigg, E. A. (1995). Cell cycle regulation of the activity and subcellular localization of Plk1, a human protein kinase implicated in mitotic spindle function. J. Cell Biol. 129,1617 -1628.[Abstract]
Gonzalez, C., Saunders, R. D., Casal, J., Molina, I., Carmena, M., Ripoll, P. and Glover, D. M. (1990). Mutations at the asp locus of Drosophila lead to multiple free centrosomes in syncytial embryos, but restrict centrosome duplication in larval neuroblasts. J. Cell Sci. 96,605 -616.[Abstract]
Gonzalez, C., Sunkel, C. E. and Glover, D. M. (1998). Interactions between mgr, asp, and polo: asp function modulated by polo and needed to maintain the poles of monopolar and bipolar spindles. Chromosoma 107,452 -460.[CrossRef][Medline]
Grallert, A. and Hagan, I. M. (2002).
Schizosaccharomyces pombe NIMA-related kinase, Fin1, regulates spindle
formation and an affinity of Polo for the SPB. EMBO J.
21,3096
-3107.
Hagan, I. and Yanagida, M. (1995). The product of the spindle formation gene sad1+ associates with the fission yeast spindle pole body and is essential for viability. J. Cell Biol. 129,1033 -1047.[Abstract]
Herrmann, S., Amorim, I. and Sunkel, C. E. (1998). The POLO kinase is required at multiple stages during spermatogenesis in Drosophila melanogaster. Chromosoma 107,440 -451.[CrossRef][Medline]
Jang, Y., Lin, C., Ma, S. and Erikson, R. L.
(2002). Functional Studies on the role of the C-terminal domain
of mammalian polo-like kinase. Proc. Natl. Acad. Sci.
USA 99,1984
-1989.
Jin, M., Fujita, M., Culley, B. M., Apolinario, E., Yamamoto,
M., Maundrell, K. and Hoffman, C. S. (1995). sck1, a high
copy number suppressor of defects in the cAMP-dependent protein kinase pathway
in fission yeast, encodes a protein homologous to the Saccharomyces cerevisiae
SCH9 kinase. Genetics
140,457
-467.
Karaiskou, A., Jessus, C., Brassac, T. and Ozon, R.
(1999). Phosphatase 2A and polo kinase, two antagonistic
regulators of cdc25 activation and MPF auto-amplification. J. Cell
Sci. 112,3747
-3756.
Kim, S. H., Lin, D. P., Matsumoto, S., Kitazono, A. and
Matsumoto, T. (1998). Fission yeast Slp1: an effector of the
Mad2-dependent spindle checkpoint. Science
279,1045
-1047.
Kumagai, A. and Dunphy, W. G. (1996). Purification and molecular cloning of Plx1, a Cdc25-regulatory kinase from Xenopus egg extracts. Science 273,1377 -1380.[Abstract]
Lane, H. A. and Nigg, E. A. (1996). Antibody microinjection reveals an essential role for human polo-like kinase 1 (Plk1) in the functional maturation of mitotic centrosomes. J. Cell Biol. 135,1701 -1713.[Abstract]
Lane, H. A. and Nigg, E. A. (1997). Cell-cycle control: POLO-like kinases join the outer circle. Trends Cell Biol. 7,63 -68.[CrossRef]
Lee, K. S. and Erikson, R. L. (1997). Plk is a functional homolog of Saccharomyces cerevisiae Cdc5, and elevated Plk activity induces multiple septation structures. Mol. Cell Biol. 17,3408 -3417.[Abstract]
Lee, K. S., Grenfell, T. Z., Yarm, F. R. and Erikson, R. L.
(1998). Mutation of the polo-box disrupts localization and
mitotic functions of the mammalian polo kinase Plk. Proc. Natl.
Acad. Sci. USA 95,9301
-9306.
Lee, K. S., Song, S. and Erikson, R. L. (1999).
The polo-box-dependent induction of ectopic septal structures by a mammalian
polo kinase, plk, in Saccharomyces cerevisiae. Proc. Natl. Acad.
Sci. USA 96,14360
-14365.
Leung, G. C., Hudson, J. W., Kozarova, A., Davidson, A., Dennnis, J. W. and Sicheri, F. (2002). The Sak polo-box comprises a structural domain sufficient for mitotic subcellular localization. Nat. Struct. Biol. 9,719 -724.[CrossRef][Medline]
Lin, C. Y., Madsen, M. L., Yarm, F. R., Jang, Y. J., Liu, X. and
Erikson, R. L. (2000). Peripheral Golgi protein GRASP65 is a
target of mitotic polo-like kinase (Plk) and Cdc2. Proc. Natl.
Acad. Sci. USA 97,12589
-12594.
Llamazares, S., Moreira, A., Tavares, A., Girdham, C., Spruce, B. A., Gonzalez, C., Karess, R. E., Glover, D. M. and Sunkel, C. E. (1991). polo encodes a protein kinase homolog required for mitosis in Drosophila. Genes Dev. 5,2153 -2165.[Abstract]
Logarinho, E. and Sunkel, C. E. (1998). The
Drosophila POLO kinase localises to multiple compartments of the mitotic
apparatus and is required for the phosphorylation of MPM2 reactive epitopes.
J. Cell Sci. 111,2897
-2909.
Maundrell, K. (1993). Thiamine-repressible expression vectors pREP and pRIP for fission yeast. Gene 123,127 -130.[CrossRef][Medline]
May, K. M., Reynolds, N., Cullen, C. F., Yanagida, M. and
Ohkura, H. (2002). Polo boxes and Cut23 (Apc8) mediate an
interaction between polo kinase and the anaphase-promoting complex for fission
yeast mitosis. J. Cell Biol.
156, 23-28.
Moreno, S., Klar, A. and Nurse, P. (1991). Molecular Genetics of fission yeast Schizosaccharomyces pombe. Methods Enzymol. 194,795 -823.[Medline]
Moutinho-Santos, T., Sampaio, P., Amorim, I., Costa, M. and Sunkel, C. E. (1999). In vivo localisation of the mitotic POLO kinase shows a highly dynamic association with the mitotic apparatus during early embryogenesis in Drosophila. Biol. Cell 91,585 -596.[CrossRef][Medline]
Muhlrad, D., Hunter, R. and Parker, R. (1992). A rapid method for localized mutagenesis of yeast genes. Yeast 8,79 -82.[Medline]
Mulvihill, D. P., Petersen, J., Ohkura, H., Glover, D. M. and
Hagan, I. M. (1999). Plo1 Kinase Recruitment to the Spindle
Pole Body and Its Role in Cell Division in Schizosaccharomyces pombe.
Mol. Biol. Cell 10,2771
-2785.
Mundt, K. E., Golsteyn, R. M., Lane, H. A. and Nigg, E. A. (1997). On the regulation and function of human polo-like kinase 1 (PLK1): effects of overexpression on cell cycle progression. Biochem. Biophys. Res. Commun. 239,377 -385.[CrossRef][Medline]
Ohkura, H., Hagan, I. M. and Glover, D. M. (1995). The conserved Schizosaccharomyces pombe kinase plo1, required to form a bipolar spindle, the actin ring, and septum, can drive septum formation in G1 and G2 cells. Genes Dev. 9,1059 -1073.[Abstract]
Ouyang, B., Pan, H., Lu, L., Li, J., Stambrook, P., Li, B. and
Dai, W. (1997). Human Prk is a conserved protein
serine/threonine kinase involved in regulating M phase functions.
J. Biol. Chem. 272,28646
-28651.
Qian, Y. W., Erikson, E., Li, C. and Maller, J. L.
(1998). Activated polo-like kinase Plx1 is required at multiple
points during mitosis in Xenopus laevis. Mol. Cell.
Biol. 18,4262
-4271.
Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press.
Sanchez, J. P., Murakami, Y., Huberman, J. A. and Hurwitz,
J. (1998). Isolation, characterization, and molecular cloning
of a protein (Abp2) that binds to a Schizosaccharomyces pombe origin of
replication (ars3002). Mol. Cell Biol.
18,1670
-1681.
Sanchez, Y., Bachant, J., Wang, H., Hu, F., Liu, D., Tetzlaff,
M. and Elledge, S. J. (1999). Control of the DNA Damage
Checkpoint by Chk1 and Rad53 Protein Kinases Through Distinct Mechanisms.
Science 286,1166
-1171.
Seong, Y. S., Kamijo, K., Lee, J. S., Fernandez, E., Kuriyama,
R., Miki, T. and Lee, K. S. (2002). A spindle checkpoint
arrest and a cytokinesis failure by the dominant-negative polo-box domain of
Plk1 in U-2 OS cells. J. Biol. Chem.
277,32282
-32293.
Shirayama, M., Zachariae, W., Ciosk, R. and Nasmyth, K.
(1998). The Polo-like kinase Cdc5p and the WD-repeat protein
Cdc20p/fizzy are regulators and substrates of the anaphase promoting complex
in Saccharomyces cerevisiae. EMBO J.
17,1336
-1349.
Smits, V. A., Klompmaker, R., Arnaud, L., Rijksen, G., Nigg, E. A. and Medema, R. H. (2000). Polo-like kinase-1 is a target of the DNA damage checkpoint. Nat. Cell Biol. 2, 672-676.[CrossRef][Medline]
Song, S. and Lee, K. S. (2001). A Novel
Function of Saccharomyces cerevisiae CDC5 in Cytokinesis. J. Cell
Biol. 152,451
-470.
Song, S., Grenfell, T. Z., Garfield, S., Erikson, R. L. and Lee,
K. S. (2000). Essential function of the polo box of Cdc5 in
subcellular localization and induction of cytokinetic structures.
Mol. Cell Biol. 20,286
-298.
Sunkel, C. E. and Glover, D. M. (1988). polo, a mitotic mutant of Drosophila displaying abnormal spindle poles. J. Cell Sci. 89,25 -38.[Abstract]
Tanaka, K., Petersen, J., MacIver, F., Mulvihill, D. P., Glover,
D. M. and Hagan, I. M. (2001). The role of Plo1 kinase in
mitotic commitment and septation in Schizosaccharomyces pombe. EMBO
J. 20,1259
-1270.
Toczyski, D. P., Galgoczy, D. J. and Hartwell, L. H. (1997). CDC5 and CKII control adaptation to the yeast DNA damage checkpoint. Cell 90,1097 -1106.[Medline]
Toyoshima-Morimoto, F., Taniguchi, E., Shinya, N., Iwamatsu, A. and Nishida, E. (2001). Polo-like kinase 1 phosphorylates cyclin B1 and targets it to the nucleus during prophase. Nature 410,215 -220.[CrossRef][Medline]
Wakefield, J. G., Bonaccorsi, S. and Gatti, M.
(2001). The drosophila protein asp is involved in microtubule
organization during spindle formation and cytokinesis. J. Cell
Biol. 153,637
-648.
Woods, A., Sherwin, T., Sasse, R., MacRae, T. H., Baines, A. J. and Gull, K. (1989). Definition of individual components within the cytoskeleton of Trypanosoma brucei by a library of monoclonal antibodies. J. Cell Sci. 93,491 -500.[Abstract]
Yarm, F. R. (2002). Plk phosphorylation
regulates the microtubule-stabilizing protein TCTP. Mol. Cell
Biol. 22,6209
-6221.