Cell Cycle-Dependent Translocation of PRC1 on the Spindle by Kif4 Is Essential for Midzone Formation and Cytokinesis

Cell cycle-dependent translocation of PRC1 on the spindle by Kif4 is essential for midzone formation and cytokinesis

Changjun Zhu and Wei Jiang*

Programs of Cancer Genetics and Epigenetics and Signal Transduction, The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037

Communicated by Erkki Ruoslahti, The Burnham Institute, La Jolla, CA, November 24, 2004 (received for review October 1, 2004) The spindle midzone, a conspicuous network of antiparallel inter- Previously, we identified the human mitotic spindle midzone- digitating nonkinetochore microtubules between separating chro- associated cyclin-dependent kinase (Cdk) substrate protein mosomes, plays a crucial role in regulating the initiation and PRC1 and showed that PRC1 plays an essential role in regulating completion of cytokinesis. In this study, we report the use of midzone formation and cytokinesis (4, 16). PRC1 has MT time-lapse microscopy and a human kinesin endoribonucleases binding and bundling activities, and Cdk phosphorylation of RNase III-prepared short interfering RNA (esiRNA) library to iden- PRC1 appears to be important for negatively regulating PRC1 tify Kif4 as a motor protein that translocates PRC1, a spindle function in early mitosis (4). In this study, we report the midzone-associated cyclin-dependent kinase substrate protein, to identification of Kif4 as a motor protein that translocates PRC1 the plus ends of interdigitating spindle microtubules during the to the plus ends of interdigitating MTs on the spindle during the metaphase-to-anaphase transition. We show that Kif4 binds to metaphase-to-anaphase transition. We also show that Cdk phos- PRC1 through its ‘‘stalk plus tail’’ domains and Kif4 and PRC1 phorylation of PRC1 controls the timing of PRC1 translocation colocalize on the spindle midzone͞midbody during anaphase and by Kif4. Our results indicate that the cell cycle-dependent cytokinesis. Suppression of Kif4 expression by Kif4 esiRNA results translocation of PRC1 by Kif4 plays an essential role in midzone in the inhibition of PRC1 translocation, a block of the midzone formation and cytokinesis. formation, and a failure of cytokinesis. PRC1 translocation and midzone formation can be restored, and the cytokinetic defects can Materials and Methods be rescued in Kif4 esiRNA-treated cells by coexpression of Kif4 but ͞ not its motor dead mutant Kif4md. Furthermore, we show that Plasmids, a Human Kinesin Dynein Endoribonucleases RNase III- cyclin-dependent kinase phosphorylation of PRC1 controls the Prepared Short Interfering RNA (esiRNA) Library, and Antibodies. The timing of PRC1 translocation by Kif4. These results, in light of the cDNAs of full-length coding region of Kif4 and its point crucial role of PRC1 in midzone formation, indicate that cell cycle- mutation or deletion mutant were generated by PCR and dependent translocation of PRC1 by Kif4 is essential for midzone subcloned into BglII–SalI sites of the mammalian expression formation and cytokinesis. vector pEGFP-C1. Fig. 1 summarizes these Kif4 constructs: (i) EGFP-Kif4; (ii) EGFP-Kif4md (motor dead), in which the Kif4 kinesin ͉ cargo ͉ cyclin-dependent kinase ͉ phosphorylation ͉ mitosis ATP-binding Walker A site GKT (amino acid residues 93–95) were changed to AAA; (iii) EGFP-motor plus stalk domains of he spindle midzone, a conspicuous network of antiparallel Kif4; (iv) EGFP-stalk plus tail domains of Kif4; (v) EGFP-motor Tinterdigitating nonkinetochore microtubules (MTs) between domain of Kif4; (vi) EGFP-stalk domain of Kif4; and (vii) separating chromosomes, has been shown to play an essential EGFP-tail of Kif4. Full-length human PRC1 cDNA (16) was role in regulating the initiation and completion of cytokinesis in subcloned into EcoRI͞SalI sites of the mammalian expression animal cells (1). Midzone assembly occurs during the met- vector pEYFP-C1 or pFLAG-1. pECFP-H2B plasmids were aphase-to-anaphase transition, at the time of cleavage furrow generated as described (17). All constructs were fully sequenced. initiation. Recent studies from cultured mammalian cells, Cae- A human kinesin͞dynein esiRNA (18) library will be described norhabditis elegans and Drosophila, have begun to reveal factors elsewhere (unpublished work). that are involved in the midzone assembly process. These factors Rabbit anti-Kif4 antibodies (481͞482) were generated against include the kinesin-like motors and the associated proteins, His-tagged Kif4-C-term fusion protein (amino acids 1000–1232). chromosomal passenger proteins, kinases, phosphatase, and the Alexa Fluor 594-conjugated anti-PRC1 antibodies were made spindle midzone bundling protein PRC1 (2–7). following the instruction for Amine-Reactive Probes (Molecular The kinesins are a family of MT-based motor proteins that Probes). Anti-Cdk-phosphorylated PRC1 (T481) antibodies generate directional movement along MTs and are involved in were purchased from Santa Cruz Biotechnology. All secondary many crucial cellular processes including cell division (8). Gene antibodies were purchased from Jackson Immunoresearch. disruption experiments in Saccharomyces cerevisiae indicated that five of six kinesins play crucial roles in regulating formation Cell Culture, Transfection, Immunoprecipitation, Immunoblotting, and of the spindle and segregation of chromosomes in mitosis (9). In Immunofluorescence Analyses. HeLa cells were cultured in 6- or higher eukaryotes, several studies have shown that a number of 24-well plates in DMEM supplemented with 10% FBS and kinesins are crucial for spindle assembly and function, chromo- transfected with 100 nM esiRNA and͞or 0.2–1 ␮g of plasmid(s) CELL BIOLOGY some segregation, mitotic checkpoint control, and cytokinesis by using Oligofectamine (Invitrogen). Two to 3 days after (10–12). For instance, the midzone-associated kinesin MKLP-1 transfection, cells were harvested or fixed for immunoprecipi- was reported to play an essential role in regulating midzone͞ midbody formation and cytokinesis in various organisms (5, 7). Klp3A, a Drosophila homologue of human kinesin-4 member Abbreviations: esiRNA, endoribonucleases RNase III-prepared short interfering RNA; MT, Kif4, was shown to be essential for midzone formation and͞or microtubule; EYFP, enhanced yellow ﬂuorescent protein; ECFP, enhanced cyan ﬂuorescent maintenance during cytokinesis (13–15). However, the precise protein; Cdk, cyclin-dependent kinase. role for Klp3A (or Kif4) in regulation of midzone formation is *To whom correspondence should be addressed. E-mail: [email protected]. not clear. © 2004 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0408438102 PNAS ͉ January 11, 2005 ͉ vol. 102 ͉ no. 2 ͉ 343–348 Downloaded by guest on October 1, 2021 with or without 0.2 ␮g of baculovirus-expressed purified Cdc2͞ cyclin B1 in 20 ␮l of Cdk kinase buffer containing 100 ␮MATP at 30°C for2hasdescribed (16). After washing with cell lysis buffer, the beads were incubated with 150 ␮g of early mitotic HeLa cell lysates at 4°C for 2 h followed by washing three times with 1 ml of cell lysis buffer. Beads-bound Kif4 protein was analyzed by immunoblotting with anti-Kif4 antibodies. Results and Discussion Kif4 Is Required for the Spindle Midzone͞Midbody Localization of PRC1 and Cytokinesis in Human Cells. PRC1 associates with the mitotic spindle poles and the mitotic spindle in early mitosis and then translocates to the spindle midzone͞midbody during anaphase and telophase (ref. 16 and Fig. 5, which is published as supporting information on the PNAS web site). Because PRC1 is not a MT-based motor protein, we hypothesized that the movement of PRC1 along the spindle during mitosis and cytokinesis would be regulated by motor proteins. To identify such motor protein(s), we screened a human kinesin͞dynein esiRNA library that we recently generated (see Materials and Methods)by time-lapse microscopy using HeLa cells expressing enhanced yellow fluorescent protein (EYFP)-PRC1 and enhanced cyan fluorescent protein (ECFP)-histone H2B fusion proteins. HeLa cells were simultaneously transfected with EYFP-PRC1 and ECFP-H2B plasmids together with individual human kinesin͞ dynein esiRNAs, and live cell images of transfected cells were obtained by time-lapse microscopy. Fig. 1A and Movie 1, which is published as supporting information on the PNAS web site, show time-lapse fluorescent and phase-contrast images of con- Fig. 1. Abnormal subcellular localization of PRC1 and failure of cytokinesis trol (luciferase) esiRNA-transfected HeLa cells expressing in HeLa cells treated with Kif4 esiRNA. HeLa cells grown on glass-bottom microwell dishes were transfected with pEYFP-PRC1 and pECFP-H2B together EYFP-PRC1 and ECFP-H2B. Consistent with the previous with 100 nM control (luciferase) (A) or Kif4 (B) esiRNA. Thirty-six hours immunofluorescence analysis (4), ectopically expressed EYFP- posttransfection, cells were placed on a 37°C heated stage, and time-lapse PRC1 localized in cytoplasmic cytoskeleton arrays in interphase images were collected every 2 min for 10 h with a Zeiss Axiovert 100M inverted and then localized on the mitotic spindle during mitosis whereas ﬂuorescence microscope and an automatic digital charge-coupled device ECFP-H2B was in the nuclei in interphase and associated with camera. The movies were edited with SLIDEBOOK 3.0 software. Representative chromosomes during mitosis. EYFP-PRC1 associated with the images of the movies are shown (EYFP-PRC1, pseudocolored red and ECFP- mitotic spindle poles and the mitotic spindle in early mitosis, H2B, pseudocolored green). Blue arrows indicate midzone͞midbody localization of PRC1 in control esiRNA-treated cells, and black arrows indicate abnor- translocated to the spindle midzone during anaphase, and then mal spindle localization of PRC1 in Kif4 esiRNA-treated cells. The numbers concentrated to the midbody during cytokinesis. Like untrans- indicate time periods in minutes. For details, see Movies 1 and 2. fected cells, cells expressing EYFP-PRC1 and ECFP-H2B pro- gressed through mitosis and cytokinesis normally. Cotransfection of the majority of kinesin esiRNAs with EYFP- tation, immunoblotting, or immunofluorescence analysis as de- PRC1 and ECFP-H2B in HeLa cells did not perturb the dynamic scribed (16). subcellular localization of EYFP-PRC1 during mitosis and cytokinesis. However, cells transfected with esiRNA against one Time-Lapse Microscopy. HeLa cells grown on 35-mm glass-bottom kinesin, Kif4, displayed striking abnormalities of subcellular microwell dishes (MatTek, Ashland, MA) were transfected with localization of EYFP-PRC1 during mitosis and cytokinesis. As 0.5 ␮g of pEYFP-PRC1 and pECFP-H2B plasmids together with shown in Fig. 1B and Movie 2, which is published as supporting 100 nM of Kif esiRNAs by using Oligofectamine (Invitrogen). information on the PNAS web site, although EYFP-PRC1 Twenty-four hours after transfection, cells were cultured with localized on the mitotic spindle in Kif4 esiRNA-treated cells in CO2-independent medium (GIBCO) containing 10% FBS over- early mitosis, it could not translocate to the spindle midzone͞ night. Then, dishes were covered with mineral oil (Sigma) and midbody during anaphase and telophase. Instead, EYFP-PRC1 transferred to a heated stage (37°C) on a Zeiss Axiovert 100M was found along the entire mitotic spindle. Kif4 esiRNA-treated microscope. Phase-contrast and fluorescence images of live cells cells formed very elongated anaphase spindles with separating were collected at 2-min intervals for 9–10 h and processed by chromosomes that moved extremely close to the spindle poles; using SLIDEBOOK 3.0 software (Intelligent Imaging Innovations, midzone formation was not evident. As the cleavage furrow Santa Monica, CA). formed and ingressed in Kif4 esiRNA-treated cells, the EYFP- PRC1-associated mitotic spindle twisted around. Ultimately, Cell Cycle Synchronization and Pull-Down Assay. For cell cycle these cells failed to complete cytokinesis and became binucle- synchronization, HeLa cells were first treated with 2 mM ated cells. Perturbation of translocation of endogenous PRC1 to thymidine for 16 h, released into fresh medium for 6 h, and then the midzone͞midbody in Kif4 esiRNA-treated cells also was blocked with medium containing 40 ng͞ml nocodazole for 12 h observed by immunofluorescence analysis (Fig. 5). Because as described (16). Mitotic cells were collected by shake-off, PRC1 is crucial for spindle midzone formation and cytokinesis washed with PBS, transferred into fresh medium, and harvested (4, 16), the results suggested that Kif4 might be the motor protein every 20 min. that translocates PRC1 along the mitotic spindle to the plus ends For pull-down assay, 1 ␮g of bacterially expressed glutathione- of interdigitating MTs, which is necessary for organizing mid- agarose beads with bound GST or GST-PRC1 were incubated zone formation and cytokinesis.

344 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0408438102 Zhu and Jiang Downloaded by guest on October 1, 2021 Kif4 and PRC1 Associate with Each Other and Colocalize on the Mitotic Spindle During Mitosis and Cytokinesis. If Kif4 is the motor protein that translocates PRC1 along the mitotic spindle during mitosis, it should interact with PRC1. To test this, human 293 cells were cotransfected with mammalian expression plasmids expressing FLAG-tagged PRC1 together with EGFP (control), EGFP- tagged Kif4 (WT), or EGFP-tagged Kif4md (a motor dead mutant in which the ATP binding Walker A consensus site was mutated from GKT to AAA) (Fig. 2A). Two days after transfection, cells were lysed, and cell lysates were immunoprecipitated with anti-FLAG antibody. Immunoblotting analysis with anti-GFP antibody showed that EGFP-Kif4 and EGFP-Kif4md coimmunoprecipitated specifically with FLAG-PRC1, indicating that FLAG-PRC1 and EGFP-Kif4 interacted with each other in vivo regardless of the motor activity of Kif4 (Fig. 2B). We also observed coimmunprecipitation of endogenous PRC1 and Kif4 in late mitosis of HeLa cells by using affinity-purified anti-PRC1 and anti-Kif4-specific antibodies (see Fig. 4). Together, the results indicated that Kif4 and PRC1 specifically associated with each other in human cells. The coimmunoprecipitation assays were used to determine the Kif4 interaction domain(s) needed for association with PRC1. FLAG-PRC1 and a series of EGFP-tagged deletion mutants of Kif4 were coexpressed in 293 cells, and cell lysates were immunoprecipitated with anti-FLAG antibody (Fig. 2A). Immunolotting analysis of these immunoprecipitates with anti- GFP antibody indicated that the stalk plus tail domains of Kif4 specifically coimmunoprecipitated with FLAG-PRC1 (Fig. 2B). These results indicated that Kif4 requires its stalk and tail domains to interact with PRC1 in vivo. We then examined the subcellular localization of Kif4 and PRC1 during mitosis and cytokinesis in HeLa cells by using purified anti-Kif4 and anti-PRC1 antibodies. Immunofluores- cence analysis showed that both endogenous Kif4 and PRC1 were nuclear proteins in interphase cells (data not shown). Kif4 and PRC1 colocalized to the mitotic spindle in early mitosis (Fig. 2D), and in anaphase translocated to the spindle midzone where they colocalized as a series of narrow MT-bundle bars at the midzone. During cytokinesis, Kif4 and PRC1 were coinciden- tally concentrated on the midbody. Thus, the association of Kif4 with PRC1 and colocalization of Kif4 with PRC1 on the mitotic spindle further support the hypothesis that Kif4 is the motor protein that translocates PRC1 along the mitotic spindle during mitosis. Previous studies reported that Kif4 also colocalized with chromosomes during mitosis (19). We found that detection of endogenous Kif4 on chromosomes was inefficient under the conditions of formaldehyde fixation. However, under the con- Fig. 2. Kif4 and PRC1 interact with each other in vivo and colocalize at the ditions of methanol fixation or ectopic expression of EGFP- mitotic spindle and spindle midzone͞midbody during mitosis and cytokinesis. (A) A schematic illustration shows EGFP-tagged Kif4, its motor dead version, tagged Kif4, we observed chromosomal association of Kif4 (Fig. and a series of deletion mutant constructs used in coimmunoprecipitation 3 and Fig. 6, which is published as supporting information on the assays (lanes a–g; for details see Materials and Methods). The asterisk indi- PNAS web site). The role of Kif4 in chromosome segregation cates triple point mutations in the ATP-binding Walker A consensus site of the was reported recently (20) although we did not observe any Kif4 motor domain (GQTGSGKTYSMG to GQTGSAAAYSMG). (B) Coimmuno- significant defects in chromosome congression, alignment, or precipitation analysis of EGFP-tagged Kif4 or EGFP-tagged Kif4md (motor segregation in cells treated with Kif4 esiRNA (Figs. 1 and 5). dead mutant) with FLAG-tagged PRC1. EGFP-tagged Kif4 (lanes a) or EGFP-

tagged Kif4md (lanes b) were coexpressed with FLAG-tagged PRC1 in 293T CELL BIOLOGY cells. Cell lysates were made and immunoprecipitated with anti-FLAG antibody. Immunoprecipitates (IP) and 1͞10 of whole-cell lysates (WCL) used in to SDS͞PAGE, transferred to poly(vinylidene difluoride) membrane, and im- immunoprecipitations were subjected to SDS͞PAGE, transferred to poly(vi- munoblotted with anti-GFP antibody (Upper) or anti-FLAG antibody (Lower). nylidene difluoride) membrane, and immunoblotted with anti-GFP antibody (D) Colocalization of Kif4 and PRC1 on the mitotic spindle and spindle mid- (Upper) or anti-FLAG antibody (Lower). (C) Coimmunoprecipitation analysis of zone͞midbody during mitosis and cytokinesis is shown. Asynchronous HeLa EGFP-tagged Kif4 or its mutant proteins with FLAG-tagged PRC1. EGFP- cells grown on coverslips were fixed with 3% formaldehyde and then stained tagged Kif4 or its mutant proteins (lanes a and c–g) were coexpressed with with rabbit anti-Kif4 antibodies (green), Alexa Fluor 594-conjugated anti- FLAG-tagged PRC1 in 293T cells. Cell lysates were made and immunoprecipi- PRC1 antibodies (red), mouse anti-␣-tubulin antibody (blue), and DAPI (DNA, tated with anti-FLAG antibody. Immunoprecipitates (IP) (Left) and 1͞10 of white). Kif4 and PRC1 colocalized on mitotic spindle in early mitosis and whole-cell lysates (WCL) used in immunoprecipitations (Right) were subjected spindle midzone and midbody in late mitosis. (Scale bar: 5 ␮m.)

Zhu and Jiang PNAS ͉ January 11, 2005 ͉ vol. 102 ͉ no. 2 ͉ 345 Downloaded by guest on October 1, 2021 Ectopic Expression of Kif4, but Not the Kif4md Mutant, Relocalizes PRC1, Reestablishes the Midzone, and Rescues the Cytokinetic Defect in Kif4 esiRNA-Treated Cells. If Kif4 is required for translocating PRC1 to the ends of interdigitating MTs and allows PRC1 to bundle them and form the midzone, one might expect that expression of exogenous Kif4, but not the Kif4md mutant, would restore the midzone localization of PRC1, reestablish the midzone, and rescue the cytokinetic defects we observed in Kif4 esiRNA-treated cells. To this end, we performed rescue experiments in Kif4 esiRNA-treated cells. HeLa cells were cotransfected with Kif4 esiRNA, which targets the 3Ј UTR of Kif4 mRNA, together with mammalian expression vectors expressing EGFP (control), EGFP-Kif4, or EGFP-Kif4md by using Kif4 cDNA lacking the native 3Ј UTR. As shown in Fig. 3A, immunoblotting analysis indicated that cotransfection of Kif4A es- iRNA with EGFP, EGFP-Kif4, or EGFP-Kif4md plasmid in HeLa cells effectively ablated the expression of endogenous Kif4. However, cotransfection of Kif4 esiRNA with EGFP-Kif4 or EGFP-Kif4md plasmid did not affect the expression of the exogenous EGFP-Kif4 or EGFP-Kif4md in these cells. These results indicated that Kif4 esiRNAs specifically and effectively suppress the expression of endogenous Kif4 but not the expression of exogenous EGFP-Kif4 or EGFP-Kif4md. Expression of EGFP-Kif4 protein, but not EGFP-Kif4md or EGFP, restored the midzone localization of PRC1, reestablished the mitotic spindle midzone, and rescued the cytokinetic defects in endogenous Kif4-depleted cells. As shown in Fig. 3 B and C, when compared with cells transfected with Kif4 esiRNA alone (GFPϪ cells) or cells cotransfected with Kif4 esiRNA and control vector EGFP, the majority of cells cotransfected with Kif4 esiRNA and EGFP-Kif4 plasmid showed normal spindle morphology during mitosis and cytokinesis, similar to that of untransfected cells (Figs. 3B and 5). In cells cotransfected with Kif4 esiRNA and EGFP-Kif4 plasmid, the midzone was reestablished, EGFP-Kif4 and PRC1 colocalized with interdigitating MT bundles in anaphase, and the number of binucleated͞ multinucleated cells was dramatically reduced (Fig. 3 B and C and Fig. 7, which is published as supporting information on the PNAS web site). By contrast, in cells cotransfected with Kif4 esiRNA and EGFP-Kif4md plasmid, the midzone localization of PRC1 was not restored and the midzone was not reestablished even though PRC1 and EGFP-Kif4md colocalized on the abnormal mitotic spindle (Fig. 3B). The percentage of binucleated͞ multinucleated cells was dramatically increased in cells cotransfected with Kif4 esiRNA and EGFP-Kif4md plasmid when compared with cells transfected with Kif4 esiRNA alone, sug- gesting that EGFP-Kif4md might have dominant-negative ef- fects (Fig. 3C). Taken together, these results indicated that the expression of EGFP-Kif4, but not its motor dead mutant EGFP- Kif4md, in Kif4 esiRNA-treated cells compensated for the depletion of endogenous Kif4, which was necessary for relocal- izing PRC1 to the plus end of interdigitating MTs of the spindle, reestablishing the midzone, and completing cytokinesis.

Cdk Phosphorylation of PRC1 Controls the Timing of Kif4-Mediated PRC1 Translocation to the Plus Ends of Interdigitating MTs at the Metaphase-to-Anaphase Transition. PRC originally was identified as a Cdk substrate in an in vitro phosphorylation screen (16). It has been suggested that Cdk phosphorylation of PRC1 nega- Fig. 3. Restoration of the localization of PRC1 and rescue of cytokinetic defects in Kif4 esiRNA-treated cells by ectopic expression of GFP-Kif4. (A) HeLa cells grown in a 6-well plate were transfected with 400 ng of pEGFP, pEGFP- (red), anti-␣-tubulin antibody (blue), and DAPI (DNA, white). The expression Kif4, or pEGFP-Kif4md. Twenty-four hours later, cells were transfected with of GFP proteins is shown in green. (Scale bar: 5 ␮m.) (C) HeLa cells grown on 100 nM of control (luciferase) esiRNA or Kif4A esiRNA. Two days after esiRNA coverslips were transfected with plasmids and͞or esiRNAs, fixed, and stained transfection, cells were lysed and lysates were subjected to SDS͞PAGE and with antibodies and DAPI as in B. The percentages of polyploidy in GFP- immunoblotted with anti-Kif4 antibody or anti-␤-actin antibody. (B) HeLa negative or GFP-positive cells transfected with the indicated esiRNAs and͞or cells grown on coverslips were transfected with plasmids and esiRNAs as in A. plasmids were scored under a fluorescence microscope. More than 300 cells After transfection, cells were fixed and stained with anti-PRC1 antibodies were counted in each experiment.

346 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0408438102 Zhu and Jiang Downloaded by guest on October 1, 2021 Fig. 4. Kif4 speciﬁcally binds to unphosphorylated PRC1. (A) HeLa cells were synchronized at the G2͞M boundary by a thymidine͞nocodazole treatment. Mitotic cells were collected by shake-off, released into fresh medium, and harvested at 10, 30, 50, 70, or 90 min after release. Percentages of cells at different stages of mitosis were determined by counting under a microscope. Whole-cell lysates (WCL) were made, subjected to SDS͞PAGE, and immunoblotted with corresponding antibodies as indicated. (B) Lysates as in A were immunoprecipitated by anti-PRC1 or anti-Kif4 antibodies. The immunoprecipitates (IP) were subjected to SDS͞PAGE and immunoblotted with anti-Kif4, anti-phospho-PRC1, or anti-PRC1 antibodies. (C) One microgram of bacterially expressed glutathione agarose- bound GST or GST-PRC1 was incubated with or without 0.5 ␮g of puriﬁed baculovirus-expressed Cdc2͞cyclin B1 (K1͞B1) in the presence of 100 ␮M ATP for 2 h at 30°C. (Middle) After washing, 1͞10th of beads were subjected to SDS͞PAGE and immunoblotted with anti-phospho-PRC1 antibodies. The rest of the beads were then incubated with 200 ␮g of early mitotic HeLa cell lysates (10 min release from a thymidine͞nocodazole block) for2hat4°C. (Top) After washing, bead-bound proteins were subjected to SDS͞PAGE and immunoblotted with anti-Kif4 antibodies. (Bottom) One microgram of GST, GST-PRC1, and Cdk- phosphorylated GST-PRC1 was used in the pull-down assay on SDS͞PAGE by Coommasie blue staining. (D) The proposed model for cell cycle-dependent translocation of PRC1 to the plus ends of interdigitating MTs by Kif4 during the metaphase-to-anaphase transition (for details, see text). APC, anaphase promoting complex.

tively regulates its function during mitosis (4, 16). One attractive creased dramatically during the metaphase-to-anaphase transi- possibility is that Cdk phosphorylation of PRC1 negatively tion, coincident with the decrease of the expression levels of regulates its interaction with Kif4. In this scenario, PRC1 is cyclin B1 (Fig. 4A). phosphorylated by Cdks (mainly Cdc2͞cyclin B) in early mitosis, Consistently, similar amounts of PRC1 from different stages and Cdk phosphorylation of PRC1 would prevent PRC1 inter- of mitosis were immunoprecipitated by anti-PRC1 antibodies action with Kif4 although both PRC1 and Kif4 associate with (Fig. 4B). High levels of phosphorylated PRC1 were detected in mitotic spindle. During metaphase-to-anaphase transition, when the early mitosis anti-PRC1 immunoprecipitates. As cells en- all cellular Cdk activity is eliminated because of the degradation tered anaphase, the levels of phosphorylated PRC1 in anti-PRC1 of cyclin B by the anaphase-promoting complex, PRC1 would be immunoprecipitates were dramatically decreased (Fig. 4B). In dephosphorylated by an active mitotic phosphatase (e.g., contrast, very low levels of Kif4 were detected in the anti-PRC1 Cdc14A or PP1␥). Dephosphorylation of PRC1 would promote immunoprecipitates from early mitosis, but high levels of Kif4 interaction of Kif4 with PRC1, allowing Kif4 to translocate proteins were detected in the anti-PRC1 immunoprecipitates unphosphorylated PRC1 to the plus ends of interdigitating MTs from late mitosis (Fig. 4B). Reciprocal immunoprecipitation where PRC1 could bundle the interdigitating MTs to organize experiments also were performed with anti-Kif4 antibodies. As formation of the midzone, which is necessary for cytokinesis. To shown in Fig. 4B, similar amounts of Kif4 from different stages test the model, we examined the interactions of Kif4 and PRC1, of mitosis were immunoprecipitated by anti-Kif4 antibodies. especially the interactions of Kif4 with phosphorylated PRC1, Very low levels of PRC1 were detected in the anti-Kif4 immu- during different stages of mitosis. noprecipitates from early mitosis but high levels of PRC1 CELL BIOLOGY HeLa cells were synchronized at the G2͞M boundary by a proteins were detected in the anti-Kif4 immunoprecipitates from thymidine͞nocodazole block and then released into mitosis. late mitosis. No Cdk-phosphorylated PRC1 was detected in any Cells at different stages of mitosis were lysed, and whole-cell anti-Kif4 immunoprecipitates. These results suggest that Kif4 lysates were subjected to immunoblotting analysis with anti-Kif4, preferentially interacts with unphosphorylated PRC1. anti-PRC1, anti-Cdk-phosphorylated PRC1 (T481), or anti- To confirm that Kif4 specifically interacts with unphosphor- cyclin B1-specific antibodies. Expression levels of Kif4 and PRC1 ylated PRC1, but not phosphorylated PRC1, we performed in remained constant during mitosis. However, the levels of Cdk- vitro pull-down analyses. Bacterially expressed GST-PRC1 or phosphorylated PRC1 fluctuated during mitosis. The levels of GST alone was bound to glutathione-agarose beads and incu- Cdk-phosphorylated PRC1 were high in early mitosis but de- bated with or without baculovirus-expressed purified Cdc2͞

Zhu and Jiang PNAS ͉ January 11, 2005 ͉ vol. 102 ͉ no. 2 ͉ 347 Downloaded by guest on October 1, 2021 cyclin B1 in the presence of ATP. As shown in Fig. 4C, that Kif4 interacts with PRC1 in vivo and that the interaction Cdc2͞cyclin B1 could efficiently phosphorylate GST-PRC1 in occurs through the Kif4 C-terminal stalk plus tail domain. As the vitro as determined by immunoblotting analysis using antiphos- translocation of PRC1 on the spindle is to the plus ends of the phorylated PRC1(T481)-specific antibodies. GST-phosphory- interdigitating MTs and dependent on Kif4, our results indicate lated PRC1, GST-PRC1, or GST controls then were incubated that PRC1 is a critical downstream cargo of Kif4 on the spindle with early mitotic HeLa cell lysates. GST-PRC1, but not Cdc2͞ during mitosis. While this article was in preparation, Kurasawa cyclin B1-phosphorylated GST-PRC1 or GST controls, was able et al. (22) reported the interaction between Kif4 and PRC1 to pull down endogenous Kif4 (Fig. 4C). These results indicated although the study did not show that Kif4 is required for that unphosphorylated PRC1, but not phosphorylated PRC1, translocation of PRC1 on the spindle during mitosis. specifically interacts with Kif4. Taken together, our results Third, cytokinesis is a carefully regulated process that must indicate that PRC1 is phosphorylated in early mitosis and coordinate precisely with other events of the cell cycle. The phosphorylated PRC1 does not associate with Kif4. During the best-known cell cycle regulators are Cdks. Previous studies metaphase-to-anaphase transition, PRC1 is dephosphorylated showed that inactivation of Cdk activity through destruction of and interacts with Kif4. mitotic cyclin B is a critical event for the metaphase-to-anaphase We conclude that Kif4 is the motor protein that translocates transition. For example, cells expressing a stable nondegradable PRC1 along mitotic spindle to the plus ends of interdigitating cyclin B mutant arrest in anaphase and fail to undergo cytoki- MTs during the metaphase-to-anaphase transition and that Cdk nesis (2). Thus, coupling cytokinesis to Cdk inactivation is a key phosphorylation of PRC1 controls the timing of the transloca- mechanism for cells to ensure that division is not initiated until tion of PRC1 by Kif4. Several lines of evidence support our chromosomes have been separated. We find that the interaction conclusion. First, previous studies showed that PRC1 is a MT of Kif4 and PRC1 depends on Cdk phosphorylation of PRC1. binding and bundling Cdk substrate protein that plays an essen- We show that PRC1 is phosphorylated in early mitosis and tial role in regulating the spindle midzone formation and cyto- phosphorylated PRC1 does not associate with Kif4. During the kinesis in human cells (4, 16). PRC1 interacts with MTs directly metaphase-to-anaphase transition, PRC1 is dephosphorylated and bundles antiparallel MTs into MT bundles in vitro. Because and interacts with Kif4. These results, together with the fact that PRC1 translocates from spindles to the spindle midzone͞ Kif4 is required for the translocation of PRC1 to the plus ends midbody, it had been proposed that PRC1 bundles the spindle of interdigitating MTs during the metaphase-to-anaphase tran- plus ends of interdigitating MTs to form the midzone that is sition, indicate that Cdk phosphorylation of PRC1 controls the necessary for cytokinesis. Our immunofluorescence and time- timing of the translocation of PRC1 by Kif4. lapse video studies, in which we monitored the movement of Taken together, we present the following model for the cell PRC1 on the mitotic spindle in live cells, demonstrated that cycle-dependent translocation of PRC1 to the plus ends of depletion of Kif4 expression by Kif4 esiRNA impaired the interdigitating MTs by Kif4 during the metaphase-to-anaphase transition (Fig. 4D). In early mitosis, PRC1 is phosphorylated by translocation of PRC1 on the spindle during the metaphase-to- ͞ anaphase transition, inhibited midzone formation and caused Cdks (mainly Cdc2 cyclin B1), and Cdk phosphorylation of cytokinetic defects. Furthermore, we have shown that ectopic PRC1 prevents PRC1 interaction with Kif4 although both PRC1 expression of Kif4, but not its motor dead Kif4md mutant, and Kif4 associate with the mitotic spindle. During the met- restores the translocation of PRC1 along the spindle, reestab- aphase-to-anaphase transition, the anaphase promoting complex lishes the midzone, and rescues the cytokinetic defects in Kif4 triggers proteolysis of cyclin B, resulting in the elimination of all esiRNA-treated cells. These results strongly indicate that Kif4 is cellular Cdk activity. In the absence of Cdk activity, PRC1 is dephosphorylated by an active mitotic phosphatase (e.g., the motor protein required for PRC1 translocation on the ␥ spindle during mitosis and that Kif4-dependent translocation Cdc14A or PP1 ). Dephosphorylation of PRC1 promotes interaction of Kif4 with PRC1. Kif4 translocates PRC1 to the plus allows PRC1 to bundle interdigitating MTs and form the mid- ends of interdigitating MTs, allowing PRC1 to bundle the zone that is necessary for cytokinesis. interdigitating MTs and organize formation of the midzone Second, based on the position of the motor domain, Kif4 is during anaphase that is necessary for cytokinesis. categorized as a N-class kinesin (21). The motor domains of N-class kinesins use ATP to fuel the movement along MTs We thank Drs. Tony Hunter, Nanxin Li, and Joel Leverson for critical anterogradely toward the plus ends. Besides the motor domains, reading of the manuscript and Ningning Sai for laboratory support. This N-class kinesins have different stalk and tail domains at the C work is supported by grants from the Edward Mallinckrodt, Jr., Foun- terminus that mediate oligomerization, regulation of motor dation and the Lisa U. Pardee Foundation and by National Institutes of activity, and interactions with their specific cargos (8). We show Health Grant GM67859 (to W.J.).

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