The Dual-Specificity Phosphatase CDC14B Bundles and Stabilizes Microtubules

The Cdc14 dual-specificity phosphatases regulate key eventsin the eukaryotic cell cycle. However, little is known aboutthe function of mammalian CDC14B family members. Here, we demonstratethat subcellular localization of CDC14B protein is cell cycleregulated. CDC14B can bind, bundle, and stabilize microtubulesin vitro independently of its catalytic activity. Basic aminoacid residues within the nucleolar targeting domain are importantfor both retaining CDC14B in the nucleolus and preventing microtubulebundling. Overexpression of CDC14B resulted in the formationof cytoplasmic CDC14B and microtubule bundles in interphasecells. These microtubule bundles were resistant to microtubuledepolymerization reagents and enriched in acetylated ${alpha}$ -tubulin.Expression of cytoplasmic forms of CDC14B impaired microtubulenucleation from the microtubule organization center. CDC14Bis thus a novel microtubule-bundling and -stabilizing protein,whose regulated subcellular localization may help modulate spindleand microtubule dynamics in mitosis.

INTRODUCTION

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Introduction

The mammalian spindle midzone or central mitotic spindle formedbetween the two separating sets of chromatids consists of bundledinterdigitating microtubules (MTs) (18). Only at this time ofthe cell cycle do mammalian cells generate stable and bundledMTs. Numerous proteins required for cell cleavage accumulateat the midzone of mitotic spindles and play important rolesfor the completion of cytokinesis in several experimental systems(18). Proteins belonging to this category include chromosomalpassenger proteins, such as the inner centromere protein (INCENP),Aurora, and survivin; MT motor proteins, such as CHO1/MKLP1and KLP3A; and the polo family kinases (75). Among these proteins,some play essential roles in regulating density and bundlingof the central mitotic spindles and are required for properformation of the spindle midzone (1, 37, 48, 51, 61, 74). Asthe cleavage furrow closes, the midbody is formed at the centerof intracellular bridging MTs (75). Although the function ofthe midbody is still not clear, studies from several experimentalsystems have suggested that the midbody helps coordinate cytokinesisand G₁ phase transition (46, 75).

The dual-specificity phosphatase Cdc14 is conserved from yeastto mammals and plays a variety of roles during cell division(64). In budding yeast Saccharomyces cerevisiae, Cdc14 functionsprimarily to inactivate cyclin-dependent kinase (CDK) activityat the end of mitosis, thereby allowing reentry into G₁ phase.Cells that lack Cdc14 arrest with elongated mitotic spindles,separated chromosomes (49), and high levels of Clb-Cdc28 kinaseactivity (67). Cdc14 inactivates CDK through dephosphorylationof the cyclin-dependent kinase inhibitor Sic1, which is therebystabilized, and dephosphorylation of Cdh1, which is then ableto activate the anaphase-promoting complex, the E3 ubiquitinligase that degrades the Clb B-type cyclins (24). The low CDKstate in G₁ phase is essential for the establishment of originsof DNA replication for the ensuing S phase (8). Incomplete inactivationof CDK activity (for example, in cells that lack the CDK inhibitorSic1) causes massive replication defects and genome instability(30, 43). From G₁ until early anaphase, Cdc14 is sequesteredin an inactive state in the nucleolus (55, 68). Release of Cdc14from the nucleolus and exit from mitosis is dictated by theCdc14 early anaphase release network and mitotic exit network(58, 59). In the fission yeast Schizosaccharomyces pombe, theCdc14 homolog Clp1/Flp1 appears to function primarily in determiningthe onset of mitosis (7, 65). In addition to its role in mitoticexit in budding yeast, activation of Cdc14 in early anaphaseleads to dephosphorylation of Sli15, which in turn directs theSli15-Ipl1 (INCENP-Aurora) kinase complex to the anaphase spindleand promotes midzone spindle assembly (45). There is also evidencethat Cdc14 helps mediate spindle assembly in Caenorhabditiselegans, where CeCDC-14 is localized to the central spindleand midbody (16). Suppression of CeCDC-14, using RNA-mediatedinterference, leads to embryonic lethality, primarily due tofailure in establishing central mitotic spindles and cytokinesis(16).

In humans, two CDC14 paralogs, CDC14A and CDC14B, have beenidentified (32). CDC14A dynamically localizes to the interphasecentrosome and plays a role in centrosome separation (27, 33).Similar to the budding yeast Cdc14, CDC14A can dephosphorylateHct1/Cdh1 (3) and INCENP (17). The Xenopus othologs of humanCDC14A, called XCdc14 ${alpha}$ and XCdc14ß, are essential forembryonic division and localize to the nucleolus and centrosome,respectively (26). In contrast to CDC14A, CDC14B has not beenstudied in detail, except that CDC14B associates with nucleoli(27), centrosomes, and intranuclear filaments, and it may beinvolved in nuclear structure maintenance (39).

Here, we show that the CDC14B protein can bind, bundle, andstabilize MTs in vitro independently of its catalytic activity.We demonstrate that CDC14B localizes to the equator of centralspindles in anaphase and to the center of the midbody duringtelophase and cytokinesis. Overexpression of cytoplasmic CDC14Bcauses MT bundling and stabilization in interphase cells. Moreover,MT nucleation from the microtubule organization center (MTOC)was delayed in cells with bundled MTs. The regulated localizationof CDC14B may therefore directly control spindle assembly anddisassembly during mitosis.

MATERIALS AND METHODS

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Materials and Methods

Cell culture and transfection. U-2OS (ATCC), Saos-2 (ATCC), and 293T (ATCC) cells were cultivatedin high-glucose Dulbecco's modified Eagle's medium (Invitrogen),supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin,and 100 µg/ml streptomycin in a 5% CO₂ atmosphere at 37°C.Cells were transfected by FuGene 6 (Roche) or LipofectaminePlus (Invitrogen) according to the manufacturer's instructions.After 21-h transfection of Cdc14B plasmids, cells were treatedwith MT-depolymerizing drugs: nocodazole (10 µg/ml, 2h; Sigma), vinblastine (1 µM, 4 h; Sigma), or Colcemid(500 ng/ml, 4 h; Sigma).

For the nocodazole washout experiment, U-2OS cells transfectedwith different Cdc14B-FLAG plasmids were exposed to nocodazoleat a final concentration of 10 µg/ml for 2 h. The drugwas removed by being rapidly washed three times in warm phosphate-bufferedsaline (PBS). Cells were fixed at 2, 4, 6, 8, 10, and 12 minafter washout in methanol at –20°C and then processedat room temperature for immunofluorescence staining of CDC14Band ${alpha}$ -tubulin.

Cloning and site-directed mutagenesis. Cdc14B open reading frame (ORF) cDNA was amplified by PCR fromthe Marathon human heart cDNA library (Clontech) using the followingprimers: forward, 5'-ACTCCCGGGTCCATGAAGCGGAAAAGCGAGC-3'; andreverse, 5'-AGTCCCGGGTTAACGCAAGACTGTTTTAGTCC-3'. The PCR productwas digested with SmaI and cloned into the SnaBI site of pMX-pievector (a kind gift from Gerry Nolan) carrying an N-terminal6-myc epitope tag. Sequencing analysis of the resulting plasmidconfirmed that the Cdc14B cDNA was identical to hCdc14B in GenBank(AF023158.1), except for a T-to-C transition at position 52from the first ATG. The BamHI/BglII fragment containing themyc-tagged Cdc14B was subcloned into pRSHisGal (MT852) yeastexpression vector. The last 68 bp of Cdc14B were deleted fromthis construct. All of the Cdc14B mutants (C314S, D287A, KKIR29-32AAIA,and KKIR29-32AAIA plus C314S) were generated by site-directedmutagenesis using the QuikChange Site-Directed Mutagenesis kit(Stratagene) and verified by sequencing analysis. Annealingprimers used for site-directed mutagenesis will be providedupon request. Cdc14B expression plasmids were generated by subcloningthe entire Cdc14B ORF into pET16b (Novagen), pGEX-3X (AmershamBiosciences), p3xFlag-CMV-14 (Sigma), and pEGFP-N3 (Clontech)vectors. The inducible expression system of Cdc14B was generatedby subcloning the C-terminal enhanced green fluorescent protein(EGFP)-tagged Cdc14B into pBI-tet vector (Clontech). Cdc14AORF cDNA was amplified by PCR from the Marathon human testiscDNA library (Clontech) using the following primers: forward,5'-CATAGAATTCCATGGCAGCGGAGTCAGGGGAAC-3'; and reverse, 5'-CGCTGCGGCCGCTTAGTAATGAACATATTCAGACTG-3'.The PCR product was cloned into pMXpie. Sequencing analysisof the resulting plasmid confirmed that the Cdc14A cDNA insertwas identical to hCdc14A in GenBank (AF122013). The BamHI/NotIfragment containing myc-tagged Cdc14A was subsequently subclonedinto pRSHisGal and the entire ORF was subcloned into pET16b.MAP4^MTB (MT binding domain, 692 to 1,151 amino acids of MT-associatedprotein 4 [MAP4]) (22) was amplified from a Marathon human braincDNA library (Clontech) using the following primers: forward,5'-GGAGCTCCCACCAAGCCCA-3'; and reverse, 5'-GATGCTTGTCTCCTGGATCTG-3'.It was then cloned into pET16b.

Antibodies and immunofluorescence. Rabbit anti-CDC14A and anti-CDC14B antibodies (affinity purifiedwith CDC14A and CDC14B peptides, respectively) were purchasedfrom Zymed Laboratories, Inc. To increase the antibody specificityfor CDC14B, the antibody was engaged for an additional roundof affinity purification using glutathione-S-transferase (GST)-CDC14Bfusion protein or CDC14B separated by factor Xa from a GST-CDC14Bfusion on a nitrocellulose membrane. The antibody was elutedwith a buffer containing 0.75 ml of 4 M MgCl₂ and 50 µg/mlbovine serum albumin (BSA). The buffer was then exchanged overan NAP-10 column (Sephadex G25 medium, DNA grade; PharmaciaBiotech) equilibrated with 50 µg/ml BSA in PBS. Each fractionwas then tested by immunocytochemical and Western blot analyses.To verify the specificity of the antibody, the CDC14B antibodywas either preabsorbed to a GST-CDC14B fusion protein on beads(depleted) or preincubated with glutathione-eluted GST-CDC14Bprotein in solution (competed). Both the depleted and the competedantibody fractions were used for Western blotting and immunostainingas controls of antibody specificity.

Mouse anti-myc (9E10) and anti ß-actin monoclonalantibodies were obtained from Santa Cruz and Cytoskeleton, Inc.,respectively. For double immunostaining, rabbit anti-CDC14Band mouse anti ${alpha}$ -tubulin (B-5-1-2; Sigma), or mouse anti-acetylated ${alpha}$ -tubulin (6-11B-1; Sigma) antibodies were used. For double stainingof CDC14B-FLAG transfected cells, mouse anti-FLAG M2 (Sigma)and rabbit anti- ${alpha}$ -tubulin (Neo Markers) antibodies were used.Alexa Fluor 568 goat anti-rabbit immunoglobulin G and AlexaFluor 488 goat anti-mouse immunoglobulin G were purchased fromMolecular Probes, Inc.

For immunostaining, U-2OS cells were either grown on coverslipsor in slide chambers. We fixed cells in 100% methanol at –20°C,followed by brief extraction with 0.5% NP-40. In some experiments,cells were also fixed with 2.5% paraformaldehyde in PBS for10 min, followed by 0.1% Triton X-100 extraction at room temperature.Both fixation protocols gave rise to the same staining patternsin this study. Immunofluorescence images were visualized witha Zeiss immunofluorescence microscope and acquired with a charge-coupleddevice camera using the Photoshop software.

Immunoblotting, recombinant protein production, and phosphatase assay. U-2OS cell pellets were lysed in buffer 3 (1% NP-40, 0.1 M Tris,pH 8.0, 0.15 M NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride).To prepare yeast cell lysates, cells propagating in log phasewere collected, and proteins were released by being vortexedin acid-washed glass beads containing 10% trichloroacetic acid.The protein pellets were resuspended in buffer 3, followed bypH adjustment. Forty micrograms of total lysates was separatedby 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE). Proteins were transferred to nitrocellulose membranewith a semidry electrophoresis transfer apparatus (Bio-Rad)and probed with antibodies according to the manufacturer's instructions.Blots were developed using the ECL system (Pharmacia AmershamBiotech).

GST-CDC14B, CDC14B-His, CDC14A-His, and MAP4^MTB-His fusion proteinswere prepared as described according to the manufacturer's instructions.Briefly, Escherichia coli cells carrying GST- or His-taggedplasmids were induced by 1 mM (final concentration) IPTG (isopropyl- $a$ -D-thiogalactopyranoside)for 4 h at 30°C, and soluble fractions were isolated withglutathione-Sepharose beads (Amersham Pharmacia) or nitrilotriaceticacid-Ni beads (QIAGEN), respectively. A phosphatase assay wasperformed at 30°C in a volume of 100 µl containing15 mM ${rho}$ -nitrophenyl phosphate, 50 mM imidazole, pH 6.9, 1 mMdithiothreitol, 1 mM EDTA, and equal amounts of each recombinantprotein ( $~$ 2 µg). The reaction was terminated by the additionof 400 µl of 0.25 N NaOH, and absorbance was read at anoptical density of 405 nm on a spectrophotometer.

In vitro microtubule-binding and -bundling assay. MT-binding assays were performed with the Microtubule AssociatedProtein Spin-Down Biochem Assay kit (Cytoskeleton, Inc.). PurifiedHis-tagged CDC14A, CDC14B, and MAP4^MTB proteins were exchangedinto PEM buffer {80 mM PIPES [piperazine-N,N'-bis(2-ethanesulfonicacid)], pH 6.8, 1 mM EGTA, and 1 mM MgCl₂} using a desaltingspin column (Pierce) immediately prior to the assay. In addition,CDC14B preparations were prespun through 50% glycerol cushion-PEMat 100,000 x g for 15 min and the supernatants were used inthe binding assays. For each assay, MTs were assembled from100 µg of pure tubulins (isolated from bovine brain; Cytoskeleton,Inc.) in 20 µl of PEM containing 1 mM GTP and 5% glycerolat 35°C for 20 min and immediately stabilized in 200 µlof warm PEM-20 µM Taxol (paclitaxel). The MTs (4 µMtubulin) were incubated with CDC14B^wt (0.2 µM), CDC14B^C314S(0.2 µM), CDC14A (0.5 µM), MAP4^MTB (0.5 µM),or BSA (2.3 µM) in a total volume of 50 µl at roomtemperature for 30 min. The reaction mixtures were then centrifugedthrough a 50% glycerol cushion-PEM-Taxol at 100,000 x g at 25°Cfor 15 min. The supernatant and pellet were resolved on 10%SDS-PAGE and the presence of CDC14B, CDC14A, and tubulin wasdetected by immunoblot analysis. Protein abundance in the controlreactions was determined by Coomassie blue staining and densitometry.

For MT-bundling assays, rhodamine-MTs were prepared using amixture of 20 µg of rhodamine-labeled tubulins ( $≥$ 99% pure,from bovine brain; Cytoskeleton, Inc.) and 100 µg of unlabeledtubulins as described above. After incubating MTs (0.4 µMtubulin) with CDC14B (0.04 µM), CDC14A (0.03 µM),MAP4^MTB (0.1 µM), or MAP2 (0.3 µM, from bovine brain;Cytoskeleton, Inc.) at room temperature for 30 min, the mixtureswere immediately dropped onto slides and examined by fluorescencemicroscopy. Each protein of the same concentration for the MT-bundlingassay was incubated with the MTs (0.2 µM tubulin) forelectron microscopic analysis. For each reaction mixture, 2µl was fixed in 60 µl of warm PEM fixative containing1% glutaraldehyde, 1 mM GTP, and 30% glycerol. Five microlitersof the fixed samples was applied to grids with carbon and negativelystained with 2% uranyl formate. All images were recorded atmagnifications of x50,000 or x70,000 under low-dose conditionswith a JEOL 1200 EX II microscope at 100 kV.

Yeast growth conditions. Conditions for growth of the temperature sensitive cdc14-3 mutantstrain were previously described (67). Transformation of yeastcells was performed by the lithium acetate-polyethylene glycolmethod (13). Transformed cells were selected on His dropoffplates with and without 2% galactose at permissive (25°C)and nonpermissive (37°C) temperatures.

RESULTS

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Results

Conservation of CDC14B function. To study the function of human CDC14B, we set out to determinewhether human CDC14B is functionally equivalent to its yeastortholog, Cdc14, by testing the ability of the human gene tocomplement a temperature sensitive (TS) cdc14-3 mutant strainof S. cerevisiae (49). Expression of human CDC14B from the inducibleGAL1 promoter efficiently restored growth of the cdc14-3 mutantstrain at 37°C (Fig. 1A). In contrast, when expressed ata level equivalent to CDC14B, human CDC14A failed to rescuethe cdc14-3 strain (Fig. 1A and B). As a control, Clp1p, a Schizosaccharomycespombe ortholog of Cdc14 also rescued the cdc14-3 defect (65).To determine whether catalytic activity of CDC14B was requiredfor complementation, the conserved cysteine (C314) and aspartate(D287) residues in the active site of CDC14B (15) were mutatedto serine and alanine, respectively. Phosphatase assays confirmedthat the CDC14B^C314S and CDC14B^D287A mutant proteins were catalyticallyinert (Table 1). Neither CDC14B^C314S nor CDC14B^D287A rescuedcdc14-3 lethality at 37°C (Fig. 1A). Human CDC14B is thereforea functional counterpart of the S. cerevisiae Cdc14 phosphatase.

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FIG. 1. (A) Expression of human CDC14B complements cdc14-3 defects at nonpermissive temperatures. The temperature-sensitive mutant cdc14-3 cells containing the indicated plasmids were tested for colony formations on yeast-peptone-dextrose plates with or without (w/o) galactose (Gal) at nonpermissive (37°C) and permissive (25°C) temperatures. Human CDC14B^C314S and CDC14B^D287A are catalytic "dead" mutants (Table 1) (15). Clp1, a Schizosaccharomyces pombe ortholog of S. cerevisiae Cdc14, was used as a positive control (65). (B) Western blot analysis of Myc-tagged human CDC14A and CDC14B expression using anti-myc antibody (9E10) in cdc14-3 cells transformed with pGAL-Cdc14A (lane 1), pGAL-Cdc14B (lane 2), and vector alone (lane 3). All plasmids were constructed in pRSHisGal (MT852) yeast expression vector.

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TABLE 1. Summary of in vivo and in vitro studies of CDC14B

Subcellular localization of CDC14B protein is cell cycle regulated. In S. cerevisiae, Cdc14 is regulated through sequestration inthe nucleolus (55, 68). To investigate whether a similar mechanismoperates for human CDC14B, we examined the subcellular localizationof CDC14B in U-2OS cells at different stages of the cell cycle.To do so, we developed a highly purified anti-CDC14B antibodyby subjecting a commercial affinity-purified anti-CDC14B antibody(Zymed Laboratory, Inc.) to an additional round of purificationagainst a GST-CDC14B fusion protein. Immunoblot analysis demonstratedthat the purified antibody recognized a 62-kDa protein in U-2OScells and that this species was not detected after predepletionor competition of the antibody with GST-CDC14B protein (Fig.2A, left). This antibody preparation specifically recognizedFLAG-tagged CDC14B expressed in transfected U-2OS cells butnot FLAG-tagged CDC14A (Fig. 2A, right). Longer exposure ofthis blot revealed a species at a molecular weight consistentwith endogenous CDC14B but not endogenous CDC14A (data not shown).In agreement with a previous report (27), immunostaining ofasynchronous U-2OS cells using the affinity-purified antibodyrevealed that CDC14B localized in nucleoli during interphaseand dispersed throughout the cell during prophase and metaphase(Fig. 2B, top). Significantly, we also found that CDC14B accumulatedat the spindle midzone in anaphase, at the dense center of themidbody in telophase, and along the intracellular bridge atthe termination of cytokinesis (Fig. 2B, top). To affirm thespecificity of the CDC14B staining, we showed that the anti-CDC14Bantibody preincubated with GST-CDC14B did not reveal any CDC14Bsignal, regardless of the cell cycle stage (Fig. 2B, panel "compete").We verified the specificity of midbody staining by colocalizationof a midbody protein, AIM-1 (data not shown) (62). Identicalsubcellular localization of CDC14B was observed with 293T andSaos-2 cells (data not shown).

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FIG. 2. Subcellular localization of human CDC14B protein in U-2OS cells at different stages of the cell cycle. (A) Characterization of anti-CDC14B antibody. (Left) Western blot analysis of endogenous CDC14B protein with anti-CDC14B antibody (original, Zymed Lab, Inc.), affinity-purified anti-CDC14B antibody with GST-CDC14B fusion protein (purified), anti-CDC14B antibody that has been preblocked with GST-CDC14B (compete) and anti-CDC14B antibody that has been predepleted with GST-CDC14B (deplete). (Right) U-2OS cells transiently transfected by FLAG-tagged-Cdc14A (lane 1) and -Cdc14B (lane 2) were analyzed by Western blotting using affinity-purified anti-CDC14B, anti-FLAG antibodies, or anti-CDC14B antibody that had been predepleted with purified GST-CDC14B protein (deplete). (B) U-2OS cells were labeled with affinity-purified antibody against CDC14B (red), monoclonal antibody against ${alpha}$ -tubulin (green), and DAPI for DNA (blue). Representative CDC14B localizations in nucleoli, spindle midzone, midbody, and intracellular bridge at different stages of a cell cycle are shown. Note that CDC14B localizes to the equator of central spindle in anaphase, is confined to the dense center of midbody flanked by bundled MTs in telophase, and stretches out along intracellular bridging MTs at the end of cytokinesis. No nucleolar, midzone, or midbody staining was evident when the same anti-CDC14B antibody was preblocked with GST-CDC14B (compete).

The spindle midzone in anaphase contains bundled central MTsand many chromosomal passenger proteins (75). To determine therelationship between CDC14B and bundled central MTs, we performeddouble immunofluorescence staining using anti-CDC14B and anti- ${alpha}$ -tubulinantibodies in U-2OS cells. In anaphase, CDC14B accumulated atthe midzone in a region with overlapping MTs, while in telophase,CDC14B localized to the center of the midbody, which was flankedby the bundled MTs (Fig. 2B, bottom). At the end of cytokinesis,when cells were approaching the final stage of separation, CDC14Bbecame dispersed and localized along the intracellular bridgeas the bundled MTs became thin and confined within the intracellularbridge (Fig. 2B, bottom).

CDC14B directly binds microtubules in vitro. The localization of CDC14B in central spindles suggests thatCDC14B may interact with MTs. To test this hypothesis, we performedan MT cosedimentation assay using His-tagged CDC14B (CDC14B-His)and Taxol-stabilized MTs assembled from purified tubulins. MTswere incubated with CDC14B-His in solution and then sedimentedthrough a glycerol cushion by centrifugation. For these assays,it was essential that the CDC14B protein be freshly prepared(data not shown). At a tubulin:CDC14B molar ratio of 20:1 (basedon the densitometry of the input proteins in Coomassie-stainedgels), approximately 60% of the input wild-type CDC14B-His cosedimentedwith MTs, whereas in the absence of MTs CDC14B-His remainedin the supernatant (Fig. 3A). Because the sedimentation efficiencyof MTs under these conditions was also approximately 60%, CDC14Bwas quantitatively bound to the purified MTs in this assay.As controls, after incubation with MTs, His-tagged MAP4^MTB (22)was recovered with similar efficiency, while BSA remained exclusivelyin the supernatant (Fig. 3B, bottom). We then determined whetherthe interaction with MTs required CDC14B catalytic activity.Similar to wild-type CDC14B, catalytically inactive CDC14B^C314Sproteins associated efficiently with MTs (Fig. 3A). Taken together,these results demonstrate that CDC14B can directly and specificallybind to MTs in vitro in a manner that appears to be independentof its catalytic activity. As a fraction of CDC14A was recentlyshown to associate with the spindle poles and the central spindle(17), we tested the possibility that CDC14A might also bindto MTs. Indeed, purified CDC14A-His was able to bind to MTswith a binding efficiency 11-fold higher than that of the backgroundlevel, although capture was somewhat less efficient than forCDC14B (Fig. 3B). The ability to interact directly with MTsmay thus be a general property of the Cdc14 phosphatase family.

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FIG. 3. CDC14B and CDC14A are MT-binding proteins. CDC14B^wt-His, CDC14B^C314S-His, or CDC14A-His proteins were incubated in the presence (+) or absence (–) of Taxol-stabilized MTs and then subjected to a glycerol cushion centrifugation. Pellets (P) and supernatants (S) were collected and separated by SDS-PAGE. The presence of CDC14B, CDC14A, and MTs were detected by Western blot analysis (A and B), and the input proteins were quantified on a Coomassie-stained gel (A and B). The input molar ratio of CDC14B^wt-His or CDC14B^C314S-His to MT (tubulin) was 1:20 the input molar ratio of CDC14A-His to tubulin was 1:8. Note that there are two bands in the CDC14A-His input (B) but only the lower one is CDC14A determined by Western blot analysis. MAP4^MTB-His and BSA were used as positive and negative controls, respectively (Coomassie-stained gel) (B, bottom).

Cytoplasmic CDC14B has microtubule-bundling activity in vivo. The direct association of CDC14B with MTs in vitro promptedus to investigate a possible role of CDC14B in MT network regulationin vivo. For this purpose, we transfected FLAG-tagged wild-typeCDC14B (CDC14B^wt-FLAG) into U-2OS cells and then immunostainedcells with anti-FLAG antibody. As reported previously (27),the majority of the transfected cells exhibited colocalizationof CDC14B with interphase nucleoli and nuclei (Table 1). However,in 14% of the cells, CDC14B^wt-FLAG also appeared to be bundledas perinuclear rings or MT-like bundled structures. Double immunostainingwith anti-FLAG and anti- ${alpha}$ -tubulin antibodies revealed that theCDC14B^wt-FLAG bundles partially colocalized with MT networks(Fig. 4A, panels a to f). To further investigate possible physicalinteractions between CDC14B and MT networks, we generated aCDC14B mutant that predominantly localizes in the cytoplasm,where MT networks are normally assembled. The first 54 aminoacids of CDC14B contain a potential nucleolar targeting domain,the removal of which results in relocalization of the majorityof CDC14B protein into the cytoplasm (27). Within this domain,amino acid residues 7 to 32 (⁷RRSSWAAAPPCSRRCSSTSPGVKKIR³²)contain a potential bipartite nuclear localization sequence(K/R₂-X_10-20-K/R₃; in boldface type in the sequence above) (9).To abrogate nuclear localization, we thus mutated the threebasic residues at positions 29, 30, and 32 to alanines (CDC14B^{KKIR29-32AAIA},abbreviated as CDC14B^KKIR). In comparison with CDC14B^wt-FLAGtransfected cells, the number of cytoplasmic CDC14B bundlesin CDC14B^KKIR-FLAG-transfected cells was significantly increased(t test; P < 0.01) (Table 1). All of the cytoplasmic CDC14B^KKIR-FLAGprotein appeared in rings or MT-like bundles and partially colocalizedwith MTs (Fig. 4A, panels a to f). This pattern of CDC14B bundlesresembled that of other MT-associated proteins, which oftenform bundled rings and irregular filaments when overexpressedin cells (2, 29, 34, 57, 63, 70, 72). CDC14B^KKIR-FLAG did notappear to colocalize with cytoskeletal intermediate filamentsor actins by double immunostaining with anti-FLAG and anti-vimentin(VIM-3B4; Chemicon) antibodies, or with anti-FLAG antibody andfluorescein-conjugated phalloidin (Molecular Probes, Inc.) (datanot shown). These results suggest that the ²⁹KKIR³² motif restrainsthe ability of CDC14B to form cytoplasmic bundles, presumablythrough nucleolar and/or nuclear sequestration of CDC14B.

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FIG. 4. Overexpressed CDC14B proteins form cytoplasmic bundles and stabilize MT network. (A) Immunofluorescence staining of U-2OS cells transfected with CDC14B^wt- and CDC14B^KKIR-FLAG (see also Table 1). Twenty-one hours after transfection, cells were fixed and immunostained with anti-FLAG and ${alpha}$ -tubulin antibodies (No treatment). Representative images of CDC14B cytoplasmic bundles or perinuclear rings (arrows) and nucleolar or nuclear expression (arrowhead) are shown (a and d). CDC14B^K&C- and CDC14B^C314S-FLAG proteins showed similar bundling structures. However, the ratio of bundles versus nucleolar expression differed (see Table 1 for details). All cytoplasmic CDC14B bundles partially colocalized with the bundled MTs (c and f). After nocodazole (10 µg/ml; 2 h) and Colcemid (500 ng/ml; 4 h) treatment, MT structures were completely disrupted in the nontransfected cells (h and, k). High dose of vinblastine (1 µM; 4 h) resulted in the formation of tubulin-vinblastine paracrystals (n). In contrast, in cells expressing cytoplasmic CDC14B bundles, MTs remained bundled (h, k, and n) and colocalized with bundled CDC14Bs (i, l, and o). Red, CDC14B; green, tubulin; and blue, DNA. (B) In vitro MT-bundling assay. The MT-bundling activities of CDC14B and CDC14A were examined by incubating purified CDC14B^wt-His, CDC14B^KKIR-His, or CDC14A-His proteins with rhodamine-labeled MTs. Each reaction mixture was then applied to slides for examination under fluorescence (top) and electron microscopes (bottom). MAP4^MTB-His (22) and MAP2 proteins (23) were used as positive and negative controls, respectively. Note that MTs in the presence of CDC14B^wt-His, CDC14B^KKIR-His, and CDC14A-His are long and thick in comparison with MTs in MAP2 reaction (top). High magnification (x50,000) of MTs showed that CDC14B^wt-His, CDC14B^KKIR, CDC14A-His, and MAP4^MTB-His caused extensive MT bundles (bottom). Scale bar, 200 nm. (C) CDC14B stabilizes MTs in vitro. Rhodamine-labeled MTs were incubated with purified CDC14B^wt-His or MAP2 for 30 min at room temperature. Subsequently, the reaction mixtures were placed on ice for 30 min, followed by fluorescence microscopic examination. Cold-resistant MT bundles were also observed when CDC14B^KKIR-His, CDC14B^C314S-His, and CDC14B^K&C-His proteins were used.

To determine whether CDC14B catalytic activity is required forits cytoplasmic translocation and MT bundling, we generateda double mutant by changing the active cysteine into serine(C314S) in a CDC14B^KKIR mutant (CDC14B^{KKIR29-32AAIA&C314S},abbreviated as CDC14B^K&C). Approximately 20% of CDC14B^K&C-FLAG-transfectedcells exhibited cytoplasmic CDC14B bundles, which partiallycolocalized with MT bundles (Table 1). The percentage of cellswith bundles appeared to vary somewhat among cells transfectedwith different Cdc14B constructs, ranging from 59% (CDC14B^KKIR),20% (CDC14B^K&C), and 14% (CDC14B^wt) to 4% (CDC14B^C314S)(Table 1). Since the extent of cytoplasmic CDC14B^K&C localizationwas about twofold less than that of CDC14B^KKIR (t test; P <0.005), it is possible that Cdc14B catalytic activity is requiredfor Cdc14B release from the nucleoli and/or its maintenancein the cytoplasm. In all cases, however, each of the CDC14Bvariants showed very similar bundled structures and colocalizationwith MTs, regardless of catalytic activity. These findings suggestthat the catalytic activity of CDC14B is not required for inductionof MT bundles in the cytoplasm or for localization of CDC14Bwithin these MT bundles. Similar results were also obtainedwhen wild-type or mutant CDC14B was expressed as GFP fusionsfrom a doxycycline-regulatable promoter (see Table S1 in thesupplemental material). We noticed that the CDC14B^wt-EGFP cellsalso formed intranuclear filamentous structures and associatedwith centrosomes, depending on the level of protein expression(see Fig. S1 in the supplemental material) in agreement witha recent report (39).

MT-bundling activity of CDC14B. The collective evidence that CDC14B localizes to midzone centralspindles (Fig. 2B), directly binds to MTs in vitro (Fig. 3),and causes MT bundling in vivo (Fig. 4A, panels a to f) suggeststhat CDC14B might be a bona fide MT-bundling protein. We thereforeexamined the effects of CDC14B in an in vitro MT-bundling assaybased on the visualization of MTs from rhodamine-labeled tubulins.Purified CDC14B^wt-His or CDC14B^KKIR-His proteins were incubatedwith rhodamine-MTs, and the reaction mixtures were examinedimmediately by fluorescence microscopy. CDC14B stimulated theformation of thick and long MTs (Fig. 4B, top), comparable tothe MTs bundled by the MAP4^MTB-His protein used as a positivecontrol (22). A similar result was obtained when CDC14A-Hiswas used in the MT-bundling assay (Fig. 4B, top). In contrast,MTs were thin and short in the presence of the MAP2 protein,which is unable to support MT bundling in vitro (5, 28). Thinand short MTs were also observed when BSA was used as a negativecontrol (data not shown). To confirm that the thick and longMTs were indeed MT bundles, we examined the reaction mixturesby electron microscopy. Compared with a single MT seen in thepresence of MAP2, ordered arrays of multiple MTs were observedin the presence of CDC14B^wt-His, CDC14B^KKIR-His, CDC14A-His,and MAP4^MTB-His (positive control) proteins (Fig. 4B, bottom).CDC14B^KKIR-bundled MTs often formed tighter bundles and containedmore MTs than CDC14B^wt-bundled MTs, suggesting that the ²⁹KKIR³²sequence may partially impair MT bundling, in addition to servingas a nuclear localization signal. All of the CDC14B proteinsthat bound to MTs also appeared to support MT bundling in vitro,even those mutants devoid of phosphatase activity (Table 1).The results obtained in this purified system suggest that bothCDC14A and CDC14B have intrinsic MT-bundling activity.

To examine whether the MT-bundling activity of CDC14B playsa role in MT regulation, we first tested MT stability by treatingU-2OS cells with MT depolymerization reagents. Twenty-one hoursafter transfection with various Cdc14B-FLAG constructs (Table1), cells were treated with nocodazole (10 µg/ml; 2 h),Colcemid (500 ng/ml; 4 h) or vinblastine (1 µM; 4 h) andthen double labeled with anti-FLAG and anti- ${alpha}$ -tubulin antibodies.In nontransfected cells, nocodazole and Colcemid completelydisrupted MT networks (Fig. 4A, panels h and k), while treatmentwith vinblastine induced multiple vinblastine-tubulin paracrystals(56) (Fig. 4A, panel n). However, in cells bearing cytoplasmicCDC14B bundles, the MT network structures were refractory todisruption by each of the drugs tested (Fig. 4A, panels h, k,and n). Importantly, in the drug-treated cells the stabilizedMTs colocalized exclusively with cytoplasmic CDC14B (Fig. 4A,panels i, l, and o), in comparison with the partial colocalizationof MTs with CDC14B in untreated cells (Fig. 4A, panel c). Totest whether CDC14B could directly stabilize MTs independentlyof any other MT-associated proteins, we examined the effectof CDC14B on MT cold sensitivity in the in vitro-purified system.Most mammalian MTs are cold sensitive and undergo rapid disassemblyat low temperatures (69). When MTs were premixed with CDC14B-Hisprotein and incubated on ice for 30 min, there was little orno depolymerization (Fig. 4C). In contrast, in the presenceof MAP2, most MTs disassembled upon incubation at the low temperature(Fig. 4C).

CDC14B stabilizes microtubules that are enriched in acetylated ${alpha}$ -tubulins. Acetylated ${alpha}$ -tubulin accumulates in stable MTs (44, 47, 52, 60,71). To further verify the MT stabilization by CDC14B, we examinedthe tubulin acetylation status of the drug-resistant MTs. Whennocodazole-treated cells expressing CDC14B bundles were examinedusing an antibody specific to acetylated ${alpha}$ -tubulin, we foundthat the nocodazole-resistant MTs were enriched with acetylatedtubulins and completely colocalized with the CDC14B bundles(Fig. 5A, top). Colocalization of acetylated ${alpha}$ -tubulin and CDC14Bwas also detected in the absence of nocodazole treatment (Fig.5A, bottom). Immunoblot analysis revealed that acetylated tubulinsaccumulated in Cdc14B^KKIR-FLAG-transfected cells (Fig. 5B, lanes4 and 9), regardless of nocodazole treatment. Because the majorityof the ectopically expressed CDC14B^wt, CDC14B^K&C, and CDC14B^C314Sproteins were constrained to the nucleoplasm and nucleoli (Table1), we were not able to detect accumulation of acetylated tubulinin these cells (Fig. 5B, lanes 2, 3, 5, 7, 8, and 10). Thesefindings strengthen the suggestion that CDC14B is an MT-stabilizingprotein.

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FIG. 5. Effect of cytoplasmic CDC14B bundles on ${alpha}$ -tubulin acetylation. (A) Colocalization of acetylated tubulins and CDC14B bundles in the presence (+ NZ, 10 µg/ml and 2 h) or absence (– NZ) of nocodazole treatment. (B) Expression of cytoplasmic CDC14B leads to accumulation of acetylated tubulins. After transient transfection, U-2OS cells were incubated with (+) or without (–) nocodazole for 2 h. Cell lysates were resolved on 10% SDS-PAGE and Western blotting was performed by using anti-acetylated tubulin, ${alpha}$ -tubulin, FLAG, and ß-actin antibodies, respectively. Mock transfection (lanes 1 and 6), transfection with CDC14B^K&C-FLAG (lanes 2 and 7), CDC14B^C314S-FLAG (lanes 3 and 8), CDC14B^KKIR-FLAG (lanes 4 and 9), and CDC14B^wt-FLAG (lanes 5 and 10).

Cytoplasmic CDC14B delays microtubule nucleation from MTOCs. MTs emanating from MTOCs in interphase cells can rapidly reorganizeto form a bipolar mitotic spindle during cell division. Thedynamic behavior of these MTs can be analyzed by measuring thekinetics of MT nucleation from MTOCs (12, 47). Analysis of MTsrecovered after nocodazole treatment revealed that while MTnucleation from MTOCs still occurred, these events were considerablydelayed in cells bearing cytoplasmic CDC14B bundles. Two minutesafter nocodazole washout, nontransfected cells exhibited longMTs radiating from MTOCs (Fig. 6, top right panel), whereasno obvious MTOC could be identified in the cytoplasmic CDC14Bexpressing cells (Fig. 6, top left panel). Similar results wereobserved at 4, 6, 8, and 10 min after nocodazole washout (datanot shown). Twelve minutes after washout, the majority of nontransfectedcells developed normal MT arrays (Fig. 6, bottom), as describedby others (47, 57). At this time point, however, only a smallfraction of cells bearing cytoplasmic CDC14B bundles had initiatedMT nucleation from MTOCs (Fig. 6, bottom, right). Despite impairmentof MT nucleation by cytoplasmic CDC14B, we did not observe anyalteration of centrosome copy numbers by immunostaining for ${gamma}$ -tubulin (data not shown). These results suggest that alterationof CDC14B subcellular localization can alter MT dynamics andthat CDC14B-mediated MT stabilization may delay MT nucleation.

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FIG. 6. Expression of cytoplasmic CDC14B bundle delays MT nucleation from microtubule organization center. Twenty-one hours after transfection with CDC14B^KKIR-FLAG, cells were exposed to the MT depolymerization drug nocodazole (10 µg/ml) for 2 h and then washed out of nocodazole, followed by 2-, 4-, 6-, 8-, and 12-min incubation in warm fresh culture medium. Representative results from 2- and 12-min time points are shown. Transfected CDC14B proteins and MTs were visualized by being stained with anti-FLAG (left) and ${alpha}$ -tubulin (right) antibodies. Two minutes after nocodazole washout, long MTs were nucleated from MTOCs in nontransfected cells (2 min; arrowhead). In contrast, MTs remained bundled and no obvious MTOC appeared in cells with CDC14B bundles at the 2-min time point. Twelve minutes after nocodazole washout, all of the nontransfected cells appeared to be repopulated by relative normal MT arrays. In contrast, only a small portion of CDC14B-bundled cells just started to nucleate MTs from semiprominent MTOCs (arrowhead).

DISCUSSION

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Discussion

Human CDC14B is the functional ortholog of budding yeast Cdc14,as judged by its ability to complement yeast strains defectivein Cdc14 function and the analogous spatial regulation evidentin both yeast and human cells. In budding yeast, Cdc14 is sequesteredin the nucleolus prior to anaphase through binding to its nucleolarinhibitor Net1/Cfi1 (55, 68). Release of Cdc14 from the nucleolusin the late stages of mitosis allows the dephosphorylation ofsubstrates and subsequent exit from mitosis (67). We suggestthat a similar nucleolar sequestration mechanism may in partserve to prevent CDC14B from directly interacting with interphaseMTs in human cells. Failure to prevent this interaction willhave dire effects on MT networks and dynamics. As there is noobvious metazoan homolog of Cfi1/Net1, it has yet to be determinedhow sequestration of CDC14B in the nucleolus is controlled.However, the potential bipartite NLS containing the ²⁹KKIR³²sequence within CDC14B could interact with importin ${alpha}$ and ßin a manner similar to that of other MT-associated proteins,such as NuMA and TPX2 (25). Alternatively, it is possible thatthis motif may mediate an interaction of CDC14B with an unknownnucleolar anchor protein(s). Regardless of the precise mechanism,reimport of CDC14B into the nucleus and/or nucleolus after mitosismust be necessary to prevent disruption of nascent MT networksin G₁ phase.

The cell cycle-dependent subcellular localization of CDC14Bto spindle structures resembles that of other midzone-midbody-associatedproteins, for example, PRC1, CHO1, and PTP-BL (20, 36, 37).Sequence analysis of CDC14B did not reveal any consensus repetitivemotifs commonly observed in the MT binding domains of otherMAPs, such as Tau, MAP2, and MAP4 (10, 41), suggesting thatCDC14B may contain novel MT binding motifs. The induction ofdrug- and cold-resistant MT bundles associated with CDC14B overexpressionindicates that CDC14B can regulate MT stability. Many otherMAPs such as Tau, MAP2, and NuSAP are capable of stabilizingMTs both in vivo and in vitro (31, 50, 60). During anaphase,central overlapping MTs become bundled to form the midzone centralspindle (35). Successful completion of cytokinesis depends onthe timely and spatially regulated assembly of the midzone (6,14, 73). Because CDC14B localizes to the midzone and can bundleand/or stabilize MTs, CDC14B may be involved in the regulationof central spindle assembly and mitotic MT dynamics. Indeed,genetic evidence suggests an analogous function for the C. elegansCdc14 ortholog (16). While the manuscript was under review,Higuchi and Uhlmann demonstrated that yeast Cdc14 indeed functionsto stabilize mitotic MT dynamics and suggested that downstreamtargets of Cdc14 are involved in this process (21). Thus, MT-bindingand -stabilizing activity may be a general and conserved featureof all Cdc14 family members. A recent study indicates that CDC14Bmay have an unexpected role in maintenance of nuclear structure(39). In agreement with this suggestion, we found that a portionof CDC14B was localized to centrosomes when expressed at a lowlevel but to intranuclear filaments and cytoplasmic bundleswhen expressed at a high level from a doxycycline-regulatablepromoter (see Fig. S1 in the supplemental material). CDC14Bmay thus play a number of roles in mitosis, including spindleand centrosome regulation and control of nuclear architecture.At this point, genetic evidence in support of this notion inmammalian cells is lacking, in part because small interferingRNA and short hairpin RNA approaches fail to completely eliminateCDC14B protein and cause little or no defect in tissue culturecells (reference 39 and data not shown). Genetic analysis ofCDC14B function must await generation of CDC14B null cell linesor CDC14B knockout mice. Interestingly, ectopic expression ofPLK1, Cdc5-like polo-like kinase 1 (38), in CDC14B knockdowncells led to severe nuclear deformation and cytokinesis defects(39). This synthetic genetic interaction between PLK1 and CDC14Bmay in part reflect the fact that the two proteins regulatecytokinesis in the same pathway, in agreement with our findingthat CDC14B may be involved in the regulation of central spindleformation.

Our observation that acetylated ${alpha}$ -tubulin accumulates in MT bundlesof CDC14B transfected cells is consistent with the fact thatoverexpression of other MT-associated proteins, such as MAP1B,MAP2, or TAU, increases MT stability and ${alpha}$ -tubulin acetylation(60). In several experimental systems, acetylated ${alpha}$ -tubulinsare most abundant in stable MTs but absent from more dynamicMT structures (44, 47, 52, 60, 71). Concordantly, we find thatCDC14B protein normally localizes in the spindle midzone andthe center of the midbody, precisely when acetylated tubulinsaccumulate in the stable MTs (47). It is possible that underphysiological conditions, CDC14B participates in the establishmentand/or maintenance of MT stabilization in the spindle midzoneeither through directly stabilizing and bundling MTs and/orthrough modulating tubulin acetylation. Recently, it has beensuggested that CDC14B dephosphorylates a tubulin deacetylase,SIRT2, which in turn triggers SIRT2 ubiquitination and degradation(11, 42). It is conceivable that CDC14B exerts its effects onMT stabilization in part through SIRT2 degradation and consequentaccumulation of acetylated tubulins. However, we cannot ruleout the possibility that CDC14B stabilizes MTs and that thesestabilized MTs accumulate acetylated tubulins, as suggestedby Palazzo et al. in drug-induced tubulin acetylation experiments(44).

Cytoskeletal MTs undergo continuous assembly and disassemblyduring the cell cycle and development (12, 19, 54). We havefound that inappropriate localization of CDC14B to the cytoplasmdelays MT nucleation from MTOCs. The timely regulated exit ofCDC14B from the nucleolus during the cell cycle may well becritical to avoid disruption of MT dynamics. Recently, Saitoet al. demonstrated that CDC14 promotes cell cycle arrest invarious tissues of C. elegans, possibly through regulating CKI-1protein stability (53). It is possible that CDC14 participatesin both cell cycle regulation (53) and microtubule organization(16) in C. elegans. Indeed, constitutive expression of cytoplasmicCDC14B causes MT bundling and stabilization and arrests cellsin G₁ (our unpublished observations). Overexpression of theMT-associated protein MAP4 also stabilizes MTs and significantlydelays G₁/S transition (40), while the anticancer agent Taxolinduces MT hyperpolymerization and blocks untransformed cellsin late G₁ through an as-yet-uncharacterized MT-dependent G₁checkpoint mechanism (66).

As a dual-specificity phosphatase, CDC14B executes its functionsin part through dephosphorylation of proteins that are phosphorylatedby proline-directed kinases (15, 27). The spectrum of Cdc14substrates encompasses a number of proteins important for CDKregulation. A spindle-associated function has been establishedfor Cdc14 in yeast, as it has been demonstrated that dephosphorylationof Sli15 by Cdc14 directs Sli15-Ipl1, an INCENP/Aurora kinase-likecomplex, to the spindle midzone in a step important for centralspindle assembly (45). Since INCENP is phosphorylated priorto mitosis in higher organisms (4), the intrinsic MT-stabilizingand -bundling properties of CDC14B may serve to direct CDC14Bto the central spindle where it coordinates central spindleassembly with INCENP dephosphorylation. Indeed, CDC14A localizesto the central spindle and can dephosphorylate mitotic INCENP(17); furthermore, we demonstrated here that CDC14A can bindand bundle MTs in vitro. Given that both CDC14A and CDC14B arepresent in the central spindle, it will be important to determinewhether the two phosphatases act in synergy to regulate INCENPdephosphorylation and central spindle formation. In additionto the catalytic functions of Cdc14 in spindle dynamics, wehave found that CDC14B can unexpectedly bind, stabilize, andbundle MTs independently of its phosphatase activity. Thus,CDC14B may directly stabilize and organize the central spindleindependently of any intermediate substrate. To our knowledge,this is also the first instance in which the biological functionof a phosphatase has been segregated from its intrinsic phosphataseactivity. Elucidation of the mechanisms that regulate CDC14Band the means whereby it alters MT dynamics will undoubtedlybe crucial for understanding mitotic transition in the normaland cancer cell cycles.

ACKNOWLEDGMENTS

We thank Lea Harrington and Angelika Amon for critical readingof the manuscript and Ashley Davis for technical support regardingthe in vitro MT-bundling assay. We also thank Angelika Amonfor the cdc14-3 strain and Kathy Gould for the pGAL-clp1 plasmid.

Y.W. acknowledges the support of the Laboratory Directed Researchand Development Program (LDRD), Oak Ridge National Laboratory,and the Office of Biological and Environmental Research, U.S.Department of Energy, under contract DE-AC05-00OR22725 withUT-Battelle, LLC. H.P.C. is a postdoctoral fellow supportedby LDRD. M.T. is supported by grants from the Canadian Institutesof Health Research and holds a Canada Research Chair in FunctionalGenomics and Bioinformatics.

FOOTNOTES

* Corresponding author. Mailing address: Life Sciences Division, Oak Ridge National Laboratory, Bethel Valley Rd., Oak Ridge, TN 37831. Phone: (865) 574-5396. Fax: (865) 574-5345. E-mail: ywa{at}ornl.gov.

Supplemental material for this article may be found at http://mcb.asm.org/.

REFERENCES

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