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.

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), 3 as well as cytoplasmic dynein, play critical roles in centrosome separation, spindle formation, and chromosome alignment and segregation (1,2). For example, Kid, a member of the chromosome-associated KIFs, is important for chromosome alignment and orientation (3)(4)(5). KIF4, together with microtubule-bundling protein PRC1, participates in the organization of central spindle midzone formation (6). Furthermore, Caenorhabditis elegans ZEN-4/MKLP1 is required for central spindle assembly and cytokinesis (7).
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). The sea urchin kinesin-2 homolog, kinesin II, is present transiently in the mitotic apparatus of dividing embryos (18). 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") (19,20). 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.
Antibodies-Rabbit polyclonal antibody to KAP3 (N1 and C2) were prepared as described previously (14). 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 ␣-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.
Phosphatase Treatment-KAP3 immunoprecipitates were washed three times with bacterial alkaline phosphatase buffer (20 mM Tris-HCl, pH 8.0, 1 mM MgCl 2 ) 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 Na 3 VO 4 and 10 mM NaF).
Cell Cycle Synchronization-HeLa cells were synchronized by thymidine at the G 1 /S boundary as described (21). 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 G 1 /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 (22,23). 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 ϫ g for 30 min. The supernatants, together with 10 g/ml polybrene (Sigma), were added to NIH3T3 cells. The infected cells were selected using 2 g/ml puromycin (Sigma) for 3 days.

RESULTS
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 G 2 /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  FEBRUARY 17, 2006 • VOLUME 281 • NUMBER 7 not observed in the presence of phosphatase inhibitors (Fig. 1B). These results suggest that KAP3 is phosphorylated during mitosis.

The Kinesin-2 Complex and 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). 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 (data not shown). Although this mutant was localized at the spindle microtubules, similar to wild-type KIF3B, cells expressing this mutant showed an unusual number of centrosomes and abnormal spindle formation (Fig. 5A). By contrast, cells expressing the full-length KIF3B fused to GFP or control retrovirus vector did not show any abnormality in mito-sis. Furthermore, flow cytometric analysis revealed that the frequency of chromosomal aneuploidy in cells expressing this mutant was about three times higher than that of cells expressing the full-length KIF3B fused to GFP or control retrovirus vector (Fig. 5B). Similar aberrant mitosis was found to occur when a fragment of KIF3A fused to GFP (Fig.  3A, GFP-KIF3A Mutant 3 (Mut 3)), which can form a complex with KIF3B but cannot interact with KAP3, was expressed in NIH3T3 cells (data not shown). These results suggest that the interaction of KIF3A/3B with KAP3 may be important for the proper progression of mitosis.

DISCUSSION
In the present study, we demonstrated that human KIF3A/3B and KAP3 form a complex in mitosis and that they localize at the mitotic apparatus, mainly at the spindle microtubules and centrosomes in HeLa cells. Furthermore, we showed that expression of a dominant-negative mutant of KIF3A/3B results in aberrant mitosis. Our findings suggested that the kinesin-2 complex plays a critical role not only in interphase but also in mitosis.
Our domain analysis revealed that amino acids 528 -614 of KAP3 may interact with the C-terminal regions of KIF3A/3B. This result is consistent with previous data obtained by low angle rotary-shadowing electron microscopy showing that KAP3 is a globular protein and is associated with the tail domain of KIF3A/3B (16). We utilized deletion constructs of KIF3A/3B used for domain analysis (Fig. 3A, Mutant 3 (Mut 3)) to assess the significance of the kinesin-2 complex in mitosis since we could not efficiently knock down expression of KIF3A/3B and KAP3 using small interfering RNA. These mutants could form a heterodimer but could not interact with KAP3. Interestingly, when expressed in NIH3T3 cells, these mutants caused chromosomal aneuploidy and aberrant spindle formation. Thus, the interaction between KIF3A/3B and KAP3 may be required for spindle formation and chromosome segregation, although one cannot exclude the possibility that a protein(s) other than KAP3 also interacts and is critical for this function. We speculate that there may be an important mitosis-specific cargo protein(s) to which KIF3A/3B associate via an interaction with KAP3. Intriguingly, it has been reported that APC localizes to the ends of microtubules embedded in kinetochores and plays a critical role in chromosome segregation (24,25). Since we have previously found that APC is associated with KIF3A/3B via an association with KAP3 (14), APC may be one of these critical cargo proteins involved in the progression of mitosis.
It was reported that mutations in the kinesin-2 motor subunit genes result in chromosome loss phenotypes in Chlamydomonas (19). However, it was suggested that these phenotypes arise due to toxic effects of the mutant gene products. Also, microinjection of a kinesin-2-specific monoclonal antibody in sea urchin embryos inhibits ciliogenesis but has no effect on mitosis or cytokinesis (20). Furthermore, homozygous KIF3A and KIF3B knock-out mice apparently undergo many cell divisions until they die during the midgestational period (26 -28), suggest- ing that kinesin-2 is not critical for the progression of mitosis. These findings appear to conflict with our results. However, it may be possible that the importance of kinesin-2 function in mitosis may differ depending on the cell type and developmental stage. To further clarify the role of kinesin-2 in mitosis, it would be necessary to establish knock-out cell lines and to scrutinize the function of kinesin-2 in mitosis.
We showed that KAP3 is phosphorylated during mitosis. In this regard, it is interesting that some members of the KIF family have been reported to be phosphorylated by mitotic kinases. For example, CENP-E and Eg5 are phosphorylated by cdc2-cyclin B1 (29,30). Xenopus Aurora kinase pEg2-mediated phosphorylation of Eg5 is required for spindle formation and stabilization (31). Also, cdc2-cyclin B1-mediated phosphorylation of Kid controls its distribution to spindle and chromosomes (32). Thus, it is possible that phosphorylation of KAP3 may play a role in the regulation of mitotic progression. Although phosphorylation of KAP3 did not affect its KIF3A/3B binding ability, it is interesting to speculate that phosphorylation of KAP3 may regulate its interaction with cargo proteins that are involved in spindle formation and chromosome segregation. Identification of mitosis-specific cargo proteins and kinases responsible for KAP3 phosphorylation may provide insights into the significance of KAP3 phosphorylation.