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Biological Mechanisms Section, Laboratory of Leukocyte Biology, National Cancer Institute—Frederick Cancer Research and Development Center, Frederick, Maryland 21702 [Y. F., D. K. F.]; National Institute on Aging, Baltimore, Maryland 21224 [D. L. L.]; and IRSP, Scientific Applications International Corp.—Frederick, National Cancer Institute—Frederick Cancer Research and Development Center, Frederick, Maryland 21702 [D. K. F.]
Abstract
The polo-like kinase (Plk) has been shown to be associated with the anaphase-promoting complex at the transition from metaphase to anaphase and to regulate ubiquitination, the process that targets proteins for degradation by proteasomes. In this study, we have identified proteasomal proteins interacting with Plk by mass spectrometry and found that Plk and 20S proteasome subunits could be reversibly immunoprecipitated from both human CA46 cells and HEK 293 cells transfected with HA-Plk. Furthermore, both coprecipitated Plk and baculovirus-expressed Plk were able to phosphorylate proteasome subunits, and metabolic labeling studies indicate that Plk is partially responsible for the phosphorylation of 20S proteasome subunits C9 and C8 in vivo. In addition, phosphorylation of proteasomes by Plk enhanced proteolytic activity toward an artificial substrate Suc-L-L-V-Y-AMC in vitro and in vivo. Finally, we were also able to detect Plk associated with 26S proteasomes under certain conditions. Together our results suggest that Plk is an important mitotic regulator of proteasome activity.
Introduction
The initiation and completion of mitosis is strictly regulated by the cell cycle regulatory machinery, mainly by the cyclin-dependent kinases (1) . In addition to cyclin-dependent kinases, another serine/threonine kinase family, the Plks,3 also play important roles at multiple stages of the cell cycle (2) . After the Drosophila polo kinase was first identified (3) , Plks have been identified in various organisms from yeast to humans (reviewed Ref. 4 ). The protein levels of Plk, its phosphorylation status, and kinase activity are tightly regulated during cell cycle progression (5, 6, 7) .
Early studies found that Plk activity is required for the establishment
of a normal bipolar spindle in Drosophila and
Schizosaccharomyces pombe (3
, 8)
.
Microinjection of Plk-specific antibodies into HeLa cells suppresses
centrosome separation and decreases levels of 
-tubulin
(9)
. Cellular localization data also support this function
for Plk. Plk is mainly localized at the centrosome in interphase cells,
whereas during mitosis, a fraction of Plk is concentrated at the
spindle poles and midzone (10)
. Other functions of Plks
have been reported. Xenopus Plx1 is able to phosphorylate
and activate CDC25c, an activator of cdc2/cyclin B complex (7
, 11)
. Finally, Plk activity is required for adaptation to DNA
damage in yeast (12)
and the completion of meiosis and
cytokinesis (13, 14, 15)
.
Recent studies have emphasized that Plk functions at late stages of the cell cycle, especially in regulating the APC or ubiquitin ligase system. It has been reported that cdc2/cyclin B-activated Plk is able to phosphorylate three components of the APC and improve the ability of the APC to ubiquitinate cyclin B in vitro (16 , 17) . In Saccharomyces cerevisiae, cdc5 mutant cells display a defect in the destruction of mitotic cyclins and a reduction in the cyclin-ubiquitin ligase activity of the APC (18) . In Xenopus egg extracts, Plx1 is required for the APC-mediated degradation of cyclin B2 and cut2, both of which are important for the M-to-I phase transition (19) . Therefore, in part, Plk regulates the completion of mitosis via its regulation of ubiquitination of target proteins, a step preceding proteolysis by proteasomes.
The 26S proteasome is composed of a 20S core catalytic complex and 19S
regulatory caps. The 20S proteasome is a Mr
700,000, barrel-shaped structure, formed by two 
-rings on the outer
side and two ß-rings on the inner side of the barrel
(20)
. Each ring is composed of seven small proteins with
molecular weights of Mr 21,000–32,000. Together
with the 19S regulator, proteasomes are responsible for proteolysis of
most ubiquitinated proteins. Substrates of proteasomes include cell
cycle regulatory proteins, antigens, and misfolded or damaged proteins.
Thus, their activity is important for cell cycle progression,
metabolism, and immune responses. However, the mechanisms regulating
the activity of proteasomes are not well understood. Phosphorylation of
proteasome components C8 and C9 seems to increase proteolytic
activity, but the kinase responsible for the phosphorylation has not
been identified (21
, 22)
. Casein kinase II was found to
copurify with 20S proteasomes and to phosphorylate a
Mr 30,000 proteasome subunit; however, whether
the phosphorylation mediated by casein kinase II affects proteasome
activity is still in question (23)
. Further work is needed
to understand the correlation of proteasome subunit phosphorylation
with proteolytic activity. Other regulatory mechanisms may include
subunit composition switches induced by IFN-, differential
association of 20S with 19S or 11S regulatory complexes, and changes in
steady-state levels of individual proteasome proteins
(24)
.
In a previous report, we showed that Plk can be detected in a
multiprotein complex, and we identified -, ß-, and -tubulins as
partners in the complex (25)
. Other groups have reported
several additional proteins interacting with Plk at M phase, including
mitotic kinesin-like protein-1 (6)
, members of the APC
(17)
, peptidyl-prolyl isomerase 1 (26
, 27)
,
and a microtubule-associated protein (28)
. It is likely
that Plk is a member of several distinct macromolecular complexes that
regulate discrete functions. Identification of such complexes should be
helpful in understanding the unknown aspects of Plk functions. In this
report, we show that Plk is stably associated with 20S proteasomes and
that Plk-mediated in vitro phosphorylation of proteasome
subunits increases proteolytic activity and that such regulation also
occurs in vivo.
Results
Identification of Proteasome Components as Plk-associated Proteins
in Human CA46 Cells.
Plk polyclonal antibody (Zymed) was raised against a COOH-terminal Plk
peptide, it recognized a Mr 64,000 protein in
CA46 cell lysates (for the antibody specificity, also refer to Ref.
6
), and preincubation of the antibody solution with the
cognate COOH-terminal peptide abolished the signal on Western blot
(Fig. 1
,Lanes 1 and 2). This Plk antibody was used to
immunoprecipitate Plk from mitotic cell lysates (Fig. 1
, Lanes
3 and 4). In addition to Plk, several other proteins
were consistently coimmunoprecipitated. More importantly, these
proteins were specifically competed when Plk COOH-terminal peptide was
included in the immunoprecipitation. Our focus was placed on several
low molecular weight proteins at the Mr
30,000–36,000 range (bands 1 to 3) that were clearly competed by the
Plk peptide. These proteins were excised from the gel, reduced and
alkylated with iodoacetic acid, digested with sequencing grade trypsin,
and analyzed by mass spectrometry. Sequences obtained matched the
following 20S proteasome subunits in the NCBI protein database: xapc7,
, 
, and C3 of -subunits; C5 and C10 of ß-subunits (Table 1)
. Among the six subunits identified, four subunits had 13% or more of
their total amino acid sequences positively identified. Several
nomenclatures have been used to name proteasome subunits; we will adopt
the system used by Kopp et al. (20)
, Affiniti
(where most of proteasome subunit antibodies used in this study were
obtained), and others (29, 30, 31)
. According to this
nomenclature, the seven -subunits of the human 20S proteasome are:
C2, C3, C8, C9, xapc7, , and . We detected all but C3 in Plk
immunoprecipitates by immunoblotting (Fig. 2A)
. The C3 antibody only detected a very weak signal in total lysates (data
not shown), and we expect that C3 is present in this complex. The seven
ß-subunits are C5, C7, C10, N3, X, Y, and Z. Among them, subunits X,
Y, and Z could individually switch to LMP7, LMP2 (or delta), and MECL-1
when induced by IFN- (24)
. Subunits C10, N3, X, Y, and
Z were detected in Plk immunoprecipitates by immunoblotting. No
antibodies specifically recognizing subunits C5 and C7 are currently
available. Therefore, 13 of 14 types of subunits were positively
identified to be associated with Plk either by sequence data or by
immunoprecipitation/immunoblotting. All 11 subunits detected by
coprecipitation were competed, along with Plk, by including excess
COOH-terminal peptide in the immunoprecipitation. Similar results were
observed in S-phase and prometaphase cells, although the amounts of Plk
were reduced in interphase cells (data not shown).
|
|
|
, C8, and C9
coprecipitated small amounts of Plk, which were only visualized after
prolonged exposure of the Western blot (Fig. 2B)
. This weak
detection of Plk in proteasome precipitates may be attributable to the
abundance of proteasome protein (up to 1% of the cell protein content;
Ref. 29
), compared with low levels of Plk in cell lysates
(<0.03% of the total cell mass). Alternatively, Plk could be
associated with only a specific subset of proteasomes.
Proteasomes have two main isoforms in cells: 20S and 26S (20S-19S). We
tested for the presence of the 19S regulator in the Plk
immunoprecipitates. No 19S regulator was detected in Plk
immunoprecipitations performed in NP40 lysis buffer. Because the 26S
proteasome requires ATP and low salt for its stability (32
, 33)
, cells were homogenized in a low salt buffer [20
mM Tris/HCl (pH 7.0), 5 mM ATP, 1
mM ß-mercaptoethanol, 0.1 mM EDTA, and 20%
glycerol], and a similar immunoprecipitation was repeated in this
buffer. Under these conditions, ATPase subunit 7 of the 19S regulator
was indeed detected by immunoblotting 20S- and Plk immuoprecipitates
(Fig. 2C)
. Therefore, it appears that given the conditions
under which 26S proteasomes remain intact, Plk is associated with the
26S particles containing the 19S regulatory caps. In this study, we
focus our attention on the Plk/20S proteasome interaction because the
20s proteasomes carry the core catalytic activity, and most 19S subunit
antibodies from Affiniti only recognize yeast proteins.
Association of HA-tagged Plk with Proteasomes in Transfected HEK
293 Cells.
To further investigate the specificity of Plk/proteasome association,
we transfected HEK 293 cells with HA-tagged wild-type Plk,
kinase-inactive mutant K82R, or kinase domain-deleted fragment. All
these constructs were highly expressed in 293 cells (Fig. 3A)
. Both wild-type and K82R were coimmunoprecipitated with the proteasome,
whereas deletion of the kinase domain nearly abolished the interaction
(Fig. 3B)
. Similarly, HA-Plk and HA-K82R were also
coprecipitated by an antibody to the proteasome "core." In the
reciprocal experiment, proteasomes were coprecipitated with HA-Plk and
HA-K82R by the HA tag antibody (Fig. 3C)
. Again, in cells
transfected with full-length Plk and K82R, coprecipitated proteasome
subunits were easy to detect, whereas it required much longer exposure
of the Western blot to detect subunits coprecipitated with 
KD (data
not shown). Therefore, with increased expression of Plk protein in
transfected HEK 293 cells, proteasomes, and full-length Plk can be
reversibly and efficiently coprecipitated. These results indicate that
the presence of proteasomes in Plk immunoprecipitates observed in Figs. 1
and 2
was not attributable to cross-reaction with the Plk antibody.
The reduction in coprecipitation seen with the Plk kinase domain
deletion mutant indicates that the interaction of Plk and proteasomes
requires the kinase domain, but not its activity, in binding to the
large 20S structure.
|
-32P]ATP,
autoradiography revealed phosphorylated proteins of
Mr 
32,000 (data not shown). To identify the
phosphorylated proteins, the kinase reaction mixture was boiled in 1%
SDS to disrupt protein associations, and antibodies specific to
individual proteasome subunits were added to the mixture for
reimmunoprecipitation. Previous reports have shown that -subunits C8
and C9 of the 20S proteasome are phosphorylated in vivo
(22)
. Reimmunoprecipitation with monoclonal antibodies
specific for C8 and C9 isolated phosphorylated proteins of the expected
sizes (Fig. 4A)
, whereas no phosphorylated or C2 were detected. The polyclonal
antibody to the 20S proteasome "core" recognizes an unnamed
proteasome protein of Mr 30,000. This "core"
antibody reprecipitated a phosphorylated Mr
30,000 protein.
|
. Profiles of Coomassie-stained proteins associated with
HA-Plk and HA-K82R were essentially identical (Fig. 4C)
.
These results suggest that Plk is directly responsible for
phosphorylation of proteasome components in vitro.
We next tested the ability of purified recombinant Plk to bind to and
phosphorylate proteasomes. His6-Plk was purified from
insect cells with nickel-chelating beads and incubated with CA46
mitotic lysates at 4°C or 37°C, and beads were washed extensively
after incubation. Proteins bound on beads were released and analyzed by
immunoblotting. The C8 subunit was detected on beads incubated at
37°C (Fig. 5
A, Lane 2), whereas it was not present in beads incubated at 4°C
(Fig. 5
A, Lane 1). Probing with other proteasome subunit
antibodies produced similar results (data not shown). Lanes
3 and 4 show that a similar amount of
His6-Plk was incubated with cell lysates at 4°C or
37°C. Thus, recombinant Plk can associate with proteasomes in
vitro but only at physiological temperature.
|
-casein (10)
, but it did
not phosphorylate purified proteasome preparations (Fig. 5
B, Lane
1). However, His6-Plk activated by okadaic acid
treatment of insect cells had higher kinase activity toward
casein and reduced mobility on SDS-PAGE (data not shown),
similar to that observed for Plk immunoprecipitated from mitotic cells
(16)
, and such activated Plk was able to phosphorylate
exogenously added proteasomes (Fig. 5
B, Lane 2). We
preincubated His6-Plk purified from nontreated insect cells
with CA46 M-phase lysates to take advantage of the fact that
proteasomes bind to His6-Plk beads at 37°C to see whether
His6-Plk could be activated by endogenous CA46 lysate
factors. It was found that when the preincubation took place at 37°C,
His6-Plk subsequently phosphorylated proteasome proteins
captured from the cell lysate (Fig. 5
B, Lane 4). Six
phosphorylated protein bands between Mr 28,000
and Mr 36,000 were detected in Lane
4, a pattern typical of purified 20S proteasome protein profiles
on Coomassie-stained gels (Fig. 5
B, Lane 6). Immunodepletion
of proteasomes from the M-phase lysates before the preincubation
removed the six phosphorylated proteins (Fig. 5
B, Lane 5).
Lanes 1 and 2 in Fig. 5C
show the
purity of His6-Plk prepared from untreated or okadaic
acid-treated insect cells. These data indicate that activated
His6-Plk is able to associate with endogenous proteasomes
from human CA46 cell lysates and to phosphorylate proteasome subunits.
Because cdc2/cyclin B has been reported to phosphorylate and activate
Plk to phosphorylate APC (17)
, we also treated
His6-Plk with purified cdc2/cyclin B. Although we found
that His6-Plk was phosphorylated by cdc2, we detected no
activation of the recombinant Plk in phosphorylation of added purified
proteasomes (data not shown).
We have quantified the amount of proteasome subunits C8 and C9
phosphorylated by His6-Plk in vitro by
two-dimension immunoblotting. The in vitro kinase reaction
mixtures containing okadaic acid-activated His6-Plk and 20S
proteasome proteins were separated on NEPHGE and 12% SDS-PAGE.
Phosphorylated proteins were detected by autoradiography, and the total
C8 and C9 proteins present in the reaction were subsequently revealed
with immunoblotting. Autoradiography revealed two phosphorylated
proteins at Mr 31,000 (Fig. 6A)
. These phosphorylated proteins were later confirmed to be proteasome
subunits C8 and C9 by immunoblotting with antibodies specific to C8 and
C9 (Fig. 6, B and C)
. Both C8 and C9 have
multiple forms migrating at pH ranges of 4.5 to 7.5, and 4.5 to 5.9, on
this NEPHGE system. We found that the vast majority of C8 was already
phosphorylated when the 20S proteasomes were purified (Fig. 6D)
, whereas phosphorylated C9 was not detected in the
purified preparation (Fig. 6E)
. Phosphatase treatment of the
proteasome proteins leads to the accumulation of basic forms (data not
shown). The immunoblots in Fig. 6, B and C
, were
scanned by a densitometer, and the areas matching the phosphorylated
proteins were calculated. About 16% of subunit C9 was phosphorylated
by His6-Plk in vitro under the experimental
conditions. We estimated that the percentage of C8 phosphorylated by
His6-Plk was <5%. This low level of phosphorylation
correlates well to the modest change in the proteasome activity induced
by Plk-mediated phosphorylation of proteasomes in vitro (see
results in Table 2
).
|
|
. A duplicate set of samples was treated with 1% SDS to disrupt protein
associations and subjected to reimmunoprecipitation with mAb specific
to proteasome subunits C9 and C8. Fig. 7B
shows that
phosphorylation of subunit C9 was completely abolished in cells
transfected with HA-K82R. In contrast, phosphorylation of subunit C8
(Fig. 7C)
in cells transfected with HA-K82R was not greatly
reduced from control cells, although the wild-type transfections showed
a substantial increase over the controls. These results suggest that
Plk plays a significant role in phosphorylating proteasomes in
vivo.
|
B, Lane 6). Purified proteasome preparations (1
µg/reaction) were subjected to phosphorylation by activated
His6-Plk, or mock reactions, followed by degradation assays
as described in "Materials and Methods." The fluorogenic artificial
substrate Suc-L-L-V-Y-AMC was used to monitor proteasome activity. As
seen in Table 2A
, phosphorylation by okadaic acid-activated
His6-Plk increased the proteolytic activity of 20S
proteasomes by 2-fold, and phosphorylation by M-phase-activated
His6-Plk increased activity by 1.5-fold. Numbers in Table 2A
represent results from five independent experiments.
Having shown that Plk is able to phosphorylate proteasome subunits
in vivo and Plk-mediated phosphorylation regulates
proteasome activities in vitro, we tested proteasome
activities in 293 cells transfected with HA-Plk or HA-K82R (Table 2B)
.
Proteasomes were immunoprecipitated with the polyclonal antibody to the
"core" and monoclonal antibody to the subunit of the proteasome
in the presence of phosphatase inhibitors in the lysis buffer. Similar
experiments were repeated five times. Proteolytic activities associated
with immunoprecipitated proteins are shown. Cells transfected with
HA-Plk displayed higher proteolytic activities than vector control
cells, whereas cells transfected with HA-K82R had lower (20%
reduction) activity than the control cells, suggesting that
Plk-mediated phosphorylation of proteasomes regulates their activity
in vivo. These modest alterations in activity probably
relate to the fact that not all of the cells in the culture are
effectively transfected, and not all proteasomes are associated with
recombinant Plk. Interestingly, we found that without the phosphatase
inhibitors, okadaic acid and calyculin A, in the lysis buffer, only a
minimal amount of proteasome activity was detected, implying that
phosphorylation is an important regulatory event for the
ubiquitin-proteasome pathway.
Discussion
We have demonstrated that Plk interacts with 20S proteasomes. This is supported by both mass spectrometry analysis of coimmunoprecipitated proteins from CA46 cells and reciprocal immunoprecipitation/immunoblotting in both CA46 and Plk-transfected HEK 293 cells. Overall, our results demonstrate that Plk functionally interacts with the 20S proteasome and positively regulates proteasome activity. This is a novel role for Plk in cell cycle regulation and a new mechanism for coordinating proteasome activity through the cell cycle. Like Plk, proteasomes are present in the nucleus as well as the cytoplasm in different types of cells (34) . Cytoplasmic proteasomes can be found in the endoplasmic reticulum, Golgi apparatus, and intermediate filaments. The distribution of proteasomes undergoes changes during cell cycle progression. In mitotic cells, proteasomes are found associated with the spindle poles and chromosomes (35 , 36) , which are also major localization sites of Plk (10) . Thus, colocalization of proteasomes and Plk is compatible with our findings that Plk interacts with proteasomes. Results from coimmunoprecipitation studies indicate that only a fraction of total proteasomes are associated with Plk; however, this small portion could be localized to critical sites, such as spindle poles and kinetochores.
Previously, mammalian Plk and yeast Cdc5 have been shown to regulate
ubiquitination of mitotic cyclins through the phosphorylation of the
APC (16
, 17)
. Both Plk and Cdc5 are substrates of the
ubiquitin pathway (18
, 37
, 38)
. In this study, association
of Plk with proteasomes was detected during S phase and prometaphase,
times when Plk protein is not degraded, indicating that it is not a
transient association occurring just before degradation of Plk.
Furthermore, the Plk we detected associated with proteasomes is not
itself ubiquitinated (Fig. 1)
. Therefore, the association we
demonstrate in this study between Plk and proteasome is not
attributable to proteasome-mediated degradation of Plk.
We have shown that coprecipitated Plk and recombinant Plk were able to
phosphorylate subunits of the 20S proteasome and have identified
subunits C8 and C9 to be two of the subunits phosphorylated by Plk
in vitro and in vivo. We observed 16% of
subunit C9 phosphorylated by recombinant Plk in vitro. This
moderate level of phosphorylation correlates well to the 1.5–2-fold
change in the proteasome activity induced by Plk-mediated
phosphorylation. Although the general change in proteolytic activity is
modest, we expect that the small fraction of proteasomes associated
with and regulated by Plk in vivo is localized to important
mitotic machineries: spindle pole and kinetochores. Thus, the effect of
Plk on proteasome activity could be significant. Both Figs. 6
and 7
indicate that Plk is more important to the phosphorylation of subunit
C9 than to that of C8. Therefore, it seems that proteasome subunit C9
is a preferred substrate for Plk. Recombinant Plk phosphorylated
multiple proteins of purified proteasomes, compared with a single
phospho-band detected that coprecipitated with Plk. This may be
attributable to: (a) proteasomes immunoprecipitated from
M-phase lysates that are already partially phosphorylated;
(b) limited amounts of Plk and proteasomes in coprecipitated
samples; and (c) the abundance of purified proteasome
proteins in kinase reactions involving recombinant Plk. In the in
vitro activity test, we observed an increased proteolytic activity
in proteasomes phosphorylated by activated Plk. Thus, it appears that
Plk is both a substrate, as shown previously (37
, 38)
, and
a regulator of proteasomes. It is noteworthy that we only detected 20S
proteasomes associated with Plk and not 26S proteasomes containing the
19S regulatory caps, when cells were lysed in the NP40 lysis buffer.
This finding is explained by the instability of 26S proteasome
particles in the high salt buffer lacking ATP. Our results indicate
that Plk does interact with the intact 26S complex under low salt
conditions. Because the 20S proteasome harbors the core catalytic
activity of proteasomes, the effect of Plk phosphorylation may have
profound physiological consequences. Direct control of proteasome
activity through Plk-mediated phosphorylation of 20S subunits may be
crucial to mitotic progression. Alternatively, phosphorylation of C8
and C9 may help the binding of the outer -ring of the 20S proteasome
to the 19S regulator and assembly of the 26S proteasome. This
speculation is supported by a previous report showing that
phosphorylation of C8 and C9 is even higher in the 26S proteasome than
in the core catalytic complex (22)
. Lastly, it is also
possible that phosphorylation of proteasomes might be required for the
translocation of proteasomes in cells.
Proteolysis of regulatory proteins at distinct times is crucial to normal cell cycle progression and organism development. An obvious example is the destruction of cyclin B1, which is required for exit from mitosis (1 , 39) . Thus, it is extremely important that the proteasome activity be properly controlled. It has been reported that in dividing cells, a dramatic increase of proteasome proteins associated with spindle microtubles is observed in early and late anaphase (35) . The transition from metaphase to anaphase requires a rapid increase in proteasome activity, which cannot be achieved easily by coordinated synthesis of multiple proteasomal proteins. A posttranslational modification seems to be a physiologically desirable way to rapidly achieve a robust proteolytic activity and relocalization of proteasomes. In this context, Plk appears to be a good candidate to carry out the phosphorylation for multiple reasons: (a) Plk protein degradation occurs later than degradation of cyclin B1 during mitosis (6) ; (b) redistribution of proteasomes from the interphase patterns to multiple sites on the mitotic machinery is similar to Plk redistribution; (c) Plk phosphorylates the same subunits of the 20S proteasome in vitro and in vivo that have been reported previously to be phosphorylated in vivo; and (d) cells transfected with wild-type Plk show increased proteasome phosphorylation and proteolytic activity, whereas cells transfected with mutant inactive Plk show decreased phosphorylation of proteasome components and decreased activity. Indeed, competing endogenous Plk activity with dominant-negative HA-K82R prolongs mitosis of 293 cells (data not shown).
Several Plk-interacting proteins have been reported, including mitotic kinesin-like protein (6) , a microtubule-associated protein (28) , tubulins (25) , components of APC (17) , and Pin1 (26 , 27) . These proteins, together with proteasomes, are likely key players in linking Plk to its functions in regulation of spindle formation and completion of mitosis. At present it is difficult to evaluate the relationships among the Plk-containing complexes that have been described. In the present studies, we found that the Plk-20S proteasome complex did not contain tubulins (data not shown). It is thus likely that Plk is a component of several multimolecular complexes allowing for its versatile functions during cell cycle progression. We are currently attempting to identify several other proteins that are consistently coimmunoprecipitated with Plk and which may represent additional types of cell cycle regulatory complexes.
Materials and Methods
Cell Culture and Lysates.
Human B-cell lymphoma cells, CA46, were maintained in RPMI 1640
supplemented with 10% FBS. Prometaphase cells were obtained by
treating cells with nocodazole (100 ng/ml) for 16 h. To
synchronize cells at the G1-S transition, cells were
blocked with aphidicolin (0.75 µM) for 16 h,
released for 8 h, and then blocked with thymidine (2.5
mM) for 16 h. Cells at G1 phase were
obtained by synchronization with mimosine (300 µM) for
16 h. Cells were lysed in NP40 lysis buffer [50 mM
Tris (pH 7.4), 1% NP40, 250 mM NaCl, 10 mM
NaF, 5 mM EDTA, 1 mM DTT, 1 µg/ml leupeptin,
1 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl
fluoride] on ice for 1 h. Lysates were centrifuged at
100,000 x g for 1 h at 4°C. The clarified
supernatant (S100) was used for immunoprecipitation experiments.
Immunoprecipitation.
Plk was immunoprecipitated with Plk polyclonal antibody (Zymed) at 5
µg/ml and protein G Sepharose (Sigma) in the NP40 lysis buffer in the
presence or absence of COOH-terminal Plk peptide
(Ac-CLSSRSASNRLKAS-OH). Proteasome subunit components were
immunoprecipitated with antibodies specific to individual proteasome
subunits (Affiniti).
Transfection of HEK 293 Cells with HA-tagged Plk.
Human embryonic kidney 293 cells were transfected with 2.5 µg of
DNA/10-cm plate according to the Qiagen SuperFect transfection protocol
(25)
. Thirty-six h after transfection, cells were
synchronized with nocodazole to maximize the expression level of
HA-Plk. Cells were lysed in the NP40 lysis buffer, and S100 was
recovered. HA-tagged Plk was immunoprecipitated with a monoclonal
antibody specific to the HA tag (BAbCo).
In Vitro Phosphorylation.
With immunoprecipitated samples, beads were washed twice in the kinase
buffer [20 mM HEPES (pH 7.4), 150 mM KCl, 10
mM MgCl2, 1 mM EGTA, 0.5
mM DTT, 5 mM NaF, and 0.1 mM
orthovanadate], and the kinase reaction was performed in 20 µl of
kinase buffer containing 10 µCi [-32P]ATP (3000
Ci/mmol; DuPont NEN) at 37°C for 1 h. The reaction was
terminated by addition of 5x Laemmli SDS sample buffer and boiled for
5 min. For reimmunoprecipitation samples, the reaction mixture was
boiled in 1% (w/v) SDS for 5 min and diluted in 1 ml of RIPA buffer
[50 mM Tris (pH 7.4), 1% NP40, 1% deoxycholate, 0.1%
SDS, 350 mM NaCl, 5 mM NaF, and 5
mM EGTA]. The supernatant was recovered for the second
immunoprecipitation.
Purification of 20S Proteasomes.
20S proteasomes were purified by multiple chromatographic steps from
asynchronous CA46 cells according to the procedure of Brown and Monaco
(40)
.
Monitoring of Proteasome Activity.
Proteasome activity was tested as described by Brown and Monaco
(40)
. Briefly, proteasome preparations were added to 100
µl of 100 mM Tris/acetate (pH 7.0) with 250
nM fluorogenic substrate Suc-Leu-Leu-Val-Tyr-AMC
(CalBiochem). The reactions were carried out at 37°C for 30 min and
terminated by the addition of 100 µl of ice-cold ethanol.
Fluorescence was measured (excitation, 380 nm; emission, 460 nm) on a
fluorometer (Turner Design).
To monitor proteasome activity during purification, 5 µl of each
fraction were used. To test changes of proteasome activity after
in vitro phosphorylation (Table 2A)
, the reaction mixture
supernatant was recovered, combined with a 25-µl wash from the beads,
and added to Tris/acetate buffer containing substrate.
To test proteasome activity in 293 cells, cells were lysed in NP40
buffer supplemented with phosphatase inhibitors (20 nM
okadaic acid, 2 nM calyculin A) 36 h after
transfection. 20S proteasomes were immunoprecipitated with antibodies
against the "core" and subunits. Immunoprecipitates were washed
extensively in the NP40 buffer and then in Tris/acetate buffer, and
proteolytic activities associated with the immunoprecipitates were
detected as described above with Suc-Leu-Leu-Val-Tyr-AMC as substrate.
Expression of His6-Plk in Insect Cells.
The XhoI/StuI fragment (encoding amino acids
10–600) of the murine Plk cDNA was excised from pBlueScript-Th10
(41)
and ligated to pAcHLT vector (Pharmingen) already
digested with XhoI and StuI. pAcHLT-mPlk was
cotransfected into SF9 insect cells with linearized BaculoGOLD virus
DNA (Pharmingen). The culture medium containing virus-carrying
recombinant Plk was amplified three times. His6-Plk was
maximally expressed 72 h after infection. The recombinant protein
His6-Plk was purified from insect cell lysates with ProBond
nickel-chelating resin (Invitrogen) under native conditions. Solubility
and kinase activity of the recombinant protein were tested. Some insect
cells were treated with 1 µM okadaic acid for 4 h
before harvest. To obtain M-phase lysate-activated
His6-Plk, purified His6-Plk on beads was
incubated with CA46 mitotic lysates with 1 mM ATP at 4°C
or 37°C for 1 h.
Electrophoresis and Immunoblotting.
Proteins were separated on 10% SDS-PAGE and transferred to Immobilon-P
(Millipore). Blots were blocked in 4% nonfat dry milk/TBST
[10mM Tris-base (pH 7.4), 0.9% NaCl, 0.05% Tween
20], incubated in the primary antibody solution for 2 h,
and rinsed, then incubated in the peroxidase-conjugated secondary
antibody solution for 1 h. Blots were rinsed, and specific signals
were detected with the Chemiluminescence Reagent (DuPont NEN).
Two-dimensional NEPHGE. The in vitro kinase reaction mixtures of 20S/His6-Plk was separated on a 6% acrylamide NEPHGE according to Farrar and Ferris (42) . The gel was then applied to the top of the second dimension gel, 12% SDS-PAGE. After the second dimension electrophoresis, the gels were transferred to Immobilon-P for autoradiography, followed by immunoblotting.
32P Metabolic Labeling.
HEK 293 cells were transfected 2 days before labeling. Cells were
washed three times with P-DMEM (Life Technologies, Inc.), and then
cultured for 3 h in P-DMEM supplemented with 10% phosphate-free
fetal bovine serum and 0.5 mCi/ml [32P]orthophosphate
(DuPont NEN). Cells were rinsed with cold P-DMEM three times and lysed
in NP40 lysis buffer supplemented with 20 nM okadaic acid
and 2 nM calyculin A. Clarified lysates were preabsorbed
with protein G-Sepharose for 3 h at 4°C. Proteasomes were
immunoprecipitated with pAb to "core" and mAb to subunit.
Reimmunoprecipitations with C9 and C8 mAb were carried out as above.
Acknowledgments
We thank Drs. Ira O. Daar and Chou-Chi Li for critical reading and valuable suggestions on the manuscript and Dr. Roger Moore of The Beckman Research Institute of The City of Hope (funding from NIH Cancer Center Support Grant CA33572) for providing technical service.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This research was funded by the National Cancer
Institute under Contract NO1CO56000 (to Science Applications
International Corp.). 
2 To whom requests for reprints should be
addressed, at Biological Mechanisms Section, Laboratory of Leukocyte
Biology, National Cancer Institute—Frederick Cancer Research and
Development Center, Frederick, MD 21702. Phone: (301) 846-1429; Fax:
(301) 846-6641; E-mail: Ferris{at}mail.ncifcrf.gov
3 The abbreviations used are: Plk, polo-like
kinase; APC, anaphasepromoting complex; NEPHGE, nonequilibrating
pH gel electrophoresis; P-DMEM, phosphate-free DMEM; mAb, monoclonal
antibody.
Received for publication 9/ 6/00. Revision received 11/28/00. Accepted for publication 11/29/00.
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