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Polo-like Kinase Is a Cell Cycle-regulated Kinase Activated during Mitosis

Polo-like Kinase Is a Cell Cycle-regulated Kinase Activated during Mitosis THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 270, No. 36, Issue of September 8, pp. 21086–21091, 1995 Printed in U.S.A. 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‡, Mark R. Smith§, Patrick M. O’Connor , Sharon Maloid§, Kelly Mihalic‡, Jerry L. Spivak , Dan L. Longo‡, and Douglas K. Ferris§** From the ‡Laboratory of Leukocyte Biology, Biological Response Modifiers Program, Division of Cancer Treatment and the §Biological Carcinogenesis and Development Program, Program Resources, Inc./DynCorp, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland 21702-1201, the Laboratory of Molecular Pharmacology, Developmental Therapeutics Program, Division of Cancer Treatment, NCI, National Institutes of Health, Bethesda, Maryland 20892, and the Division of Hematology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 22). In addition to such architectural proteins, the tumor sup- Previously, we demonstrated that expression of polo- like kinase (PLK) is required for cellular DNA synthesis pressor gene products p53 (23) and RB (24–28) are both subject and that overexpression of PLK is sufficient to induce to regulatory phosphorylation by CdK family members (7). DNA synthesis. We now report that the endogenous Thus, it is clear that reversible phosphorylation reactions play levels of PLK, its phosphorylation status, and protein a major role in regulating cell cycle progression, and the list of kinase activity are tightly regulated during cell cycle enzymes known to be involved is rapidly expanding. progression. PLK protein is low in G , accumulates dur- 1 A number of cell cycle-regulated kinases unrelated to the ing S and G M, and is rapidly reduced after mitosis. CdKs have also been identified in lower eukaryotes, including During mitosis, PLK is phosphorylated on serine, and its the homologous yeast CDC5 (29) and Drosophila polo (30). Polo serine threonine kinase function is activated at a time was first identified as an embryonic lethal Drosophila mutation cdc2 close to that of p34 . The phosphorylated form of PLK causing formation of monopolar and multipolar mitotic spin- migrates with reduced mobility on SDS-polyacrylamide dles and abnormal segregation of chromosomes (31). Saccharo- gel electrophoresis, and dephosphorylation by purified myces cerevisiae CDC5 mutants likewise are impaired in mi- protein phosphatase 2A converts it to the more rapidly totic spindle formation, but the CDC5 kinase also appears to migrating form and reduces the total amount of PLK cdc2 have a function during S phase (29). Recently, we and others kinase activity. Purified p34 -cyclin B complex can (32–36) have cloned a putative mammalian polo homologue, phosphorylate PLK protein in vitro but causes little in- polo-like kinase (PLK) (32–36). We previously showed that crease in PLK kinase activity. 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- The processes of cell growth and division are stringently length PLK antisense RNA reduced serum-stimulated tritiated regulated to ensure fidelity of DNA replication and correct thymidine incorporation. On the basis of those results, we segregation of genetic information (1, 2). So essential are these suggested that PLK had an important S phase function (34). In processes to cellular homeostasis that many of the gene prod- the present study, we investigate the cell cycle regulation of ucts regulating passage through the cell cycle are highly con- PLK by measuring endogenous PLK protein levels, phospho- served both in amino acid sequence and function between or- rylation state, and kinase activity. We show that PLK protein ganisms as evolutionarily divergent as yeast and man (3, 4). levels are low in G , increase during S, remain high through Within the last few years, a variety of related enzymes known G M, and are rapidly decreased after mitosis. During the G to as cyclin-dependent protein kinases (CdKs) have been identi- 2 2 M phase transition, PLK protein is phosphorylated on serine, fied that are activated and inactivated at specific times during and its kinase function is stimulated. Dephosphorylation of cell cycle progression (5–8). The CdKs must form complexes PLK reduces its activity by 5–10-fold and converts the slower with a cyclin family member to be activated (9, 10) and are also migrating form to the faster migrating form. We further dem- subject to both positive and negative phosphorylation by other onstrate that Cdc2/cyclin B can phosphorylate PLK but has protein kinases and dephosphorylation by protein phospha- little effect on PLK kinase activity, suggesting that mitotic tases (11–17). Together, the family of CdKs constitutes critical activation of PLK will not be explained solely by Cdc2. components of the engine that propels the cell through the cycle. Many proteins that are phosphorylated in a cell cycle- EXPERIMENTAL PROCEDURES specific fashion have been identified as putative targets of CdK Cell Culture—Murine NIH3T3 and human Hela cells were cultured regulation including nuclear lamins (18, 19), nucleolin (20, 21), in Dulbecco’s modified minimum essential medium (Life Technologies, and other matrix proteins, as well as cytoskeletal proteins (7, Inc.) supplemented with 10% calf serum (Life Technologies, Inc.). CA46, a human B-cell lymphoma line, was cultured in RPMI 1640 (Life Tech- nologies, Inc.) supplemented with 15% fetal calf serum. * The costs of publication of this article were defrayed in part by the 35 32 Materials—[ S]Methionine and [g- P]ATP were obtained from payment of page charges. This article must therefore be hereby marked DuPont NEN. [ H]Thymidine was from Amersham. All other reagents “advertisement” in accordance with 18 U.S.C. Section 1734 solely to were from Sigma unless otherwise specified. indicate this fact. PLK Antisera—Two polyclonal PLK antisera designated 8845 and ** To whom correspondence should be addressed. Tel.: 301-846-1427; 8847 were raised in New Zealand White rabbits immunized with syn- Fax: 301-846-5651. thetic peptides coupled to KLH. Antiserum 8845 was generated against The abbreviations used are: CdK, cyclin-dependent protein kinases; PLK, polo-like kinase; PP2A, protein phosphatase 2A; PAGE, polyacryl- a peptide TAGKLPRAPADPGKAGVPG corresponding to amino acids amide gel electrophoresis. 6–24 of human PLK, and 8847 was generated against the homologous This is an Open Access article under the CC BY license. Activation of PLK during Mitosis 21087 murine peptide KAGKLARAPADLGKGGVPG. cyclin B (Promega). Samples to be further processed by in vitro phos- Drug Treatments and Cell Cycle Analysis—Cells were synchronized phorylation in the PLK kinase buffer were washed twice in 1 ml of by treatment for 10–15 h with either mimosine (300 mM), aphidicolin kinase buffer to remove Cdc2/cyclin, and the PLK casein kinase reac- (0.75 mM), or nocodazole (100 ng/ml). Double aphidicolin synchroniza- tion was carried out as described above. tion was performed by drug treatment overnight followed by a 10-h Gel Electrophoresis and Immunoblot Analysis—SDS-polyacrylamide release and retreatment overnight. Cells to be released from drug gel electrophoresis and transfer to Immobilon (Millipore) was carried treatment were washed three times in media without drug and resus- out by standard methods, except that gels were prepared using a 120:1 pended in fresh media. For cell cycle analysis, cells were harvested by acrylamide:bisacrylamide mixture to increase separation of the mitotic centrifugation, washed in ice-cold phosphate-buffered saline, lysed in PLK doublet. Membranes were blocked2hin5% nonfat dry milk, 2% 138 mM NaCl, 5 mM KCl, 440 mM KH PO , 335 mM Na HPO ,1mM goat serum, 0.5% Tween 20, 150 mM NaCl, 20 mM Tris base, pH 8.2 2 4 2 4 CaCl , 500 mM MgCl , 400 mM MgSO ,24mM Hepes, 0.2% w/v bovine (blocking buffer). Probing with PLK antiserum (1:2000) was carried out 2 2 4 serum albumin, 0.4% (w/v) Nonidet P-40 (detergent buffer). Cells were 2 h or overnight at room temperature in the same buffer containing then treated with RNase, and cellular DNA was stained with propidium 0.02% sodium azide. After washing four times in 20 mM Tris, pH 8.2, iodide. Cell cycle determination was performed using a Becton Dickin- 150 mM NaCl, 0.05% Tween 20 (TBST), the membranes were probed son fluorescence-activated cell analyzer, and data were interpreted with 200 ng/ml peroxidase-labeled goat anti-rabbit IgG for2hin block- using the SFIT model program provided by the manufacturer. Meas- ing buffer. After extensive washing in TBST, the membranes were urements of mitotic index were recorded after harvesting cells and processed for enhanced chemiluminescence using ECL (Amersham) washing once with ice-cold phosphate-buffered saline. Cells were resus- reagents according to the manufacturer’s instructions. Autoradiogra- pended in 0.5 ml of half-strength phosphate-buffered saline for 10 min phy of gels or blots and ECL exposures were carried out using Kodak at room temperature, then stored overnight at 4 °C in 6 ml of a 2% XAR film (Rochester, NY). solution of 3:1 ethanol:glacial acetic acid. Samples were then resus- RESULTS pended 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, Characterization of PLK Antisera—To study PLK protein at least 500 cells were randomly counted, and mitotic cells were scored expression and function, we generated two synthetic PLK pep- by their lack of nuclear membrane and evidence of chromosome tide polyclonal antisera in rabbits designated 8847 and 8845. condensation. Antiserum 8845 was generated against a human PLK peptide For mitotic shakeoff experiments, Hela or NIH3T3 cells were serum sequence located near the N terminus of PLK, and 8847 was 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 generated against the homologous murine PLK peptide. This flask vigorously several times and harvested by centrifugation. The region of PLK was chosen on the basis of its hydrophilicity and remaining adherent cells were collected by scraping and were used to because the N-terminal region of PLK is the least highly evo- prepare interphase extracts. lutionarily conserved portion at the amino acid sequence level Metabolic Labeling—Cells to be metabolically labeled were washed (34) and therefore, we reasoned, would be more likely to be three times in either phosphate-free media or methionine-free media, antigenic. Fig. 1A demonstrates immunoprecipitation of in then labeled for 2–14 h in the appropriate media containing 0.5 mCi/ml 32 35 35 plk of either ortho[ P]phosphate or [ S]methionine and 5% calf serum vitro translated [ S]methionine-labeled murine p64 using that had been dialyzed against phosphate-free or methionine-free immune 8845 or 8847 antisera. Specificity of the two antisera media. toward the in vitro translated PLK was confirmed by compar- Immunoprecipitation and Kinase Reactions—All procedures during ison with pre-immune serum. Fig. 1B shows immunoprecipita- lysis, clarification, and immunoprecipitation were performed at 4 °C. tion from [ S]methionine-labeled human CA46 cells and com- Cells were lysed for 30 min in buffer 1, 50 mM Tris (pH 7.5), 1% Brij 96, petition with cognate peptide. The major specifically competed 10 mM NaF, 10 mM sodium pyrophosphate, 1 mg/ml aprotinin, 1 mg/ml plk leupeptin, 1 mM phenylmethylsulfonyl fluoride, and lysates were clar- protein, p64 , migrates at the same position as in vitro trans- ified by centrifugation at 100,000 3 g for 20 min. One ml of supernatant lated PLK, and its identity was confirmed by Western blot was added to each 1.5-ml Eppendorf tube containing 3 mlofPLK analysis. Several other proteins, indicated by arrows, were also antisera prebound to 120 ml of a 20% solution of protein A-Sepharose. specifically competed by the peptide and could represent either Lysates were immunoprecipitated for 2–3 h on a rotator, then washed cross-reactive proteins or proteins that are complexed with 4–5 times with buffer 1 and either treated with SDS sample buffer or, PLK. Since these other proteins are not reactive with the for PLK kinase reactions, washed an additional time in buffer 2 (10 mM Hepes, pH 7.4, 10 mM MnCl ,5mM MgCl ). In vitro PLK kinase reac- peptide antiserum by immunoblot analysis (C), we favor the 2 2 tions were performed for 20 min at 37 °C using a 40-ml reaction mixture latter possibility. At present, we have not identified any of composed of 38 ml of buffer 2 and 2 ml of 3000 Ci/mmol [g- P]ATP either these other proteins. For reference, C shows a typical immu- with or without dephosphorylated casein (20 mg/reaction) added as an noblot of total CA46 cell protein using either immune or pre- exogenous substrate. Assays of Cdc2 kinase activity were performed for immune 8845 antisera. Immunoblotting revealed a single pro- 20 min at 37 °C, using a reaction mixture consisting of 38 mlof20mM tein of 64 kDa was recognized by the 8845 antiserum. Tris-HCl, 10 mM MgCl , pH 7.5, containing 5 mM unlabeled ATP, 10 mCi of [g- P]ATP (3000 Ci/mmol), and 3 mg of histone H1 (Boehringer Essentially identical results were obtained using antisera 8847 Mannheim). Kinase reactions were terminated by the addition of 30 ml to detect endogenous PLK protein in murine cells (not shown). of 3 3 SDS sample buffer, boiled, and loaded onto polyacrylamide gels. In conclusion, the 8845 and 8847 antisera were able to immu- Phosphatase Assays—Immunoprecipitations were washed once in 0.5 plk noprecipitate and immunoblot p64 from either human or M Tris, pH 7.8, pelleted by centrifugation, and all wash buffer was murine cells. removed using a Hamilton syringe. 100 ml of purified protein phospha- Cell Cycle Regulation of PLK Expression—We investigated tase 2A (PP2A) dilution buffer (Upstate Biotechnology Inc.) was added whether PLK protein levels were cell cycle regulated by immu- either with or without 5 units of PP2A and either with or without 20 nM (final) okadaic acid (Sigma) dissolved in 5 mlofMe SO. Samples were 2 noblotting. CA46 cells synchronized at G S with mimosine, in S incubated for 20–40 min at 30 °C. After completion of phosphatase phase with aphidicolin, or at G M with nocodazole (Fig. 2A) reactions, the reaction mixture was removed, and the reaction was were lysed and clarified; proteins were separated by SDS- stopped by the addition of 50 mlof3 3 SDS buffer, or the reactions were PAGE and analyzed by immunoblotting (Fig. 2B). We found washed twice in kinase buffer and further processed for casein kinase that PLK protein was substantially reduced in G S mimosine- immune complex kinase reactions by methods described above. In Vitro Phosphorylation of PLK by Purified Cdc2/Cyclin B—CA46 blocked cells (M) compared to unsynchronized cells (U); in cells were synchronized in S phase by blocking overnight with aphidi- contrast, much higher levels were detected in both S phase (A) colin, and cell lysates were prepared. PLK immunoprecipitations were and prometaphase-blocked cells (N). Two closely migrating performed using 8845 antiserum in the presence or absence of cognate PLK-specific bands were apparent in the extract made from the peptide. Precipitates were then subjected to in vitro phosphorylation in prometaphase-arrested cells (N), and the majority of PLK pro- 50 ml of Cdc2 buffer (20 mM Hepes, pH 7.5, 1 mM dithiothreitol, 5 mM tein was present in the slower migrating band. These results MgCl ,50 mM ATP, 20 mCi [g- P]ATP) for 30 min at 30 °C in the presence or absence of 100 ng of purified mitotic starfish oocyte Cdc2/ suggested that PLK protein levels are regulated during the cell 21088 Activation of PLK during Mitosis FIG.1. Characterization of PLK an- tisera. A, murine PLK was [ S]methi- onine labeled during translation in vitro, and aliquots of the translated protein were immunoprecipitated with preim- mune or immune PLK peptide antisera and analyzed by 7.5% acrylamide SDS- PAGE and autoradiography. Lane 1, pre- immune 8847; lane 2, preimmune 8845; lane 3, sample of total; lane 4, immune 8847; lane 5, immune 8845. B, asynchro- nously growing human CA46 lymphoma cells were metabolically labeled with [ S]methionine, lysed, and immunopre- cipitated with 8845 antisera in the pres- ence (1) or absence (2) of cognate peptide and analyzed by 7.5% acrylamide SDS- PAGE and autoradiography. C, samples containing 75 mg each of total CA46 cell proteins were separated by 7.5% SDS- PAGE, transferred to Immobilon, and probed with immune (lane 1) or preim- mune PLK antisera 8845 (lane 2). Pri- mary antibody was detected using a per- oxidase-labeled goat anti-rabbit and ECL. 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 phospho- rylated 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 precipi- tates is due to PLK and not a coprecipitating enzyme. To substantiate the kinase activity results obtained with drug-synchronized cells, a mitotic shake off experiment was performed with murine NIH3T3 cells (Fig. 3B). Immunopre- cipitations from equivalent numbers of mitotic or the inter- phase adherent cells were performed, and immune complex kinase assays were performed using casein as exogenous sub- strate. No kinase activity was detected in the preimmune or FIG.2. Cell cycle changes in PLK protein. CA 46 cells were immune competition assays performed from mitotic extracts, treated 16 h with nothing (U), mimosine (M), aphidicolin (A), or nocoda- zole (N). A, cells were harvested and prepared for cell cycle fax analysis. and only very low amounts of activity were detected in the B, cell lysates were analyzed by SDS-PAGE and immunoblotting with immune interphase assay. Consistent with the results shown 8845 antiserum. in A, PLK isolated from mitotic cells showed increased kinase activity. To directly assess PLK phosphorylation state during mitosis, cycle, as has previously been observed (33), and further suggest we compared in vivo phosphorylated PLK from NIH3T3 cells, that a portion of PLK undergoes a mitotic modification that isolated by mitotic shake off, to [ S]methionine-labeled PLK reduces its mobility. immunoprecipitated from unsynchronized cells in the presence PLK Is Phosphorylated and Activated during Mitosis—Di- or absence of cognate competitor peptide (C). We found that rect assessment of PLK phosphorylation status and kinase PLK was indeed labeled with P during mitosis (C) but was activity was performed using immunoprecipitated PLK. Fig. undetected in extracts from adherent cells (not shown). Fur- 3A, upper panel, shows immunoblot analysis of PLK immuno- thermore, the phosphorylated form appeared to migrate more precipitated with preimmune or immune 8845 antisera from slowly in SDS gels compared to the [ S]methionine-labeled unsynchronized, S phase, or mitotic human CA46 cells. The blot shows that a large fraction of PLK isolated from mitotic PLK protein from unsynchronized cells. These results con- firmed our speculation that PLK is phosphorylated in a cell cells migrates slower than PLK isolated from S phase cells or from unsynchronized cells, in which only a relatively small cycle-specific manner and could arise either through an auto- phosphorylation reaction or through the action of a separate proportion (10–15%) of mitotic cells are present. The middle and lower panels in A represent the corresponding immune kinase. complex kinase assays showing in vitro phosphorylation of PLK To confirm that the mitotic specific phosphorylation of PLK itself and casein, respectively. Although approximately equal was responsible for its reduced mobility, immunoprecipitated Activation of PLK during Mitosis 21089 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 dem- onstrated serine/threonine phosphorylation of casein and also autophosphorylation of serine and threonine residues in im- mune 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 phos- phorylated on serine, has increased phosphotransferase activ- ity, 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 arti- ficial conditions. It might also be that a very active phospha- tase specifically removes phosphate from threonine in vivo during mitosis as a means of regulating PLK activity. At pres- ent, we cannot distinguish between these possibilities. PLK Phosphorylation Occurs with Kinetics Similar to Cdc2 Activation—We wished to compare the timing of PLK phospho- cdc2 rylation with activation of p34 , a kinase known to be re- FIG.3. Mitotic PLK is phosphorylated on serine and has re- quired for promotion of mitosis (5, 11, 14) and which is acti- duced mobility and increased kinase activity. A, CA46 cells were vated by Cdc25-mediated dephosphorylation at the G to M either unsynchronized (U) or synchronized with aphidicolin in S phase phase transition (17). Human CA46 cells were synchronized at (S) or with nocodazole in prometaphase of mitosis (M). Lysates were prepared and immunoprecipitated (I.P.) with preimmune (P)orim- the G S transition by a double aphidicolin block and then were mune (I) 8845 PLK antiserum. Immunoprecipitates were subjected to released into nocodazole to trap the cells in prometaphase of immune complex kinase assays with dephosphorylated casein added as mitosis. After various periods of release from the aphidicolin an exogenous substrate and analyzed by 8845 immunoblot and autora- block, the cells were harvested and subjected to cell cycle FACS diography. Upper panel, immunoblot; middle panel, autoradiography of immune complex autophosphorylation of PLK; bottom panel, autora- analysis, mitotic index analysis, and immunoblot analysis to cdc2 diography of immune complex phosphorylation of casein. B, NIH3T3 examine PLK and p34 (Fig. 4A). The slower migrating form cells were serum starved for 36 h and then stimulated with serum for of PLK was first detected 7.5 h after aphidicolin release (upper 18–20 h. Mitotic cells (M) were collected by mechanical shake off, and panel) when 90% of the cells were in G M and 40% had entered adherent interphase cells (I) were harvested by scraping. Lysates pre- 2 pared from equal numbers (1 3 10 ) of interphase or mitotic cells were M phase as determined by mitotic index analysis. As cells immunoprecipitated (I.P.) with preimmune (P) or immune 8847 PLK continued to accumulate in mitosis, we observed a steadily antiserum with (IC) or without (I) competing cognate peptide, and increasing amount of PLK present in the slower migrating immune complex kinase assays were performed using casein as sub- form. The kinetics with which PLK became phosphorylated strate. Autoradiography is shown. C, asynchronously growing NIH3T3 35 35 cells were labeled metabolically with [ S]methionine ( S) or with look similar to the timing of Cdc2 activation detected either by orthophosphate ( P). Methionine-labeled cells were harvested with a loss of the slower migrating hyperphosphorylated form of scraper, while orthophosphate-labeled mitotic cells were harvested by cdc2 p34 (lower panel), or by activation of Cdc2 kinase activity. mechanical shake off. Lysates were immunoprecipitated using 8847 Panel C shows PLK casein kinase and Cdc2 histone kinase antiserum with (1) or without (2) cognate peptide and analyzed by SDS-PAGE and autoradiography. D and E, nocodazole-synchronized activity during a shorter time course after release from aphidi- CA46 extracts were prepared and immunoprecipitated with PLK anti- colin into nocodazole. The largest increases in the kinase ac- serum. Immunoprecipitates were treated with nothing (lane 1), PP2A tivity of both PLK and Cdc2 occurred between 3 and 6 h, near (lane 2), PP2A and Me SO (lane 3), or PP2A, Me SO, and okadaic acid 2 2 the time when electrophoretic mobility changes of each became (lane 4) and subjected to in vitro casein kinase assays. Reactions were analyzed by SDS-PAGE and immunoblotting (D) and autoradiography apparent. To try to determine whether phosphorylation of PLK (E). F, phosphoamino acid analysis of in vitro phosphorylated PLK and was more closely associated with G or M phase cells, the same casein isolated from A and in vivo phosphorylated PLK from C. O, experiment was repeated except that after release from aphidi- origin; S, serine; T, threonine; Y, tyrosine. colin and just before nocodazole addition, the cells were treated with nitrogen mustard (0.7 mM, 30 min), a DNA damaging mitotic PLK was treated with purified protein phosphatase 2A agent that causes a prolonged delay at the G check point (2, in the presence or absence of okadaic acid and the effects of the 2 38, 39) (B). Although by 17.5 h after the release 90% of the cells dephosphorylation on mobility and casein kinase activity de- were in G M, less than 15% were in mitosis, and no mobility termined (Fig. 3, D and E). Immunoprecipitated PLK not shifts of either PLK or Cdc2 were detected. In contrast to the treated with phosphatase (D, lane 1) migrated as a doublet and results shown in A and C, when the synchronized cells were had substantial kinase activity (E, lane 1), while PLK treated with PP2A, with (lane 3) or without (lane 2)Me SO, the carrier treated with nitrogen mustard prior to release into nocodazole, there was a gradual accumulation of kinase activity during the for okadaic acid, was mostly converted to the faster migrating form and had 5–10-fold less kinase activity (E). The effects of time course that paralleled the increase in PLK protein but no abrupt increase (D). We conclude that phosphorylation and the PP2A treatment on PLK mobility and kinase activity could be completely blocked by the addition of okadaic acid (lane 4) activation of PLK occurs as cells enter mitosis at about the demonstrating that the effects were due to the phosphatase same time that Cdc2 is activated. activity and not to some contaminating activity present in the In Vitro Phosphorylation of PLK by Cdc2—Since PLK is 21090 Activation of PLK during Mitosis FIG.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 mus- tard 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 show- ing FACS and mitotic index results. C, immune complex kinase assay. Cells were double blocked with aphidicolin and re- leased into nocodazole. At the indicated times, lysates were prepared and immu- noprecipitated in the presence (1) and absence (2) 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 mus- tard, and released into nocodazole. At the indicated times, lysates were prepared and immunoprecipitated with 8845 anti- serum. Autoradiography is shown. activated at about the same time as Cdc2, we tested whether purified activated Cdc2/cyclin B could phosphorylate and acti- vate 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 autophospho- rylate (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, stand- ard PLK immune complex casein kinase reactions were per- formed (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). Phosphoryla- tion of PLK by Cdc2/cyclin B followed by autophosphorylation of PLK also did not cause a shift in PLK mobility (not shown). DISCUSSION In our initial report on the cloning of human and murine FIG.5. In vitro phosphorylation of PLK by Cdc2/cyclin B. PLK PLK, we showed evidence that PLK has a function in S phase was immunoprecipitated from aphidicolin-synchronized CA46 cell ex- (34) similar to the finding for the yeast PLK homologue cdc5, tracts in the absence or presence of cognate peptide and subjected to in which apparently has roles during S phase and during mitosis vitro phosphorylation in a Cdc2 reaction mixture in the presence or (29). More recently, it has been shown that PLK steady state absence of purified Cdc2-cyclin B complex. Samples were then sepa- rated by SDS-PAGE, transferred to Immobilon, and analyzed by auto- message (35, 36) and protein levels (33) are cell cycle regulated, radiography (B) and immunoblotting (A) or were washed with PLK with low levels of each detected in G and higher levels in S and reaction mixture to remove the Cdc2-cyclin B complex and reaction G . Our findings support and extend these earlier observations mixture; samples were then subjected to a PLK casein kinase assay (C) by showing that 1) PLK kinase activity is cell cycle regulated, and analyzed by SDS-PAGE and autoradiography. 2) PLK activation involves a phosphorylation reaction, 3) mi- totic PLK is phosphorylated on serine, 4) activation of PLK activated at the anaphase-telophase transition, while our data occurs with kinetics similar to Cdc2, and 5) PLK kinase acti- suggest an earlier activation of PLK, probably during the G to vation, like Cdc2 activation, is integrated into the G check- prophase transition. Our data must also be considered in light point response to DNA damage. of the recent publication by Golsteyn and co-workers (33), Fenton and Glover (40) have shown that in synchronously showing that in interphase cells, PLK is diffusely distributed dividing Drosophila cells, polo kinase is tightly regulated and throughout both the cytoplasm and nucleus, whereas just after Activation of PLK during Mitosis 21091 cell division, PLK localized to the midbody of the post-mitotic fore may represent members of a PLK complex (and potential bridge, which is suggestive of a function during late mitosis/ substrates). Identification of these proteins is likely to help cytokinesis. The earlier activation of PLK at the G M phase reveal the role of PLK in regulating cell cycle progression. transition suggested by our data, however, is not incompatible REFERENCES with a role later in mitosis. Together, our results suggest that 1. Hartwell, L. H., Mortimer, R. K., Culotti, J., and Culotli, M. (1973) Genetics 74, while PLK is similar to Drosophila polo in structure and may 267–286 overlap functionally, there may also be some differences. 2. Hartwell, L., and Weinert, T. (1989) Science 246, 629–634 We found that PLK was phosphorylated on one or more 3. Lee, M. G., and Nurse, P. (1987) Nature 327, 31–35 4. Norbury, C., and Nurse, P. (1992) Annu. Rev. Biochem. 61, 441–470 serine residues during mitosis and that its kinase activity was 5. Heuvel, v.d. H., and Harlow, E. (1993) Science 262, 2050–2054 increased severalfold compared to equal amounts of interphase 6. Meyerson, M., Enders, G. H., Wu, C. L., Su, L. K., Gorka, C., Nelson, C., PLK. In addition, we found that a slower migrating form of Harlow, E., and Tsai, L. H. (1992) EMBO J. 11, 2909–2917 7. Nigg, E. A. (1993) Curr. Opin. Cell Biol. 5, 187–193 PLK was detected in mitotic extracts. Since dephosphorylation 8. Pines, J., and Hunter, T. (1990) New Biol. 2, 389–401 of PLK by PP2A converts the slower migrating form to the 9. Evans, T., Rosenthal, E. T., Youngblom, J., Distel, D., and Hunt, T. (1983) Cell 33, 389–396 faster migrating species and reduces the kinase activity asso- 10. Murray, A. W. (1989) Nature 339, 280–286 ciated with the precipitate, it is tempting to speculate that the 11. Draetta, G., and Beach, D. (1988) Cell 54, 17–26 reduced mobility form of PLK represents the activated enzyme. 12. Draetta, G., Piwnica-Worms, H., Morrison, D., Druker, B., Roberts, T., and Beach, D. (1988) Nature 336, 738–744 However, our present data are not sufficient to prove it, since it 13. Gautier, J., Matsukawa, T., Nurse, P., and Maller, J. (1989) Nature 339, is possible that PLK, like Cdc2, contains both positive and 626–629 14. Gould, K. L., Moreno, S., Owen, D. J., Sazer, S., and Nurse, P. (1991) EMBO negative phosphorylation sites, and the reduced mobility, fully J. 10, 3297–3309 phosphorylated form might well be completely inactive, while 15. Krek, W., and Nigg, E. A. (1991) EMBO J. 10, 305–316 an intermediately phosphorylated faster migrating form could 16. Krek, W., and Nigg, E. A. (1992) New Biol. 4, 323–329 17. Sebastian, B., Kakizuka, A., and Hunter, T. (1993) Proc. Natl. Acad. Sci. be the most active. The identity of the kinase or kinases re- U. S. A. 90, 3521–3524 sponsible for mitotic phosphorylation of PLK is unknown, but 18. Peter, M., Nakagawa, J., Doree, M., Labbe, J. C., and Nigg, E. A. (1990) Cell 61, 591–602 the reduced mobility associated with mitotic phosphorylated 19. Ward, G. E., and Kirschner, M. W. (1990) Cell 61, 561–577 PLK is not achieved by autophosphorylation in vitro by either 20. Belenguer, P., Caizergues-Ferrer, M., Labbe, J., Doree, M., and Amalric, F. PLK precipitated from interphase cells (Fig. 2, upper panel)or (1990) Mol. Cell. Biol. 10, 3607–3618 21. Peter, M., Nakagawa, J., Doree, M., Labbe, J. C., and Nigg, E. A. (1990) Cell by autophosphorylation of in vitro translated PLK, nor does 60, 791–801 autophosphorylation seem to increase casein kinase activity 22. Lewin, B. (1990) Cell 61, 743–752 (data not shown). Thus, activation of PLK probably involves 23. Bischoff, J. R., Friedman, P. N., Marchak, D. R., Prives, C., and Beach, D. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 4766–4770 phosphorylation by some other kinase(s), one obvious possibil- 24. Dowdy, S. F., Hinds, P. W., Louie, K., Reed, S. I., Arnold, A., and Weinberg, cdc2 ity being p34 ; however, PLK does not have any Cdc2 con- R. A. (1993) Cell 73, 499–511 25. Ewen, M. E., Sluss, H. K., Sherr, C. J., Matsushime, H., Kato, J., and sensus-like sequences, (S/T)PX(K/R) (41), containing serine, Livingston, D. M. (1993) Cell 73, 487–497 nor did in vitro phosphorylation of PLK by purified Cdc2/cyclin 26. Hu, Q., Lees, J. A., Buchkovich, K. J., and Harlow, E. (1992) Mol. Cell. Biol. 12, B cause a shift in PLK mobility or substantial increases in its 971–980 27. Kato, J-Y., Matsushime, H., Hiebert, S. W., Ewen, M. E., and Sherr, C. J. activity. On the basis of these results, we conclude that while (1993) Genes & Dev. 7, 331–342 Cdc2 may be involved in regulating PLK activity, it cannot 28. Templeton, D. J. (1992) Mol. Cell. Biol. 12, 435–443 29. Kitada, K., Johnson, A. L., Johnston, L. H., and Sugino, A. (1993) Mol. Cell. alone account for the mitotic modification and activation of Biol. 13, 4445–4457 PLK. It should be noted, however, that interphase (Fig. 3), and 30. Llamazares, S., Moreira, A., Tavares, A., Girdham, C., Spruce, B. A., Gonzalez, even in vitro translated PLK (data not shown), does have some C., Karess, R. E., Glover, D. M., and Sunkel, C. E. (1991) Genes & Dev. 5, 2153–2165 basal level of immune complex kinase activity, and therefore, 31. Sunkel, C., and Glover, D. M. (1988) J. Cell Sci. 89, 25–38 mitotic phosphorylation is not a requirement for activity, al- 32. Clay, F. J., McEwen, S. J., Bertoncello, I., Wilks, A., and Dunn, A. R. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 4882–4886 though it does increase it. This basal activity during interphase 33. Golsteyn, R. M., Schultz, S. J., Bartek, J., Ziemiecki, A., Ried, T., and Nigg, E. may account for our earlier observation that inhibition of PLK A. (1994) J. Cell Sci. 107, 1509–1517 expression by antisense caused a decrease in DNA replication 34. Hamanaka, R., Maloid, S., Smith, M. R., O’Connell, C. D., Longo, D. L., and Ferris, D. K. (1994) Cell Growth & Differ. 5, 249–257 (34). Determination of phosphorylation site(s) within the PLK 35. Holtrich, U., Wolf, G., Brauninger, A., Karn, T., Bohme, B., Rubsamen- protein and their relationship to kinase activity may help un- Waigmann, H., and Strebhardt, K. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 1736–1740 derstand the regulation of PLK and suggest potential kinases 36. Lake, R. J., and Jelinek, W. R. (1993) Mol. Cell. Biol. 13, 7793–7801 that phosphorylate PLK. 37. Hanks, S., and Quinn, A. M. (1991) Methods Enzymol. 200, 3862 The identification of substrates is an important issue regard- 38. Murray, A. W. (1992) Nature 359, 599–604 39. O’Connor, P. M., Ferris, D. K., Hoffmann, I., Jackman, J., Draetta, G., and ing the specific roles of PLK during cell cycle progression. PLK Kohn, K. W. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 9480–9484 coprecipitates with several other proteins that are not cross- 40. Fenton, B., and Glover, D. M. (1993) Nature 363, 637–640 reactive with the antisera by immunoblot analysis and there- 41. Nigg, E. A. (1991) Semin. Cell Biol. 2, 261–270 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Polo-like Kinase Is a Cell Cycle-regulated Kinase Activated during Mitosis

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 270, No. 36, Issue of September 8, pp. 21086–21091, 1995 Printed in U.S.A. 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‡, Mark R. Smith§, Patrick M. O’Connor , Sharon Maloid§, Kelly Mihalic‡, Jerry L. Spivak , Dan L. Longo‡, and Douglas K. Ferris§** From the ‡Laboratory of Leukocyte Biology, Biological Response Modifiers Program, Division of Cancer Treatment and the §Biological Carcinogenesis and Development Program, Program Resources, Inc./DynCorp, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland 21702-1201, the Laboratory of Molecular Pharmacology, Developmental Therapeutics Program, Division of Cancer Treatment, NCI, National Institutes of Health, Bethesda, Maryland 20892, and the Division of Hematology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 22). In addition to such architectural proteins, the tumor sup- Previously, we demonstrated that expression of polo- like kinase (PLK) is required for cellular DNA synthesis pressor gene products p53 (23) and RB (24–28) are both subject and that overexpression of PLK is sufficient to induce to regulatory phosphorylation by CdK family members (7). DNA synthesis. We now report that the endogenous Thus, it is clear that reversible phosphorylation reactions play levels of PLK, its phosphorylation status, and protein a major role in regulating cell cycle progression, and the list of kinase activity are tightly regulated during cell cycle enzymes known to be involved is rapidly expanding. progression. PLK protein is low in G , accumulates dur- 1 A number of cell cycle-regulated kinases unrelated to the ing S and G M, and is rapidly reduced after mitosis. CdKs have also been identified in lower eukaryotes, including During mitosis, PLK is phosphorylated on serine, and its the homologous yeast CDC5 (29) and Drosophila polo (30). Polo serine threonine kinase function is activated at a time was first identified as an embryonic lethal Drosophila mutation cdc2 close to that of p34 . The phosphorylated form of PLK causing formation of monopolar and multipolar mitotic spin- migrates with reduced mobility on SDS-polyacrylamide dles and abnormal segregation of chromosomes (31). Saccharo- gel electrophoresis, and dephosphorylation by purified myces cerevisiae CDC5 mutants likewise are impaired in mi- protein phosphatase 2A converts it to the more rapidly totic spindle formation, but the CDC5 kinase also appears to migrating form and reduces the total amount of PLK cdc2 have a function during S phase (29). Recently, we and others kinase activity. Purified p34 -cyclin B complex can (32–36) have cloned a putative mammalian polo homologue, phosphorylate PLK protein in vitro but causes little in- polo-like kinase (PLK) (32–36). We previously showed that crease in PLK kinase activity. 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- The processes of cell growth and division are stringently length PLK antisense RNA reduced serum-stimulated tritiated regulated to ensure fidelity of DNA replication and correct thymidine incorporation. On the basis of those results, we segregation of genetic information (1, 2). So essential are these suggested that PLK had an important S phase function (34). In processes to cellular homeostasis that many of the gene prod- the present study, we investigate the cell cycle regulation of ucts regulating passage through the cell cycle are highly con- PLK by measuring endogenous PLK protein levels, phospho- served both in amino acid sequence and function between or- rylation state, and kinase activity. We show that PLK protein ganisms as evolutionarily divergent as yeast and man (3, 4). levels are low in G , increase during S, remain high through Within the last few years, a variety of related enzymes known G M, and are rapidly decreased after mitosis. During the G to as cyclin-dependent protein kinases (CdKs) have been identi- 2 2 M phase transition, PLK protein is phosphorylated on serine, fied that are activated and inactivated at specific times during and its kinase function is stimulated. Dephosphorylation of cell cycle progression (5–8). The CdKs must form complexes PLK reduces its activity by 5–10-fold and converts the slower with a cyclin family member to be activated (9, 10) and are also migrating form to the faster migrating form. We further dem- subject to both positive and negative phosphorylation by other onstrate that Cdc2/cyclin B can phosphorylate PLK but has protein kinases and dephosphorylation by protein phospha- little effect on PLK kinase activity, suggesting that mitotic tases (11–17). Together, the family of CdKs constitutes critical activation of PLK will not be explained solely by Cdc2. components of the engine that propels the cell through the cycle. Many proteins that are phosphorylated in a cell cycle- EXPERIMENTAL PROCEDURES specific fashion have been identified as putative targets of CdK Cell Culture—Murine NIH3T3 and human Hela cells were cultured regulation including nuclear lamins (18, 19), nucleolin (20, 21), in Dulbecco’s modified minimum essential medium (Life Technologies, and other matrix proteins, as well as cytoskeletal proteins (7, Inc.) supplemented with 10% calf serum (Life Technologies, Inc.). CA46, a human B-cell lymphoma line, was cultured in RPMI 1640 (Life Tech- nologies, Inc.) supplemented with 15% fetal calf serum. * The costs of publication of this article were defrayed in part by the 35 32 Materials—[ S]Methionine and [g- P]ATP were obtained from payment of page charges. This article must therefore be hereby marked DuPont NEN. [ H]Thymidine was from Amersham. All other reagents “advertisement” in accordance with 18 U.S.C. Section 1734 solely to were from Sigma unless otherwise specified. indicate this fact. PLK Antisera—Two polyclonal PLK antisera designated 8845 and ** To whom correspondence should be addressed. Tel.: 301-846-1427; 8847 were raised in New Zealand White rabbits immunized with syn- Fax: 301-846-5651. thetic peptides coupled to KLH. Antiserum 8845 was generated against The abbreviations used are: CdK, cyclin-dependent protein kinases; PLK, polo-like kinase; PP2A, protein phosphatase 2A; PAGE, polyacryl- a peptide TAGKLPRAPADPGKAGVPG corresponding to amino acids amide gel electrophoresis. 6–24 of human PLK, and 8847 was generated against the homologous This is an Open Access article under the CC BY license. Activation of PLK during Mitosis 21087 murine peptide KAGKLARAPADLGKGGVPG. cyclin B (Promega). Samples to be further processed by in vitro phos- Drug Treatments and Cell Cycle Analysis—Cells were synchronized phorylation in the PLK kinase buffer were washed twice in 1 ml of by treatment for 10–15 h with either mimosine (300 mM), aphidicolin kinase buffer to remove Cdc2/cyclin, and the PLK casein kinase reac- (0.75 mM), or nocodazole (100 ng/ml). Double aphidicolin synchroniza- tion was carried out as described above. tion was performed by drug treatment overnight followed by a 10-h Gel Electrophoresis and Immunoblot Analysis—SDS-polyacrylamide release and retreatment overnight. Cells to be released from drug gel electrophoresis and transfer to Immobilon (Millipore) was carried treatment were washed three times in media without drug and resus- out by standard methods, except that gels were prepared using a 120:1 pended in fresh media. For cell cycle analysis, cells were harvested by acrylamide:bisacrylamide mixture to increase separation of the mitotic centrifugation, washed in ice-cold phosphate-buffered saline, lysed in PLK doublet. Membranes were blocked2hin5% nonfat dry milk, 2% 138 mM NaCl, 5 mM KCl, 440 mM KH PO , 335 mM Na HPO ,1mM goat serum, 0.5% Tween 20, 150 mM NaCl, 20 mM Tris base, pH 8.2 2 4 2 4 CaCl , 500 mM MgCl , 400 mM MgSO ,24mM Hepes, 0.2% w/v bovine (blocking buffer). Probing with PLK antiserum (1:2000) was carried out 2 2 4 serum albumin, 0.4% (w/v) Nonidet P-40 (detergent buffer). Cells were 2 h or overnight at room temperature in the same buffer containing then treated with RNase, and cellular DNA was stained with propidium 0.02% sodium azide. After washing four times in 20 mM Tris, pH 8.2, iodide. Cell cycle determination was performed using a Becton Dickin- 150 mM NaCl, 0.05% Tween 20 (TBST), the membranes were probed son fluorescence-activated cell analyzer, and data were interpreted with 200 ng/ml peroxidase-labeled goat anti-rabbit IgG for2hin block- using the SFIT model program provided by the manufacturer. Meas- ing buffer. After extensive washing in TBST, the membranes were urements of mitotic index were recorded after harvesting cells and processed for enhanced chemiluminescence using ECL (Amersham) washing once with ice-cold phosphate-buffered saline. Cells were resus- reagents according to the manufacturer’s instructions. Autoradiogra- pended in 0.5 ml of half-strength phosphate-buffered saline for 10 min phy of gels or blots and ECL exposures were carried out using Kodak at room temperature, then stored overnight at 4 °C in 6 ml of a 2% XAR film (Rochester, NY). solution of 3:1 ethanol:glacial acetic acid. Samples were then resus- RESULTS pended 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, Characterization of PLK Antisera—To study PLK protein at least 500 cells were randomly counted, and mitotic cells were scored expression and function, we generated two synthetic PLK pep- by their lack of nuclear membrane and evidence of chromosome tide polyclonal antisera in rabbits designated 8847 and 8845. condensation. Antiserum 8845 was generated against a human PLK peptide For mitotic shakeoff experiments, Hela or NIH3T3 cells were serum sequence located near the N terminus of PLK, and 8847 was 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 generated against the homologous murine PLK peptide. This flask vigorously several times and harvested by centrifugation. The region of PLK was chosen on the basis of its hydrophilicity and remaining adherent cells were collected by scraping and were used to because the N-terminal region of PLK is the least highly evo- prepare interphase extracts. lutionarily conserved portion at the amino acid sequence level Metabolic Labeling—Cells to be metabolically labeled were washed (34) and therefore, we reasoned, would be more likely to be three times in either phosphate-free media or methionine-free media, antigenic. Fig. 1A demonstrates immunoprecipitation of in then labeled for 2–14 h in the appropriate media containing 0.5 mCi/ml 32 35 35 plk of either ortho[ P]phosphate or [ S]methionine and 5% calf serum vitro translated [ S]methionine-labeled murine p64 using that had been dialyzed against phosphate-free or methionine-free immune 8845 or 8847 antisera. Specificity of the two antisera media. toward the in vitro translated PLK was confirmed by compar- Immunoprecipitation and Kinase Reactions—All procedures during ison with pre-immune serum. Fig. 1B shows immunoprecipita- lysis, clarification, and immunoprecipitation were performed at 4 °C. tion from [ S]methionine-labeled human CA46 cells and com- Cells were lysed for 30 min in buffer 1, 50 mM Tris (pH 7.5), 1% Brij 96, petition with cognate peptide. The major specifically competed 10 mM NaF, 10 mM sodium pyrophosphate, 1 mg/ml aprotinin, 1 mg/ml plk leupeptin, 1 mM phenylmethylsulfonyl fluoride, and lysates were clar- protein, p64 , migrates at the same position as in vitro trans- ified by centrifugation at 100,000 3 g for 20 min. One ml of supernatant lated PLK, and its identity was confirmed by Western blot was added to each 1.5-ml Eppendorf tube containing 3 mlofPLK analysis. Several other proteins, indicated by arrows, were also antisera prebound to 120 ml of a 20% solution of protein A-Sepharose. specifically competed by the peptide and could represent either Lysates were immunoprecipitated for 2–3 h on a rotator, then washed cross-reactive proteins or proteins that are complexed with 4–5 times with buffer 1 and either treated with SDS sample buffer or, PLK. Since these other proteins are not reactive with the for PLK kinase reactions, washed an additional time in buffer 2 (10 mM Hepes, pH 7.4, 10 mM MnCl ,5mM MgCl ). In vitro PLK kinase reac- peptide antiserum by immunoblot analysis (C), we favor the 2 2 tions were performed for 20 min at 37 °C using a 40-ml reaction mixture latter possibility. At present, we have not identified any of composed of 38 ml of buffer 2 and 2 ml of 3000 Ci/mmol [g- P]ATP either these other proteins. For reference, C shows a typical immu- with or without dephosphorylated casein (20 mg/reaction) added as an noblot of total CA46 cell protein using either immune or pre- exogenous substrate. Assays of Cdc2 kinase activity were performed for immune 8845 antisera. Immunoblotting revealed a single pro- 20 min at 37 °C, using a reaction mixture consisting of 38 mlof20mM tein of 64 kDa was recognized by the 8845 antiserum. Tris-HCl, 10 mM MgCl , pH 7.5, containing 5 mM unlabeled ATP, 10 mCi of [g- P]ATP (3000 Ci/mmol), and 3 mg of histone H1 (Boehringer Essentially identical results were obtained using antisera 8847 Mannheim). Kinase reactions were terminated by the addition of 30 ml to detect endogenous PLK protein in murine cells (not shown). of 3 3 SDS sample buffer, boiled, and loaded onto polyacrylamide gels. In conclusion, the 8845 and 8847 antisera were able to immu- Phosphatase Assays—Immunoprecipitations were washed once in 0.5 plk noprecipitate and immunoblot p64 from either human or M Tris, pH 7.8, pelleted by centrifugation, and all wash buffer was murine cells. removed using a Hamilton syringe. 100 ml of purified protein phospha- Cell Cycle Regulation of PLK Expression—We investigated tase 2A (PP2A) dilution buffer (Upstate Biotechnology Inc.) was added whether PLK protein levels were cell cycle regulated by immu- either with or without 5 units of PP2A and either with or without 20 nM (final) okadaic acid (Sigma) dissolved in 5 mlofMe SO. Samples were 2 noblotting. CA46 cells synchronized at G S with mimosine, in S incubated for 20–40 min at 30 °C. After completion of phosphatase phase with aphidicolin, or at G M with nocodazole (Fig. 2A) reactions, the reaction mixture was removed, and the reaction was were lysed and clarified; proteins were separated by SDS- stopped by the addition of 50 mlof3 3 SDS buffer, or the reactions were PAGE and analyzed by immunoblotting (Fig. 2B). We found washed twice in kinase buffer and further processed for casein kinase that PLK protein was substantially reduced in G S mimosine- immune complex kinase reactions by methods described above. In Vitro Phosphorylation of PLK by Purified Cdc2/Cyclin B—CA46 blocked cells (M) compared to unsynchronized cells (U); in cells were synchronized in S phase by blocking overnight with aphidi- contrast, much higher levels were detected in both S phase (A) colin, and cell lysates were prepared. PLK immunoprecipitations were and prometaphase-blocked cells (N). Two closely migrating performed using 8845 antiserum in the presence or absence of cognate PLK-specific bands were apparent in the extract made from the peptide. Precipitates were then subjected to in vitro phosphorylation in prometaphase-arrested cells (N), and the majority of PLK pro- 50 ml of Cdc2 buffer (20 mM Hepes, pH 7.5, 1 mM dithiothreitol, 5 mM tein was present in the slower migrating band. These results MgCl ,50 mM ATP, 20 mCi [g- P]ATP) for 30 min at 30 °C in the presence or absence of 100 ng of purified mitotic starfish oocyte Cdc2/ suggested that PLK protein levels are regulated during the cell 21088 Activation of PLK during Mitosis FIG.1. Characterization of PLK an- tisera. A, murine PLK was [ S]methi- onine labeled during translation in vitro, and aliquots of the translated protein were immunoprecipitated with preim- mune or immune PLK peptide antisera and analyzed by 7.5% acrylamide SDS- PAGE and autoradiography. Lane 1, pre- immune 8847; lane 2, preimmune 8845; lane 3, sample of total; lane 4, immune 8847; lane 5, immune 8845. B, asynchro- nously growing human CA46 lymphoma cells were metabolically labeled with [ S]methionine, lysed, and immunopre- cipitated with 8845 antisera in the pres- ence (1) or absence (2) of cognate peptide and analyzed by 7.5% acrylamide SDS- PAGE and autoradiography. C, samples containing 75 mg each of total CA46 cell proteins were separated by 7.5% SDS- PAGE, transferred to Immobilon, and probed with immune (lane 1) or preim- mune PLK antisera 8845 (lane 2). Pri- mary antibody was detected using a per- oxidase-labeled goat anti-rabbit and ECL. 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 phospho- rylated 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 precipi- tates is due to PLK and not a coprecipitating enzyme. To substantiate the kinase activity results obtained with drug-synchronized cells, a mitotic shake off experiment was performed with murine NIH3T3 cells (Fig. 3B). Immunopre- cipitations from equivalent numbers of mitotic or the inter- phase adherent cells were performed, and immune complex kinase assays were performed using casein as exogenous sub- strate. No kinase activity was detected in the preimmune or FIG.2. Cell cycle changes in PLK protein. CA 46 cells were immune competition assays performed from mitotic extracts, treated 16 h with nothing (U), mimosine (M), aphidicolin (A), or nocoda- zole (N). A, cells were harvested and prepared for cell cycle fax analysis. and only very low amounts of activity were detected in the B, cell lysates were analyzed by SDS-PAGE and immunoblotting with immune interphase assay. Consistent with the results shown 8845 antiserum. in A, PLK isolated from mitotic cells showed increased kinase activity. To directly assess PLK phosphorylation state during mitosis, cycle, as has previously been observed (33), and further suggest we compared in vivo phosphorylated PLK from NIH3T3 cells, that a portion of PLK undergoes a mitotic modification that isolated by mitotic shake off, to [ S]methionine-labeled PLK reduces its mobility. immunoprecipitated from unsynchronized cells in the presence PLK Is Phosphorylated and Activated during Mitosis—Di- or absence of cognate competitor peptide (C). We found that rect assessment of PLK phosphorylation status and kinase PLK was indeed labeled with P during mitosis (C) but was activity was performed using immunoprecipitated PLK. Fig. undetected in extracts from adherent cells (not shown). Fur- 3A, upper panel, shows immunoblot analysis of PLK immuno- thermore, the phosphorylated form appeared to migrate more precipitated with preimmune or immune 8845 antisera from slowly in SDS gels compared to the [ S]methionine-labeled unsynchronized, S phase, or mitotic human CA46 cells. The blot shows that a large fraction of PLK isolated from mitotic PLK protein from unsynchronized cells. These results con- firmed our speculation that PLK is phosphorylated in a cell cells migrates slower than PLK isolated from S phase cells or from unsynchronized cells, in which only a relatively small cycle-specific manner and could arise either through an auto- phosphorylation reaction or through the action of a separate proportion (10–15%) of mitotic cells are present. The middle and lower panels in A represent the corresponding immune kinase. complex kinase assays showing in vitro phosphorylation of PLK To confirm that the mitotic specific phosphorylation of PLK itself and casein, respectively. Although approximately equal was responsible for its reduced mobility, immunoprecipitated Activation of PLK during Mitosis 21089 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 dem- onstrated serine/threonine phosphorylation of casein and also autophosphorylation of serine and threonine residues in im- mune 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 phos- phorylated on serine, has increased phosphotransferase activ- ity, 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 arti- ficial conditions. It might also be that a very active phospha- tase specifically removes phosphate from threonine in vivo during mitosis as a means of regulating PLK activity. At pres- ent, we cannot distinguish between these possibilities. PLK Phosphorylation Occurs with Kinetics Similar to Cdc2 Activation—We wished to compare the timing of PLK phospho- cdc2 rylation with activation of p34 , a kinase known to be re- FIG.3. Mitotic PLK is phosphorylated on serine and has re- quired for promotion of mitosis (5, 11, 14) and which is acti- duced mobility and increased kinase activity. A, CA46 cells were vated by Cdc25-mediated dephosphorylation at the G to M either unsynchronized (U) or synchronized with aphidicolin in S phase phase transition (17). Human CA46 cells were synchronized at (S) or with nocodazole in prometaphase of mitosis (M). Lysates were prepared and immunoprecipitated (I.P.) with preimmune (P)orim- the G S transition by a double aphidicolin block and then were mune (I) 8845 PLK antiserum. Immunoprecipitates were subjected to released into nocodazole to trap the cells in prometaphase of immune complex kinase assays with dephosphorylated casein added as mitosis. After various periods of release from the aphidicolin an exogenous substrate and analyzed by 8845 immunoblot and autora- block, the cells were harvested and subjected to cell cycle FACS diography. Upper panel, immunoblot; middle panel, autoradiography of immune complex autophosphorylation of PLK; bottom panel, autora- analysis, mitotic index analysis, and immunoblot analysis to cdc2 diography of immune complex phosphorylation of casein. B, NIH3T3 examine PLK and p34 (Fig. 4A). The slower migrating form cells were serum starved for 36 h and then stimulated with serum for of PLK was first detected 7.5 h after aphidicolin release (upper 18–20 h. Mitotic cells (M) were collected by mechanical shake off, and panel) when 90% of the cells were in G M and 40% had entered adherent interphase cells (I) were harvested by scraping. Lysates pre- 2 pared from equal numbers (1 3 10 ) of interphase or mitotic cells were M phase as determined by mitotic index analysis. As cells immunoprecipitated (I.P.) with preimmune (P) or immune 8847 PLK continued to accumulate in mitosis, we observed a steadily antiserum with (IC) or without (I) competing cognate peptide, and increasing amount of PLK present in the slower migrating immune complex kinase assays were performed using casein as sub- form. The kinetics with which PLK became phosphorylated strate. Autoradiography is shown. C, asynchronously growing NIH3T3 35 35 cells were labeled metabolically with [ S]methionine ( S) or with look similar to the timing of Cdc2 activation detected either by orthophosphate ( P). Methionine-labeled cells were harvested with a loss of the slower migrating hyperphosphorylated form of scraper, while orthophosphate-labeled mitotic cells were harvested by cdc2 p34 (lower panel), or by activation of Cdc2 kinase activity. mechanical shake off. Lysates were immunoprecipitated using 8847 Panel C shows PLK casein kinase and Cdc2 histone kinase antiserum with (1) or without (2) cognate peptide and analyzed by SDS-PAGE and autoradiography. D and E, nocodazole-synchronized activity during a shorter time course after release from aphidi- CA46 extracts were prepared and immunoprecipitated with PLK anti- colin into nocodazole. The largest increases in the kinase ac- serum. Immunoprecipitates were treated with nothing (lane 1), PP2A tivity of both PLK and Cdc2 occurred between 3 and 6 h, near (lane 2), PP2A and Me SO (lane 3), or PP2A, Me SO, and okadaic acid 2 2 the time when electrophoretic mobility changes of each became (lane 4) and subjected to in vitro casein kinase assays. Reactions were analyzed by SDS-PAGE and immunoblotting (D) and autoradiography apparent. To try to determine whether phosphorylation of PLK (E). F, phosphoamino acid analysis of in vitro phosphorylated PLK and was more closely associated with G or M phase cells, the same casein isolated from A and in vivo phosphorylated PLK from C. O, experiment was repeated except that after release from aphidi- origin; S, serine; T, threonine; Y, tyrosine. colin and just before nocodazole addition, the cells were treated with nitrogen mustard (0.7 mM, 30 min), a DNA damaging mitotic PLK was treated with purified protein phosphatase 2A agent that causes a prolonged delay at the G check point (2, in the presence or absence of okadaic acid and the effects of the 2 38, 39) (B). Although by 17.5 h after the release 90% of the cells dephosphorylation on mobility and casein kinase activity de- were in G M, less than 15% were in mitosis, and no mobility termined (Fig. 3, D and E). Immunoprecipitated PLK not shifts of either PLK or Cdc2 were detected. In contrast to the treated with phosphatase (D, lane 1) migrated as a doublet and results shown in A and C, when the synchronized cells were had substantial kinase activity (E, lane 1), while PLK treated with PP2A, with (lane 3) or without (lane 2)Me SO, the carrier treated with nitrogen mustard prior to release into nocodazole, there was a gradual accumulation of kinase activity during the for okadaic acid, was mostly converted to the faster migrating form and had 5–10-fold less kinase activity (E). The effects of time course that paralleled the increase in PLK protein but no abrupt increase (D). We conclude that phosphorylation and the PP2A treatment on PLK mobility and kinase activity could be completely blocked by the addition of okadaic acid (lane 4) activation of PLK occurs as cells enter mitosis at about the demonstrating that the effects were due to the phosphatase same time that Cdc2 is activated. activity and not to some contaminating activity present in the In Vitro Phosphorylation of PLK by Cdc2—Since PLK is 21090 Activation of PLK during Mitosis FIG.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 mus- tard 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 show- ing FACS and mitotic index results. C, immune complex kinase assay. Cells were double blocked with aphidicolin and re- leased into nocodazole. At the indicated times, lysates were prepared and immu- noprecipitated in the presence (1) and absence (2) 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 mus- tard, and released into nocodazole. At the indicated times, lysates were prepared and immunoprecipitated with 8845 anti- serum. Autoradiography is shown. activated at about the same time as Cdc2, we tested whether purified activated Cdc2/cyclin B could phosphorylate and acti- vate 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 autophospho- rylate (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, stand- ard PLK immune complex casein kinase reactions were per- formed (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). Phosphoryla- tion of PLK by Cdc2/cyclin B followed by autophosphorylation of PLK also did not cause a shift in PLK mobility (not shown). DISCUSSION In our initial report on the cloning of human and murine FIG.5. In vitro phosphorylation of PLK by Cdc2/cyclin B. PLK PLK, we showed evidence that PLK has a function in S phase was immunoprecipitated from aphidicolin-synchronized CA46 cell ex- (34) similar to the finding for the yeast PLK homologue cdc5, tracts in the absence or presence of cognate peptide and subjected to in which apparently has roles during S phase and during mitosis vitro phosphorylation in a Cdc2 reaction mixture in the presence or (29). More recently, it has been shown that PLK steady state absence of purified Cdc2-cyclin B complex. Samples were then sepa- rated by SDS-PAGE, transferred to Immobilon, and analyzed by auto- message (35, 36) and protein levels (33) are cell cycle regulated, radiography (B) and immunoblotting (A) or were washed with PLK with low levels of each detected in G and higher levels in S and reaction mixture to remove the Cdc2-cyclin B complex and reaction G . Our findings support and extend these earlier observations mixture; samples were then subjected to a PLK casein kinase assay (C) by showing that 1) PLK kinase activity is cell cycle regulated, and analyzed by SDS-PAGE and autoradiography. 2) PLK activation involves a phosphorylation reaction, 3) mi- totic PLK is phosphorylated on serine, 4) activation of PLK activated at the anaphase-telophase transition, while our data occurs with kinetics similar to Cdc2, and 5) PLK kinase acti- suggest an earlier activation of PLK, probably during the G to vation, like Cdc2 activation, is integrated into the G check- prophase transition. Our data must also be considered in light point response to DNA damage. of the recent publication by Golsteyn and co-workers (33), Fenton and Glover (40) have shown that in synchronously showing that in interphase cells, PLK is diffusely distributed dividing Drosophila cells, polo kinase is tightly regulated and throughout both the cytoplasm and nucleus, whereas just after Activation of PLK during Mitosis 21091 cell division, PLK localized to the midbody of the post-mitotic fore may represent members of a PLK complex (and potential bridge, which is suggestive of a function during late mitosis/ substrates). Identification of these proteins is likely to help cytokinesis. The earlier activation of PLK at the G M phase reveal the role of PLK in regulating cell cycle progression. transition suggested by our data, however, is not incompatible REFERENCES with a role later in mitosis. Together, our results suggest that 1. Hartwell, L. H., Mortimer, R. K., Culotti, J., and Culotli, M. 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Published: Sep 1, 1995

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