Role of the Kinesin-2 Family Protein, KIF3, during Mitosis

During mitosis, kinesin and dynein motor proteins play critical roles in the equal segregation of chromosomes between two daughter cells. Kinesin-2 is composed of two microtubule-based motor subunits, KIF3A/3B, and a kinesin-associated protein known as KAP3, which links KIF3A/3B to cargo that is carried to cellular organelles along microtubules in interphase cells. We have shown here that the kinesin-2 complex is localized with components of the mitotic apparatus such as spindle microtubules and centrosomes. Furthermore, we found that expression of a mutant KIF3B, which is able to associate with KIF3A but not KAP3 in NIH3T3 cells, caused chromosomal aneuploidy and abnormal spindle formation. Our data suggested that the kinesin-2 complex plays an important role not only in interphase but also in mitosis. During mitosis, kinesin and dynein motor proteins play critical roles in the equal segregation of chromosomes between two daughter cells. Kinesin-2 is composed of two microtubule-based motor subunits, KIF3A/3B, and a kinesin-associated protein known as KAP3, which links KIF3A/3B to cargo that is carried to cellular organelles along microtubules in interphase cells. We have shown here that the kinesin-2 complex is localized with components of the mitotic apparatus such as spindle microtubules and centrosomes. Furthermore, we found that expression of a mutant KIF3B, which is able to associate with KIF3A but not KAP3 in NIH3T3 cells, caused chromosomal aneuploidy and abnormal spindle formation. Our data suggested that the kinesin-2 complex plays an important role not only in interphase but also in mitosis. Chromosomes are equally segregated into two daughter cells during mitosis, and errors in this process may result in chromosomal aneuploidy, cancer, or cell death. The kinesin superfamily of proteins (KIFs), 3The abbreviations used are: KIF, kinesin superfamily of proteins; APC, adenomatous polyposis coli; GFP, green fluorescent protein; PBS, phosphate-buffered saline. as well as cytoplasmic dynein, play critical roles in centrosome separation, spindle formation, and chromosome alignment and segregation (1.Heald R. Cell. 2000; 102: 399-402Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 2.Mountain V. Compton D.A. Anat. Rec. 2000; 261: 14-24Crossref PubMed Scopus (30) Google Scholar). For example, Kid, a member of the chromosome-associated KIFs, is important for chromosome alignment and orientation (3.Antonio C. Ferby I. Wilhelm H. Jones M. Karsenti E. Nebreda A.R. Vernos I. Cell. 2000; 102: 425-435Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 4.Funabiki H. Murray A.W. Cell. 2000; 102: 411-424Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 5.Levesque A.A. Compton D.A. J. Cell Biol. 2001; 154: 1135-1146Crossref PubMed Scopus (175) Google Scholar). KIF4, together with microtubule-bundling protein PRC1, participates in the organization of central spindle midzone formation (6.Kurasawa Y. Earnshaw W.C. Mochizuki Y. Dohmae N. Todokoro K. EMBO J. 2004; 23: 3237-3248Crossref PubMed Scopus (248) Google Scholar). Furthermore, Caenorhabditis elegans ZEN-4/MKLP1 is required for central spindle assembly and cytokinesis (7.Mishima M. Pavicic V. Gruneberg U. Nigg E.A. Glotzer M. Nature. 2004; 430: 908-913Crossref PubMed Scopus (216) Google Scholar). The heterotrimeric kinesin-2 complex was first identified by Cole et al. (8.Cole D.G. Cande W.Z. Baskin R.J. Skoufias D.A. Hogan C.J. Scholey J.M. J. Cell Sci. 1992; 101: 291-301PubMed Google Scholar, 9.Cole D.G. Chinn S.W. Wedaman K.P. Hall K. Vuong T. Scholey J.M. Nature. 1993; 366: 268-270Crossref PubMed Scopus (222) Google Scholar). Kinesin-2 is one of the most ubiquitously expressed KIFs (10.Kondo S. Sato-Yoshitake R. Noda Y. Aizawa H. Nakata T. Matsuura Y. Hirokawa N. J. Cell Biol. 1994; 125: 1095-1107Crossref PubMed Scopus (210) Google Scholar, 11.Yamazaki H. Nakata T. Okada Y. Hirokawa N. J. Cell Biol. 1995; 130: 1387-1399Crossref PubMed Scopus (252) Google Scholar) and has been implicated in the intracellular transport of membrane-bound organelles and protein complexes in various tissues such as neurons, melanosomes, and epithelial cells (12.Tuma M.C. Zill A. Le Bot N. Vernos I. Gelfand V. J. Cell Biol. 1998; 143: 1547-1558Crossref PubMed Scopus (154) Google Scholar, 13.Takeda S. Yamazaki H. Seog D.H. Kanai Y. Terada S. Hirokawa N. J. Cell Biol. 2000; 148: 1255-1265Crossref PubMed Scopus (157) Google Scholar, 14.Jimbo T. Kawasaki Y. Koyama R. Sato R. Takada S. Haraguchi K. Akiyama T. Nat. Cell Biol. 2002; 4: 323-327Crossref PubMed Scopus (250) Google Scholar). Kinesin-2 is a heterotrimeric complex composed of a KIF3A/3B heterodimer and KAP3 (8.Cole D.G. Cande W.Z. Baskin R.J. Skoufias D.A. Hogan C.J. Scholey J.M. J. Cell Sci. 1992; 101: 291-301PubMed Google Scholar, 9.Cole D.G. Chinn S.W. Wedaman K.P. Hall K. Vuong T. Scholey J.M. Nature. 1993; 366: 268-270Crossref PubMed Scopus (222) Google Scholar, 11.Yamazaki H. Nakata T. Okada Y. Hirokawa N. J. Cell Biol. 1995; 130: 1387-1399Crossref PubMed Scopus (252) Google Scholar, 15.Wedaman K.P. Meyer D.W. Rashid D.J. Cole D.G. Scholey J.M. J. Cell Biol. 1996; 132: 371-380Crossref PubMed Scopus (121) Google Scholar, 16.Yamazaki H. Nakata T. Okada Y. Hirokawa N. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8443-8448Crossref PubMed Scopus (169) Google Scholar). The KIF3A/3B heterodimer possesses a plus-end-directed microtubule sliding activity that uses energy derived from ATP hydrolysis (11.Yamazaki H. Nakata T. Okada Y. Hirokawa N. J. Cell Biol. 1995; 130: 1387-1399Crossref PubMed Scopus (252) Google Scholar). On the other hand, KAP3 links KIF3A/3B with various cargo proteins, including fodrin and the tumor suppressor adenomatous polyposis coli (APC). By binding to fodrin, the kinesin-2 heterotrimer transports fodrin-associating vesicles that are important for neurite building (13.Takeda S. Yamazaki H. Seog D.H. Kanai Y. Terada S. Hirokawa N. J. Cell Biol. 2000; 148: 1255-1265Crossref PubMed Scopus (157) Google Scholar). Also, the kinesin-2 heterotrimer transports APC along microtubules to the tips of membrane protrusions (14.Jimbo T. Kawasaki Y. Koyama R. Sato R. Takada S. Haraguchi K. Akiyama T. Nat. Cell Biol. 2002; 4: 323-327Crossref PubMed Scopus (250) Google Scholar). Although kinesin-2 has mainly been reported to participate in intracellular transport in interphase cells, it has been shown to localize at the mitotic apparatus. In Chlamydomonas, the KIF3A homolog FLA10 protein is most abundant near the centrioles and the mitotic spindle during mitosis (17.Vashishtha M. Walther Z. Hall J.L. J. Cell Sci. 1996; 109: 541-549PubMed Google Scholar). The sea urchin kinesin-2 homolog, kinesin II, is present transiently in the mitotic apparatus of dividing embryos (18.Henson J.H. Cole D.G. Terasaki M. Rashid D. Scholey J.M. Dev. Biol. 1995; 171: 182-194Crossref PubMed Scopus (37) Google Scholar). However, detailed functional analyses have revealed that kinesin-2 is not critical for the progression of mitosis in both Chlamydomonas and sea urchin embryos (see “Discussion”) (19.Miller M.S. Esparza J.M. Lippa A.M. Lux III, F.G. Cole D.G. Dutcher S.K. Mol. Biol. Cell. 2005; 16: 3810-3820Crossref PubMed Scopus (47) Google Scholar, 20.Morris R.L. Scholey J.M. J. Cell Biol. 1997; 138: 1009-1022Crossref PubMed Scopus (147) Google Scholar). In the present study, we have attempted to investigate the function and regulation of kinesin-2 during mitosis in mammalian cells and have found that kinesin-2 localizes to the mitotic apparatus and contributes to molecular events important for the progression of mitosis. Cell Culture—HeLa, HEK293T and Plat-E cells were cultured in Dulbecco's modified Eagle's medium (Nissui Pharmaceuticals, Taito, Tokyo, Japan) supplemented with 10% (v/v) fetal bovine serum. NIH3T3 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) calf serum. Antibodies—Rabbit polyclonal antibody to KAP3 (N1 and C2) were prepared as described previously (14.Jimbo T. Kawasaki Y. Koyama R. Sato R. Takada S. Haraguchi K. Akiyama T. Nat. Cell Biol. 2002; 4: 323-327Crossref PubMed Scopus (250) Google Scholar). Rabbit polyclonal antibodies to KIF3A and KIF3B were prepared by immunizing rabbits with peptides containing amino acids 563–671 of KIF3A and 657–747 of KIF3B, respectively. Antibodies were purified by affinity chromatography using columns to which the antigens used for immunization had been linked. Monoclonal antibody to KIF3A/3B (Kinesin II) was obtained from Covance (Princeton, NJ). Monoclonal antibodies to α-tubulin and cyclin B1 were from Oncogene Research (Boston, MA) and Santa Cruz Biotechnology (sc-245, Santa Cruz, CA), respectively. Monoclonal and polyclonal antibodies to GFP were from Quantum (Montreal, Quebec, Canada) and Clontech, respectively. Immunoprecipitation and Immunoblotting—Cells were lysed in buffer A (50 mm Tris-HCl at pH 7.5, 150 mm NaCl, 5 mm EDTA at pH 7.5, 2 mm Na3VO4, 10 mm NaF) containing 1% Triton X-100. Lysates were incubated with antibodies for 1 h at 4° C. The immuno complexes were adsorbed to protein G-Sepharose 4B (Amersham Biosciences) for 1 h at 4 °C. Blocking of antibodies was performed by preincubating antibodies (1–2 μg) for 1 h at4°C with a 100-fold molar excess of the antigens used for immunization. After washing extensively with buffer A containing 0.1% Triton X-100, samples were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane filter (Immobilon P, Millipore, Bedford, MA). The blot was analyzed by immunoblotting with alkaline phosphatase-conjugated mouse anti-rabbit IgG or goat anti-mouse IgG (Promega BioSciences, San Luis Obispo, CA) as a secondary antibody. Primary antibodies were diluted as follows: anti-KAP3 C2 polyclonal antibody, 1:1,000; anti-KIF3A/3B monoclonal antibody, 1: 1,000; anti-KIF3A and KIF3B polyclonal antibody, 1:1,000; anticyclin B1 monoclonal antibody, 1:250; anti-GFP monoclonal antibody, 1:250; anti-α-tubulin monoclonal antibody, 1:1,000. Immunostaining—HeLa cells were fixed with 4% paraformaldehyde in PBS for 20 min at 37 °C and then fixed with methanol-acetone (1:1, v/v) for 10 min at –20 °C. Fixed cells were incubated with blocking solution (1% bovine serum albumin in PBS) and were double-stained with antibodies against KAP3 C2 (polyclonal) and α-tubulin (monoclonal), KIF3A or KIF3B (polyclonal) and α-tubulin (monoclonal), or GFP (polyclonal) and α-tubulin (monoclonal). Staining patterns obtained with these antibodies were visualized by incubation with secondary antibodies: fluorescein-5-isothiocyanate-labeled anti-rabbit IgG (for KAP3, KIF3A/3B, and GFP) and tetramethylrhodamine-5 (and -6)-isothiocyanate-labeled anti-mouse IgG (for α-tubulin). Chromosomes were stained with TOTO3 (Molecular Probes, Eugene, OR). Dilutions of primary antibodies were as follows: anti-KAP3 C2 polyclonal antibody, 1:100; anti-KIF3A or KIF3B polyclonal antibody, 1:50; anti-GFP polyclonal antibody, 1:100, anti-α-tubulin monoclonal antibody, 1:100. Cells were photographed with a Carl Zeiss LSM510 laser microscope (Göttingen, Germany). Phosphatase Treatment—KAP3 immunoprecipitates were washed three times with bacterial alkaline phosphatase buffer (20 mm Tris-HCl, pH 8.0, 1 mm MgCl2) and incubated at 50 °C for 30 min with 0.2 units of bacterial alkaline phosphatase (TAKARA, Kyoto, Japan) in the absence or presence of phosphatase inhibitors (2 mm Na3VO4 and 10 mm NaF). Cell Cycle Synchronization—HeLa cells were synchronized by thymidine at the G1/S boundary as described (21.Heintz N. Sive H.L. Roeder R.G. Mol. Cell. Biol. 1983; 3: 539-550Crossref PubMed Scopus (312) Google Scholar). In brief, cells were treated with 2.5 mm thymidine (Sigma) for 12 h, washed three times with PBS, and incubated for an additional 12 h under normal growth conditions. Finally, thymidine was added again for 12 h to block cells at the G1/S boundary. Cells were then washed three times with PBS and placed under fresh growth medium. Cells were harvested at various time points as indicated. To obtain more enriched M phase cells, exponentially growing HeLa cells were treated with 100 ng/ml nocodazole (Sigma) for 24 h. Mitotic rounded-up cells were collected by gentle pipetting. Retroviral Infection—DNA fragments encoding GFP-KIF3A or GFP-KIF3B were cloned into the retroviral vector pMX-puro (22.Onishi M. Kinoshita S. Morikawa Y. Shibuya A. Phillips J. Lanier L.L. Gorman D.M. Nolan G.P. Miyajima A. Kitamura T. Exp. Hematol. 1996; 24: 324-329PubMed Google Scholar, 23.Morita S. Kojima T. Kitamura T. Gene Ther. 2000; 7: 1063-1066Crossref PubMed Scopus (1361) Google Scholar). The retroviral vectors were transiently transfected into Plat-E cells using Effectene (Qiagen, Hilden, Germany). After 24 h, cells were changed into fresh medium. After an additional 24 h, culture medium was collected and centrifuged at 2,000 × g for 30 min. The supernatants, together with 10 μg/ml polybrene (Sigma), were added to NIH3T3 cells. The infected cells were selected using 2 μg/ml puromycin (Sigma) for 3 days. KAP3 Is Phosphorylated during Mitosis—To investigate the role of kinesin-2 in mitosis, we first examined the expression and modification of KIF3A/3B and KAP3 using HeLa cells released from double thymidine block. Immunoblotting analysis revealed that the levels of KIF3A/3B and KAP3 expression increased slightly during G2/M progression (Fig. 1A). Furthermore, a protein larger than the normal KAP3 was found to appear concomitant with the start of cyclin B destruction. Treatment of KAP3 immunoprecipitates with bacterial alkaline phosphatase resulted in the disappearance of this protein and a concomitant increase in the normal migrating form of KAP3, and this conversion was not observed in the presence of phosphatase inhibitors (Fig. 1B). These results suggest that KAP3 is phosphorylated during mitosis. KIF3A/3B and KAP3 Form a Complex during Mitosis—We next examined whether KIF3A/3B is associated with KAP3 in HeLa cells that were synchronized at mitosis by nocodazole treatment. Lysates from nocodazole-treated cells were subjected to immunoprecipitation with anti-KAP3 antibody followed by immunoblotting with anti-KIF3A/3B antibodies. We found that both phosphorylated and unphosphorylated forms of KAP3 coprecipitated with KIF3A/3B and that coprecipitation was inhibited by preincubation of anti-KAP3 antibody with the antigen used for immunization (Fig. 2). Thus, KIF3A/3B are associated with KAP3 during mitosis. Regions Required for KIF3A/3B and KAP3 Interaction—To determine the regions of KIF3A/3B responsible for their interaction with KAP3, we generated various deletion mutants of KIF3A/3B. When ectopically expressed in HEK293T cells, the two KIF3A/3B mutants lacking the C-terminal regions failed to coprecipitate with KAP3 (Fig. 3A). In contrast, KIF3A/3B mutants containing the C-terminal regions coprecipitated with KAP3. Thus, the C-terminal regions of KIF3A/3B are important for their interaction with KAP3. Similar experiments with various deletion mutants of KAP3 revealed that a fragment containing amino acids 528–694 is able to interact with KIF3A/3B (Fig. 3B). In addition, a fragment of KAP3 containing amino acids 1–614 was found to retain KIF3A/3B binding activity. Thus, amino acids 528–614 of KAP3 may be important for its interaction with KIF3A/3B. KIF3A/3B and KAP3 Localize to the Mitotic Apparatus—We next examined the subcellular localization of KIF3A/3B and KAP3 in mitosis. Since commercially available anti-KIF3A/3B monoclonal antibodies were not suitable for immunostaining analysis, we generated polyclonal antibodies against the C-terminal regions of KIF3A and KIF3B, respectively. Immunoblotting analysis using HeLa cell lysates revealed that each antibody specifically recognizes KIF3A and KIF3B, respectively (Fig. 4A). This result is consistent with the fact that there is little amino acid sequence similarity among the C-terminal regions of the KIF family of proteins. Immunostaining analysis with these antibodies revealed intense KIF3A staining localized at the centrosomes in interphase and prophase cells but only weak staining at the centrosomes after prometaphase (Fig. 4B). When chromosomes began to condense and the mitotic spindle was formed in prometaphase, KIF3A was localized mainly at the spindle microtubules. From metaphase through telophase, KIF3A was concentrated at the midzone and was present mainly at the centrosomes during cytokinesis. KIF3A was also localized around the cellular cortex throughout mitosis. We also examined KAP3 localization during mitosis using a polyclonal antibody against the C-terminal region of KAP3 (C2) (14.Jimbo T. Kawasaki Y. Koyama R. Sato R. Takada S. Haraguchi K. Akiyama T. Nat. Cell Biol. 2002; 4: 323-327Crossref PubMed Scopus (250) Google Scholar). When cells entered prometaphase, KAP3 was found to be localized at the centrosomes and spindle microtubules (Fig. 4C). In metaphase, KAP3 was also detected on the chromosomes, and this localization was especially prominent on the chromosomes. From metaphase to telophase, KAP3 was concentrated intensively at centrosomes and midzone and remained at the centrosome during cytokinesis. Thus, the immunostaining patterns of KAP3 were partially similar to those of KIF3A. These results suggested that a certain population of KIF3A/3B and KAP3 colocalizes at the mitotic apparatus. Effects of KIF3 Mutants on Mitotic Progression—To elucidate the function of kinesin-2 in mitosis, we expressed a fragment of KIF3B fused to GFP (Fig. 3A, GFP-KIF3B Mutant 3 (Mut 3)) in NIH3T3 cells by retrovirus and examined its effects on the progression of mitosis. This fragment lacks the C-terminal one-third of KIF3B and is unable to form a complex with KAP3 (Fig. 3A) but can still associate with KIF3A not Although this mutant was localized at the spindle similar to KIF3B, cells this mutant an of centrosomes and abnormal spindle formation (Fig. By contrast, cells the KIF3B fused to GFP or retrovirus vector not in mitosis. Furthermore, analysis revealed that the of chromosomal aneuploidy in cells this mutant was three times than that of cells the KIF3B fused to GFP or retrovirus vector (Fig. Similar mitosis was found to a fragment of KIF3A fused to GFP (Fig. 3A, GFP-KIF3A Mutant 3 (Mut which can form a complex with KIF3B but interact with KAP3, was expressed in NIH3T3 cells not These results suggest that the interaction of KIF3A/3B with KAP3 may be important for the progression of mitosis. In the present study, we that KIF3A/3B and KAP3 form a complex in mitosis and that localize at the mitotic mainly at the spindle microtubules and centrosomes in HeLa cells. Furthermore, we that expression of a mutant of KIF3A/3B results in mitosis. Our suggested that the kinesin-2 complex plays a critical role not only in interphase but also in mitosis. Our analysis revealed that amino acids 528–614 of KAP3 may interact with the C-terminal regions of KIF3A/3B. This result is consistent with data obtained by that KAP3 is a protein and is associated with the of KIF3A/3B H. Nakata T. Okada Y. Hirokawa N. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8443-8448Crossref PubMed Scopus (169) Google Scholar). We deletion of KIF3A/3B used for analysis (Fig. 3A, Mutant 3 (Mut 3)) to the of the kinesin-2 complex in mitosis we not expression of KIF3A/3B and KAP3 using These mutants form a heterodimer but not interact with KAP3. expressed in NIH3T3 cells, these mutants caused chromosomal aneuploidy and spindle formation. Thus, the interaction between KIF3A/3B and KAP3 may be required for spindle formation and chromosome one the that a other than KAP3 also and is critical for this We that there may be an important cargo to which KIF3A/3B associate an interaction with KAP3. it has been reported that APC localizes to the of microtubules in and plays a critical role in chromosome segregation R. J. C. R. M. C. J.H. C. J. H. Nat. Cell Biol. 2001; 3: PubMed Scopus Google Scholar, A.A. Nat. Cell Biol. 2001; 3: PubMed Scopus Google Scholar). Since we have previously found that APC is associated with KIF3A/3B an with KAP3 (14.Jimbo T. Kawasaki Y. Koyama R. Sato R. Takada S. Haraguchi K. Akiyama T. Nat. Cell Biol. 2002; 4: 323-327Crossref PubMed Scopus (250) Google APC may be one of these critical cargo proteins in the progression of mitosis. was reported that in the kinesin-2 motor result in chromosome in Chlamydomonas (19.Miller M.S. Esparza J.M. Lippa A.M. Lux III, F.G. Cole D.G. Dutcher S.K. Mol. Biol. Cell. 2005; 16: 3810-3820Crossref PubMed Scopus (47) Google Scholar). However, it was suggested that these to effects of the mutant Also, of a monoclonal antibody in sea urchin embryos but has on mitosis or cytokinesis R.L. Scholey J.M. J. Cell Biol. 1997; 138: 1009-1022Crossref PubMed Scopus (147) Google Scholar). Furthermore, KIF3A and KIF3B cell during the S. Y. Okada Y. S. A. Kanai Y. M. Hirokawa N. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, S. Y. Y. Okada Y. S. Hirokawa N. J. Cell Biol. PubMed Scopus Google Scholar, E. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google that kinesin-2 is not critical for the progression of mitosis. These appear to with However, it may be that the of kinesin-2 function in mitosis may on the cell and To the role of kinesin-2 in mitosis, it be to cell and to the function of kinesin-2 in mitosis. We that KAP3 is phosphorylated during mitosis. In this it is that of the KIF family have been reported to be phosphorylated by mitotic For example, and are phosphorylated by B1 H. 1994; PubMed Scopus Google Scholar, A. M. M. Nigg E.A. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar). of is required for spindle formation and R. R. Le K. C. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). Also, of its to spindle and chromosomes M. N. K. J. T. EMBO J. PubMed Scopus Google Scholar). Thus, it is that of KAP3 may play a role in the regulation of mitotic Although of KAP3 not its KIF3A/3B binding it is to that of KAP3 may its interaction with cargo proteins that are in spindle formation and chromosome of cargo proteins and responsible for KAP3 may into the of KAP3 We Y. Y. and T. for and T. Kitamura for Plat-E cells.

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