(Received for publication, April 5, 1995; and in revised form, June 12, 1995)
From the
Previously, we demonstrated that expression of polo-like kinase
(PLK) is required for cellular DNA synthesis and that overexpression of
PLK is sufficient to induce DNA synthesis. We now report that the
endogenous levels of PLK, its phosphorylation status, and protein
kinase activity are tightly regulated during cell cycle progression.
PLK protein is low in G, accumulates during S and
G
M, 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
p34
. 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 p34
-cyclin B complex can
phosphorylate PLK protein in vitro but causes little increase
in PLK kinase activity.
The processes of cell growth and division are stringently
regulated to ensure fidelity of DNA replication and correct segregation
of genetic information(1, 2) . So essential are these
processes to cellular homeostasis that many of the gene products
regulating passage through the cell cycle are highly conserved both in
amino acid sequence and function between organisms as evolutionarily
divergent as yeast and man(3, 4) . Within the last few
years, a variety of related enzymes known as cyclin-dependent protein
kinases (CdKs) ()have been identified that are activated and
inactivated at specific times during cell cycle
progression(5, 6, 7, 8) . The CdKs
must form complexes with a cyclin family member to be activated (9, 10) and are also subject to both positive and
negative phosphorylation by other protein kinases and dephosphorylation
by protein
phosphatases(11, 12, 13, 14, 15, 16, 17) .
Together, the family of CdKs constitutes critical components of the
engine that propels the cell through the cycle. Many proteins that are
phosphorylated in a cell cycle-specific fashion have been identified as
putative targets of CdK regulation including nuclear
lamins(18, 19) , nucleolin(20, 21) ,
and other matrix proteins, as well as cytoskeletal
proteins(7, 22) . In addition to such architectural
proteins, the tumor suppressor gene products p53 (23) and RB (24, 25, 26, 27, 28) are
both subject to regulatory phosphorylation by CdK family
members(7) . Thus, it is clear that reversible phosphorylation
reactions play a major role in regulating cell cycle progression, and
the list of enzymes known to be involved is rapidly expanding.
A
number of cell cycle-regulated kinases unrelated to the CdKs have also
been identified in lower eukaryotes, including the homologous yeast
CDC5 (29) and Drosophilapolo(30) .
Polo was first identified as an embryonic lethal Drosophila mutation causing formation of monopolar and multipolar mitotic
spindles and abnormal segregation of chromosomes(31) . Saccharomyces cerevisiae CDC5 mutants likewise are impaired in
mitotic spindle formation, but the CDC5 kinase also appears to have a
function during S phase(29) . Recently, we and others (32, 33, 34, 35, 36) have
cloned a putative mammalian polo homologue, polo-like kinase
(PLK)(32, 33, 34, 35, 36) .
We previously showed that microinjection of full-length in vitro transcribed PLK message into serum-deprived NIH3T3 cells induced
tritiated thymidine incorporation and that conversely microinjection 1
of full-length PLK antisense RNA reduced serum-stimulated tritiated
thymidine incorporation. On the basis of those results, we suggested
that PLK had an important S phase function (34) . In the
present study, we investigate the cell cycle regulation of PLK by
measuring endogenous PLK protein levels, phosphorylation state, and
kinase activity. We show that PLK protein levels are low in
G, increase during S, remain high through G
M,
and are rapidly decreased after mitosis. During the G
to M
phase transition, PLK protein is phosphorylated on serine, and its
kinase function is stimulated. Dephosphorylation of PLK reduces its
activity by 5-10-fold and converts the slower migrating form to
the faster migrating form. We further demonstrate that Cdc2/cyclin B
can phosphorylate PLK but has little effect on PLK kinase activity,
suggesting that mitotic activation of PLK will not be explained solely
by Cdc2.
For mitotic shakeoff experiments, Hela or NIH3T3 cells were serum starved (0.5% fetal calf serum) for 36 h, then stimulated with 10% fetal calf serum. After 18 h, round mitotic cells were detached by rapping the flask vigorously several times and harvested by centrifugation. The remaining adherent cells were collected by scraping and were used to prepare interphase extracts.
Figure 1:
Characterization of PLK antisera. A, murine PLK was [S]methionine labeled
during translation in vitro, and aliquots of the translated
protein were immunoprecipitated with preimmune or immune PLK peptide
antisera and analyzed by 7.5% acrylamide SDS-PAGE and autoradiography. Lane 1, preimmune 8847; lane 2, preimmune 8845; lane 3, sample of total; lane 4, immune 8847; lane 5, immune 8845. B, asynchronously growing human
CA46 lymphoma cells were metabolically labeled with
[
S]methionine, lysed, and immunoprecipitated
with 8845 antisera in the presence (+) or absence(-) of
cognate peptide and analyzed by 7.5% acrylamide SDS-PAGE and
autoradiography. C, samples containing 75 µg each of total
CA46 cell proteins were separated by 7.5% SDS-PAGE, transferred to
Immobilon, and probed with immune (lane 1) or preimmune PLK
antisera 8845 (lane 2). Primary antibody was detected using a
peroxidase-labeled goat anti-rabbit and
ECL.
Figure 2: Cell cycle changes in PLK protein. CA 46 cells were treated 16 h with nothing (U), mimosine (M), aphidicolin (A), or nocodazole (N). A, cells were harvested and prepared for cell cycle fax analysis. B, cell lysates were analyzed by SDS-PAGE and immunoblotting with 8845 antiserum.
Figure 3:
Mitotic PLK is phosphorylated on serine
and has reduced mobility and increased kinase activity. A,
CA46 cells were either unsynchronized (U) or synchronized with
aphidicolin in S phase (S) or with nocodazole in prometaphase
of mitosis (M). Lysates were prepared and immunoprecipitated (I.P.) with preimmune (P) or immune (I) 8845
PLK antiserum. Immunoprecipitates were subjected to immune complex
kinase assays with dephosphorylated casein added as an exogenous
substrate and analyzed by 8845 immunoblot and autoradiography. Upper panel, immunoblot; middle panel,
autoradiography of immune complex autophosphorylation of PLK; bottom panel, autoradiography of immune complex
phosphorylation of casein. B, NIH3T3 cells were serum starved
for 36 h and then stimulated with serum for 18-20 h. Mitotic
cells (M) were collected by mechanical shake off, and adherent
interphase cells (I) were harvested by scraping. Lysates
prepared from equal numbers (1 10
) of interphase or
mitotic cells were immunoprecipitated (I.P.) with preimmune (P) or immune 8847 PLK antiserum with (IC) or without (I) competing cognate peptide, and immune complex kinase
assays were performed using casein as substrate. Autoradiography is
shown. C, asynchronously growing NIH3T3 cells were labeled
metabolically with [
S]methionine (
S) or with orthophosphate (
P). Methionine-labeled cells were
harvested with a scraper, while orthophosphate-labeled mitotic cells
were harvested by mechanical shake off. Lysates were immunoprecipitated
using 8847 antiserum with (+) or without(-) cognate peptide
and analyzed by SDS-PAGE and autoradiography. D and E, nocodazole-synchronized CA46 extracts were prepared and
immunoprecipitated with PLK antiserum. Immunoprecipitates were treated
with nothing (lane 1), PP2A (lane 2), PP2A and
Me
SO (lane 3), or PP2A, Me
SO, and
okadaic acid (lane 4) and subjected to in vitro casein kinase assays. Reactions were analyzed by SDS-PAGE and
immunoblotting (D) and autoradiography (E). F, phosphoamino acid analysis of in vitro phosphorylated PLK and casein isolated from A and in
vivo phosphorylated PLK from C. O, origin; S, serine; T, threonine; Y,
tyrosine.
To substantiate the kinase activity results obtained with drug-synchronized cells, a mitotic shake off experiment was performed with murine NIH3T3 cells (Fig. 3B). Immunoprecipitations from equivalent numbers of mitotic or the interphase adherent cells were performed, and immune complex kinase assays were performed using casein as exogenous substrate. No kinase activity was detected in the preimmune or immune competition assays performed from mitotic extracts, and only very low amounts of activity were detected in the immune interphase assay. Consistent with the results shown in A, PLK isolated from mitotic cells showed increased kinase activity.
To directly assess
PLK phosphorylation state during mitosis, we compared in vivo phosphorylated PLK from NIH3T3 cells, isolated by mitotic shake
off, to [S]methionine-labeled PLK
immunoprecipitated from unsynchronized cells in the presence or absence
of cognate competitor peptide (C). We found that PLK was
indeed labeled with
P during mitosis (C) but was
undetected in extracts from adherent cells (not shown). Furthermore,
the phosphorylated form appeared to migrate more slowly in SDS gels
compared to the [
S]methionine-labeled PLK
protein from unsynchronized cells. These results confirmed our
speculation that PLK is phosphorylated in a cell cycle-specific manner
and could arise either through an autophosphorylation reaction or
through the action of a separate kinase.
To confirm that the mitotic
specific phosphorylation of PLK was responsible for its reduced
mobility, immunoprecipitated mitotic PLK was treated with purified
protein phosphatase 2A in the presence or absence of okadaic acid and
the effects of the dephosphorylation on mobility and casein kinase
activity determined (Fig. 3, D and E).
Immunoprecipitated PLK not treated with phosphatase (D, lane 1) migrated as a doublet and had substantial kinase
activity (E, lane 1), while PLK treated with PP2A,
with (lane 3) or without (lane 2) MeSO,
the carrier for okadaic acid, was mostly converted to the faster
migrating form and had 5-10-fold less kinase activity (E). The effects of the PP2A treatment on PLK mobility and
kinase activity could be completely blocked by the addition of okadaic
acid (lane 4) demonstrating that the effects were due to the
phosphatase activity and not to some contaminating activity present in
the PP2A.
Phosphoamino acid analysis was performed to determine which amino acids in PLK are phosphorylated during mitosis and which amino acids are PLK phosphorylated in immune complex kinase assays with casein (F). As predicted by its catalytic domain sequence(37) , immunoprecipitated PLK demonstrated serine/threonine phosphorylation of casein and also autophosphorylation of serine and threonine residues in immune complex kinase assays. The phosphorylation of the two amino acids appeared roughly equal in the in vitro reactions. In contrast, we could only detect phosphoserine in mitotic PLK labeled in vivo. Thus, PLK isolated from mitotic cells is phosphorylated on serine, has increased phosphotransferase activity, and has reduced mobility on SDS gels.
Several mechanisms could account for the failure to detect phosphothreonine in orthophosphate-labeled mitotic PLK. The labeling in vitro of both serine and threonine could represent an artifact of the in vitro activity in which residues that are not phosphorylated in vivo become phosphorylated due to the artificial conditions. It might also be that a very active phosphatase specifically removes phosphate from threonine in vivo during mitosis as a means of regulating PLK activity. At present, we cannot distinguish between these possibilities.
Figure 4: Comparison of the timing of PLK and Cdc2 activation. A, CA46 cells were double aphidicolin blocked and released into nocodazole. B, CA46 cells were double blocked and released as in A, except just before release into nocodazole the cells were treated with nitrogen mustard for 30 min. At the indicated time points, cells were harvested for cell cycle FACS, mitotic index, and immunoblot analysis for PLK and Cdc2. Upper panels, PLK immunoblot; middle panels, Cdc2 immunoblot; bottom panels, graphs showing FACS and mitotic index results. C, immune complex kinase assay. Cells were double blocked with aphidicolin and released into nocodazole. At the indicated times, lysates were prepared and immunoprecipitated in the presence (+) and absence(-) of competing peptides, with 8845 PLK antisera and a Cdc2 specific antisera. Immune complex kinase assays with casein for PLK and histone for Cdc2 were performed. Autoradiography is shown. D, PLK immune complex kinase assay. Cells were double blocked with aphidicolin, treated with nitrogen mustard, and released into nocodazole. At the indicated times, lysates were prepared and immunoprecipitated with 8845 antiserum. Autoradiography is shown.
Figure 5: In vitro phosphorylation of PLK by Cdc2/cyclin B. PLK was immunoprecipitated from aphidicolin-synchronized CA46 cell extracts in the absence or presence of cognate peptide and subjected to in vitro phosphorylation in a Cdc2 reaction mixture in the presence or absence of purified Cdc2-cyclin B complex. Samples were then separated by SDS-PAGE, transferred to Immobilon, and analyzed by autoradiography (B) and immunoblotting (A) or were washed with PLK reaction mixture to remove the Cdc2-cyclin B complex and reaction mixture; samples were then subjected to a PLK casein kinase assay (C) and analyzed by SDS-PAGE and autoradiography.
In our initial report on the cloning of human and murine PLK,
we showed evidence that PLK has a function in S phase (34) similar to the finding for the yeast PLK homologue cdc5,
which apparently has roles during S phase and during
mitosis(29) . More recently, it has been shown that PLK steady
state message (35, 36) and protein levels (33) are cell cycle regulated, with low levels of each detected
in G and higher levels in S and G
. Our findings
support and extend these earlier observations by showing that 1) PLK
kinase activity is cell cycle regulated, 2) PLK activation involves a
phosphorylation reaction, 3) mitotic PLK is phosphorylated on serine,
4) activation of PLK occurs with kinetics similar to Cdc2, and 5) PLK
kinase activation, like Cdc2 activation, is integrated into the G
checkpoint response to DNA damage.
Fenton and Glover (40) have shown that in synchronously dividing Drosophila cells, polo kinase is tightly regulated and activated at the
anaphase-telophase transition, while our data suggest an earlier
activation of PLK, probably during the G to prophase
transition. Our data must also be considered in light of the recent
publication by Golsteyn and co-workers(33) , showing that in
interphase cells, PLK is diffusely distributed throughout both the
cytoplasm and nucleus, whereas just after cell division, PLK localized
to the midbody of the post-mitotic bridge, which is suggestive of a
function during late mitosis/cytokinesis. The earlier activation of PLK
at the G
M phase transition suggested by our data, however,
is not incompatible with a role later in mitosis. Together, our results
suggest that while PLK is similar to Drosophila polo in
structure and may overlap functionally, there may also be some
differences.
We found that PLK was phosphorylated on one or more
serine residues during mitosis and that its kinase activity was
increased severalfold compared to equal amounts of interphase PLK. In
addition, we found that a slower migrating form of PLK was detected in
mitotic extracts. Since dephosphorylation of PLK by PP2A converts the
slower migrating form to the faster migrating species and reduces the
kinase activity associated with the precipitate, it is tempting to
speculate that the reduced mobility form of PLK represents the
activated enzyme. However, our present data are not sufficient to prove
it, since it is possible that PLK, like Cdc2, contains both positive
and negative phosphorylation sites, and the reduced mobility, fully
phosphorylated form might well be completely inactive, while an
intermediately phosphorylated faster migrating form could be the most
active. The identity of the kinase or kinases responsible for mitotic
phosphorylation of PLK is unknown, but the reduced mobility associated
with mitotic phosphorylated PLK is not achieved by autophosphorylation in vitro by either PLK precipitated from interphase cells (Fig. 2, upper panel) or by autophosphorylation of in vitro translated PLK, nor does autophosphorylation seem to
increase casein kinase activity (data not shown). Thus, activation of
PLK probably involves phosphorylation by some other kinase(s), one
obvious possibility being p34; however, PLK
does not have any Cdc2 consensus-like sequences,
(S/T)PX(K/R)(41) , containing serine, nor did in
vitro phosphorylation of PLK by purified Cdc2/cyclin B cause a
shift in PLK mobility or substantial increases in its activity. On the
basis of these results, we conclude that while Cdc2 may be involved in
regulating PLK activity, it cannot alone account for the mitotic
modification and activation of PLK. It should be noted, however, that
interphase (Fig. 3), and even in vitro translated PLK
(data not shown), does have some basal level of immune complex kinase
activity, and therefore, mitotic phosphorylation is not a requirement
for activity, although it does increase it. This basal activity during
interphase may account for our earlier observation that inhibition of
PLK expression by antisense caused a decrease in DNA
replication(34) . Determination of phosphorylation site(s)
within the PLK protein and their relationship to kinase activity may
help understand the regulation of PLK and suggest potential kinases
that phosphorylate PLK.
The identification of substrates is an important issue regarding the specific roles of PLK during cell cycle progression. PLK coprecipitates with several other proteins that are not cross-reactive with the antisera by immunoblot analysis and therefore may represent members of a PLK complex (and potential substrates). Identification of these proteins is likely to help reveal the role of PLK in regulating cell cycle progression.