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Originally published In Press as doi:10.1074/jbc.M400482200 on March 15, 2004 Originally published In Press as doi:10.1074/jbc.M400482200 on March 11, 2004

J. Biol. Chem., Vol. 279, Issue 20, 21367-21373, May 14, 2004
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The Polo Box Is Required for Multiple Functions of Plx1 in Mitosis*

Junjun Liu{ddagger}, Andrea L. Lewellyn, Lin G. Chen, and James L. Maller§

From the Howard Hughes Medical Institute and Department of Pharmacology, University of Colorado School of Medicine, Denver, Colorado 80262

Received for publication, January 15, 2004 , and in revised form, March 11, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Polo-like kinases comprise a family of evolutionarily conserved serine/threonine protein kinases that play multiple roles in cell cycle regulation. In addition to the N-terminal catalytic domain, polo-like kinases have one or two highly conserved C-terminal non-catalytic regions, termed polo boxes. These motifs are required for targeting these kinases to subcellular mitotic structures. Here we report that kinase-dead Xenopus polo-like kinase (Plx1NA) functions as a competitor of endogenous Plx1 for polo box binding site(s) and inhibits the activation of Cdc25C and the G2-M transition in vivo. However, kinase-dead Plx1 with a point mutation in the polo box region (Plx1NAWF) did not have inhibitory effects. The ability of Plx1NA to block activation of the anaphase-promoting complex/cyclosome also requires an intact polo box. Microinjection of Plx1NA but not Plx1NAWF mRNA into Xenopus embryos caused cleavage arrest and formation of monopolar spindles, an effect previously seen in embryos injected with anti-Plx1 antibody. Spindle assembly experiments in vitro also showed that only monopolar spindles formed in Xenopus egg extracts supplemented with recombinant Plx1NA and that the spindle assembly process was delayed. Taken together, these results indicate that the polo box is required for Plx1 function in both the G2-M and the metaphase/anaphase transitions during the cell cycle.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Polo-like kinases (Plks)1 are a family of conserved mitotic serine/threonine kinases. Several members of this kinase family have been found in both mammalian cells and Xenopus, including mammalian Plk1 (Xenopus Plx1), Plk2/Snk (Xenopus Plx2), Plk3/Prk/Fnk (Xenopus Plx3), and Sak (1-4). In other species, however, only one form of the kinase has been found, including Polo in Drosophila, Plo1 in Schizosaccharomyces pombe, and Cdc5 in Saccharomyces cerevisiae. Plks participate in several stages of mitosis, including mitotic entry, mitotic exit, and cytokinesis (reviewed in Ref. 4).

All Plks share a closely related catalytic domain in the N terminus and one or two characteristic sequence motifs, the polo boxes, in the non-catalytic C terminus (reviewed in Ref. 4). The polo boxes are thought to localize the respective kinase to various mitotic structures during cell cycle progression, including centrosomes in early M phase, the spindle midzone in early and late anaphase, and the midbody in cytokinesis, presumably to promote interaction of the kinase with specific substrates and effectors (5, 6). The same motifs may also regulate Plk activity by binding to and inhibiting the kinase domain (6). Recently, the C-terminal motifs were implicated in stabilizing the protein level of polo-like kinases by interacting with Hsp90 (7). Sak has only one polo box, and the crystal structure of murine Sak revealed the formation of intermolecular homodimers both in vitro and in vivo (8), suggesting that Plks with two polo boxes might also form heterodimers. A single point mutation, W414F in Plk1, which most likely destroys the integrity of the polo box domain structure, dramatically affects the ability of human Plk1 to complement the cdc5-1 defect in yeast and disrupts Plk localization at spindle poles and septin ring structures (5).

Plx1 positively regulates the G2-M transition by phosphorylating and activating Cdc25C, which in turn dephosphorylates and activates Cdc2 (9, 10). Both Plx1 and the related kinase, Prk, have been shown to physically associate with Cdc25C (10, 11). At the beginning of mitosis, various proteins including Plks are recruited to the centrosomes during the "maturation" process (12, 13). Centrosomal localization may underline the requirement of Plks for establishment of a bipolar spindle (13, 14), a function first observed in a Drosophila polo mutant (12, 15). This is a conserved function, inasmuch as microinjection of Plx1 or Plk1 antibody into Xenopus embryos or HeLa cells results in the formation of monopolar spindles (13, 14). Several lines of evidence indicate the important role of Plks in mitotic exit, possibly by participating in activation and inactivation of the anaphase-promoting complex/cyclosome (APC/C). The vertebrate APC/C is a ubiquitin-protein isopeptide ligase (E3), a 20S multiprotein complex consisting of at least 10 core subunits, which, in combination with a ubiquitin-activating enzyme (E1), a ubiquitin carrier protein (E2 or UBC), and an activator protein, Cdc20/Fizzy, promotes degradation of substrate proteins by the 26S proteasome (16-18). Of the three ubiquitination components, the APC/C seems to be the only one whose activity oscillates during the cell cycle. It has been reported that in some systems Plks are able to phosphorylate APC/C subunits, including Cdc16 and Cdc27, jointly with Cdc2-cyclin B (18-20), and this phosphorylation may be required for Fizzy to fully activate the APC/C (21). The activated APC/C subsequently ubiquitinates and promotes degradation of mitotic regulators, including cyclin B, leading to the inactivation of Cdc2 and exit from mitosis. The polo box of Plo1 interacts with Cut23 (APC/C8), a subunit of the APC/C in fission yeast, during mitosis (22), and the disruption of this interaction results in metaphase arrest. This finding suggests that the interaction is crucial for mitotic exit by directing Plo1 activity toward the APC/C and activating the complex. This idea is supported by the ability of kinase-dead Plx1 to block APC/C activation in Xenopus egg extracts (23, 24). Plks also appear to promote the onset of cytokinesis in a polo box-dependent manner. Cdc5 with conservative mutations in the polo box is unable to localize to cytokinetic neck filaments and fails to induce elongated cells with ectopic septin ring structures (5, 25). Consistent with this notion, Cdc11 and Cdc12, two septins thought to be the major structural components of the neck filaments (26), have been identified as polo box-interacting proteins (27).

Although Plks play a pivotal role in the cell cycle, especially during mitosis, so far much of the work about the role of the polo box has focused on subcellular localization and cytokinesis, and little is known about the role of the polo box in mitotic entry and exit. To better understand the role of the polo box for Plk function, we have used the Xenopus system to examine the involvement of the polo box in mitotic entry and exit. For this purpose, we generated three Xenopus Plx1 mutants (Fig. 1): Plx1NA, a kinase-dead Plx1 whose kinase activity is abolished by a N172A point mutation in its kinase domain (14); Plx1NAWF, a kinase-dead Plx1 with a point mutation, W408F, in its first polo box, equivalent to the W414F mutation in Plk1, which has been demonstrated to dramatically abolish the localization capability of the motifs (5); and Plx1WF, a protein with the same polo box point mutation but with intact kinase activity. In this report, we show that the activation of Cdc25C and the G2-M transition is dependent on the polo box of Plx1 and that Plx1-dependent regulation of APC/C activity during exit from mitosis requires an intact polo box.



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  		FIG. 1.
Structures of Plx1 and Plx1 mutants. Schematic representation of the Plx1 constructs used in this study: Plx1, wild-type Plx1; Plx1WF, Plx1 with a point mutation, W408F, in polo box 1; Plx1NA, Plx1 with a point mutation, N172A, in the kinase domain, which encodes a kinase-dead Plx1; and Plx1NAWF, Plx1 with both point mutations, N172A and W408F. The black box represents the catalytic domain, and the gray boxes represent polo box 1 and polo box 2 (left and right gray box, respectively). Recombinant proteins produced in Sf9 cells are tagged with His6 residues at the C termini, and mRNAs encode a FLAG tag at the C termini.

 

   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Constructs—A W408F mutation in polo box 1 was introduced into Plx1 and Plx1-N172A by PCR using pOTV-Plx1 (Plx1) and pOTV-Plx1-N172A (Plx1NA) (14) as templates, with primers having the following sequences: 5'-ATATTCTGGATCAGCAAATTCGTGGATTACTCGGACAAATACG-3' and 5'-CGTATTTGTCCGAGTAATCCACGAATTTGCTGATCCAGAATAT-3'. These constructs, which encode proteins with a FLAG tag at the C terminus, were used for transcription of mRNA for Plx1NA and Plx1NAWF. Baculovirus expression constructs were created by PCR using the above-mentioned four constructs as templates, with pairs of primers having the following sequences: 5'-GACGACGACAAGATGGCTCAAGTGGCCGGTAAGAAAC-3' and 5'-GAGGAGAAGCCCGGTTTAGTGGTGGTGGTGGTGGTGTGCCGAGGCCTTTACGTGTG-3'. The underlined sequences are Plx1 sequences, and the non-underlined sequences are Sf9 expression vector pBac-2cp LIC (Novagen) overhang-compatible sequences. In the case of the second primer, the non-underlined sequence also includes a His6 tag and a stop codon. The full-length Plx1, Plx1WF, Plx1NA, or Plx1NAWF cDNAs were then subcloned into pBac-2cp.

Generation of mRNAs and Recombinant Proteins—Plx1NA and Plx1NAWF mRNAs were transcribed with the T7 mMESSAGE mMACHINE kit (Ambion), phenol/chloroform purified, and dissolved in nuclease-free H2O (Ambion) at a final concentration of 1 mg/ml. The Plx1, Plx1WF, Plx1NA, or Plx1NAWF recombinant proteins were expressed in Sf9 cells by the University of Colorado Cancer Center Core facility for tissue culture, and the proteins were purified as described previously (14).

Oocyte Microinjection and Detection of Cdc25C—Stage VI oocytes were injected with 40 nl of mRNA encoding FLAG-tagged Plx1NA or Plx1NAWF proteins (1 mg/ml) and incubated in 2% Ficoll and 0.1x MMR (10 mM NaCl, 0.2 mM KCl, 0.1 mM MgSO4, 0.2 mM CaCl2, and 0.5 mM HEPES) at 17 °C overnight. After addition of progesterone (final concentration, 10 µM) to the medium, groups of three oocytes were harvested at the indicated times, frozen on dry ice, and stored at -80 °C until further analysis. Extracts were prepared, and Cdc25C was detected with rabbit anti-Cdc25C antibody as described previously (14).

Preparation and Manipulation of CSF Extracts, Immunoblotting, and H1 Kinase Assay—Metaphase II-arrested (CSF) extracts were prepared as described previously (28). Due to the activity of CSF, these extracts are arrested at meiosis II with a high level of cyclin B/Cdc2 activity. The arrest can be released by addition of calcium, causing exit from metaphase arrest and proteolysis of cyclins by the APC/C. Each individual reaction was made by transferring 100 µl of extract into a 1.5-ml microcentrifuge tube on ice and then adding 7 µl of buffer or recombinant Plx1 proteins (1.5 mg/ml), as indicated. After a 20-min incubation on ice, CSF arrest was released by addition of 1 µl of 50 mM CaCl2, and incubation was carried out at 21 °C. Samples of 2.5 µl were taken at the indicated times, mixed with 22.5 µl of extraction buffer (14), frozen on dry ice, and stored at -80 °C. For immunoblotting, 8 µl of the above-mentioned diluted sample was used. Cyclin B1 and B2 were detected with sheep anti-cyclin B1 and anti-cyclin B2 antibodies prepared as described previously (28), Cdc27 was blotted with anti-Cdc27 monoclonal antibody (BD Sciences), and histone H1 kinase activity was determined as described previously (14).

In Vitro Spindle Assembly, Microinjection, Immunofluorescence, and Microscopy—To assess the effect of Plx1NA on mitotic cyclin degradation, 30 µl of CSF extract was supplemented with 1.2 µl of buffer or Plx1NA or Plx1NAWF recombinant protein (1.5 mg/ml) on ice for 20 min. Xenopus sperm nuclei (final concentration, 100-300/µl) were then added, followed by addition of calcium to release CSF arrest. After 60 min, 1 µl of extract was mixed with 4 µl of fixation/stain buffer (48% glycerol, 11% formaldehyde, 1x MMR (100 mM NaCl, 2 mM KCl, 1 mM MgSO4, 2 mM CaCl2, and 5 mM HEPES), and 1 µg/ml DAPI), and the sample was monitored with an Olympus BH2 fluorescence microscope. The remaining extract was centrifuged onto a coverslip through 5 ml of BRB80 (80 mM PIPES, 1 mM MgCl2, 1 mM EGTA, 40% glycerol) in a HB-4 rotor in a Sorvall centrifuge at 12,000 rpm for 10 min. The sample on the coverslip was fixed with methanol at -20 °C for 5 min, blocked with 3% bovine serum albumin in BRB80 for 1 h, and blotted with anti-{alpha}-tubulin monoclonal antibody for 1 h, followed by three 5-min washes with PBS and staining with donkey anti-mouse Cy3-conjugated antibody and DAPI (Molecular Probes). The sample was observed with a Nikon microscope.

To assess the effect of Plx1NA on spindle assembly during re-entry into mitosis in a CSF extract, 20 µl of CSF extract was supplemented with Xenopus sperm nuclei (final concentration, 100-300/µl), and CSF arrest was released by the addition of 0.2 µl of 50 mM CaCl2. Forty-five min after the release, 1.5 µl of buffer or Plx1NA or Plx1NAWF recombinant protein was added, and the extract was further incubated at room temperature for another 35 min. Then, 20 µl of fresh CSF extract was added to drive the extract back into mitosis. Sixty min later, 1 µl of the extract was subjected to DAPI staining, and nuclear morphology was assessed as described above. Seventy min after the addition of the fresh CSF extract, the spindles were spun down onto a coverslip (29), stained, and observed as described above.

To assess the effect of Plx1NA on cell division, embryos at the two-cell stage were injected in both blastomeres with 40 nl of mRNA encoding FLAG-tagged Plx1NA or Plx1NAWF proteins (1 mg/ml), cultured in 0.1x MMR for 5 h, and fixed with Dent's solution (20% Me2SO in methanol). {alpha}-Tubulin was detected with anti-{alpha}-tubulin monoclonal antibody (Sigma) and visualized by Cy3-conjugated donkey anti-mouse IgG antibody (Molecular Probes), and DNA was stained with SYTOX Green (Molecular Probes). Confocal microscopy was performed with a PCM2000 microscope (Nikon).

Immunoprecipitation—In each individual immunoprecipitation, 15 µl of Dynabeads protein G (Dynal) was washed and equilibrated with PBS and then blocked with 5% bovine serum albumin in PBS for 2 h at 4 °C. The Dynabeads protein G was conjugated with 10 µl of anti-His monoclonal antibody (200 µg/ml; Sigma) by rotation in 1 ml of PBS at 4 °C for 1.5 h, followed by three 5-min washes with cold PBS at 4 °C. In the case of Cdc27 immunoprecipitation, 5 µl of His6-tagged recombinant Plx1 protein (1.5 mg/ml) was incubated with the protein G beads-antibody complex, followed by three 5-min washes with cold PBS at 4 °C. The protein G immunocomplex was then incubated with 100 µl of CSF extract in 1 ml of cold PBS in the presence of 1x protease inhibitor mixture (Roche Applied Science) at 4 °C for 2 h. The immunoprecipitates were washed three times (15 min each) with 1 ml of cold PBS at 4 °C, resuspended in 30 µl of SDS-PAGE sample buffer, and analyzed by gel electrophoresis and Western blotting.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The Activation of Cdc25C by Plx1 in Xenopus Oocytes Is Polo Box-dependent—Entry into mitosis depends on phosphorylation and activation of Cdc25C, a dual-specificity phosphatase that dephosphorylates and activates the Cdc2-cyclin B complex (28, 30-32). A variety of evidence indicates that Plx1 functions upstream of Cdc25C and triggers the activation of the phosphatase, although Cdc2-cyclin B can also phosphorylate Cdc25C in a positive feedback loop (9, 14, 33-35). The observation that microinjection of anti-Plx1 antibody or Plx1NA recombinant protein (see Fig. 2) into Xenopus oocytes significantly delays the activation of Cdc25C (14) led us to investigate whether the phosphorylation of Cdc25C by Plx1 is polo box-dependent. We reasoned that if Plx1NA competes with endogenous Plx1 for binding to Cdc25C, then it likely would inhibit the phosphorylation of this substrate by endogenous Plx1 and delay or inhibit the maturation of Xenopus oocytes. On the other hand, Plx1NAWF should have a minimal effect on oocyte maturation because the W408F point mutation abolishes the binding capability of the polo box. We injected either Plx1NA or Plx1NAWF mRNA into G2-arrested stage VI Xenopus oocytes and sampled groups of three oocytes at the indicated times. The activation of Cdc25C is evident as slower-migrating phosphorylated forms on SDS-PAGE (28, 36, 37). As shown by immunoblotting (Fig. 2A), the activation of Cdc25C from oocytes injected with Plx1NA mRNA was delayed for 2.5 h compared with control oocytes injected with buffer. The delay in Cdc25C activation also delayed the G2-M transition as monitored by nuclear envelope breakdown (germinal vesicle breakdown). Oocytes injected with Plx1NAWF mRNA, however, were only slightly delayed relative to the buffer control.



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  		FIG. 2.
Plx1NA significantly delays the activation of Cdc25C. A, stage VI Xenopus oocytes were microinjected with 40 nl of either buffer (top two panels), Plx1NA mRNA (1 mg/ml; middle two panels), or Plx1NAWF mRNA (1 mg/ml; bottom two panels). After an overnight incubation at 17 °C, the oocytes were activated by the addition of progesterone (final concentration, 10 µM), and groups of three oocytes were frozen at the indicated times (numbers at top, hours after progesterone addition). Samples of extracts were immunoblotted for Cdc25C, and the expression of Plx1 was monitored by blotting with anti-FLAG horseradish peroxidase (Sigma). At 6.5 h after progesterone treatment, the percentage of oocytes that had undergone germinal vesicle breakdown was assessed by white spot formation indicative of nuclear envelope breakdown (right panel). The percentage of germinal vesicle breakdown was 90%, 5%, and 80% for oocytes injected with buffer, Plx1NA, and Plx1NAWF, respectively. Similar results were obtained in three independent experiments. B, to assess the effect of Plx1NA on entry into mitosis in vitro, 45 min after calcium addition, a CSF extract containing sperm nuclei was supplemented with either buffer, Plx1NA recombinant protein, or Plx1NAWF recombinant protein for 35 min, and then the extract was driven back into mitosis by addition of an equal volume of fresh CSF extract (29). Sixty min later, 1 µl of each extract was stained with DAPI and examined by fluorescence microscopy. Based on the morphology of sperm nuclei, the extracts supplemented with buffer (a) and Plx1NAWF recombinant protein (c) had entered mitosis, whereas the extract supplemented with Plx1NA recombinant protein (b) was in interphase. Similar results were observed in three independent experiments.

 
Plx1NA, but not Plx1NAWF, Delays Entry into Mitosis in Vitro—To further investigate whether a functional polo box is needed for mitotic entry, we used an in vitro system to monitor the effect of Plx1NA or Plx1NAWF on entry into mitosis. Xenopus egg extracts arrested at meiotic metaphase II by CSF activity can be released by addition of calcium, leading to entry into interphase (38). At room temperature, the extracts replicate DNA, and upon addition of fresh CSF extract, they reenter mitosis (29, 38). To observe the effect of Plx1NA and Plx1NAWF on mitotic entry, we added the recombinant proteins to the extracts in interphase after calcium release. Sixty min after addition of fresh CSF extract, which drove the extracts back into mitosis (29), both extract incubated with buffer and extract incubated with Plx1NAWF recombinant protein had re-entered mitosis (Fig. 2B, a and c), as evidenced by the morphology of the sperm nuclei. However, mitotic entry in extracts incubated with Plx1NA was significantly delayed, and interphase nuclei were observed. This result strongly suggests that the polo box is required for Plx1 to regulate mitotic entry, presumably by mediating interaction between Plx1 and Cdc25C.

Both Kinase Activity and the Polo Box Are Needed for Plx1 to Regulate APC/C Activity at Mitotic Exit—At the end of mitosis, the APC/C is activated and cyclin B is destroyed, leading to reduction of histone H1 kinase activity and exit from mitosis. Plks have been reported to be involved in the regulation of APC/C activity in two different ways. First, they are thought to phosphorylate the APC/C jointly with Cdc2-cyclin B to enable Fzy/Cdc20 to activate the APC/C (19). Second, their activity is required to prevent premature inactivation of the APC/C, possibly by antagonizing the effect of an unidentified microcystin-sensitive phosphatase, which dephosphorylates the APC/C and inactivates it (39). Kinase-dead Plx1 inhibits the reduction of histone H1 kinase activity and the degradation of cyclin B during CSF release initiated by addition of calcium (23, 24), thereby blocking mitotic exit. The inhibitory effect of Plx1NA on cyclin B degradation was thought to be due to the suppression of Plx1 activity that keeps APC/C active. However, subsequent immunoprecipitation experiments showed that Plx1NA did not irreversibly reduce endogenous Plx1 activity.2 The discovery that the polo box of Plo1 interacts with Cut23 (22), a subunit of APC/C in fission yeast, led us to examine the possibility that Xenopus Plx1 might regulate APC/C activity in a polo box-dependent way. The previous observation that kinase-dead Plx1 inhibited the release of CSF activity might reflect competition with endogenous Plx1 for polo box-binding site(s), as in the case of Cdc25C activation. To test this hypothesis, we compared the effect of Plx1NA and Plx1NAWF on CSF release. We reasoned that because Plx1NAWF has no polo box-dependent localization/binding capability, it should not compete with endogenous Plx1 for its substrates and therefore should have a minimal effect on CSF release. CSF-arrested Xenopus egg extracts containing either Plx1NA or Plx1NAWF recombinant protein were released by the addition of calcium. Samples taken at the times indicated were immunoblotted for cyclin B1 and B2 levels (Fig. 3A). Because the mobility of Cdc27 is another indicator of APC/C activation and cyclin B degradation (40, 41), we also blotted Cdc27 for its mobility change reflective of phosphorylation/dephosphorylation. Extracts not released by calcium served as a negative control showing stabilized cyclin B levels and unchanged Cdc27 mobility (Fig. 3A, left panel). Consistent with previous reports (23, 24), Plx1NA protein inhibited the degradation of both cyclin B1 and B2 (Fig. 3A, right panel) in the first 20 min after calcium addition. The Plx1NAWF protein, however, had no effect on cyclin B degradation. Although Plx1NAWF did not affect the mobility change of Cdc27 compared with the control, Plx1NA delayed the shift of Cdc27, suggesting that its phosphorylation was regulated directly or indirectly by Plx1. The histone H1 kinase activity of Cdc2-cyclin B correlated well with the cyclin B levels in each sample (Fig. 3B). To verify the mitotic status of the extracts, we added Xenopus sperm nuclei to the extracts and examined their morphology 20 min after the addition of calcium. Nuclei in Plx1NA-supplemented extracts showed mitotic morphology (Fig. 3C, b), whereas the nuclei from control and Plx1NAWF-supplemented extracts exhibited interphase morphology (Fig. 3C, a and c). This result was further verified by examining the sperm nuclei spun down onto a coverslip. As shown in Fig. 3C, bottom panels, metaphase spindles were recovered from the Plx1NA-supplemented extract (e), and interphase nuclei were recovered from the other two extracts (d and f). These results suggest that the inhibition of CSF release by Plx1NA is through competition with endogenous Plx1 for substrate(s) whose phosphorylation appears to be important for activating the mitotic cyclin proteolysis pathway.



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  		FIG. 3.
Plx1NA, but not Plx1NAWF, inhibits the degradation of cyclin B and delays activation of the APC/C. A metaphase-arrested Xenopus egg extract (CSF extract) is arrested at meiosis II due to the activity of CSF, with a high level of cyclin B protein and a high level of cyclin B/Cdc2 kinase activity. Upon the release of CSF arrest by addition of calcium, anaphase commences, and B-type cyclins are destroyed by the APC/C; consequently, histone H1 kinase activity is reduced, and the extract exits mitosis. We compared the effect of Plx1NA and Plx1NAWF recombinant proteins on this process. A, analysis of cyclin B and Cdc27. In a CSF extract supplemented with buffer without calcium, the cyclin B1 and B2 levels and Cdc27 phosphorylation status remained unchanged (left panel). When a CSF extract was supplemented with either buffer or Plx1NAWF, and the CSF activity was subsequently released by addition of calcium, the cyclin B1 and B2 levels in the extract decreased, and Cdc27 changed to a faster-moving form (right panel). However, in the extract supplemented with Plx1NA, both cyclin B1 and B2 remained stable after addition of calcium, and the Cdc27 mobility change was delayed, indicative of delayed APC/C activation. Similar results were obtained in four independent experiments. B, histone H1 kinase activity. The stable histone H1 kinase activity of an unreleased CSF extract without calcium is shown in the left panel, and the histone H1 kinase activity of the CSF extracts supplemented with either buffer, Plx1NA, or Plx1NAWF as described in A is shown in the right panel. Similar results were obtained in three independent experiments. C, nuclei and spindle morphology. After incubation of CSF extracts with buffer, Plx1NA recombinant protein, or Plx1NAWF recombinant protein, Xenopus sperm nuclei were added. Sixty min after the addition of calcium, 1 µl of extract was fixed and stained with DAPI and observed by fluorescence microscopy. Top panels (a-c), morphology of the sperm nuclei. Bottom panels (d-f), sperm nuclei of 25 µl of each extract were centrifuged onto a coverslip, stained for DNA and {alpha}-tubulin, and observed by immunofluorescence microscopy. Similar results were observed in two independent experiments. Blue, DNA; red, {alpha}-tubulin. D, Plx1 association with the APC/C is polo box-dependent. A pull-down experiment was carried out using either Plx1 or Plx1WF recombinant protein as bait in CSF extracts (as described under "Experimental Procedures"). The recovered complexes were analyzed by SDS-PAGE and immunoblotting for Cdc27 or Plx1 as indicated.

 
Plx1 Association with the APC/C Is Polo Box-dependent—Because the regulation of APC/C-dependent cyclin proteolysis by Plx1 appears to be polo box-dependent, we wanted to know whether Plx1 is associated with the APC/C, and, if so, whether the association is polo box-dependent. There is evidence that the polo box interacts with Cut23, one of the APC/C subunits in fission yeast (22). This interaction may be necessary to recruit polo kinase to the APC/C to facilitate phosphorylation of other subunits, because there is no evidence to date showing the phosphorylation of Cut23 by Plks. To address this issue, we used immobilized Plx1 or Plx1WF recombinant protein as pull-down bait with CSF extracts, and the complexes were recovered and blotted with an antibody to Cdc27. Western blotting revealed that Cdc27 associated with Plx1 but not with Plx1WF or beads alone (Fig. 3D). Other experiments showed that Plx1NA also co-immunoprecipitates with Cdc27, whereas Plx1NAWF does not (data not shown). These results suggest that the polo box is required for Plx1 to interact with APC/C subunits, either directly or indirectly, to regulate APC/C activity.

Plx1-dependent Reversal of APC/C Inhibition by Plx1NA Requires the Polo Box—If the inhibition of cyclin B degradation by Plx1NA reflects competition for endogenous Plx1 substrate(s), then the addition of an excess amount of wild-type Plx1 recombinant protein should shift the balance of competition in favor of active Plx1 and re-activate cyclin B proteolysis. On the other hand, polo box-deficient Plx1WF, although still kinase active, should not compete with Plx1NA for the binding site(s) and should not significantly restore cyclin B degradation. The point mutation in the polo box does not affect the kinase activity of Plk (5), and the activities in these experiments of both Plx1 and Plx1WF recombinant proteins toward casein are similar (data not shown). This ensures that any difference in effects of Plx1 and Plx1WF are due to solely the polo box mutation. To test this hypothesis, we first incubated a CSF extract with Plx1NA recombinant protein for 20 min, and then an equal amount of Plx1 or Plx1WF recombinant protein was added to the extract, followed by calcium addition. As shown Fig. 4A, left panel, 20 min after CSF release, both cyclin B1 and B2 levels in the Plx1-supplemented extract were reduced to the same level as in the control, and Cdc27 was dephosphorylated at that time in both cases. In contrast, both cyclin B1 and B2 levels in the Plx1WF-supplemented extract remained high 20 min after CSF release, and the mobility of Cdc27 was similar to that of Plx1NA-inhibited extracts, indicating a failure to significantly rescue APC/C activation. The histone H1 kinase activity in the extract was consistent with the level of degradation of cyclin B in each case (Fig. 4A, right panel). The fact that wild-type Plx1 can rescue the cyclin B degradation after Plx1NA has bound to Plx1 substrate(s) suggests that polo box-dependent binding to target molecules is reversible.



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  		FIG. 4.
Plx1, but not Plx1WF recombinant protein, restores cyclin B degradation inhibited by Plx1NA. A, to a CSF extract in which release was inhibited by Plx1NA, Plx1 or Plx1WF recombinant protein was added. In the Plx1-supplemented extract, the cyclin B level was reduced to the same level relative to the control 20 min after the addition of calcium (left panel), and the dephosphorylation of Cdc27 was facilitated. In contrast, the cyclin B level in the Plx1WF-supplemented extract remained relatively stable, and the dephosphorylation of Cdc27 was delayed, similar to the effects seen in the extract supplemented with Plx1NA protein alone. Right panel, the histone H1 kinase activity of each sample at the indicated times correlated with the cyclin B level. Similar results were obtained in three independent experiments. B, the inhibitory effect of Plx1NA on CSF release is partially alleviated by xFzy. When xFzy was added to a CSF extract supplemented with Plx1NA, both cyclin B1 and B2 levels were significantly reduced 20 min after calcium release. The dephosphorylation of Cdc27 was also facilitated, compared with that in the extract with Plx1NA alone. Similar results were obtained in four independent experiments. Right panel, histone H1 kinase activity also indicates partial rescue of CSF release by xFzy.

 
xFzy Significantly Alleviates the Inhibition of Cyclin B Degradation Imposed by Plx1NA—It has been shown that in addition to phosphorylation of the APC/C, two WD-40 repeat-containing proteins, Fizzy (Fzy/Cdc20) and Fizzy-related (Fzr/Cdh1/Hct1), are required for the degradation of mitotic cyclins (42). In both yeast and the somatic cells of higher eukaryotes, the APC/C-dependent proteolytic pathway remains active from anaphase phase to late G1, at which time it is turned off. During this time, APC/C switches from a mitotically phosphorylated form that uses Fzy as activator to a dephosphorylated form that uses Fzr (43-47). However, Fzr is not expressed during early embryonic cell cycles, at least in Drosophila and Xenopus (48, 49), which shortens the window of APC/C activity.

Fzy has been reported to cause CSF release in the absence of calcium, with 3 µM Fzy causing complete degradation of cyclin B2 (50). Inasmuch as Fzy may activate APC/C in the absence of calcium, it was important to determine whether it could also activate the APC/C in the presence of Plx1NA. When we added bacterially expressed Xenopus Fzy (xFzy) at a final concentration of 3 µM to extracts supplemented with Plx1NA, we found that cyclin B degradation was partially restored (Fig. 4B, left panel). Twenty min after release of CSF activity, the level of cyclin B1 was reduced to about one-tenth of the original level, twice the level in the control, and cyclin B2 was reduced to about one-fifth of the original level. This result correlates with the H1 kinase activity in each extract (Fig. 4B, right panel). Even when the dose of xFzy was increased to 4.5 µM, cyclin B was never reduced to the same level as in the control (data not shown). The cyclin B levels in Plx1NA-supplemented extracts, however, remained unchanged. The dephosphorylation of Cdc27 in the xFzy-supplemented extract was delayed compared with the control, but it was noticeably faster than that in the extracts supplemented with Plx1NA alone. This result suggests that Plx1NA may inhibit the APC/C upstream of xFzy.

Plx1NA, but not Plx1NAWF, Causes Cleavage Arrest in Xenopus Embryos and Formation of Monopolar Spindles—The preceding results indicate that the polo box is required at both mitosis entry and exit in Xenopus, by affecting the activation of Cdc25C in oocytes and the activity of the APC/C in egg extracts, respectively. To examine the role of the polo box for mitotic exit in vivo, we injected Plx1NA and Plx1NAWF mRNAs into early Xenopus embryos. As shown in Fig. 5A, one blastomere of a two-cell-stage embryo injected with Plx1NA mRNA showed a dramatic cleavage arrest (Fig. 5A, i), whereas the embryos injected with Plx1NAWF mRNA divided normally relative to the buffer control (Fig. 5A, a and e). Confocal microscopy of the arrested cells revealed the formation of monopolar spindles (Fig. 5A, j-l); the {alpha}-tubulin exhibits an astral pattern with radially distributed chromosomes around the pole, an effect also observed in Plx1 antibody-injected embryos (14). In both buffer and Plx1NAWF controls, however, normal bipolar spindles were formed (Fig. 5A, b-d and f-h). To further study effects on spindles, we carried out a spindle assembly experiment in vitro. As shown in Fig. 5B, b, monopolar spindles were formed in the extracts supplemented with Plx1NA recombinant protein, whereas normal bipolar spindles were observed in both control and Plx1NAWF-supplemented extracts (Fig. 5B, a and c). Plks have been demonstrated to be required for the establishment of bipolar spindles in Xenopus and Drosophila (12-15). The same phenotype observed in embryos injected with both Plx1 antibody and Plx1NA mRNA could be due to the same effect: loss of or interference with Plx1 function. In the former case, the activity of endogenous Plx1 was neutralized by Plx1 antibody (14), whereas in the latter case, the function of endogenous Plx1 was likely suppressed due to loss of polo box-dependent substrate binding.



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  		FIG. 5.
Plx1NA causes cleavage arrest in Xenopus embryos and formation of monopolar spindles. A, one blastomere of a two-cell Xenopus embryo was microinjected with 40 nl of buffer (a), Plx1NAWF mRNA (e), or Plx1NA mRNA (i) and monitored for 5 h. Embryos injected with buffer or with Plx1NAWF mRNA divided normally and formed normal bipolar spindles (b-d and f-h, respectively). The Plx1NA mRNA, however, caused cleavage arrest (i) and formation of monopolar spindles (j-l). Immunoblotting experiments showed that both Plx1 proteins were translated to equal extents (data not shown). Similar results were observed in several experiments. Green, DNA; red, {alpha}-tubulin. B,to further study the formation of monopolar spindles, we examined the spindles assembled in vitro in a CSF extract. Forty-five min after calcium addition to a CSF extract containing Xenopus sperm nuclei, either buffer, Plx1NA recombinant protein, or Plx1NAWF recombinant protein was added to the extract and incubated for an additional 35 min. Then, an equal volume of fresh CSF extract was added to drive the extract back into mitosis. Seventy min later, the spindles formed in the extracts were spun down onto a coverslip, stained for DNA and {alpha}-tubulin, and observed by fluorescence microscopy. As shown, normal bipolar mitotic spindles were formed in the extract supplemented with buffer (a) or Plx1NAWF recombinant protein (c), whereas monopolar spindles were formed in the extracts supplemented with Plx1NA recombinant protein (b). Similar results were obtained in two independent experiments. Blue, DNA; red, {alpha}-tubulin.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Plks play pivotal roles throughout mitosis, and the polo box, a unique domain of kinases in this family, has been shown to be important for Plk function in cytokinesis (25, 51). However, the role of the polo box in earlier stages of mitosis has remained uncharacterized. We show in this report that the polo box of Xenopus Plx1 plays a crucial role in several important mitotic events. Plx1 has been shown to be absolutely required for the activation of Cdc25C during the G2-M transition in Xenopus, and no other kinase appears to be able to substitute for this function of Plx1 (33). However, once activated, Cdc2-cyclin B is capable of activating Cdc25C in a positive feedback loop (52). In this study, Plx1NA delayed but did not completely inhibit the activation of Cdc25C. Because endogenous Plx1 activity is not inhibited by Plx1NA,2 activation of Cdc25C by endogenous Plx1 may be one reason. This result is consistent with evidence that injection of an inhibitory antibody to Plx1 does not completely block Cdc25C activation. It appears that as little as 10% of normal Plx1 activity may eventually cause Cdc25C activation in vivo (14, 33), probably because Plx1 binds to Cdc25C (10). Recently, polo box domains of human, Xenopus, and yeast Plks were shown to recognize similar phosphoserine/threonine-containing motifs, and this motif is present in Cdc25C (53, 54). Mutations in the C-terminal polo box disrupted the interaction of Plk1 with Cdc25C in vivo (54), consistent with the reduced effect of Plx1NAWF on inhibition of Cdc25C activation (Fig. 2).

In this work, we showed that the interaction of Plx1 with the APC/C is polo box-dependent. At this stage, we cannot determine which APC/C subunit the polo box of Plx1 interacts with, and the possibility of indirect interaction with APC/C through other associated proteins cannot be excluded. However, the result further supports the hypothesis that the polo box targets the catalytic domain of Plx1 to its substrate(s), in this case, possibly subunits of APC/C or APC/C regulator(s), to regulate APC/C activity. Our experiments demonstrate that Plx1NA significantly inhibits the activation of APC/C, as judged by the degradation of cyclin B1 (Fig. 4B). One possible mechanism is by affecting the phosphorylation of APC/C subunits, a prerequisite for Fzy to activate the APC/C (21). Plk has been suggested to directly phosphorylate APC/C subunits jointly with Cdc2-cyclin B (19). The phosphorylation of APC/C by either Plk or Cdc2-cyclin B alone does not enable Fzy to fully activate the APC/C. However, after phosphorylation by both kinases, a nearly complete degradation of cyclin B was observed (19). Consistent with these observations, our result showed that when Plx1 activity was suppressed by Plx1NA, possibly through polo box-dependent binding to APC/C subunit(s), xFzy could not fully restore the degradation of cyclin B. Another possibility accounting for APC/C inhibition by Plx1NA is regulation of the association of other inhibitory proteins with the APC/C. One such candidate is Mad2, which associates with Fzy and inhibits its activation (55). Recently, it was shown that this association is regulated through phosphorylation of Mad2 (56), and only unphosphorylated Mad2 associates with Fzy (56). Our unpublished data3 show that Plx1 phosphorylates Mad2 in vitro. It is therefore possible that at mitotic exit, besides the phosphorylation of APC/C subunits, Plx1 activity is also required to phosphorylate Mad2 and dissociate it from Fzy to allow full activation of the APC/C.

During the preparation of this manuscript, Chung and Chen (57) reported that wild-type xFzy failed to trigger CSF release, even if a 16-fold excess of xFzy over the endogenous level was added. However, when a phosphorylation site mutant of xFzy was used, CSF release was achieved (57). This result correlates with previous reports that addition of CaMKII to CSF extracts or calcium ionophore treatment of eggs caused dephosphorylation of xFzy and CSF release (49, 58). This finding suggests that our inability to completely restore cyclin B degradation inhibited by Plx1NA by addition of xFzy might also reflect phosphorylation of the added xFzy.

Plx1 is required for the establishment of bipolar spindles (13, 14), and we showed in this study that this function of Plx1 is polo box-dependent. The detailed mechanism of how Plks regulate bipolar spindle formation is not clear, but the spindle effects may reflect the involvement of Plks in both centrosome duplication and maturation (13, 59). Depletion of Plk by siRNA significantly reduces centrosome amplification in hydroxyurea-treated U2OS cells (59). In HeLa cells, the formation of monoastral microtubule arrays is a predicted result of impaired centrosome maturation, a process that requires Plk activity (13). In HeLa cells injected with anti-Plk1 antibody, each monoastral microtubule array contains two centrosomes that have duplicated but failed to separate sufficiently to form bipolar spindles (13). Both centrosome size and MPM-2 marker acquisition in these monoastral structures are significantly smaller, indicating the necessity of Plk1 function for centrosome maturation/duplication, possibly by recruiting specific proteins necessary for maturation to centrosomes. In support of this, it has been found that Plk is involved in recruiting {gamma}-tubulin and associated molecules to the centrosome (12, 13, 60, 61). The identity of the exact Plk substrate(s) involved in centrosome maturation/duplication remains to be determined.

In conclusion, our data suggest that the polo box targets the catalytic activity of Plx1 to Cdc25C, whose phosphorylation by Plx1 is required to trigger the G2-M transition (33); the polo box is required for Plx1 to execute its function in bipolar spindle formation; and at late mitosis, the polo box directs Plx1 to the APC/C, where it is required for APC/C activation at the metaphase/anaphase transition. This suggests that the functions of Plx1 in mitosis are largely polo box-dependent. The interaction of the polo box with substrate/adaptor molecules provides an important mechanism to ensure the activity of Plks is directed toward its relevant substrates.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grant GM26743-24 and the Howard Hughes Medical Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} An associate of the Howard Hughes Medical Institute. Back

§ An Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed. Tel.: 303-315-7075; Fax: 303-315-7160; E-mail: jim.maller{at}uchsc.edu.

1 The abbreviations used are: Plk, polo-like kinase; APC/C, anaphase-promoting complex/cyclosome; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; CSF, cytostatic factor; DAPI, 4',6-diamidino-2-phenylindole; PBS, phosphate-buffered saline. Back

2 Y. W. Qian and J. L. Maller, unpublished data. Back

3 B. J. Tunquist, J. Liu, and J. L. Maller, unpublished data. Back


   ACKNOWLEDGMENTS
 
We are very grateful to Eleanor Erikson for critical reading of the manuscript and help with chromatographic purification of recombinant proteins, Dr. Yutaka Matsumoto for help with in vitro spindle assembly experiments, and the University of Colorado Cancer Center Core facility for DNA sequencing and recombinant protein expression in Sf9 cells (National Institutes of Health Grant CA46934).



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