©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Polo-like Kinase Is a Cell Cycle-regulated Kinase Activated during Mitosis (*)

(Received for publication, April 5, 1995; and in revised form, June 12, 1995)

Ryoji Hamanaka (1) Mark R. Smith (2) Patrick M. O'Connor (3) Sharon Maloid (2) Kelly Mihalic (1) Jerry L. Spivak (4) Dan L. Longo (1) Douglas K. Ferris (2)(§)

From the  (1)Laboratory of Leukocyte Biology, Biological Response Modifiers Program, Division of Cancer Treatment and the (2)Biological Carcinogenesis and Development Program, Program Resources, Inc./DynCorp, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland 21702-1201, the (3)Laboratory of Molecular Pharmacology, Developmental Therapeutics Program, Division of Cancer Treatment, NCI, National Institutes of Health, Bethesda, Maryland 20892, and the (4)Division of Hematology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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(1), accumulates during S and G(2)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.


INTRODUCTION

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) (^1)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(1), increase during S, remain high through G(2)M, and are rapidly decreased after mitosis. During the G(2) 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.


EXPERIMENTAL PROCEDURES

Cell Culture

Murine NIH3T3 and human Hela cells were cultured in Dulbecco's modified minimum essential medium (Life Technologies, Inc.) supplemented with 10% calf serum (Life Technologies, Inc.). CA46, a human B-cell lymphoma line, was cultured in RPMI 1640 (Life Technologies, Inc.) supplemented with 15% fetal calf serum.

Materials

[S]Methionine and [-P]ATP were obtained from DuPont NEN. [^3H]Thymidine was from Amersham. All other reagents were from Sigma unless otherwise specified.

PLK Antisera

Two polyclonal PLK antisera designated 8845 and 8847 were raised in New Zealand White rabbits immunized with synthetic peptides coupled to KLH. Antiserum 8845 was generated against a peptide TAGKLPRAPADPGKAGVPG corresponding to amino acids 6-24 of human PLK, and 8847 was generated against the homologous murine peptide KAGKLARAPADLGKGGVPG.

Drug Treatments and Cell Cycle Analysis

Cells were synchronized by treatment for 10-15 h with either mimosine (300 µM), aphidicolin (0.75 µM), or nocodazole (100 ng/ml). Double aphidicolin synchronization was performed by drug treatment overnight followed by a 10-h release and retreatment overnight. Cells to be released from drug treatment were washed three times in media without drug and resuspended in fresh media. For cell cycle analysis, cells were harvested by centrifugation, washed in ice-cold phosphate-buffered saline, lysed in 138 mM NaCl, 5 mM KCl, 440 µM KH(2)PO(4), 335 µM Na(2)HPO(4), 1 mM CaCl(2), 500 µM MgCl(2), 400 µM MgSO(4), 24 mM Hepes, 0.2% w/v bovine serum albumin, 0.4% (w/v) Nonidet P-40 (detergent buffer). Cells were then treated with RNase, and cellular DNA was stained with propidium iodide. Cell cycle determination was performed using a Becton Dickinson fluorescence-activated cell analyzer, and data were interpreted using the SFIT model program provided by the manufacturer. Measurements of mitotic index were recorded after harvesting cells and washing once with ice-cold phosphate-buffered saline. Cells were resuspended in 0.5 ml of half-strength phosphate-buffered saline for 10 min at room temperature, then stored overnight at 4 °C in 6 ml of a 2% solution of 3:1 ethanol:glacial acetic acid. Samples were then resuspended in 0.5 ml of 3:1 ethanol:glacial acetic acid for 10 min, dropped onto glass slides, air dried, and stained with giemsa. For each sample, at least 500 cells were randomly counted, and mitotic cells were scored by their lack of nuclear membrane and evidence of chromosome condensation.

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.

Metabolic Labeling

Cells to be metabolically labeled were washed three times in either phosphate-free media or methionine-free media, then labeled for 2-14 h in the appropriate media containing 0.5 mCi/ml of either ortho[P]phosphate or [S]methionine and 5% calf serum that had been dialyzed against phosphate-free or methionine-free media.

Immunoprecipitation and Kinase Reactions

All procedures during lysis, clarification, and immunoprecipitation were performed at 4 °C. Cells were lysed for 30 min in buffer 1, 50 mM Tris (pH 7.5), 1% Brij 96, 10 mM NaF, 10 mM sodium pyrophosphate, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, and lysates were clarified by centrifugation at 100,000 g for 20 min. One ml of supernatant was added to each 1.5-ml Eppendorf tube containing 3 µl of PLK antisera prebound to 120 µl of a 20% solution of protein A-Sepharose. Lysates were immunoprecipitated for 2-3 h on a rotator, then washed 4-5 times with buffer 1 and either treated with SDS sample buffer or, for PLK kinase reactions, washed an additional time in buffer 2 (10 mM Hepes, pH 7.4, 10 µM MnCl(2), 5 mM MgCl(2)). In vitro PLK kinase reactions were performed for 20 min at 37 °C using a 40-µl reaction mixture composed of 38 µl of buffer 2 and 2 µl of 3000 Ci/mmol [-P]ATP either with or without dephosphorylated casein (20 µg/reaction) added as an exogenous substrate. Assays of Cdc2 kinase activity were performed for 20 min at 37 °C, using a reaction mixture consisting of 38 µl of 20 mM Tris-HCl, 10 mM MgCl(2), pH 7.5, containing 5 µM unlabeled ATP, 10 µCi of [-P]ATP (3000 Ci/mmol), and 3 µg of histone H1 (Boehringer Mannheim). Kinase reactions were terminated by the addition of 30 µl of 3 SDS sample buffer, boiled, and loaded onto polyacrylamide gels.

Phosphatase Assays

Immunoprecipitations were washed once in 0.5 M Tris, pH 7.8, pelleted by centrifugation, and all wash buffer was removed using a Hamilton syringe. 100 µl of purified protein phosphatase 2A (PP2A) dilution buffer (Upstate Biotechnology Inc.) was added either with or without 5 units of PP2A and either with or without 20 nM (final) okadaic acid (Sigma) dissolved in 5 µl of Me(2)SO. Samples were incubated for 20-40 min at 30 °C. After completion of phosphatase reactions, the reaction mixture was removed, and the reaction was stopped by the addition of 50 µl of 3 SDS buffer, or the reactions were washed twice in kinase buffer and further processed for casein kinase immune complex kinase reactions by methods described above.

In Vitro Phosphorylation of PLK by Purified Cdc2/Cyclin B

CA46 cells were synchronized in S phase by blocking overnight with aphidicolin, and cell lysates were prepared. PLK immunoprecipitations were performed using 8845 antiserum in the presence or absence of cognate peptide. Precipitates were then subjected to in vitro phosphorylation in 50 µl of Cdc2 buffer (20 mM Hepes, pH 7.5, 1 mM dithiothreitol, 5 mM MgCl(2), 50 µM ATP, 20 µCi [-P]ATP) for 30 min at 30 °C in the presence or absence of 100 ng of purified mitotic starfish oocyte Cdc2/cyclin B (Promega). Samples to be further processed by in vitro phosphorylation in the PLK kinase buffer were washed twice in 1 ml of kinase buffer to remove Cdc2/cyclin, and the PLK casein kinase reaction was carried out as described above.

Gel Electrophoresis and Immunoblot Analysis

SDS-polyacrylamide gel electrophoresis and transfer to Immobilon (Millipore) was carried out by standard methods, except that gels were prepared using a 120:1 acrylamide:bisacrylamide mixture to increase separation of the mitotic PLK doublet. Membranes were blocked 2 h in 5% nonfat dry milk, 2% goat serum, 0.5% Tween 20, 150 mM NaCl, 20 mM Tris base, pH 8.2 (blocking buffer). Probing with PLK antiserum (1:2000) was carried out 2 h or overnight at room temperature in the same buffer containing 0.02% sodium azide. After washing four times in 20 mM Tris, pH 8.2, 150 mM NaCl, 0.05% Tween 20 (TBST), the membranes were probed with 200 ng/ml peroxidase-labeled goat anti-rabbit IgG for 2 h in blocking buffer. After extensive washing in TBST, the membranes were processed for enhanced chemiluminescence using ECL (Amersham) reagents according to the manufacturer's instructions. Autoradiography of gels or blots and ECL exposures were carried out using Kodak XAR film (Rochester, NY).


RESULTS

Characterization of PLK Antisera

To study PLK protein expression and function, we generated two synthetic PLK peptide polyclonal antisera in rabbits designated 8847 and 8845. Antiserum 8845 was generated against a human PLK peptide sequence located near the N terminus of PLK, and 8847 was generated against the homologous murine PLK peptide. This region of PLK was chosen on the basis of its hydrophilicity and because the N-terminal region of PLK is the least highly evolutionarily conserved portion at the amino acid sequence level (34) and therefore, we reasoned, would be more likely to be antigenic. Fig. 1A demonstrates immunoprecipitation of in vitro translated [S]methionine-labeled murine p64 using immune 8845 or 8847 antisera. Specificity of the two antisera toward the in vitro translated PLK was confirmed by comparison with pre-immune serum. Fig. 1B shows immunoprecipitation from [S]methionine-labeled human CA46 cells and competition with cognate peptide. The major specifically competed protein, p64, migrates at the same position as in vitro translated PLK, and its identity was confirmed by Western blot analysis. Several other proteins, indicated by arrows, were also specifically competed by the peptide and could represent either cross-reactive proteins or proteins that are complexed with PLK. Since these other proteins are not reactive with the peptide antiserum by immunoblot analysis (C), we favor the latter possibility. At present, we have not identified any of these other proteins. For reference, C shows a typical immunoblot of total CA46 cell protein using either immune or preimmune 8845 antisera. Immunoblotting revealed a single protein of 64 kDa was recognized by the 8845 antiserum. Essentially identical results were obtained using antisera 8847 to detect endogenous PLK protein in murine cells (not shown). In conclusion, the 8845 and 8847 antisera were able to immunoprecipitate and immunoblot p64 from either human or murine cells.


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.



Cell Cycle Regulation of PLK Expression

We investigated whether PLK protein levels were cell cycle regulated by immunoblotting. CA46 cells synchronized at G(1)S with mimosine, in S phase with aphidicolin, or at G(2)M with nocodazole (Fig. 2A) were lysed and clarified; proteins were separated by SDS-PAGE and analyzed by immunoblotting (Fig. 2B). We found that PLK protein was substantially reduced in G(1)S mimosine-blocked cells (M) compared to unsynchronized cells (U); in contrast, much higher levels were detected in both S phase (A) and prometaphase-blocked cells (N). Two closely migrating PLK-specific bands were apparent in the extract made from the prometaphase-arrested cells (N), and the majority of PLK protein was present in the slower migrating band. These results suggested that PLK protein levels are regulated during the cell cycle, as has previously been observed(33) , and further suggest that a portion of PLK undergoes a mitotic modification that reduces its mobility.


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.



PLK Is Phosphorylated and Activated during Mitosis

Direct assessment of PLK phosphorylation status and kinase activity was performed using immunoprecipitated PLK. Fig. 3A, upper panel, shows immunoblot analysis of PLK immunoprecipitated with preimmune or immune 8845 antisera from unsynchronized, S phase, or mitotic human CA46 cells. The blot shows that a large fraction of PLK isolated from mitotic cells migrates slower than PLK isolated from S phase cells or from unsynchronized cells, in which only a relatively small proportion (10-15%) of mitotic cells are present. The middle and lower panels in A represent the corresponding immune complex kinase assays showing in vitro phosphorylation of PLK itself and casein, respectively. Although approximately equal amounts of PLK protein were present in the three immune precipitates, autophosphorylation of PLK and phosphorylation of exogenous casein were increased more than 5-fold in the mitotic precipitate compared to the S phase precipitate, and intermediate amounts of casein kinase activity were present in precipitates from unsynchronized cells. Virtually no kinase activity was detected in the preimmune precipitate. Immune precipitated, in vitro translated wild type PLK also phosphorylated casein, while an in vitro translated ATP binding site mutant PLK did not (data not shown). That result suggests that the kinase activity detected in the PLK immune precipitates is due to PLK and not a coprecipitating enzyme.


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 10^6) 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(2)SO (lane 3), or PP2A, Me(2)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) Me(2)SO, 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.

PLK Phosphorylation Occurs with Kinetics Similar to Cdc2 Activation

We wished to compare the timing of PLK phosphorylation with activation of p34, a kinase known to be required for promotion of mitosis (5, 11, 14) and which is activated by Cdc25-mediated dephosphorylation at the G(2) to M phase transition(17) . Human CA46 cells were synchronized at the G(1)S transition by a double aphidicolin block and then were released into nocodazole to trap the cells in prometaphase of mitosis. After various periods of release from the aphidicolin block, the cells were harvested and subjected to cell cycle FACS analysis, mitotic index analysis, and immunoblot analysis to examine PLK and p34(Fig. 4A). The slower migrating form of PLK was first detected 7.5 h after aphidicolin release (upper panel) when 90% of the cells were in G(2)M and 40% had entered M phase as determined by mitotic index analysis. As cells continued to accumulate in mitosis, we observed a steadily increasing amount of PLK present in the slower migrating form. The kinetics with which PLK became phosphorylated look similar to the timing of Cdc2 activation detected either by loss of the slower migrating hyperphosphorylated form of p34 (lower panel), or by activation of Cdc2 kinase activity. Panel C shows PLK casein kinase and Cdc2 histone kinase activity during a shorter time course after release from aphidicolin into nocodazole. The largest increases in the kinase activity of both PLK and Cdc2 occurred between 3 and 6 h, near the time when electrophoretic mobility changes of each became apparent. To try to determine whether phosphorylation of PLK was more closely associated with G(2) or M phase cells, the same experiment was repeated except that after release from aphidicolin and just before nocodazole addition, the cells were treated with nitrogen mustard (0.7 µM, 30 min), a DNA damaging agent that causes a prolonged delay at the G(2) check point (2, 38, 39) (B). Although by 17.5 h after the release 90% of the cells were in G(2)M, less than 15% were in mitosis, and no mobility shifts of either PLK or Cdc2 were detected. In contrast to the results shown in A and C, when the synchronized cells were treated with nitrogen mustard prior to release into nocodazole, there was a gradual accumulation of kinase activity during the time course that paralleled the increase in PLK protein but no abrupt increase (D). We conclude that phosphorylation and activation of PLK occurs as cells enter mitosis at about the same time that Cdc2 is activated.


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.



In Vitro Phosphorylation of PLK by Cdc2

Since PLK is activated at about the same time as Cdc2, we tested whether purified activated Cdc2/cyclin B could phosphorylate and activate PLK immunoprecipitated from S phase synchronized CA46 cells (Fig. 5). Panel B shows phosphorylation of PLK by Cdc2/cyclin B in vitro (lane 2) and demonstrates that under the Cdc2 reaction conditions, PLK did not efficiently autophosphorylate (lane 1). Panel A shows immunoblot analysis of the immunoprecipitated PLK demonstrating, that although Cdc2 was able to phosphorylate PLK in vitro, it did not cause any mobility shift comparable to that seen in vivo as cells enter mitosis. The affect of Cdc2/cyclin B phosphorylation on PLK casein kinase activity was also tested. After removing the Cdc2/cyclin B and reaction mixture from duplicate samples, standard PLK immune complex casein kinase reactions were performed (C). In five separate experiments, we did not detect any PLK mobility shifts caused by in vitro phosphorylation by Cdc2/cyclin B; however, we did consistently see small increases in PLK casein kinase activity (less than 2-fold). Phosphorylation of PLK by Cdc2/cyclin B followed by autophosphorylation of PLK also did not cause a shift in PLK mobility (not 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.




DISCUSSION

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(1) and higher levels in S and G(2). 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(2) 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(2) 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(2)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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 301-846-1427; Fax: 301-846-5651.

(^1)
The abbreviations used are: CdK, cyclin-dependent protein kinases; PLK, polo-like kinase; PP2A, protein phosphatase 2A; PAGE, polyacrylamide gel electrophoresis.


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