The Plk (polo-like kinase) family is involved in cell-cycle machinery. Despite the possible overlapping involvement of Plk1 and Plk3 in cell-cycle distribution, the precise role of each Plk might be different. To investigate mechanisms that may differentiate their physiological roles, we compared the substrate specificities of Plk1 and Plk3 using synthetic peptides. Among these substrate peptides, topoisomerase IIα EKT1342DDE-containing synthetic peptide was strongly phosphorylated by Plk3 but not by Plk1. By modulating the topoisomerase IIα peptide, we identified residues at positions +1, +2 and +4 as determinants of differential substrate recognition between Plk1 and Plk3. Acidic residues at positions +2 and +4 appear to be a positive determinant for Plk3 but not Plk1. Variation at position +1 appears to be tolerated by Plk3, while a hydrophobic residue at +1 is critical for Plk1 activity. The direct phosphorylation of Thr1342 of topoisomerase IIα by Plk3 was demonstrated with an in vitro kinase assay, and overexpression of Plk3 induced the phosphorylation of Thr1342 in cellular topoisomerase IIα. Furthermore, the physical interaction between Plk3 and topoisomerase IIα was also demonstrated in cells in addition to phosphorylation. These data suggest that topoisomerase IIα is a novel physiological substrate for Plk3 and that Plk1 and Plk3 play different roles in cell-cycle regulation.

INTRODUCTION

Plk (polo-like kinase), which originates from the Drosophila polo gene, is a serine/threonine kinase involved in cell-cycle regulation [1,2] and is characterized by a conserved N-terminal kinase domain and a homologous C-terminal domain called the polo-box. In mammalian cells, four Plks have been identified: Plk1, Plk2, Plk3 and Plk4 [3].

Plk1, which is the best-characterized mammalian Plk, plays important roles in G2/M-phase progression, covering multiple steps such as centrosome maturation [4,5], entry into mitosis [6], sister chromatid separation [7], exit from mitosis [8] and cytokinesis [9], through phosphorylation of various substrates. Plk1 is essential for mitotic transition, as functional interventions with siRNA (small interfering RNA) [10,11] and small-molecule inhibitors [12,13] caused mitotic arrest followed by the induction of apoptosis.

Plk3 is also involved in cell-cycle regulation at the G2/M-phase, although it is not as well characterized as Plk1. Plk3 appears to be involved in the entry into mitosis through phosphorylation of Cdc25C, which controls the Cdc2/cyclinB complex [14], and Plk3 has also been implicated in the regulation of Golgi fragmentation during mitosis [15]. In addition to its role in cell-cycle machinery, Plk3 may also function in checkpoint activation and apoptosis induction. Plk3 is activated in response to DNA damage in an ATM (ataxia telangiectasia mutated)-dependent fashion [16], and it physically interacts with and phosphorylates p53 [17]. Overexpression of Plk3 induces apoptosis in mammalian cells, which is mediated in part via p53 activation [18].

Despite the possible overlapping involvement of Plk1 and Plk3 in G2/M progression, the precise role of each Plk might be different. One possible mechanism for the differentiation of the physiological roles of these two enzymes is differential recognition of their substrates. With respect to the substrate specificity of Plk1, D/E-X-S/T-hydrophobic-X-D/E is a reported consensus phosphorylation sequence [19]. A recent study of the substrate specificity of the Plk family showed that both Plk1 and Plk3 recognize acidic substrate peptides and that the substrate specificity of Plk3 was slightly different from that of Plk1 [20]. However, a precise comparison of substrate specificity between Plk1 and Plk3 has not yet been done, and few candidates for Plk3-specific substrates have been identified.

Topoisomerase II is a nuclear enzyme that regulates DNA topology by transiently breaking and rejoining double-stranded DNA; thus, it catalyses the decatenation and unknotting of topologically-linked DNA circles and the relaxation of supercoiled DNA [21]. Two isoenzymes, topoisomerase IIα and topoisomerase IIβ, exist in mammalian cells. The cell-cycle distribution of topoisomerase IIα is greatest in the G2/M-phase, whereas that of topoisomerase IIβ remains constant [22,23]. Topoisomerase IIα has been implicated in essential functions in the cell cycle, such as chromosome decatenation after DNA replication [24] and chromosome condensation and segregation in the G2/M-phase [25]. These cell-cycle specific roles of topoisomerase II may be regulated by phosphorylation by various kinases [2633]; however, the functional relevance of most of these phosphorylations has not been fully elucidated.

In the present study, we compared the substrate specificities of Plk1 and Plk3 by using various substrate peptides. We found that a peptide sequence from topoisomerase IIα is strongly phosphorylated by Plk3 but not Plk1 and that Thr1342 in topoisomerase IIα is efficiently phosphorylated by the exogenous expression of Plk3 in HEK-293T cells [HEK-293 cells (human embryonic kidney cells) expressing the large T-antigen of SV40 (simian virus 40)]. Our results suggest that topoisomerase IIα is a novel physiological substrate for Plk3.

MATERIALS AND METHODS

Preparation of proteins and synthetic peptides

The human Plk1 and Plk3 genes were amplified by PCR from human testis cDNA (Clontech). The primers for Plk1 amplification were 5′-CCGAATTCATGAGTGCTGCAGTGACTGCAGGGA-3′ and 5′-CCGAATTCTTAGGAGGCCTTGAGACGGTTGCTG-3′. The primers for Plk3 amplification were 5′-CCGGATCCATGGAGCCTGCCGCCGGTTTCCTGT-3′ and 5′-CCGAATTCCTAGGCTGGGCTGCGGTCCCGGAGC-3′. The resultant PCR products were cloned into pBlueScript SKII(+) and confirmed by DNA sequencing. For expression and purification purposes, Plk1 and Plk3 cDNA were cloned into the pGEX6P-1 vector (GE Healthcare). GST (glutathione transferase)-fused Plk proteins were produced in BL21(DE3) by incubation with 0.1 mM IPTG (isopropyl-β-D-thiogalactopyranoside) at 25 °C for 12 h. GST–Plk was absorbed onto glutathione–Sepharose 4B (GE Healthcare) and eluted with PreScission protease. Topoisomerase IIα protein was obtained from TopoGEN (Port Orange, FL, U.S.A.). Substrate peptides were synthesized and purified by HPLC.

Antibodies

The following antibodies were used in this study: anti-Plk1 antibody (Zymed), anti-Plk3 antibody (BD Pharmingen), anti-topoisomerase II α antibody (MBL, Nagoya, Japan) and anti-phospho-Thr1342 topoisomerase IIα antibody (MBL).

In vitro kinase assay

Substrate proteins were incubated with 150 nM Plk1 or 60 nM Plk3 at 30 °C for 30 min in R buffer (20 mM Tris/HCl, pH 7.4, 10 mM MgCl2, 4.5 mM 2-mercaptoethanol, 1 mM EGTA) that contained 50 μM ATP and 10 μCi [γ-33P]ATP (3000 Ci/mmol; GE Healthcare). Phosphorylated proteins were analysed by SDS/PAGE with subsequent autoradiography.

Biotinylated synthetic peptides were incubated with 150 nM Plk1 or 60 nM Plk3 at 30 °C for 60 min in R buffer containing 50 μM ATP and 5 μCi [γ-33P]ATP in a final volume of 20 μl. Assays were terminated by adding 10 μl of H3PO4. Biotinylated peptides were trapped on SAM2 biotin capture membranes (Promega) that were washed three times with 2 M NaCl, four times with 2 M NaCl in 1% H3PO4, and then monitored for radioactivity in a liquid-scintillation counter.

Plasmid construction

For expression in mammalian cells, Plk1 and Plk3 cDNA were cloned into the pCMVTag2B vector (Stratagene). To generate kinase-inactive Plk3, the QuickChange site-directed mutagenesis kit (Stratagene) was used to replace the lysine residue at position 91 with methionine and the aspartic acid residue at position 185 with alanine.

Cell culture and transfection

HEK-293T cells were grown in Dulbecco's modified Eagle's medium (Gibco BRL Life Technologies) supplemented with 10% heat-inactivated FBS (fetal bovine serum; Moregate BioTech, Hamilton, Australia). HEK-293T cells were transfected with the indicated expression plasmids using FuGENE™6 transfection reagent (Roche Molecular Biochemicals) in 6-well dishes according to the manufacturer's instructions.

Immunoprecipitation

For immunoprecipitation of the transient transfection, HEK-293T cells transfected with expression plasmid were suspended in extraction buffer (50 mM Hepes, pH 7.4, 250 mM NaCl, 0.2% NP40, 5 mM EDTA, 10% glycerol) with protease inhibitor cocktail [1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, 0.8 μM aprotinin from bovine lung, 15 μM E-64, 20 μM leupeptin hemisulfate monohydrate, 50 μM bestatin, 10 μM pepstatin A; Nacalai Tesque, Kyoto, Japan] on ice for 30 min. Soluble cell extracts were obtained by centrifugation (12000 g) at 4 °C for 30 min. Cell extracts were incubated with antibodies [40 μl of anti-FLAG M2 Affinity Gel Freezer-Safe (Sigma) for 106 cells and 3 μl of anti-topoisomerase IIα monoclonal antibody for 107 cells] on ice for 60 min, followed by the addition of protein A agarose and rotation for 2 h at 4°C. Samples were washed three times with extraction buffer and eluted with Laemmli SDS sample buffer.

Western blot analysis

For preparation of whole cell lysates, cells were lysed using extraction buffer and protein concentrations were determined with the BCA protein assay (Pierce). Equal amounts of protein were resolved by SDS/PAGE (7.5% gel) and transferred to PVDF membrane. Western blots were visualized by ECL® (enhanced chemiluminescence) plus (GE Healthcare).

RESULTS

Plk1 and Plk3 recognize different amino acid sequences

To investigate the differences in the substrate specificity of Plk1 and Plk3, we examined the phosphorylation of synthetic peptides by an in vitro kinase assay of recombinant Plks produced in Escherichia coli. As shown in Figure 1(A), recombinant Plks were confirmed with Coomassie Brilliant Blue staining and also from Western blotting with antibodies against Plk1 and Plk3.

Phosphorylation of topoisomerase IIα by Plk3 in vitro

Figure 1
Phosphorylation of topoisomerase IIα by Plk3 in vitro

(A) Plk1and Plk3 were expressed in E. coli and purified. These Plks were subjected to SDS/PAGE and then to Coomassie Brilliant Blue (CBB) staining and Western blotting (WB) with antibodies against Plk1 and Plk3. (B) Phosphorylation of topoisomerase IIα protein by purified Plks in vitro. Plk1 and Plk3 were incubated with topoisomerase IIα in R buffer with [γ-33P]ATP at 30 °C for 30 min. Phosphorylated proteins in the reaction mixture were separated by SDS/PAGE and detected by autoradiography or Western blots (WB) with anti-phospho-topoisomerase IIα Thr1342 antibody.

Figure 1
Phosphorylation of topoisomerase IIα by Plk3 in vitro

(A) Plk1and Plk3 were expressed in E. coli and purified. These Plks were subjected to SDS/PAGE and then to Coomassie Brilliant Blue (CBB) staining and Western blotting (WB) with antibodies against Plk1 and Plk3. (B) Phosphorylation of topoisomerase IIα protein by purified Plks in vitro. Plk1 and Plk3 were incubated with topoisomerase IIα in R buffer with [γ-33P]ATP at 30 °C for 30 min. Phosphorylated proteins in the reaction mixture were separated by SDS/PAGE and detected by autoradiography or Western blots (WB) with anti-phospho-topoisomerase IIα Thr1342 antibody.

Peptides from known phosphorylation sites of Plk1, Cdc25C Ser198, cyclinB1 Ser133, and cyclinB1 Ser147 [3436], and peptides containing acidic residues, α-casein Ser56 and topoisomerase IIα Thr1342 [37], were tested for phosphorylation (Table 1). Plk1 more efficiently phosphorylated α-casein Ser56-containing peptide and Cdc25C Ser198-containing peptide, as compared to the other peptides. These two peptides have acidic residues at the −2 and +3 positions and a hydrophobic residue at the +1 position; thus, the recognition by Plk1 is consistent with the previously reported Plk1 consensus motif, D/E-X-S/T-hydrophobic-X-D/E [19].

Table 1
Phosphorylation of synthetic peptides of various proteins

Synthetic peptides that contained acidic sequences from various proteins were incubated with purified Plk1 and Plk3. The levels of incorporation of [γ-33P]ATP are calculated from the mean of triplicate 60 min assays. The phosphorylation rate is expressed as a percentage of the rate of phosphorylation of Cdc25C Ser198 substrate peptide by each kinase. The rate of phosphorylation of the Cdc25C peptide by Plk1 and Plk3 was 5.4×102 pmol/min/mg protein and 5.7×103 pmol/min/mg protein respectively. Acidic residues are shown in bold, and hydrophobic amino acids important for Plk1 recognition are underlined. The reported consensus phosphorylation motif for Plk1 is also shown [19]. φ, hydrophobic residue; ND, not detectable; topo, topoisomerase.

 Amino acid position relative to phosphorylated residue Phosphorylation rate by 
Peptide (100 μM) −6 −5 −4 −3 −2 −1 Plk1 Plk3 
Topo IIα Thr1342 D D E D D E D ND 3700 
α-Casein Ser56 E E L D 190 250 
Cdc25C Ser198 D E E L D E 100 100 
Cyclin B1 Ser147 D D ND ND 
Cyclin B1 Ser133  E 30 10 
Plk1 consensus     D/E S/T φ D/E      
 Amino acid position relative to phosphorylated residue Phosphorylation rate by 
Peptide (100 μM) −6 −5 −4 −3 −2 −1 Plk1 Plk3 
Topo IIα Thr1342 D D E D D E D ND 3700 
α-Casein Ser56 E E L D 190 250 
Cdc25C Ser198 D E E L D E 100 100 
Cyclin B1 Ser147 D D ND ND 
Cyclin B1 Ser133  E 30 10 
Plk1 consensus     D/E S/T φ D/E      

Similar to Plk1, Plk3 also efficiently phosphorylated α-casein Ser56-containing peptide and Cdc25C Ser198-containing peptide, but not the cyclin B1 peptides. This recognition tendency appears to be similar to that of Plk1, suggesting that Plk3 also has a preference for acidic residues at the −2 and +3 positions. However, Plk3 had a strikingly different ability from Plk1 to phosphorylate the topoisomerase IIα Thr1342 peptide, which is the recognition site for CK2 [38]. Although it was not phosphorylated by Plk1, the topoisomerase IIα peptide was efficiently phosphorylated by Plk3 at a rate that was about 40-fold the rate of Cdc25C peptide phosphorylation. This topoisomerase IIα peptide has acidic residues at positions −3, +1, +2 and +4, which are absent in other peptides such as Cdc25C and casein, suggesting that these acidic residues might contribute to the increased efficiency of phosphorylation by Plk3. In addition, while an acidic residue at the +1 position is completely detrimental to Plk1, Plk3 tolerated this acidic residue well, which strongly suggests a differential recognition preference at the +1 position between Plk1 and Plk3.

Recognition preference at the +1, +2 and +4 positions differs between Plk1 and Plk3

To investigate the importance of each amino acid residue in the topoisomerase IIα peptide for Plk3 recognition, we examined the effect of alanine substitutions from positions −5 to +4. As shown in Table 2, alanine substitutions at position −2, +2, +3 or +4 reduced the Plk3-mediated phosphorylation levels to less than half. Thus, Plk3 appears to prefer acidic residues at positions −2, +2, +3 and +4. Of these positions, Plk1 is also known to have a preference for acidic residues at positions −2 and +3; thus, these sites are common determinants for both Plk1 and Plk3. In contrast, the preference for acidic residues at positions +2 and +4 was a rather specific characteristic for Plk3. This preference could explain why Plk3 has a 40-fold higher phosphorylation of topoisomerase IIα peptide as compared to the Cdc25C peptide, in which positions +2 and +4 are not acidic residues, but are lysine and glutamine respectively.

Table 2
Effect of minor modifications of topoisomerase ll peptides on the phosphorylation by Plk3

Topoisomerase IIα-derived peptides were incubated with purified Plk3. The levels of incorporation of [γ-33P]ATP are calculated from the mean of triplicate 60 min assays. The phosphorylation rate is expressed as a percentage of the rate of phosphorylation of Cdc25C Ser198 substrate peptide by each kinase. Acidic residues are shown in bold, and substituted alanine residues are shown in italics. The reported consensus phosphorylation motif for Plk1 is also shown [19]. φ, hydrophobic residue; topo, topoisomerase.

 Amino acid position relative to phosphorylated residue Phosphorylation rate by 
TopoII peptide (100 μM) −6 −5 −4 −3 −2 −1 Plk3 
TopoII WT D D E D D E D 3700 
TopoII−5A A D E D D E D 3100 
TopoII−4A D A D E D D E D 3500 
TopoII−3A D A E D D E D 2600 
TopoII−2A D D A D D E D 1200 
TopoII−1A D D E A D D E D 3400 
TopoII+1A D D E A D E D 2500 
TopoII+2A D D E D A E D 900 
TopoII+3A D D E D D A D 1000 
TopoII+4A D D E D D E A 1600 
Cdc25C Ser198 D E E D E  100 
Plk1 consensus     D/E S/T φ D/E     
 Amino acid position relative to phosphorylated residue Phosphorylation rate by 
TopoII peptide (100 μM) −6 −5 −4 −3 −2 −1 Plk3 
TopoII WT D D E D D E D 3700 
TopoII−5A A D E D D E D 3100 
TopoII−4A D A D E D D E D 3500 
TopoII−3A D A E D D E D 2600 
TopoII−2A D D A D D E D 1200 
TopoII−1A D D E A D D E D 3400 
TopoII+1A D D E A D E D 2500 
TopoII+2A D D E D A E D 900 
TopoII+3A D D E D D A D 1000 
TopoII+4A D D E D D E A 1600 
Cdc25C Ser198 D E E D E  100 
Plk1 consensus     D/E S/T φ D/E     

Next, we compared the effect of substitution of position +1 on the phosphorylation of topoisomerase IIα peptide by Plk1 and Plk3, since differential recognition preference at the +1 position was suggested between Plk1 and Plk3. As shown in Table 3, the replacement of aspartic acid at position +1 with the hydrophobic residue phenylalanine did not affect the phosphorylation by Plk3. On the other hand, this replacement resulted in dramatically increased phosphorylation by Plk1, consistent with a previous report [19]. These results suggest that a hydrophobic residue at the +1 position is not important for Plk3, whereas Plk1 strongly prefers hydrophobic amino acids at the +1 position. Collectively, these results indicate differential substrate recognition between Plk1 and Plk3, suggesting that Plk3 prefers substrates that have acidic residues at positions −2, +2, +3 and +4.

Table 3
Effect of minor modifications of topoisomerase ll peptides on the phosphorylation by Plk1 and Plk3

Topoisomerase IIα-derived peptides were incubated with purified Plk1 and Plk3. The levels of incorporation of [γ-33P]ATP are calculated from the mean of triplicate 60 min assays. The phosphorylation rate is expressed as a percentage of the rate of phosphorylation of Cdc25C Ser198 substrate peptide by each kinase. Hydrophobic amino acids important for Plk1 recognition are underlined, and substituted residues are shown in italics. The reported consensus phosphorylation motif for Plk1 is also shown [19]. φ, hydrophobic residue; ND, not detected; topo, topoisomerase; WT, wild-type.

 Amino acid position relative to phosphorylated residue Phosphorylation rate by 
TopoII Peptide (100 μM) -6 -5 -4 -3 -2 -1 Plk1 Plk3 
TopoII WT ND 3700 
TopoII+1A A ND 2500 
TopoII+1F F 270 3700 
Cdc25C Ser198 L  100 100 
Plk1 consensus     D/E S/T φ D/E      
 Amino acid position relative to phosphorylated residue Phosphorylation rate by 
TopoII Peptide (100 μM) -6 -5 -4 -3 -2 -1 Plk1 Plk3 
TopoII WT ND 3700 
TopoII+1A A ND 2500 
TopoII+1F F 270 3700 
Cdc25C Ser198 L  100 100 
Plk1 consensus     D/E S/T φ D/E      

Differential phosphorylation preference of topoisomerase IIα protein at Thr1342 between Plk1 and Plk3

The strong phosphorylation of the topoisomerase IIα peptide by Plk3 suggested that topoisomerase IIα is a potential substrate for Plk3. Therefore, we tested the phosphorylation of topoisomerase IIα by recombinant Plk3 in an in vitro enzyme assay. Purified topoisomerase IIα was incubated in the presence of [γ-33P]ATP with recombinant Plk3 and Plk1, and the phosphorylation was analysed with autoradiography after SDS/PAGE. As shown in Figure 1(B), topoisomerase IIα was efficiently phosphorylated by Plk3 but not by Plk1. Western blot analysis with anti-phospho-topoisomerase IIα Thr1342 antibody showed a strong signal in topoisomerase IIα after incubation with Plk3 but not with Plk1. These results suggest that topoisomerase IIα was a direct substrate for Plk3 and that Thr1342 is a potential phosphorylation site for Plk3.

Substrate specificity in cells

In the enzyme assay, we demonstrated that topoisomerase IIα was phosphorylated by Plk3. We then investigated if this phosphorylation occurs in cells. HEK-293T cells were transfected with Plk3 and Plk1, and phosphorylation of endogenous topoisomerase IIα was examined with anti-phospho-topoisomerase IIα Thr1342 antibody (Figure 2A). Overexpression of Plk3 strongly induced the signal detected by anti-phospho-topoisomerase IIα Thr1342 antibody, while Plk1 did not increase the signal strength. In addition, the expression of kinase dead mutants Plk3 K91M and D185A as controls did not cause phosphorylation of topoisomerase IIα Thr1342 (Figure 2B), which rules out the possibility of an indirect effect of Plk3 overexpression. Thus, these results indicate that Plk3 phosphorylates the Thr1342 residue of topoisomerase IIα in cells.

Phosphorylation of topoisomerase IIα by Plk3 but not Plk1 in cells

Figure 2
Phosphorylation of topoisomerase IIα by Plk3 but not Plk1 in cells

(A) FLAG–Plk1 or –Plk3 was expressed in HEK-293T cells. The extracts were subjected to immunoblotting with anti-phospho-topoisomerase IIα Thr1342 antibody (anti-P-Thr1342), anti-topoisomerase IIα antibody and anti-FLAG M2 antibody. Positions of molecular-mass markers (kDa) are indicated on the left. (B) FLAG–Plk3 K91M or D185A was expressed in HEK-293T cells. The extracts were subjected to immunoblotting with anti-phosphotopoisomerase IIα Thr1342 antibody (anti-P-Thr1342), anti-topoisomerase IIα antibody and anti-FLAG M2 antibody. pTopoIIα, topoisomerase IIα phosphorylated at Thr1342; WB, Western blot.

Figure 2
Phosphorylation of topoisomerase IIα by Plk3 but not Plk1 in cells

(A) FLAG–Plk1 or –Plk3 was expressed in HEK-293T cells. The extracts were subjected to immunoblotting with anti-phospho-topoisomerase IIα Thr1342 antibody (anti-P-Thr1342), anti-topoisomerase IIα antibody and anti-FLAG M2 antibody. Positions of molecular-mass markers (kDa) are indicated on the left. (B) FLAG–Plk3 K91M or D185A was expressed in HEK-293T cells. The extracts were subjected to immunoblotting with anti-phosphotopoisomerase IIα Thr1342 antibody (anti-P-Thr1342), anti-topoisomerase IIα antibody and anti-FLAG M2 antibody. pTopoIIα, topoisomerase IIα phosphorylated at Thr1342; WB, Western blot.

Given the possibility that topoisomerase IIα is a substrate for Plk3, we tested whether Plk3 physically interacts with topoisomerase IIα. HEK-293T cells were transfected with FLAG-tagged Plk3, and proteins were immunoprecipitated with anti-FLAG antibody. The associated topoisomerase IIα was analysed by Western blotting. As shown in Figure 3(A), endogenous phosphorylated topoisomerase IIα was detected by the anti-phospho-Thr1342 antibody in immunoprecipitates from the lysate. Conversely, FLAG–Plk3 was co-immunoprecipitated with topoisomerase IIα after immunoprecipitation with anti-topoisomerase IIα antibody from the same lysate (Figure 3B). These results suggest a possible interaction between Plk3 and topoisomerase IIα. Taken together, our results suggest that topoisomerase IIα is a substrate for Plk3 in cells.

Interaction between topoisomerase IIα and Plk3 in cells.

Figure 3
Interaction between topoisomerase IIα and Plk3 in cells.

(A) FLAG–Plk3 was expressed in HEK-293T cells. The extracts were immunoprecipitated with anti-FLAG antibody and immunoblotted with anti-phospho-topoisomerase IIα Thr1342 antibody (anti-P-Thr1342) or anti-FLAG M2 antibody. (B) FLAG–Plk3 was expressed in HEK-293T cells. The extracts were immunoprecipitated with anti-topoisomerase II antibody and immunoblotted with anti-FLAG M2 antibody or anti-topoisomerase IIα antibody. IP, immunoprecipitation; pTopoIIα, topoisomerase IIα phosphorylated at Thr1342; WB, Western blot. Positions of molecular-mass markers (kDa) are indicated on the left.

Figure 3
Interaction between topoisomerase IIα and Plk3 in cells.

(A) FLAG–Plk3 was expressed in HEK-293T cells. The extracts were immunoprecipitated with anti-FLAG antibody and immunoblotted with anti-phospho-topoisomerase IIα Thr1342 antibody (anti-P-Thr1342) or anti-FLAG M2 antibody. (B) FLAG–Plk3 was expressed in HEK-293T cells. The extracts were immunoprecipitated with anti-topoisomerase II antibody and immunoblotted with anti-FLAG M2 antibody or anti-topoisomerase IIα antibody. IP, immunoprecipitation; pTopoIIα, topoisomerase IIα phosphorylated at Thr1342; WB, Western blot. Positions of molecular-mass markers (kDa) are indicated on the left.

DISCUSSION

In the present study, we compared the substrate specificities of Plk1 and Plk3. We found that topoisomerase IIα EKT1342DDE-containing synthetic peptides were efficiently phosphorylated by Plk3 but not by Plk1. By modulating the topoisomerase IIα peptides, we identified two potential determinants for differential recognition between Plk1 and Plk3. One determinant is acidic residues at positions +2 and +4, which appear to be a positive determinant for Plk3 but not for Plk1. The other determinant is variation at position +1: this variation appears to be tolerated by Plk3, while a hydrophobic residue at +1 is critical for Plk1 activity. Plk1 strongly selects hydrophobic residues such as phenylalanine at position +1, while Plk3 appears to allow variant amino acids such as aspartic acid at position +1. This difference is supported by the report of the phosphorylation sites in Cdc25C for Plk1 and Plk3. Plk3 phosphorylates Ser191 of Cdc25C, where position +1 is aspartic acid (the surrounding sequence is EIS191DELMEFS198LK), while Plk1 does not phosphorylate Ser191 but does phosphorylate Ser198 [14,20,35].

This recognition difference is similar to that of the CK (casein kinase) family. CKI selects isoleucine at the +1 position whereas CK2 selects glutamic acid/aspartic acid at this position [39]. This difference results from the different characteristics of the +1 pocket of the enzymes. The +1 pocket of CK2 is composed of basic amino acids containing lysine and arginine residues, while that of CKI is hydrophobic. The different recognition preference at the +1 position between Plk1 and Plk3 could also be explained by the difference in the structure of the +1 pocket as in the case of the CK family.

Although there are many reports about the putative substrates for CK2, the functional relationship between CK2 and these potential substrates is still not clear in some cases. Since our finding suggests that the substrate specificity of Plk3 is similar to that of CK2, it is possible that these proteins are also cellular substrates for Plk3. Therefore characterization of these proteins will bring new insights into cell mechanisms, in particular those related to the cell cycle and checkpoints.

The topoisomerase IIα Thr1342-containing sequence is a consensus recognition site for CK2; thus, it was reported that Thr1342 in topoisomerase IIα was phosphorylated by CK2; however, the data regarding phosphorylation of Thr1342 by CK2 remains in vitro data [38]. In the present study, we demonstrated direct phosphorylation of Thr1342 in cells expressing Plk3 as well as the interaction between Plk3 and topoisomerase IIα. These data suggest that Plk3 contributes to the phosphorylation of topoisomerase IIα.

The activity of topoisomerase IIα is essential for separating catenated chromosomes after replication. Thus, the regulation of its activity is important during the cell cycle [25,40]. Phosphorylation is one possible mechanism for controlling the decatenation activity of topoisomerase IIα. The phosphorylation level of Thr1342 in the G2/M-phase is twice as much as in the G1- or S-phase [37]. The catalytic activity of topoisomerase IIα is increased by phosphorylation in vitro [29] and the decatenation activity of topoisomerase IIα is regulated by BRCA1 (breast-cancer susceptibility gene 1) in a phosphorylation-dependent manner [41]. Because Plk3 is phosphorylated and activated in G2/M-phase [42], it is possible that Plk3 regulates the activity of topoisomerase IIα by phosphorylation in a cell-cycle dependent manner. Another possibility is that Plk3 regulates the activity of topoisomerase IIα when the checkpoint is activated. Insufficiently decatenated chromatids induce a checkpoint system called the decatenation checkpoint [43]. This checkpoint system induces cell-cycle arrest at G2-phase and prevents entry into mitosis until the chromatids are sufficiently decatenated by topoisomerase IIα. Although the mechanism of the decatenation checkpoint still needs to be clarified, it is possible that the checkpoint signal might activate topoisomerase IIα to facilitate the completion of decatenation. Interestingly, Plk3 is activated at G2-phase by various signals that induce checkpoints such as DNA damage [16] or oxidative stress [44], and Plk3 activates downstream factors involved in checkpoint signalling such as chk2 [45]. Therefore, it will be interesting to investigate the role of Plk3-mediated phosphorylation of topoisomerase IIα in the regulation of the G2-phase decatenation checkpoint.

In conclusion, we demonstrated the differential substrate recognition between Plk1 and Plk3, and we identified topoisomerase IIα as a potential cellular substrate for Plk3. Our results will help clarify the differential roles of Plk1 and Plk3 in the control of cell-cycle progression and checkpoints, as well as elucidate novel substrates for Plk3.

We thank M. Kobayashi (Department of Oncology, Tsukuba Research Institute, Banyu Pharmaceutical Co. Ltd, Okubo 3, Tsukuba, Ibaraki 300-2611, Japan) for donating the Plk1 and Plk3 cDNAs used in this study and T. Arai for helpful suggestions.

Abbreviations

     
  • CK

    casein kinase

  •  
  • GST

    glutathione transferase HEK-293T cells, HEK-293 cells (human embryonic kidney cells) expressing the large T-antigen of SV40 (simian virus 40)

  •  
  • Plk

    polo-like kinase

References

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