polo
Human and murine homologs of the Drosophila
serine/threonine polo kinase have been cloned. Both the human and
murine clones are about 2.1 kilobases, with open reading frames of 1.8 kilobases; they encode proteins of 603
amino acids with a predicted size of 66 kilodaltons and an apparent size of 67 kilodaltons. During embryonic development of the mouse, the Polo like kinase
mRNA is expressed in all tissues examined, whereas in adult tissues, expression is limited to thymus and
ovaries. All cell lines examined also express mRNAs of similar size. Injection of
sense mRNA into serum-starved cultured cells induces DNA synthesis, whereas
microinjection of antisense RNA into growing cells blocks DNA synthesis. When
cultured cells are induced to differentiate, gene expression of PLK is greatly
reduced. Together, these results suggest that PLK expression is restricted to, and is perhaps required by,
proliferating cells (Hamanaka, 1994).
A human protein kinase, Plk1 (for polo-like kinase 1), displays extensive sequence similarity to
Drosophila polo and S. cerevisiae Cdc5.
A single Plk1 mRNA of 2.3 kb is
highly expressed in tissues with a high mitotic index, consistent with a possible function of Plk1 in
cell proliferation. Both the protein levels and distribution of Plk1
change during the cell cycle, in a manner consistent with Plk1 having a role in mitosis. Thus, like
Drosophila polo and S. cerevisiae Cdc5, human Plk1 is likely to function in cell cycle progression (Golsteyn, 1994).
Mouse and human Polo kinase mRNA is regulated during terminal erythrodifferentiation and during the cell cycle.
Within the precommitment period of murine erythroleukemia cell terminal differentiation, most of the poly(A)
tail is lost from the Polo mRNA tail, but the amount of coding mRNA remains unchanged; this
poly(A) loss does not occur in mutant erythroleukemia cells that fail to commit to terminal differentiation.
During cell cycle, there is a fluctuation in the amount of the Polo mRNA body. The mRNA is present in
growing cells, but not in nongrowing cells. It reaches maximum abundance during G2/M phase, is absent or
present at only low levels during G1 phase, and begins to reaccumulate at approximately the middle of S
phase. The cell cycle-associated accumulation and loss of the Polo mRNA could cause a similar
fluctuation in abundance of its encoded protein kinase, thereby providing a maximum amount during M
phase, when the kinase is thought to function, and little or none at other times during the cell cycle.
Posttranscriptional regulation must be responsible for the cell cycle-associated fluctuations because
transcription rates are relatively constant during different times of the cell cycle when there are large
differences in mRNA abundance (Lake, 1993).
Plk (polo-like kinase) is closely related to Drosophila Polo. Plk is also related to the products of the Saccharomyces cerevisiae cell cycle gene MSD2 (CDC5)
and the recently described early growth response gene Snk. Together, Plk, polo, Snk, and MSD2 define a
subfamily of serine/threonine protein kinases. Plk is expressed at high levels in a number of fetal and
newborn mouse tissues but is not expressed in the corresponding adult organs. With the exception of adult
hemopoietic tissues, the only adult tissues in which Plk expression can be detected are ovaries and testes.
The patterns of Plk expression suggest an association with proliferating cells. Since Polo is
required for mitosis in Drosophila it is possible that Plk is involved in some aspect of cell cycle regulation in
mammalian cells (Clay, 1993).
Polo kinase is preferentially expressed in germ line cells. Polo is a nonreceptor type serine/threonine kinase and is predominantly
expressed in the testis, ovary, and spleen of adult mouse. The nucleotide sequence of the entire coding
regions shows Polo kinase is identical with STPK13 previously
cloned from murine erythro-leukemia cells. The protein encoded by Plk1 is closely related to the product of
Drosophila polo that plays a role in mitosis and meiosis. The Plk1 gene
is specifically expressed in spermatocytes of diplotene and diakinesis stage, in secondary spermatocytes,
and in round spermatids in testes. It is also expressed in growing oocytes and ovulated eggs. The pattern of
expression of the Plk1 gene suggests that the gene product is involved in completion of meiotic division, and
like the Drosophila Polo protein, is a maternal factor active in embryos at the early cleavage stage (Matsubara, 1995).
Mammalian polo-like kinase 1 (Plk1) is structurally related to the Polo gene product of Drosophila, Cdc5p of Saccharomyces cerevisiae, and plo1+ of Schizosaccharomyces pombe. Based on data obtained for its putative homologs in invertebrates and yeasts, human Plk1 is suspected to regulate some fundamental aspect(s) of mitosis, but no direct experimental evidence in support of this hypothesis has previously been reported. In this study, a cell duplication, microinjection assay was used to investigate the in vivo function of Plk1 in both immortalized (HeLa) and nonimmortalized (Hs68) human cells. Injection of anti-Plk1 antibodies (Plk1+) at various stages of the cell cycle has no effect on the kinetics of DNA replication but severely impairs the ability of cells to divide. Analysis of Plk1(+)-injected, mitotically arrested HeLa cells by fluorescence microscopy revealed abnormal distributions of condensed chromatin and monoastral microtubule arrays that were nucleated from duplicated but unseparated centrosomes. Most strikingly, centrosomes in Plk1(+)-injected cells were drastically reduced in size, and the accumulation of both gamma-tubulin and MPM-2 immunoreactivity was impaired. These data indicate that Plk1 activity is necessary for the functional maturation of centrosomes in late G2/early prophase and, consequently, for the establishment of a bipolar spindle. Additional roles for Plk1 at later stages of mitosis are not excluded, although injection of Plk1+ after the completion of spindle formation did not interfere with cytokinesis. Injection of Plk1+ into nonimmortalized Hs68 cells produced qualitatively similar phenotypes, but the vast majority of the injected Hs68 cells arrested in G2 as single, mononucleated cells. This latter observation hints at the existence, in nonimmortalized cells, of a centrosome-maturation checkpoint sensitive to the impairment of Plk1 function (Lane, 1996).
A single double-stranded DNA (dsDNA) break will cause yeast cells to arrest in G2/M at the DNA
damage checkpoint. If the dsDNA break cannot be repaired, cells will eventually override (that is,
adapt to) this checkpoint, even though the damage that elicited the arrest is still present. Two adaptation-defective mutants have been identified that remain permanently arrested as
large-budded cells when faced with an irreparable dsDNA break in a nonessential chromosome. This
adaptation-defective phenotype is entirely relieved by deletion of RAD9, a gene required for the
G2/M DNA damage checkpoint arrest. One mutation resides in CDC5, which encodes a
polo-like kinase, whereas a second, less penetrant, adaptation-defective mutant is affected at the
CKB2 locus, which encodes a nonessential specificity subunit of casein kinase II (see Drosophila CKII). It is likely that Cdc5p promotes checkpoint adaptation by inhibiting or bypassing the checkpoint pathway. The Cdc5p polo-like kinase has a role in activating Cdc25C, a conserved tyrosine phosphatase that removes an inhibitory phosphate on Cdc2. It may be that CKII acts to inhibit some part of the cell cycle arrest machinery that is not essential for maintaining the checkpoint arrest but is extremely important for maintaining viability during arrest (Toczyski, 1997).
PLK is
found expressed in the nuclei of tumor cells from lung and breast cancers as well as in several tumor cell lines. In
peripheral lymphocytes treated with phytohemagglutinin, elevated proliferative activity of the cells correlates with the up-regulation of
PLK protein expression. In contrast, in U937 and HL-60 cells after induction of differentiation with phorbol ester, PLK
immunostaining disappears under conditions of terminal differentiation. Most of the PLK protein is found in the nucleus of
proliferating cells with diffuse but distinct staining also in the cytoplasm. Taken together, high levels of PLK protein are associated
with cellular proliferation. Combined with other proliferative and oncogene markers, PLK may be useful for improved prediction of the
clinical prognosis of cancer patients and for early cancer diagnosis. Due to its activity late in the cell cycle, it may be a target for
cancer chemotherapy (Yuan, 1997).
Human prk encodes a novel protein serine/threonine kinase capable of strongly phosphorylating casein
but not histone H1 in vitro. prk expression is tightly regulated at various levels during different stages of
the cell cycle in lung fibroblasts. The Prk kinase activity is relatively low during mitosis, G1, and G1/S,
and peaks during late S and G2 stages of the cell cycle. Recombinant human Prk expressed through
the baculoviral vector system is capable of phosphorylating Cdc25C, a positive regulator for the G2/M
transition. Human prk shares significant sequence homology with Saccharomyces cerevisiae CDC5
and Drosophila melanogaster polo, both of which are essential for mitosis and meiosis. Full-length prk
transcripts greatly potentiate progesterone-induced meiotic maturation of Xenopus laevis oocytes. In contrast, antisense prk transcripts significantly delay and reduce the rate of oocyte maturation.
When expressed in a CDC5 mutant strain of S. cerevisiae, human Prk, but not a deletional mutant
protein, fully rescues the temperature-sensitive phenotype of the budding yeast. When taken together, these data suggest that prk
may represent a new protein kinase, playing an important role in regulating the onset and/or progression
of mitosis in mammalian cells (Ouyang 1997).
Plk (polo-like kinase) is a serine-threonine kinase that appears to function in mitotic control in
mammalian cells. PLK mRNA expression is low at the G1-S
transition, increases during S phase, and is maximally expressed during G2-M. In the present study, the human PLK gene has been cloned and the structure and function of 2 kilobases of its
5'-flanking region has been analyzed. Using synchronized cultures of HeLa cells transfected with PLK
promoter/luciferase constructs, it has been shown that the promoter of PLK is activated at S phase and is
maximal at G2-M phase. Three activating regions are located between 35 and 93 base pairs upstream of the transcription initiation site. A repressor element (CDE/CHR) is identified in the region of the transcription start site, and mutations within this element diminish cell cycle regulation of transcription. The CDE (cell cycle dependent element) is known to be a target of E2F (Liu, 1996 and Uchiumi, 1997).
In order to stabilize changes in synaptic strength, neurons activate a program of gene
expression that results in alterations of their molecular composition and structure. Fnk and Snk, two members of the polo family of cell cycle associated
kinases, are co-opted by the brain to serve in this program.
All members of
this family are characterized by the same domain topology; alignment in a phylogenetic tree indicates that the
polo-like kinases diverged before the subfamily of calmodulin kinase-related genes developed. Comparison of the deduced amino acid sequences of human Prk, rat Fnk and mouse Fnk shows that the three polypeptides are ~90% identical except for a 17-amino-acid insertion present in rat
Fnk and human Prk. Sequences encoding the 17-amino-acid insertion of human and rat Fnk are also present in
mouse mRNA from NIH-3T3 fibroblasts and are flanked by two
alternative 5' consensus splice sites in the mouse genomic sequence. This suggests that the published sequence of mouse
Fnk most likely represents a splice variant. Rat Fnk shares ~50% sequence identity with rat Snk which is ~90%
identical to the mouse homolog. The N-terminal half of Fnk and Snk harbors a
serine/threonine-specific kinase domain including all 11 subdomains described as specific for serine/threonine kinases. The C-terminal half contains a 30-amino-acid domain referred to as the polo-box,
which is highly conserved in all family members. This motif has not been described in any other protein and its
function has not been determined (Kauselmann, 1999).
Stimuli that produce synaptic
plasticity, including those that evoke long-term potentiation (LTP), dramatically increase
levels of both Fnk and Snk mRNAs. Induced Fnk and Snk proteins are targeted to the dendrites of activated neurons,
suggesting that they mediate phosphorylation of proteins in this compartment. Moreover, a conserved C-terminal
domain in these kinases is shown to interact specifically with Cib, a Ca2+ and integrin-binding protein. Cib shares greater than 27% amino acid identity with calcineurin B, the regulatory subunit of calcineurin (phosphatase 2B), and has
a similar identity to calmodulin. Like calcineurin B and calmodulin, Cib contains EF-hand motifs
responsible for Ca2+ binding. The structural similarity of Cib to calmodulin and to calcineurin B raises the possibility that
Cib might be a regulatory subunit of Snk or Fnk or, alternatively, the regulatory subunit of a phosphatase whose catalytic
subunit has not yet been identified. In this case, the association of a kinase-phosphatase complex could facilitate rapid
cycles of phosphorylation or result in an autocatalytic loop of kinase-phosphatase activation. Together, these
studies suggest a novel signal transduction mechanism in the stabilization of long-term synaptic plasticity (Kauselmann, 1999).
Polo-like kinase 1 (plk1) has been characterized in mouse oocytes during meiotic maturation and after parthenogenetic activation until entry
into the first mitotic division. Plk1 protein expression remains unchanged during maturation. However, two different
isoforms can be identified by SDS-PAGE. A fast migrating form, present in the germinal vesicle, seems characteristic of
interphase. A slower form appears as early as 30 min before germinal vesicle breakdown (GVBD); is maximal at GVBD, and
is maintained throughout meiotic maturation. This form gradually disappears after exit from meiosis. The slow form
corresponds to a phosphorylation since it disappears after alkaline phosphatase treatment. Plk1 activation, therefore, takes
place before GVBD and MAPK activation since plk1 kinase activity correlates with its slow migrating phosphorylated form.
However, plk1 phosphorylation is inhibited after treatment with two specific p34cdc2 inhibitors, roscovitine and butyrolactone,
suggesting plk1 involvement in the MPF autoamplification loop. During meiosis plk1 undergoes a cellular
redistribution consistent with its putative targets. At the germinal vesicle stage, plk1 is found diffusely distributed in the
cytoplasm and enriched in the nucleus; during prometaphase, it localizes to the spindle poles. At anaphase it relocates
to the equatorial plate and is restricted to the postmitotic bridge at telophase. After parthenogenetic activation, plk1
becomes dephosphorylated and its activity drops progressively. Upon entry into the first mitotic M-phase at nuclear
envelope breakdown plk1 is phosphorylated and there is an increase in its kinase activity. At the two-cell stage, the fast
migrating form with weak kinase activity is present. Thus plk1 is present in mouse oocytes during
meiotic maturation and the first mitotic division. The variation of plk1 activity and subcellular localization during this
period suggest its implication in the organization and progression of M-phase (Pahlavan, 2000).
The cis/trans peptidyl-prolyl isomerase, Pin1, is a regulator of mitosis that is well conserved from yeast to man. Depletion of Pin1-binding proteins from Xenopus egg extracts results in hyperphosphorylation and inactivation of the key mitotic regulator, Cdc2/cyclin B. This phenotype is a consequence of Pin1 interaction with critical upstream regulators of Cdc2/cyclin B, including the Cdc2-directed phosphatase, Cdc25, and its known regulator, Plx1. Although Pin1 could interact with Plx1 during interphase and mitosis, only the phosphorylated, mitotically active form of Cdc25 is able to bind Pin1, an event that has been recapitulated using in vitro phosphorylated Cdc25. Taken together, these data suggest that Pin1 may modulate cell cycle control through interaction with Cdc25 and its activator, Plx1 (Crenshaw, 1998).
Polo-like kinases are key regulators of mitotic structures and cell cycle progression. The kinase domain of Sak is most closely related to that of the polo-like kinases, but Sak appears to have diverged from a primordial polo-like kinase early in the radiation of metazoans. Additional complexities in the regulation of cell division during development may have driven the expansion of polo-like kinase genes in metazoans. In any case, functions of polo-like kinases appear to be highly conserved, since the expression of either mammalian Plk or PRK in cdc5-1 (a yeast polo-like kinase) mutant yeast cells rescues the mitotic defects. The mammalian polo-like kinases Plk, Snk, and PRK/Fnk have polo box domains pb1 and pb2 near the C terminus. The mutation of pb1 in mammalian Plk disrupts the localization of the enzyme to mitotic structures and blocks the rescue of the cdc5-1 mitotic defect in yeast. Targeting of the enzyme to specific subcellular locations presumably regulates timely and efficient interaction with substrates to drive multiple events in the cell cycle. Alignment of available polo-like kinase sequences reveals a single polo box domain in Sak at the C terminus, with its position and sequence similarity suggesting the designation pb2. Sak also has three PEST sequences commonly found in proteins with short half-lives, and Sak is ubiquitinated and destroyed in G1 (Hudson, 2001).
Sak kinase has a functional pb domain that localizes the enzyme to the nucleolus
during G2, to the centrosomes in G2/M, and to the cleavage furrow during cytokinesis. To study the role of Sak in embryo development, a Sak null allele was generated, the first polo-like kinase to be mutated in mice. Sak-/- embryos arrest after gastrulation at E7.5, with a marked increase in mitotic and apoptotic cells. Sak-/- embryos display cells in late anaphase or telophase that continue to
express cyclin B1 and phosphorylated histone H3. These results suggest that Sak is required for the APC-dependent destruction of cyclin B1 and for exit from mitosis in the postgastrulation embryo (Hudson, 2001).
Polo-like kinases play multiple roles in different phases of mitosis. The mammalian polo-like kinase, Plk1, is inhibited in response to DNA damage and this inhibition may lead to cell cycle arrests at multiple points in mitosis. The role of the checkpoint kinases ATM and ATR in DNA damage-induced inhibition of Plk1 has been investigated. Inhibition of Plk1 kinase activity is efficiently blocked by the radio-sensitizing agent caffeine. Using ATM(-/-) cells it has been shown that under certain circumstances, inhibition of Plk1 by DNA-damaging agents critically depends on ATM. In addition, UV radiation also causes inhibition of Plk1, and evidence is presented that this inhibition is mediated by ATR. Taken together, these data demonstrate that ATM and ATR can regulate Plk1 kinase activity in response to a variety of DNA-damaging agents (van Vugt, 2001).
The separation of sister chromatids in anaphase depends on the dissociation of cohesin from chromosomes. In vertebrates, some cohesin is removed from chromosomes at the onset of anaphase by proteolytic cleavage. In contrast, the bulk of cohesin is removed from chromosomes already in prophase and prometaphase by an unknown mechanism that does not involve cohesin cleavage. Polo-like kinase is required for the cleavage-independent dissociation of cohesin from chromosomes in Xenopus. Cohesin phosphorylation depends on Polo-like kinase and reduces the ability of cohesin to bind to chromatin. These results suggest that Polo-like kinase regulates the dissociation of cohesin from chromosomes early in mitosis (Sumara, 2002).
Cohesin dissociation from chromatin in prophase coincides with chromosome condensation and the partial separation of sister chromatid arms, but it remains unknown if cohesin dissociation is required for these or possibly for other events. The observation that chromatin containing both cohesin and
condensin can be generated by depletion of PLX1 represents a first step toward addressing these questions. In all experiments, such chromatin had an amorphous appearance in which no individual chromosomes could be distinguished. These observations are consistent with the possibility that cohesin dissociation is required for proper condensation and/or resolution of chromatid arms. However, it is presently not known if this effect is caused by blocking cohesin dissociation or by disabling some other process when PLX1 is immunodepleted. In the future it will therefore be important to generate nondissociatable cohesin mutants and to analyze how their expression influences the morphology and behavior of chromosomes in mitosis (Sumara, 2002).
Polo kinases play critical roles for proper M-phase progression. They are characterized by the presence of two regions of homology in the C-terminal non-catalytic domain, termed polo-box 1 (PB1) and polo-box 2 (PB2). Both PB1 and PB2 are required for targeting the catalytic activity of Plk1 to centrosomes, midbody, and kinetochores. Expression of either kinase-inactive PLK1/K82M or the C-terminal plk1 Delta N induces a pre-anaphase arrest with elevated Cdc2 and Plk1 activity. Prophase-arrested cells exhibit randomly oriented spindle structures, whereas metaphase cells exhibit aberrant bipolar spindles with Mad2 localization at kinetochores of misaligned chromosomes. Microtubule nucleation activity of centrosomes is not compromised. In vivo time-lapse studies reveal that expression of plk1 Delta N results in repeated cycles of bipolar spindle formation and disruption, suggestive of a defect in spindle stability. A prolonged arrest frequently leads to the generation of micronucleated cells in the absence of sister chromatid separation and centrosome duplication, indicating that micronucleation is not a result of accumulated cytokinesis failures. Interestingly, bypass of the mitotic arrest by dominant-negative spindle checkpoint components leads to a failure in completion of cytokinesis. It is proposed that, in mammalian cells, the polo-box-dependent Plk1 activity is required for proper metaphase/anaphase transition and for cytokinesis (Seong, 2002).
Chk2 (see Drosophila loki) is a protein kinase intermediary in DNA damage checkpoint pathways. DNA damage induces phosphorylation of Chk2 at multiple sites concomitant with activation. Chk2 phosphorylated at Thr-68 is found in nuclear foci at sites of DNA damage. Chk2 phosphorylated at Thr-68 and Thr-26 or Ser-28 is localized to centrosomes and midbodies in the absence of DNA damage. In a search for interactions between Chk2 and proteins with similar subcellular localization patterns, it was found that Chk2 coimmunoprecipitates with Polo-like kinase 1, a regulator of chromosome segregation, mitotic entry, and mitotic exit. Plk1 overexpression enhances phosphorylation of Chk2 at Thr-68. Plk1 phosphorylates recombinant Chk2 in vitro. Indirect immunofluorescence (IF) microscopy revealed the co-localization of Chk2 and Plk1 to centrosomes in early mitosis and to the midbody in late mitosis. These findings suggest lateral communication between the DNA damage and mitotic checkpoints (Tsvetkov, 2003).
Polo-like kinases (PLKs) have an important role in several stages of mitosis. They contribute to the activation of cyclin B/Cdc2 and are involved in centrosome maturation and bipolar spindle formation at the onset of mitosis. PLKs also control mitotic exit by regulating the anaphase-promoting complex (APC) and have been implicated in the temporal and spatial coordination of cytokinesis. Experiments in budding yeast have shown that the PLK Cdc5 may be controlled by the DNA damage checkpoint. This study reports the effects of DNA damage on Polo-like kinase-1 (Plk1) in a variety of human cell lines. Plk1 is inhibited by DNA damage in G2 and in mitosis. In line with this, it has been shown that DNA damage blocks mitotic exit. DNA damage does not inhibit the kinase activity of Plk1 mutants in which the conserved threonine residue in the T-loop has been changed to aspartic acid, suggesting that DNA damage interferes with the activation of Plk1. Significantly, expression of these mutants can override the G2 arrest induced by DNA damage. On the basis of these data it is proposed that Plk1 is an important target of the DNA damage checkpoint, enabling cell-cycle arrests at multiple points in G2 and mitosis (Smits, 2002).
The anaphase-promoting complex (APC) or cyclosome is a ubiquitin ligase that initiates anaphase and mitotic exit. APC activation is thought to depend on APC phosphorylation and Cdc20 binding. Forty-three phospho-sites on APC have been identified of which at least 34 are mitosis specific. Of these, 32 sites are clustered in parts of Apc1 and the tetratricopeptide repeat (TPR) subunits Cdc27, Cdc16, Cdc23 and Apc7. In vitro, at least 15 of the mitotic phospho-sites can be generated by cyclin-dependent kinase 1 (Cdk1), and 3 by Polo-like kinase 1 (Plk1). APC phosphorylation by Cdk1, but not by Plk1, is sufficient for increased Cdc20 binding and APC activation. Immunofluorescence microscopy using phospho-antibodies indicates that APC phosphorylation is initiated in prophase during nuclear uptake of cyclin B1. In prometaphase, phospho-APC accumulates on centrosomes where cyclin B ubiquitination is initiated, and then appears throughout the cytosol and disappears during mitotic exit. Plk1 depletion neither prevents APC phosphorylation nor cyclin A destruction in vivo. These observations imply that APC activation is initiated by Cdk1 already in the nuclei of late prophase cells (Kraft, 2003).
Centriole duplication initiates at the G1-to-S transition in
mammalian cells and is completed during the S and G2 phases. The
localization of a number of protein kinases to the centrosome has
revealed the importance of protein phosphorylation in controlling the
centriole duplication cycle. The human Polo-like kinase 2 (Plk2) is
activated near the G1-to-S transition of the cell cycle. Endogenous
and overexpressed HA-Plk2 localize with centrosomes, and this
interaction is independent of Plk2 kinase activity. In contrast, the
kinase activity of Plk2 is required for centriole duplication.
Overexpression of a kinase-deficient mutant under S-phase arrest
blocks centriole duplication. Downregulation of endogenous Plk2 with
small hairpin RNAs interferes with the ability to reduplicate
centrioles. Furthermore, centrioles fail to duplicate during the cell
cycle of human fibroblasts and U2OS cells after overexpression of a
Plk2 dominant-negative mutant. These results show that Plk2 is a
physiological centrosomal protein and that its kinase activity is
likely to be required for centriole duplication near the G1-to-S
phase transition (Warnke, 2004).
Cyclin B1-Cdk1 is the key initiator of mitosis, but when and where activation occurs has not been precisely determined in mammalian cells. Activation may occur in the nucleus or cytoplasm; just before nuclear envelope breakdown Polo-like kinase1 (Plk1) is proposed to phosphorylate cyclin B1 in its nuclear export sequence (NES), to trigger rapid nuclear import. Phospho-specific antibodies were raised against cyclin B1 that primarily recognise the active form of the complex. Cyclin B1 was shown to be initially phosphorylated on centrosomes in prophase; Plk1 phosphorylates cyclin B1, but not in the NES. Furthermore, phosphorylation by Plk1 does not cause cyclin B1 to move into the nucleus. It is concluded that cyclin B1-Cdk1 is first activated in the cytoplasm and that centrosomes may function as sites of integration for the proteins that trigger mitosis (Jackman, 2003).
Error-free chromosome segregation depends on the precise regulation of phosphorylation to stabilize kinetochore-microtubule attachments (K-fibres) on sister chromatids that have attached to opposite spindle poles (bi-oriented). In many instances, phosphorylation correlates with K-fibre destabilization. Consistent with this, multiple kinases, including Aurora B and Plk1, are enriched at kinetochores of mal-oriented chromosomes when compared with bi-oriented chromosomes, which have stable attachments. Paradoxically, however, these kinases also target to prometaphase chromosomes that have not yet established spindle attachments and it is therefore unclear how kinetochore-microtubule interactions can be stabilized when kinase levels are high. This study shows, using human retinal pigment epithelial cells and HeLa cells, that the generation of stable K-fibres depends on the B56-PP2A phosphatase, which is enriched at centromeres/kinetochores of unattached chromosomes. When B56-PP2A is depleted, K-fibres are destabilized and chromosomes fail to align at the spindle equator. Strikingly, B56-PP2A depletion increases the level of phosphorylation of Aurora B and Plk1 kinetochore substrates as well as Plk1 recruitment to kinetochores. Consistent with increased substrate phosphorylation, it was found that chemical inhibition of Aurora or Plk1 restores K-fibres in B56-PP2A-depleted cells. These findings reveal that PP2A, an essential tumour suppressor, tunes the balance of phosphorylation to promote chromosome-spindle interactions during cell division (Foley, 2011).
The mitotic kinases, Cdk1, Aurora A/B, and Polo-like kinase 1 (Plk1) have been characterized extensively to further understanding of mitotic mechanisms and as potential targets for cancer therapy. Cdk1 and Aurora kinase studies have been facilitated by small-molecule inhibitors, but few if any potent Plk1 inhibitors have been identified.
This study describes the cellular effects of a novel compound, BI 2536, a potent and selective inhibitor of Plk1. The fact that BI 2536 blocks Plk1 activity fully and instantaneously enabling study study of controversial and unknown functions of Plk1. Cells treated with BI 2536 are delayed in prophase but eventually import Cdk1-cyclin B into the nucleus, enter prometaphase, and degrade cyclin A, although BI 2536 prevents degradation of the APC/C inhibitor Emi1. BI 2536-treated cells lack prophase microtubule asters and thus polymerize mitotic microtubules only after nuclear-envelope breakdown and form monopolar spindles that do not stably attach to kinetochores. Mad2 accumulates at kinetochores, and cells arrest with an activated spindle-assembly checkpoint. BI 2536 prevents Plk1's enrichment at kinetochores and centrosomes, and when added to metaphase cells, it induces detachment of microtubules from kinetochores and leads to spindle collapse. These results suggest that Plk1's accumulation at centrosomes and kinetochores depends on its own activity and that this activity is required for maintaining centrosome and kinetochore function. The data also show that Plk1 is not required for prophase entry, but delays transition to prometaphase, and that Emi1 destruction in prometaphase is not essential for APC/C-mediated cyclin A degradation (Lénárt, 2006).
Previous experiments have clearly established that Plk1 and its orthologs have a variety of essential roles in mitosis. However, description of Plk1 inactivation phenotypes has been limited by the possibility that late mitotic functions could have been obscured by earlier defects. Furthermore, in many experimental systems, it has been difficult to assess whether Plk1 had been inactivated completely. Therefore the small-molecule inhibitor BI 2536 was used to reinvestigate functions previously ascribed to Plk1 and to gain novel insights to mitotic roles of Plk1. The data imply that Plk1 has specific functions in Emi1 degradation, activation of Cdk1-cyclin B, cohesin release, centrosome function, and regulation of microtubule-kinetochore attachments. A comparison of these results with previous data from RNAi and biochemical experiments indicate strongly that BI 2536 is a potent and specific inhibitor of Plk1 in live human cells. BI 2536 will therefore be likely to serve as a powerful tool in mitosis research (Lénárt, 2006).
The Polo-like kinase, Plk, has multiple roles in regulating mitosis. In particular, Plk1 has been postulated to function as a trigger kinase that phosphorylates and activates Cdc25C prior to the activation of cyclin B-Cdc2 and thereby initiates its activation. However, the upstream regulation of Plk1 activation remains unclear. The interplay between Plk1 and Cdc2 through meiotic and early embryonic cycles was studied in starfish. Distinct kinases, cyclin B-Cdc2, MAPK along with cyclin B- and/or cyclin A-Cdc2 and cyclin A-Cdc2, are unique upstream regulators for Plk1 activation at meiosis I, meiosis II and embryonic M-phase, respectively, indicating that Plk1 is not the trigger kinase at meiotic reinitiation. When Plk1 is required for cyclin B-Cdc2 activation, the action of Plk1 is mediated primarily through suppression of Myt1 (see Drosophila Myt-1) rather than through activation of Cdc25. It is proposed that Plk1 can be activated by either cyclin A- or cyclin B-Cdc2, and its primary target is Myt1 (Okano-Uchida, 2003).
Plk1 (Polo-like kinase 1), an evolutionarily conserved serine/threonine kinase,
is crucially involved in multiple events during the M phase. A consensus phosphorylation sequence for Plk1 has been identified by testing the ability of systematically mutated peptides derived from human Cdc25C to serve as a substrate for Plk1. The obtained results show that a hydrophobic amino acid at position +1 carboxyl-terminal of phosphorylated Ser/Thr and an acidic amino acid at position -2 are important for optimal phosphorylation by Plk1. Myt1, an inhibitory kinase for MPF, has a number of putative phosphorylation sites for Plk1 in its COOH-terminal portion. While wild-type Myt1 (Myt1-WT) serves as a good substrate for Plk1 in vitro, a mutant Myt1 (Myt1-4A), in which the four putative phosphorylation sites are replaced by alanines, does not. In nocodazole-treated cells, Myt1-WT, but not Myt1-4A, displays its mobility shift in gel electrophoresis, due to phosphorylation. These results suggest that Plk1 phosphorylates Myt1 during M phase. Thus, this study identifies a novel substrate for Plk1 by determining a consensus phosphorylation sequence by Plk1 (Nakajima, 2003).
GRASP65, a structural protein of the Golgi apparatus, has been linked
to the sensing of Golgi structure and the integration of this information with
the control of mitotic entry in the form of a Golgi checkpoint.
Cdk1-cyclin B is the major kinase phosphorylating GRASP65 in mitosis, and
phosphorylated GRASP65 interacts with the polo box domain of the polo-like
kinase Plk1. GRASP65 is phosphorylated in its C-terminal domain at four
consensus sites by Cdk1-cyclin B, and mutation of these residues to alanine
essentially abolishes both mitotic phosphorylation and Plk1 binding. Expression
of the wild-type GRASP65 C-terminus but not the phosphorylation defective mutant
in normal rat kidney cells causes a delay but not the block in mitotic entry
expected if this were a true cell cycle checkpoint. These findings identify a
Plk1-dependent signalling mechanism potentially linking Golgi structure and cell
cycle control, but suggest that this may not be a cell cycle checkpoint in the
classical sense (Preisinger, 2005 ).
It has been proposed that separase-dependent centriole disengagement at anaphase licenses centrosomes for duplication in the next cell cycle. This study tests whether such a mechanism exists in intact human cells. Loss of separase blocked centriole disengagement during mitotic exit and delayed assembly of new centrioles during the following S phase; however, most engagements were eventually dissolved. Polo-like kinase 1 (Plk1) was identified as a parallel activator of centriole disengagement. Timed inhibition of Plk1 mapped its critical period of action to late G2 or early M phase, i.e., prior to securin destruction and separase activation at anaphase onset. Crucially, when cells exited mitosis after downregulation of both separase and Plk1, centriole disengagement failed completely, and subsequent centriole duplication in interphase was also blocked. These results indicate that Plk1 and separase act at different times during M phase to license centrosome duplication, reminiscent of their roles in removing cohesin from chromosomes (Tsou, 2009).
Expression of polo-like kinase (PLK) is required for cellular
DNA synthesis and overexpression of PLK is sufficient to induce DNA synthesis.
The endogenous levels of PLK, its phosphorylation status, and protein kinase activity
are tightly regulated during cell cycle progression. PLK protein is low in G1, accumulates during S
and G2M, and is rapidly reduced after mitosis. During mitosis, PLK is phosphorylated on serine,
and its serine threonine kinase function is activated at a time close to that of p34cdc2. The
phosphorylated form of PLK migrates with reduced mobility on SDS-polyacrylamide gel
electrophoresis, and dephosphorylation by purified protein phosphatase 2A converts it to the more
rapidly migrating form and reduces the total amount of PLK kinase activity. Purified
p34cdc2-cyclin B complex can phosphorylate PLK protein in vitro but causes little increase in
PLK kinase activity (Hamanaka, 1995).
Cdc2, the cyclin-dependent kinase that controls mitosis (see Cyclin A and Cyclin B) is negatively regulated by phosphorylation on its threonine-14 and tyrosine-15 residues. Cdc25 (Drosophila homolog: String), the phosphatase that dephosphorylates both of these residues, undergoes activation and phosphorylation by multiple kinases at mitosis. Plx1, a kinase that associates with and phosphorylates the amino-terminal domain of Cdc25, has been purified from Xenopus. Cloning reveals the Plx1 is related to the Polo family of protein kinases. Recombinant Plx1 phosphorylates Cdc25 and stimulates its activity in a purified system. It is likely that Plx1 participates in the control of mitotic progression by targeting Cdc25 (Kamagai, 1996).
Proteolysis mediated by the anaphase promoting complex (APC) has a crucial role in regulating the
passage of cells through anaphase. APC is composed of the tetratricopeptide repeat (TPR) proteins
Cdc16p/Cdc23p/Cdc27p and other proteins and is required for B-type cyclin
ubiquitination in both anaphase and during G1 phase. Destruction of the anaphase inhibitor Pds1p, a novel protein, is necessary for
separation of sister chromatids, whereas destruction of the mitotic B type cyclin Clb2p is important for
disassembly of the mitotic spindle, cytokinesis and re-replication of the genome. Pds1p proteolysis
precedes that of Clb2p by at least 15 min, which helps to ensure that cells never re-replicate their
genome before they have separated sister chromatids at the previous mitosis. What triggers Pds1p
proteolysis and why does it not also trigger that of Clb2p? Apart from sharing a dependence on the
APC, these two proteolytic events differ in their dependence on other cofactors. Pds1p proteolysis
depends on a WD-repeat protein called Cdc20p, whereas Clb2p proteolysis depends on another,
related WD protein called Hct1/Cdh1p. On the other hand, destruction of Clb2p, but not that of Pds1p,
depends on the Polo-like kinase, Cdc5p. Cdc20p is essential for separation of sister chromatids,
whereas Cdc5p is not. Both Cdc5p and Cdc20p are unstable proteins whose proteolysis
is regulated by the APC. Both proteins accumulate during late G2/M phase and disappear at a late
stage of anaphase. Accumulation of Cdc20p contributes to activation of Pds1p proteolysis in
metaphase, whereas accumulation of Cdc5p facilitates the activation of Clb2p proteolysis (Shirayama, 1998).
Ubiquitin-mediated proteolysis is the key to cell cycle control. Anaphase-promoting complex/cyclosome (APC) is a ubiquitin
ligase that targets cyclin B and factors regulating sister chromatid separation for proteolysis by the proteasome. As a consequence, this proteolysis
regulates metaphase-anaphase transition and exit from mitosis. Cdc2-cyclin B-activated
Polo-like kinase (Plk) specifically phosphorylates at least three components of APC and activates APC to ubiquitinate cyclin B
in the in vitro-reconstituted system. Conversely, protein kinase A (PKA) phosphorylates two subunits of APC but suppresses
APC activity. PKA is superior to Plk in its regulation of APC; at metaphase, Plk activity peaks whereas PKA activity is falling. These results indicate that Plk and PKA regulate mitosis progression by controlling APC activity (Kotani. 1998).
The auto-catalytic activation of the cyclin-dependent kinase Cdc2 or MPF (M-phase promoting factor) is an irreversible process responsible for the entry into M phase. In Xenopus oocyte, a positive feed-back loop between Cdc2 kinase and its activating
phosphatase Cdc25 allows the abrupt activation of MPF and the entry into the first meiotic division. The
Cdc2/Cdc25 feed-back loop was studied using cell-free systems derived from Xenopus prophase-arrested oocyte. The findings support the following two-step model for MPF amplification: during the first step, Cdc25 acquires a basal catalytic activity resulting in a linear activation of Cdc2 kinase. In turn, Cdc2 partially phosphorylates Cdc25 but no amplification takes place; under this condition Plx1
kinase and its activating kinase Plkk1 are activated. However, their activity is not required for the partial phosphorylation of Cdc25. This first step occurs independent of PP2A or Suc1/Cks-dependent Cdc25/Cdc2 association. On the contrary, the second step involves the full phosphorylation and activation of Cdc25 and the initiation of the amplification loop. It depends both on PP2A inhibition and Plx1 kinase activity. Suc1-dependent Cdc25/Cdc2 interaction is required for this process (Karaiskou, 1999).
The cyclosome/anaphase-promoting complex is a multisubunit ubiquitin ligase that targets for degradation mitotic cyclins and some other cell cycle
regulators in exit from mitosis. It becomes enzymatically active at the end of mitosis. The activation of the cyclosome is initiated by its phosphorylation, a process necessary for its conversion to an active form by the ancillary protein Cdc20/Fizzy. Previous reports have implicated either cyclin-dependent kinase 1-cyclin B or polo-like kinase as the major protein kinase that directly phosphorylates and activates the cyclosome. These conflicting results could be due to the use of partially purified cyclosome preparations or of immunoprecipitated cyclosome, whose interactions with protein kinases or ancillary factors may be hampered by binding to immobilized antibody. To examine this problem, cyclosome has been purified from HeLa cells by a combination of affinity chromatography and ion exchange procedures. With the use of purified preparations, it was found that both cyclin-dependent kinase 1-cyclin B and polo-like kinase directly phosphorylate the cyclosome, but the pattern of the phosphorylation of the different cyclosome subunits by the two protein kinases is not similar.
Plk1 and Cdk1/cyclin-B have additive effects in phosphorylating and activating the APC/C; the former preferentially phosphorylates Cdc16 and Cdc23, and the latter preferentially phosphorylates Cdc27.
Each protein kinase can restore only partially the cyclin-ubiquitin ligase activity of dephosphorylated cyclosome. However, following phosphorylation by both protein kinases, an additive and nearly complete restoration of cyclin-ubiquitin ligase activity is observed. It is suggested that this joint activation may be due to the complementary phosphorylation of different cyclosome subunits by the two protein kinases (Golan, 2002).
BRCA2 is a breast tumor susceptibility gene encoding a 390-kDa protein with functions in maintaining genomic stability and cell cycle progression. Evidence has been accumulated to support the concept that BRCA2 has a critical role in homologous recombination of DNA double-stranded breaks by interacting with RAD51. In addition, BRCA2 may have chromatin modifying activity through interaction with a histone acetyltransferase protein, p300/CBP-associated factor (P/CAF). To explore how the functions of BRCA2 may be regulated, the post-translational modifications of BRCA2 throughout the cell cycle were examined. It was found that BRCA2 is hyperphosphorylated specifically in M phase and becomes dephosphorylated as cells exit M phase and enter interphase. This specific phosphorylation of BRCA2 was not observed in cells treated with DNA-damaging agents. Systematic mapping of the potential mitosis specific phosphorylation sites revealed the N-terminal 284 amino acids of BRCA2 (BR-N1) as the major region of phosphorylation and mass spectrometric analysis identified two phosphopeptides that contain 'phosphorylation consensus motifs' for Polo-like kinase 1 (Plk1). Phosphorylation of BR-N1 with Plk1 recapitulates the electrophoretic mobility change as seen in BR-N1 isolated from M phase cells. Plk1 interacts with BRCA2 in vivo, and mutation of Ser193, Ser205/206, and Thr203/207 to Ala in BR-N1 abolishes Plk1 phosphorylation, suggesting that BRCA2 is the substrate of Plk1. Furthermore, both the hyperphosphorylated and hypophosphorylated forms of BRCA2 bind to RAD51, whereas the M phase hyperphosphorylated form of BRCA2 no longer associates with the P/CAF, suggesting that the dissociation of P/CAF-BRCA2 complex is regulated by phosphorylation. Taken together, these results implicate a potential role of BRCA2 in modulating M phase progression (Lin, 2003).
Polo-like kinase 1 (Plk1) plays essential roles at multiple events during cell division, yet little is known about its physiological substrates. In a cDNA phage display screen using Plk1 C-terminal affinity columns, NudC (nuclear distribution gene C) has been identified as a Plk1 binding protein. The interaction between Plk1 and NudC has been characterized: Plk1 phosphorylates NudC at conserved S274 and S326 residues in vitro. Evidence is presented that NudC is also a substrate for Plk1 in vivo. Downregulation of NudC by RNA interference results in multiple mitotic defects, including multinucleation and cells arrested at the midbody stage, that are rescued by ectopic expression of wild-type NudC, but not by NudC with mutations in the Plk1 phosphorylation sites. These results suggest that Plk1 phosphorylation of NudC may influence cytokinesis (Zhou, 2003).
Metaphase-to-anaphase transition is a fundamental step in cell cycle progression where duplicated sister-chromatids segregate to the future daughter cells. The anaphase-promoting complex/cyclosome (APC/C) is a highly regulated ubiquitin-ligase that triggers anaphase onset and mitotic exit by targeting securin and mitotic cyclins for destruction. The Xenopus polo-like kinase Plx1 is essential to activate APC/C upon release from cytostatic factor (CSF) arrest in Xenopus egg extract. Although the mechanism by which Plx1 regulates APC/C activation has remained unclear, the existence of a putative APC/C inhibitor has been postulated whose activity would be neutralized by Plx1 upon CSF release. XErp1, a novel Plx1-regulated inhibitor of APC/C activity, has been identified, and XErp1 is shown to be required to prevent anaphase onset in CSF-arrested Xenopus egg extract. Inactivation of XErp1 leads to premature APC/C activation. Conversely, addition of excess XErp1 to Xenopus egg extract prevents APC/C activation. Plx1 phosphorylates XErp1 in vitro at a site that targets XErp1 for degradation upon CSF release. Thus, these data lead to a model of APC/C activation in Xenopus egg extract in which Plx1 targets the APC/C inhibitor XErp1 for degradation (Schmidt, 2005).
In response to DNA damage in G2, mammalian cells must avoid entry into mitosis and instead initiate DNA repair. This study shows that in response to genotoxic stress in G2, the phosphatase Cdc14B translocates from the nucleolus to the nucleoplasm and induces the activation of the ubiquitin ligase APC/CCdh1, with the consequent degradation of Plk1, a prominent mitotic kinase. This process induces the stabilization of Claspin, an activator of the DNA-damage checkpoint, and Wee1, an inhibitor of cell-cycle progression, and allows an efficient G2 checkpoint. As a by-product of APC/CCdh1 reactivation in DNA-damaged G2 cells, Claspin, which is shown in this study to be an APC/CCdh1 substrate in G1, is targeted for degradation. However, this process is counteracted by the deubiquitylating enzyme Usp28 to permit Claspin-mediated activation of Chk1 in response to DNA damage. These findings define a novel pathway that is crucial for the G2 DNA-damage-response checkpoint (Bassermann, 2008).
In animal cells, most microtubules are nucleated at centrosomes. At the onset of mitosis, centrosomes undergo a structural reorganization, termed maturation, which leads to increased microtubule nucleation activity. Centrosome maturation is regulated by several kinases, including Polo-like kinase 1 (Plk1). A centrosomal Plk1 substrate has been identified, termed Nlp (ninein-like protein), whose properties suggest an important role in microtubule organization. Nlp interacts with two components of the gamma-tubulin ring complex and stimulates microtubule nucleation. Plk1 phosphorylates Nlp and disrupts both its centrosome association and its gamma-tubulin interaction. Overexpression of an Nlp mutant lacking Plk1 phosphorylation sites severely disturbs mitotic spindle formation. It is proposed that Nlp plays an important role in microtubule organization during interphase, and that the activation of Plk1 at the onset of mitosis triggers the displacement of Nlp from the centrosome, allowing the establishment of a mitotic scaffold with enhanced microtubule nucleation activity (Casenghi, 2003).
MT nucleation from the animal centrosome clearly depends on γ-TuRCs, but the mechanisms regulating the recruitment of these complexes to the centrosome remain poorly understood. A 156 kDa centrosomal protein, Nlp, has been identified whose properties suggest that it functions as a docking protein for γ-TuRCs during interphase of the cell cycle. Nlp displays significant structural similarity to ninein, a protein implicated in the capping and anchoring of MT minus ends at both centrosomal and noncentrosomal sites. Thus, the mammalian ninein family comprises at least two members, both of which appear to play important roles in the organization of MT arrays. The centrosome association of Nlp is regulated during the cell cycle. Nlp is a substrate of Plk1 and dissociates from centrosomes in response to phosphorylation, suggesting that Plk1 triggers an exchange of gamma tubulin binding proteins at the centrosome. Such an exchange of critical PCM components is likely to constitute a key aspect of centrosome maturation (Casenghi, 2003).
With Spc110p and Spc72p, two gamma tubulin binding proteins have been identified and characterized in S. cerevisiae, but the identification of gamma tubulin binding proteins in other organisms has proven difficult. Spc110 displays some sequence similarity with kendrin/pericentrin-B, but this similarity is largely restricted to a putative calmodulin binding domain. Additional mammalian proteins, including members of the pericentrin/kendrin/CG-NAP family, Cep135 and CPAP (centrosomal P4.1-associated protein), have been proposed to bind to γ-tubulin, but their precise contributions to MT organization remain to be clarified. The present study identifies Nlp (the product of cDNA KIAA0980) as a candidate gamma tubulin binding proteins in human cells. Nlp recruits both γ-tubulin and hGCP4, both in vitro and in vivo, suggesting that Nlp binds the entire γ-TuRC. Indeed, Nlp assemblies promoted MT nucleation both in mammalian cells and in Xenopus egg extracts. Conversely, microinjection of antibodies against Nlp severely suppressed MT nucleation (Casenghi, 2003).
Over its N-terminal half, Nlp shares 37% identity with ninein. However, ninein is substantially larger than Nlp, and the C termini of the two proteins show no structural homology, except for the presence of predicted coiled-coil domains. Ninein has been proposed to function in MT anchoring rather than MT nucleation. It is difficult to rigorously distinguish various MT minus end-associated activities and it would be premature to exclude that Nlp may also contribute to MT anchoring. Immunolocalization data suggest a preferential association of Nlp with one of the two centrioles, which might be consistent with an anchoring function. However, the data strongly indicate that Nlp plays an important role in the recruitment of γ-TuRCs to the centrosome. Thus, while it is clear that both Nlp and ninein play important roles in the organization of MT networks in mammalian cells, the two proteins may have functionally diverged during evolution (Casenghi, 2003).
Both yeast two-hybrid and direct biochemical data identify Nlp as a physiological substrate of Plk1. Furthermore, the results suggest that phosphorylation by Plk1 regulates the interaction of Nlp with both centrosomes and γ-TuRCs. In contrast, there is no evidence that Plk1 phosphorylates ninein, suggesting that Nlp and ninein are regulated differently. The analysis of Nlp mutants with in vitro Plk1 phosphorylation sites altered to alanine strongly suggests that Nlp is a direct substrate of Plk1 not only in vitro but also in vivo. These results also strengthen the view that the motif [E/DxS/T] constitutes a consensus for Plk1 phosphorylation sites. Although still tentative, the availability of such a consensus sequence may facilitate the future analysis of Plk1 substrates (Casenghi, 2003).
The abrupt increase in the MT nucleation activity of centrosomes at the onset of mitosis is expected to require substantial changes in pericentriolar material composition. These are apparently controlled by several protein kinases, including Plk1, Aurora-A, and Nek2, but only a few substrates of these kinases have so far been identified. Nlp properties suggest that it functions in MT nucleation at the centrosome. Remarkably, however, Nlp is displaced from the centrosome at the onset of mitosis, when centrosomal MT nucleation activity increases dramatically. It follows that Nlp functions in centrosomal MT nucleation specifically during interphase (G1, S, and G2) of the cell cycle, but not during M phase. This implies that structurally distinct gamma tubulin binding proteins function as interphasic and mitotic scaffolds for γ-TuRC recruitment. These data further suggest that the activation of Plk1 at the G2/M transition results in the displacement of Nlp from the maturing centrosome and that this event is important for mitotic spindle formation. It is envisioned that the removal of Nlp from the centrosome constitutes a prerequisite for the recruitment of an as yet unidentified mitotic gamma tubulin binding proteins, which then confers enhanced microtubule nucleation capacity to the centrosome. According to this model, the activation of Plk1 at the onset of mitosis triggers the replacement of the interphasic MT nucleation scaffold by the mitotic scaffold (Casenghi, 2003).
Centrosomes in mammalian cells have been implicated in cytokinesis; however, their role in this process is poorly defined. A human coiled-coil protein, Cep55 (centrosome protein 55 kDa), is described that localizes to the mother centriole during interphase. Despite its association with TuRC anchoring proteins CG-NAP and Kendrin, Cep55 is not required for microtubule nucleation. Upon mitotic entry, centrosome dissociation of Cep55 is triggered by Erk2/Cdk1-dependent phosphorylation at S425 and S428. Furthermore, Cep55 locates to the midbody and plays a role in cytokinesis, as evidenced by the observation that its depletion by siRNA results in failure of this process. S425/428 phosphorylation is required for interaction with Plk1, enabling phosphorylation of Cep55 at S436. Cells expressing phosphorylation-deficient mutant forms of Cep55 undergo cytokinesis failure. These results highlight the centrosome as a site to organize phosphorylation of Cep55, enabling it to relocate to the midbody to function in mitotic exit and cytokinesis (Fabbro, 2005).
The centrosome is the principle microtubule organizing center of the mammalian cell, consisting of a pair of barrel-shaped microtubule assemblies which are nonidentical and are described as the mother and daughter centrioles. They (mainly the mother) are surrounded by pericentriolar material (PCM), which consists of a matrix of predominantly coiled-coil proteins. PCM is the main site for nucleation of cytoplasmic microtubules and microtubules that form the meiotic and mitotic spindles. The centrosome is structurally and functionally regulated in a cell cycle-dependent manner to form a bipolar spindle to ensure the proper segregation of replicated chromosomes into two daughter cells. Defects in the number, structure, and function of centrosomes can generate mono- or multipolar mitotic spindles and cytokinesis defects resulting in aneuploidy and chromosome instability: these are common characteristics of tumor cells. Therefore, it is not surprising that these centrosome abnormalities are frequently found in tumors and are usually associated with high cytological grade. Thus, it is critical to understand regulators of the centrosome cycle because it must be carefully coordinated with the cell cycle to complete cell division precisely (Fabbro, 2005).
Microtubule nucleation by the PCM requires the conserved complex, gamma-tubulin ring complex (gamma-TuRC), in metazoan organisms. In yeast, the large coiled-coil spindle pole body (SPB) protein Spc110p anchors gamma-tubulin ring complex, providing sites for microtubule nucleation. In mammalian cells, Kendrin, like its yeast homolog Spc110p, complexes with CG-NAP to provide a structural scaffold for gamma-TuRC (Takahashi, 2002). Nlp has also been implicated in gamma-TuRC anchorage (Casenghi, 2003). CG-NAP and Kendrin associate with several protein kinases and phosphatases, suggesting that microtubule nucleation may be regulated through phosphorylation of CG-NAP/Kendrin complexes or associated proteins that remain to be defined (Fabbro, 2005).
In recent years, several studies have provided a link between centrosomes and cytokinesis. Acentrosomal cells have been shown to form mitotic spindles and progress through mitosis but fail to complete cytokinesis. The molecular understanding of centrosome function in cytokinesis is only beginning to emerge. During cytokinesis, the mother centriole has been shown to transiently reposition to the midbody correlating with bridge narrowing and microtubule depolymerization, while movement away from the midbody correlates with cell cleavage. It is proposed that the mother centriole regulates an as yet unidentified pathway anchored at the centrosome that is analogous to the mitotic exit pathways in budding yeast called the 'mitotic exit network,' which is anchored at the SPB and controls mitotic exit and cytokinesis. The siRNA silencing of a recently identified mother centriole component, centriolin, produces cytokinesis failure (Gromley, 2003), suggesting that it is a component of this pathway in mammalian cells. However, additional components and pathways that control cytokinesis will need to be identified to understand the precise role of centrosomes in this process (Fabbro, 2005).
This study reports the molecular characterization of a coiled-coil protein called Cep55. Cep55 localizes to the centrosome of interphase cells and to the midbody during cytokinesis. Characterization of Cep55-depleted cells reveals that Cep55 participates in membrane abscission to form two daughter cells. Furthermore, Cdk1, Erk2, and Plk1 cooperate in the mitotic phosphorylation of Cep55, and this modification is required for its correct mitotic localization and cytokinesis function to maintain genomic stability (Fabbro, 2005).
Interestingly, Cep55 does not remain associated with the centrosome during mitosis, and its displacement from the centrosome is triggered by its phosphorylation. Therefore, a model is proposed whereby the centrosome acts as a regulatory site to organize phosphorylation of Cep55 upon mitotic entry. This phosphorylated Cep55 is then able to locate to the midbody during the final stages of cell division to function in the signal transduction pathway(s) that results in mitotic exit and cytokinesis. Therefore, it is proposed that dephosphorylation of Cep55 may allow it to relocate to the centrosome upon entry into G1 (Fabbro, 2005).
Fluctuations in the activity of Cdks drive cells to progress through mitosis. Specifically, mitotic entry is promoted by elevated activity of Cdk1 when complexed with cyclin B1, while the exit from mitosis requires the inactivation of Cdk1 and the dephosphorylation of at least a subset of Cdk1 substrates. This study demonstrates that Cep55 is phosphorylated at S425 and S428 by Cdk1/cyclin B1 upon mitotic entry, which is when this kinase is found at the centrosome. Another kinase, Erk2, has been shown to localize to the mitotic centrosomes. S425 and S428 can also be phosphorylated by Erk2 upon mitotic entry. In addition to Cdk1 and Erk2, Cep55 is also phosphorylated by Plk1 and this phosphorylation event is dependent on prior phosphorylation by Cdk1 and/or Erk2 at S425 and S428. Plk1 locates to the midbody and its role during cytokinesis has been demonstrated in yeast, Drosophila, and mammals. These data strongly indicate that Cep55 and Plk1 colocalize at the midbody and that Plk1-dependent phosphorylation of Cep55 is required for completion of cytokinesis. In addition, the Plk1-dependent phosphorylation mutant S436A causes cytokinesis failure to the same extent as the Cdk1/Erk2-dependent mutant S425/428A, indicating that phosphorylation at S436 is absolutely required for the function of Cep55 during cytokinesis, whereas phosphorylation at S425 and S428 is not required for Cep55 cytokinesis function directly but is essential for Plk1-dependent phosphorylation at S436. These findings indicate that Cep55 and Plk1 may cooperate at the midbody to coordinate mitotic exit and cytokinesis. Thus, it is proposed that unphosphorylatable Cep55 may fail to bind or generate the required signal to downstream components of the mitotic exit pathway (Fabbro, 2005).
Cep55 localizes to the centrosome via its C-terminus and this same region associates in vivo with the centrosome proteins CG-NAP and Kendrin. In contrast to CG-NAP and Kendrin, which gain affinity for the centrosome to participate in gamma-TuRC anchorage (Takahashi, 2002), Cep55 loses affinity for the centrosome upon mitotic entry coinciding with its dissociation from CG-NAP and Kendrin. Thus, it is not surprising that no requirement is found for Cep55 for microtubule nucleation. Loss of Cep55 from the centrosome coincides with its phosphorylation at the C-terminal residues S425 and S428. It is plausible to suggest that phosphorylation of Cep55 by either Erk2 or Cdk1 causes a conformational change in the protein causing it to lose affinity for CG-NAP and Kendrin, consequently becoming displaced from the centrosome at the G2/M boundary. This in turn may enable S436 of Cep55 to be accessible to Plk1 for phosphorylation. It is hypothesized that the displacement of Cep55 from the centrosome enables CG-NAP and Kendrin to strongly anchor themselves to the centrosome by binding calmodulin (Takahashi, 2002). Consistent with this idea, Cep55 and calmodulin bind the same region of CG-NAP, and the CG-NAP/Kendrin/calmodulin interaction is thought to occur only during mitosis, which is when calmodulin is observed at the centrosome (Fabbro, 2005).
Outer dense fiber 2 (ODF2) was initially identified as a major component of the sperm tail cytoskeleton, and was later suggested to be localized to somatic centrosomes and required for the formation of primary cilia. A splice variant of hODF2 called hCenexin1, but not hODF2 itself, efficiently localizes to somatic centrosomes via a variant-specific C-terminal extension and recruits Plk1 through a Cdc2-dependent phospho-S796 motif within the extension. This interaction and Plk1 activity were important for proper recruitment of pericentrin and gamma-tubulin, and, ultimately, for formation of normal bipolar spindles. Earlier in the cell cycle, hCenexin1, but again not hODF2, also contributed to centrosomal recruitment of ninein and primary cilia formation independent of Plk1 interaction. These findings provide a striking example of how a splice-generated C-terminal extension of a sperm tail-associating protein mediates unanticipated centrosomal events at distinct stages of the somatic cell cycle (Soung, 2009).
This study demonstrates that Cenexin1, a 93 kDa ODF2-splicing variant, is abundantly expressed in various somatic cells and tissues and associates with centrosomes, whereas ODF2 is richly expressed in adult testis and appeared to be largely associated with sperm tails. Unlike ODF2, Cenexin1 possesses a unique C-terminal extension that plays multiple roles at distinct stages of the cell cycle. Early in the cell cycle, hCenexin1 is required for normal ninein recruitment and primary cilia formation in a Plk1-independent manner. During late G2 and M phases, Cdc2 generates a PBD-docking site within the C-terminal extension of hCenexin1, thereby allowing proper Plk1 recruitment to the centrosomes and normal mitotic progression. It is proposed that the alternative splicing-generated C-terminal extension of hCenexin1 plays a critical role in targeting the protein to the mother centriole, thus promoting both Plk1-dependent mitotic functions and Plk1-independent ninein recruitment and ciliogenesis at distinct stages of the somatic cell cycle (Soung, 2009).
The human Polo-like kinase 1 (PLK1) and its functional homologues that are present in other eukaryotes have multiple, crucial roles in meiotic and mitotic cell division. By contrast, the functions of other mammalian Polo family members remain largely unknown. Plk4 is the most structurally divergent Polo family member; it is maximally expressed in actively dividing tissues and is essential for mouse embryonic development. This study identifies Plk4 as a key regulator of centriole duplication. Both gain- and loss-of-function experiments demonstrate that Plk4 is required -- in cooperation with Cdk2, CP110 (see Drosophila Cp110) and Hs-SAS6 -- for the precise reproduction of centrosomes during the cell cycle. These findings provide an attractive explanation for the crucial function of Plk4 in cell proliferation and have implications for the role of Polo kinases in tumorigenesis (Habedanck, 2005).
Aurora-A and Plk1 are centrosomal kinases involved in centrosome maturation and spindle assembly. The microtubule-binding protein TPX2 interacts with, and activates, Aurora-A. RNA interference-mediated inactivation was used to investigate whether Aurora-A, Plk1 and TPX2 act independently or are part of one signaling cascade in spindle formation in mammalian cells. Both specific, and over- lapping, roles of each single regulator has been identified in centrosome maturation and spindle formation: (1) Aurora-A and TPX2 are required for centriole cohesion and spindle bipolarity; (2) TPX2, besides its known role in microtubule organization, is also involved in centrosome maturation; (3) finally, Plk1 controls the localization of Aurora-A to centrosomes, as well as TPX2 recruitment to microtubules. Based on these results therefore a hierarchical functional relation between Plk1 and the Aurora-A/TPX2 pathway emerges (De Luca, 2005).
Polo-like kinase-1 (PLK1) is an essential mitotic kinase regulating multiple aspects of the cell division process1. Activation of PLK1 requires phosphorylation of a conserved threonine residue (Thr 210) in the T-loop of the PLK1 kinase domain, but the kinase responsible for this has not yet been affirmatively identified. This study shows that in human cells PLK1 activation occurs several hours before entry into mitosis, and requires aurora A (AURKA, also known as STK6)-dependent phosphorylation of Thr 210. Aurora A can directly phosphorylate PLK1 on Thr 210, and activity of aurora A towards PLK1 is greatly enhanced by Bora (also known as C13orf34 and FLJ22624), a known cofactor for aurora A. Bora/aurora-A-dependent phosphorylation is a prerequisite for PLK1 to promote mitotic entry after a checkpoint-dependent arrest. Importantly, expression of a PLK1-T210D phospho-mimicking mutant partially overcomes the requirement for aurora A in checkpoint recovery. Taken together, these data demonstrate that the initial activation of PLK1 is a primary function of aurora A (Macurek, 2008).
Mitotic chromosomal dynamics is regulated by the coordinated activities of many mitotic kinases, such as cyclin-dependent kinase 1 (Cdk1), Aurora-B or Polo-like kinase 1 (Plk1), but the mechanisms of their coordination remain unknown. Cdk1 phosphorylates Thr 59 and Thr 388 on inner centromere protein (INCENP), which regulates the localization and kinase activity of Aurora-B from prophase to metaphase. INCENP depletion disrupts Plk1 localization specifically at the kinetochore. This phenotype is rescued by the exogenous expression of INCENP wild type and INCENP mutated at Thr 59 to Ala (T59A), but not at Thr 388 to Ala (T388A). The replacement of endogenous INCENP with T388A resulted in the delay of progression from metaphase to anaphase. It is proposed that INCENP phosphorylation by Cdk1 is necessary for the recruitment of Plk1 to the kinetochore, and that the complex formation of Plk1 and Aurora-B on INCENP may play crucial roles in the regulation of chromosomal dynamics (Goto, 2006).
The epithelial cell transforming gene 2 (ECT2) protooncogene encodes a Rho exchange factor, and regulates cytokinesis. ECT2 is phosphorylated in G2/M phases, but its role in the biological function is not known. This study shows that two mitotic kinases, Cdk1 and polo-like kinase 1 (Plk1), phosphorylate ECT2 in vitro. An in vitro Cdk1 phosphorylation site (T412) has been identified in ECT2, which comprises a consensus phosphospecific-binding module for the Plk1 polo-box domain (PBD). Endogenous ECT2 in mitotic cells strongly associated with Plk1 PBD, and this binding is inhibited by phosphatase treatment. A phosphorylation-deficient mutant form of ECT2, T412A, does not exhibit strong association with Plk1 PBD compared with wild-type (WT) ECT2. Moreover, ECT2 T412A, but not phosphomimic T412D, displays a diminished accumulation of GTP-bound RhoA compared with WT ECT2, suggesting that phosphorylation of Thr-412 is critical for the catalytic activity of ECT2. Moreover, while overexpression of WT ECT2 or the T412D mutant causes cortical hyperactivity in U2OS cells during cell division, this activity is not observed in cells expressing ECT2 T412A. These results suggest that ECT2 is regulated by Cdk1 and Plk1 in concert (Niiya, 2006).
Mitotic phosphorylation of the spindle checkpoint component BubR1 is highly conserved throughout evolution. This study demonstrates that BubR1 is phosphorylated on the Cdk1 site T620, which triggers the recruitment of Plk1 and phosphorylation of BubR1 by Plk1 both in vitro and in vivo. Phosphorylation does not appear to be required for spindle checkpoint function but instead is important for the stability of kinetochore-microtubule (KT-MT) interactions, timely mitotic progression, and chromosome alignment onto the metaphase plate. By quantitative mass spectrometry, S676 was identified as a Plk1-specific phosphorylation site on BubR1. Furthermore, using a phospho-specific antibody, this site was shown to be phosphorylated during prometaphase, but dephosphorylated at metaphase upon establishment of tension between sister chromatids. These findings describe the first in vivo verified phosphorylation site for human BubR1, identify Plk1 as the kinase responsible for causing the characteristic mitotic BubR1 upshift, and attribute a KT-specific function to the hyperphosphorylated form of BubR1 in the stabilization of KT-MT interactions (Elowe, 2007).
This study describes the identification of S676 as a highly conserved Plk1-specific target site on BubR1. Several lines of evidence indicate that phosphorylation of this site correlates with lack of tension. (1) KTs stained with anti-pS676 antibody preferentially during prometaphase. Staining was gradually lost as sister chromatids reached metaphase, concomitant with the generation of tension in response to bipolar attachment. (2) Prometaphase cells were equally phosphorylated on S676 in the presence of Taxol or nocodazole, indicating that loss of tension rather than lack of attachment efficiently induced phosphorylation. (3) BubR1 phosphorylation was enhanced at KTs of misaligned chromosomes in CenpE-depleted cells, presumed to be attached but not under tension. (4) Taxol treatment of metaphase cells led to rapid reacquisition of the anti-pS676 signal and concomitant loss of RanGAP1 binding to KTs, indicative of destabilized KT-MT interactions due to loss of tension. These results raise the question of how pS676 relates to the
tension-related 3F3/2 phospho-epitope. A recent study in Xenopus suggested that loading of the 3F3/2 epitope onto KTs is dependent on prior assembly of checkpoint proteins and that simultaneous recruitment of BubR1 and Plk1 represents the final step in KT loading of the 3F3/2 antigen. Although these observations made it tempting to speculate that pS676 could make a major contribution to the 3F3/2 epitope, preliminary experiments have failed to substantiate this possibility (Elowe, 2007).
At present, the exact biochemical role of phosphorylation of BubR1 on S676 (and presumably other Plk1 sites) is not known, but it is emphasized that this phosphorylation is transient during M-phase progression. In prophase, before MTs approach KTs, there is virtually no phosphorylation at S676, which is in striking contrast to 3F3/2 staining and indicates that S676 phosphorylation requires KT-MT interactions. As cells progress through prometaphase, KTs begin to associate with spindle MTs, but as long as sister chromosomes have not undergone bipolar attachment, tension is absent and KT-MT interactions are unstable. At this stage, S676 on KT-associated BubR1 is maximally phosphorylated by Plk1, and it is postulated that the highly phosphorylated BubR1 contributes to the establishment of stable KT-MT interactions during chromosome congression. However, as soon as KT-MT interactions come under tension (due to bipolar attachment), phosphate is lost from S676. It is thus proposed that transient phosphorylation of BubR1 on S676 (and perhaps other Plk1 sites) is required for stabilizing KT-MT interactions. Whether this involves a conformational change in BubR1 or the recruitment of additional proteins remains to be determined in the future. Finally, it will be interesting to explore whether this tension-sensitive phosphorylation is regulated primarily at the level of the substrate, the upstream kinase (Plk1), or an as-yet-unidentified antagonistic phosphatase (Elowe, 2007).
Cytokinesis of animal cells requires ingression of the actomyosin-based contractile ring between segregated sister genomes. Localization of the RhoGEF Ect2 to the central spindle at anaphase promotes local activation of the RhoA GTPase, which induces assembly and ingression of the contractile ring. This study used BI 2536, an inhibitor of the mitotic kinase Plk1, to analyze the functions of this enzyme during late mitosis in human cells. It is shown that Plk1 acts after Cdk1 inactivation and independently from Aurora B to promote RhoA accumulation at the equator, contractile ring formation, and cleavage furrow ingression. Inhibition of Plk1 abolishes the interaction of Ect2 with its activator and midzone anchor, HsCyk-4 (also known as Rac GTPase activating protein 1), thereby preventing localization of Ect2 to the central spindle. It is proposed that late mitotic Plk1 activity promotes recruitment of Ect2 to the central spindle, triggering the initiation of cytokinesis and contributing to cleavage plane specification in human cells (Petronczki, 2007).
Pharmacological analysis of Plk1 during late mitosis has revealed a previously unknown function for this key mitotic kinase: triggering the initiation of cytokinesis in human cells. This work was facilitated by the use of a specific and highly potent small-molecule inhibitor that provided sufficient temporal control over Plk1 activity to pinpoint essential later mitotic roles of this kinase, which have hitherto been obscured by its earlier functions. In addition, this work emphasizes the advantages and growing importance of small-molecule inhibitors for cell biological research (Petronczki, 2007).
Although it cannot be ruled out that inhibition of other kinases contributes to some of the phenotypes observed, two key points strongly support the hypothesis that inhibition of Plk1 by BI 2536 is responsible for the cytokinesis defect. First and foremost, depletion of Plk1 using a gene-specific method reproduced one crucial hallmark of the inhibitor phenotype. Second, the conclusions are substantiated by two independent studies, which came to similar conclusions using a structurally distinct small-molecule inhibitor of Plk1 and a genetically engineered ATP analog-sensitive allele of Plk1 (Petronczki, 2007 and references therein).
The results identify Plk1 as a key regulator of the Ect2/HsCyk-4 complex that lies at the heart of cleavage furrow induction in animal cells. It is proposed that Plk1's key role in triggering the initiation of cytokinesis in human cells is to induce complex formation between Ect2 and HsCyk-4. Formation of this complex is required for RhoA activation and serves to localize the RhoGEF protein Ect2 to the central spindle. Chemical epistasis experiments have shown that this function of Plk1 is required after inactivation of Cdk1. Once bound to HsCyk-4, Ect2 could then locally activate the GTPase RhoA, which would culminate in contractile ring formation and cleavage furrow ingression at the equatorial cortex. Thus, Plk1 likely plays a vital role in specifying the cleavage plane and instructing human cells where and when to divide (Petronczki, 2007).
Consistent with the above model, Plk1 inhibition and depletion of either Ect2 or HsCyk-4 result in a very similar spectrum of early cytokinesis defects: absence of contractile ring formation, failure of equatorial RhoA accumulation and cleavage furrow ingression, and, finally, failure in ectopic furrowing during forced mitotic exit. The Plk1 inhibition phenotype closely resembles the one obtained by depletion of Ect2 or HsCyk-4 and is more severe than that observed upon mere delocalization of the Ect2/HsCyk-4 complex. Hence, in addition to controlling GEF localization, Plk1-mediated complex formation presumably contributes to the activation of Ect2's GEF function. The finding that BI 2536 blocks Ect2 binding to HsCyk-4, and Ect2 but not centralspindlin recruitment to the midzone, indicates that Plk1 kinase activity might directly regulate this interaction. Recruitment of the kinase to the midzone at anaphase might increase the local concentration of the enzyme and thereby direct Ect2/HsCyk-4 complex formation to this subcellular location (Petronczki, 2007).
Ect2 activity itself and the Ect2/HsCyk-4 complex are subject to phosphoregulation. Phosphorylation of Ect2 by Cdk1 inhibits complex formation and likely contributes to the inhibition of cytokinesis before anaphase onset. In contrast, phosphorylation of HsCyk-4 at unknown residues by an uncharacterized kinase might be crucial for complex formation. These HsCyk-4 modifications might provide a docking site for the amino-terminal BRCT domains of Ect2, which mediate HsCyk-4 binding and can act as phosphopeptide binding. Thus, it is tempting to speculate that Plk1 is responsible for these phosphorylation events on HsCyk-4. However, there is also evidence supporting an alternative model. Ect2, similar to other GEFs, can be regulated by intramolecular inhibition during which the amino-terminal BRCT region binds to and inhibits the carboxy-terminal DH/GEF domain. Phosphorylation of Ect2 by Plk1 during anaphase might alleviate this intramolecular inhibition by dissociating the Ect2 amino from the carboxyl terminus. This would set free the HsCyk-4 interaction domain and the GEF domain of Ect2 and could lead to both targeting of Ect2 to the midzone and activation of its GEF function. Consistent with this model, recent data have suggested that phosphorylation of Ect2 might induce a conformational change. Furthermore, Plk1 has the ability to bind to Ect2 via the PBD and to phosphorylate the GEF protein. Future work will be required to clarify whether Plk1 directly regulates Ect2/HsCyk-4 complex formation, identify Plk1-dependent phosphorylation sites on these proteins in vivo, and analyze the molecular mechanism by which these modifications might promote complex formation (Petronczki, 2007).
Homeostatic plasticity keeps neuronal spiking output within an optimal range in the face of chronically altered levels of network activity. Little is known about the underlying molecular mechanisms, particularly in response to elevated activity. In hippocampal neurons experiencing heightened activity, the activity-inducible protein kinase Polo-like kinase 2 (Plk2, also known as SNK) is required for synaptic scaling-a principal mechanism underlying homeostatic plasticity. Synaptic scaling also requires CDK5, which acts as a 'priming' kinase for the phospho-dependent binding of Plk2 to its substrate SPAR, a postsynaptic RapGAP and scaffolding molecule that is degraded following phosphorylation by Plk2. RNAi knockdown of SPAR weakens synapses, and overexpression of a SPAR mutant resistant to Plk2-dependent degradation prevents synaptic scaling. Thus, priming phosphorylation of the Plk2 binding site in SPAR by CDK5, followed by Plk2 recruitment and SPAR phosphorylation-degradation, constitutes a molecular pathway for neuronal homeostatic plasticity during chronically elevated activity (Seeburg, 2008).
Myosin phosphatase-targeting subunit 1 (MYPT1) binds to the catalytic subunit of protein phosphatase 1 (PP1C). This binding is believed to target PP1C to specific substrates including myosin II, thus controlling cellular contractility. Surprisingly, it was found that during mitosis, mammalian MYPT1 binds to polo-like kinase 1 (PLK1). MYPT1 is phosphorylated during mitosis by proline-directed kinases including cdc2, which generates the binding motif for the polo box domain of PLK1. Depletion of PLK1 by small interfering RNAs is known to result in loss of gamma-tubulin recruitment to the centrosomes, blocking centrosome maturation and leading to mitotic arrest. It was found that codepletion of MYPT1 and PLK1 reinstates gamma-tubulin at the centrosomes, rescuing the mitotic arrest. MYPT1 depletion increases phosphorylation of PLK1 at its activating site (Thr210) in vivo, explaining, at least in part, the rescue phenotype by codepletion. Taken together, these results identify a previously unrecognized role for MYPT1 in regulating mitosis by antagonizing PLK1 (Yamashiro, 2008).
Shugoshin 1 (Sgo1) functions as a protector of centromeric cohesion of sister chromatids in higher eukaryotes. This study provides evidence for a previously unrecognized role for Sgo1 in centriole cohesion. Sgo1 depletion via RNA interference induces the formation of multiple centrosome-like structures in mitotic cells that result from the separation of paired centrioles. Sgo1+/- mitotic murine embryonic fibroblasts display split centrosomes. Localization study of two major endogenous splice variants of Sgo1 indicates that the smaller variant, sSgo1, is found at the centrosome in interphase and at spindle poles in mitosis. sSgo1 interacts with Plk1 and its spindle pole localization is Plk1 dependent. Centriole splitting induced by Sgo1 depletion or expression of a dominant negative mutant is suppressed by ectopic expression of sSgo1 or by Plk1 knockdown. These studies strongly suggest that sSgo1 plays an essential role in protecting centriole cohesion, which is partly regulated by Plk1 (Wang, 2008).
While DNA replication and mitosis occur in a sequential manner, precisely
how cells maintain their temporal separation and order remains elusive.
This study unveils a double-negative feedback loop between replication
intermediates and an M-phase-specific structure-selective endonuclease, MUS81-SLX4
(see Drosophila Mus81
and Mus312),
which renders DNA replication and mitosis mutually exclusive. MUS81
nuclease is constitutively active throughout the cell cycle (see Drosophila
cell cycle) but requires association
with SLX4 for efficient substrate targeting. To preclude toxic processing
of replicating chromosomes, WEE1
(see Drosophila Wee1)
kinase restrains CDK1
(see Drosophila Cdk1) and PLK1
(see Drosophila polo)-mediated
MUS81-SLX4 assembly during S phase. Accordingly, WEE1 inhibition triggers
widespread nucleolytic breakage of replication intermediates, halting DNA
replication and leading to chromosome pulverization. Unexpectedly,
premature entry into mitosis-licensed by unrestrained CDK1 activity during
S phase-requires MUS81-SLX4, which inhibits DNA replication. This suggests
that ongoing replication assists WEE1 in delaying entry into M phase and,
indirectly, in preventing MUS81-SLX4 assembly. Conversely, MUS81-SLX4
activation during mitosis promotes targeted resolution of persistent
replication intermediates, which safeguards chromosome segregation (Duda, 2016).
As a pivotal mitotic regulator, polo-like kinase 1 (PLK1) is under highly coordinated and multi-layered regulation. However, the pathways that control PLK1's activity and function have just begun to be elucidated. PLK1 has recently been shown to be functionally modulated by post-translational modifications (PTMs), including phosphorylation and ubiquitination. This study reports that SUMOylation plays an essential role in regulating PLK1's mitotic function. Ubc9 was found to be recruited to PLK1 upon initial phosphorylation and activation by CDK1/cyclin B. By in vivo and in vitro SUMOylation assays, PLK1 was identified as a physiologically relevant small ubiquitin-related modifier (SUMO)-targeted protein, preferentially modified by SUMO-1. It was further shown that K492 on PLK1 is essential for SUMOylation. SUMOylation causes PLK1's nuclear import and significantly increases its protein stability, both of which are critical for normal mitotic progression and genomic integrity. These findings suggest that SUMOylation is an important regulatory mechanism governing PLK1's mitotic function (Wen, 2017).
Adriaans, I. E., Basant, A., Ponsioen, B., Glotzer, M. and Lens, S. M. A. (2019). PLK1 plays dual roles in centralspindlin regulation during cytokinesis. J Cell Biol 218(4): 1250-1264. PubMed ID: 30728176
Cytokinesis drives the physical separation of daughter cells at the end of mitosis. Failure to complete cytokinesis gives rise to tetraploid cells with supernumerary centrosomes. Depending on the cell type and cellular context, cytokinesis failure can either result in a G1 arrest or allow cell cycle progression of the tetraploid cells into the next mitosis. These dividing tetraploid cells are at risk of becoming aneuploid, owing to, for example, the extra number of centrosomes that can cause the missegregation of chromosomes during mitosis. Hence, proper execution and completion of cytokinesis is essential for genomic stability (Adriaans, 2019).
In animal cells, cytokinesis starts in anaphase with the formation of an actomyosin-based contractile ring at the equatorial cortex that drives ingression of the cleavage furrow. Before membrane furrowing, interpolar microtubules are bundled between the separating sister chromatids to form the spindle midzone (also referred to as central spindle). As the furrow ingresses, these microtubule bundles are compacted into a cytoplasmic bridge, with the midbody in its center. The midbody attaches the ingressed cell membrane to the intercellular bridge and promotes the final phase of cytokinesis, known as abscission. Formation of the contractile ring requires activation of the small GTPase RhoA by the guanine nucleotide exchange factor (GEF), ECT2. Active, GTP-bound RhoA activates components of the actomyosin-based ring, such as diaphanous-related formin that facilitates the assembly of actin filaments and Rho-kinase (ROCK), which activates nonmuscle myosin II to power ring constriction. Optogenetic manipulation of RhoA activity showed that local activation of RhoA on the cell membrane is sufficient to drive cleavage furrow initiation independent of cell cycle stage. Hence, strict spatial and temporal regulation of RhoA activity is essential to coordinate the onset of cytokinesis with nuclear division (Adriaans, 2019).
Current models for local RhoA activation and cleavage furrow initiation describe at least two anaphase spindle-derived stimulatory signals: one originating from the spindle midzone and another derived from astral microtubules that end at the equatorial cortex. Experiments in large echinoderm embryos suggest a stimulatory role of astral microtubules in the initiation of cleavage furrow ingression (Su et al., 2014; Mishima, 2016), while data in smaller (mostly mammalian) cells emphasized a role for the spindle midzone. The overlapping antiparallel microtubules of the spindle midzone serve as a platform for the localization of a variety of proteins that promote RhoA activation and cleavage furrow ingression directly parallel to the microtubule overlap. In addition, astral microtubules convey inhibitory signals at cell poles (Adriaans, 2019).
Protein regulator of cytokinesis 1 (PRC1) is essential for the assembly of a fully functional spindle midzone. PRC1 is a homodimeric microtubule-binding protein that is directly involved in bundling antiparallel microtubules. Its microtubule-bundling activity is required for spindle midzone formation, thereby indirectly contributing to the recruitment of other spindle midzone-localized proteins, such as centralspindlin and the chromosomal passenger complex. Furthermore, through interaction with the kinesin KIF4A and Polo-like kinase 1 (PLK1), PRC1 also directly recruits regulatory proteins to the spindle midzone (Adriaans, 2019).
Centralspindlin is a heterotetramer consisting of two molecules of the kinesin-6 MKLP1 (KIF23) and two molecules of RACGAP1 (hsCyk4 and MgcRacGAP). Oligomerization of the complex is needed to bundle microtubules and organize the spindle midzone. In addition to microtubule bundling, centralspindlin promotes RhoA activation and cleavage furrow initiation. This latter function of centralspindlin appears to rely on PLK1-dependent binding of RACGAP1 to ECT2. Mutation of PLK1 phosphorylation sites in the noncatalytic N terminus of RACGAP1 disrupts its interaction with the N-terminal BRCT domain in ECT2 and disturbs the initiation of cytokinesis. In line, inhibition of PLK1 activity at anaphase onset prevents RhoA activation at the equatorial cortex and cleavage furrow ingression. Because inhibition of PLK1 activity or expression of a RACGAP1 PLK1-phosphorylation site mutant disrupt the spindle midzone localization of ECT2, it was proposed that the spindle midzone localization of ECT2 is a determining factor for the spatial activation of RhoA at the equatorial cortex (Adriaans, 2019).
The chromosomal passenger complex, consisting of inner centromere protein (INCENP), Survivin, Borealin, and Aurora B kinase, relocates from chromosomes to the spindle midzone and equatorial cortex at anaphase onset in an MKLP2 (KIF20A)-dependent manner. One of the spindle midzone targets of Aurora B is the centralspindlin subunit MKLP1. Phosphorylation of S708 of MKLP1 disrupts the interaction between MKLP1 and 14-3-3 proteins, permitting centralspindlin oligomerization. In Caenorhabditis elegans embryos, Aurora B-induced oligomerization of centralspindlin promotes RhoA activation and cleavage furrow ingression, and Aurora B activity is largely dispensable when centralspindlin constitutively oligomerizes. However, in mammalian cells, inhibition of Aurora B kinase activity at anaphase onset does not prevent initiation of cytokinesis, though it does prevent completion of cytokinesis. This suggests that RhoA activation can occur in the absence of Aurora B activity in mammalian cells and that, in these cells, Aurora B activity appears to be more relevant at later stages of cytokinesis most likely by promoting the formation of the spindle midzone and midbody (Adriaans, 2019).
While the spindle midzone is considered to provide important cues for RhoA activation at the equatorial cortex in mammalian cells, knock-down of PRC1, which clearly disrupts the spindle midzone, does not impair RhoA activation and cleavage furrow ingression. This implies that spindle midzone-independent cues can also locally activate RhoA in small, mammalian cells. This study demonstrates that in the absence of PRC1, RhoA is activated at the equatorial cortex, at least in part, through centralspindlin oligomerization induced by cortical Aurora B activity. Remarkably, it was found that in PRC1-deficient cells, cytokinesis initiation can occur in the absence of PLK1 activity. This alternative PLK1-independent route to RhoA activation has been overlooked because of an unrecognized inhibitory effect of PLK1 on PRC1 in anaphase. Specifically, it is proposed that PLK1 activity limits PRC1-dependent hyperbundling of spindle midzone microtubules and reduces centralspindlin sequestration at the spindle midzone, making it available for Aurora B to activate RhoA at the equatorial cortex (Adriaans, 2019).
Cytokinesis begins upon anaphase onset. An early step involves local activation of the small GTPase RhoA, which triggers assembly of an actomyosin-based contractile ring at the equatorial cortex. This study delineated the contributions of PLK1 and Aurora B to RhoA activation and cytokinesis initiation in human cells. Knock-down of PRC1, which disrupts the spindle midzone, revealed the existence of two pathways that can initiate cleavage furrow ingression. One pathway depends on a well-organized spindle midzone and PLK1, while the other depends on Aurora B activity and centralspindlin at the equatorial cortex and can operate independently of PLK1. It was further shown that PLK1 inhibition sequesters centralspindlin onto the spindle midzone, making it unavailable for Aurora B at the equatorial cortex. It is proposed that PLK1 activity promotes the release of centralspindlin from the spindle midzone through inhibition of PRC1, allowing centralspindlin to function as a regulator of spindle midzone formation and as an activator of RhoA at the equatorial cortex (Adriaans, 2019).
This study uncovered a PLK1-independent, Aurora B-dependent route to RhoA activation and cytokinesis initiation in human cells through knock-down of the microtubule-bundling protein PRC1. This PLK1-independent route to cytokinesis initiation has been missed, due to an unrecognized inhibitory effect of PLK1 on PRC1 in anaphase. It was demonstrate that PLK1 constrains PRC1 in anaphase, which serves two purposes: first, it limits PRC1's microtubule-bundling activity; and second, it promotes the release of a (small) pool of centralspindlin from the spindle midzone. It is argued that this allows centralspindlin to function both as a regulator of spindle midzone formation and as an activator of RhoA at the equatorial cortex. How PLK1 constrains PRC1 remains to be fully determined. PRC1 is a direct substrate of PLK1, and its phosphorylation by PLK1 is needed to bind PLK1 and to localize the kinase on the spindle midzone. This makes it inherently difficult to discriminate how PLK1 affects PRC1 function in anaphase, because mutation of the PLK1 phosphosites in PRC1 impairs PLK1 recruitment to the spindle midzone . PRC1 may directly sequester centralspindlin on the spindle midzone after PLK1 inhibition, as RACGAP1 can interact with PRC1, or it may do so indirectly by creating hyperbundled microtubules in the midzone. Distinguishing these possibilities through structure-function analysis of PRC1 is also challenging because its overexpression results in precocious spindle binding during metaphase (Adriaans, 2019).
An important implication from this work is that ECT2 and RhoA activation can occur in the absence of PLK1 activity. PLK1 was shown to activate the RhoA GEF, ECT2, through phosphorylation of RACGAP1, which promotes RACGAP1 binding to the autoinhibitory N terminus of ECT2. This relieves the intramolecular inhibition on the ECT2 GEF domain. The N terminus of RACGAP1 harbors four evolutionary conserved PLK1 phosphorylation sites, and mutating these sites to alanine (RACGAP1-4A) attenuates complex formation between centralspindlin and ECT2, fails to activate RhoA, and leads to loss of ECT2 from the spindle midzone. These results point to a crucial role for PLK1 in activating ECT2 and RhoA. How can ECT2 and RhoA become activated in PRC1-depleted cells when PLK1 is inhibited? These results are complementary to other work that demonstrates that mutations in the N-terminal BRCT domains of ECT2 that strongly reduce its binding to RACGAP1 and its localization to the spindle midzone do not prevent equatorial RhoA activity and cleavage furrow ingression and supported cytokinesis. Importantly, knock-down of RACGAP1 still impaired furrow ingression in cells reconstituted with the RACGAP1 binding-deficient ECT2 mutant, as it does in PLK1-inhibited cells depleted of PRC1. Thus, RhoA activation during cytokinesis appears to be highly dependent on ECT2 activation by centralspindlin. Notably, the ECT2-RACGAP1 interaction is enhanced by, but does not require, RACGAP1 phosphorylation by PLK1. RACGAP1 contains a C-terminal GAP domain that also interacts with ECT2, which may contribute to formation and function of this complex. This mode of ECT2 activation (and thereby RhoA activation) might require, or be strongly promoted by, localized centralspindlin oligomerization at the equatorial cortex, driven by Aurora B-dependent disengagement of 14-3-3 proteins from centralspindlin (Adriaans, 2019).
This view is supported by optogenetic experiments with a C-terminally truncated ECT2 that only activates RhoA at the equator, presumably due to the presence of centralspindlin at this site. Moreover, active Aurora B is present at the equatorial cortex in PRC1-depleted cells and may provide a local environment for centralspindlin oligomerization. Indeed, in C. elegans, centralspindlin oligomerization obviates the requirement for Aurora B activity. In cultured human cells, Aurora B activity is required for furrow ingression in PRC1-depleted cells and expression of MKLP1-S708E, mimicking Aurora B phosphorylation, partly restores ingression defects caused by Aurora B inhibition. This supports the idea that centralspindlin oligomerization can drive RhoA activation independent of PLK1. However, MKLP1-S708E, only rescues furrow ingression in small fraction of the PRC1-depleted and Aurora B-inhibited HeLa cells. This infers that Aurora B-dependent furrow ingression in PRC1-depleted cells is not solely explained by the phosphorylation of a single residue in a single Aurora B substrate (i.e., MKLP1-S708). In fact, Aurora B phosphorylates several other substrates during anaphase, such as RACGAP1, vimentin, SHCBP1 (SHC binding and spindle associated 1), and possibly myosin light chain and the myosin-binding subunit of myosin phosphatase, which all contribute to furrow ingression and cytokinesis in human cells (Adriaans, 2019).
One could argue that the main role of PLK1 in cytokinesis initiation is to limit PRC1 activity to make centralspindlin available for Aurora B-dependent activation at the equatorial cortex. However, in such a scenario, inhibition of Aurora B would always impair cytokinesis initiation, and this is not the case: in PRC1-proficient cells, furrow ingression takes place when Aurora B is inhibited. This implies that PLK1-dependent RACGAP1 phosphorylation and activation of ECT2 at the spindle midzone, and Aurora B at the equatorial cortex, can in principle function as two separate pathways to centralspindlin and RhoA activation, and cytokinesis initiation. It is proposed that in wild-type cells, the 'PLK1 brake' on PRC1 will also support the release of PLK1-phosphorylated centralspindlin bound to ECT2, from the spindle midzone, allowing it to reach and activate RhoA at the equatorial cortex. Together, the PLK1- and Aurora B-dependent pathways to centralspindlin and RhoA activation may confer robustness to and proper timing of the processs of cleavage furrow ingression in mammalian cells (Adriaans, 2019).
In cells expressing the ECT2 binding-deficient RACGAP-4A mutant, PLK1 is active and expected to act on PRC1, allowing the release of a fraction of centralspindlin from the spindle midzone that can then become activated at the equatorial cortex. In other words, the Aurora B-dependent route to furrow ingression should be operational. However, cleavage furrow ingression does not take place in the RACGAP1-4A-expressing cells. Interestingly, the RACGAP1-4A mutant appears more concentrated at the spindle midzone , similar to what is observed for endogenous RACGAP1 after PLK1 inhibition. This raises the question whether RACGAP1 phosphorylation by PLK1 might also promote the release of centralspindlin from the spindle midzone (Adriaans, 2019).
In conclusion, this study provides evidence for the existence of two pathways resulting in centralspindlin and RhoA activation and cytokinesis initiation in human cells. One pathway depends on PLK1 and originates at the spindle midzone, and the other pathway depends on Aurora B activity at the equatorial cortex. It is argued that this latter pathway has gone unnoticed due to an unrecognized inhibitory effect of PLK1 on PRC1 in anaphase. It is proposed that the PLK1-dependent 'brake' on PRC1 is necessary to release a fraction of centralspindlin from the spindle midzone that can activate RhoA at the equatorial cortex. The finding that these two routes to centralspindlin and RhoA activation could operate independent from each other highlights the robustness and plasticity of centralspindlin-induced cleavage furrow formation (Adriaans, 2019).
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