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First published online July 9, 2004; 10.1104/pp.104.044818 Plant Physiology 135:1417-1429 (2004) © 2004 American Society of Plant Biologists A Plant-Specific Subclass of C-Terminal Kinesins Contains a Conserved A-Type Cyclin-Dependent Kinase Site Implicated in Folding and Dimerization1Department of Plant Systems Biology, Flanders Interuniversity Institute for Biotechnology (VIB), Ghent University, B–9052 Gent, Belgium
Cyclin-dependent kinases (CDKs) control cell cycle progression through timely coordinated phosphorylation events. Two kinesin-like proteins that interact with CDKA;1 were identified and designated KCA1 and KCA2. They are 81% identical and have a similar three-partite domain organization. The N-terminal domain contains an ATP and microtubule-binding site typical for kinesin motors. A green fluorescent protein (GFP) fusion of the N-terminal domain of KCA1 decorated microtubules in Bright Yellow-2 cells, demonstrating microtubule-binding activity. During cytokinesis the full-length GFP-fusion protein accumulated at the midline of young and mature expanding phragmoplasts. Two-hybrid analysis and coimmunoprecipitation experiments showed that coiled-coil structures of the central stalk were responsible for homo- and heterodimerization of KCA1 and KCA2. By western-blot analysis, high molecular mass KCA molecules were detected in extracts from Bright Yellow-2 cells overproducing the full-length GFP fusion. Treatment of these cultures with the phosphatase inhibitor vanadate caused an accumulation of these KCA molecules. In addition to dimerization, interactions within the C-terminally located tail domain were revealed, indicating that the tail could fold onto itself. The tail domains of KCA1 and KCA2 contained two adjacent putative CDKA;1 phosphorylation sites, one of which is conserved in KCA homologs from other plant species. Site-directed mutagenesis of the conserved phosphorylation sites in KCA1 resulted in a reduced binding with CDKA;1 and abolished intramolecular tail interactions. The data show that phosphorylation of the CDKA;1 site provokes a conformational change in the structure of KCA with implications in folding and dimerization.
Although cell division is elementary to growth, the process itself only claims a small part of the complete plant cell cycle period. During that short time, the microtubular cytoskeleton undergoes major transitions and, consecutively, a preprophase band, spindle, and phragmoplast are formed (Vantard et al., 2000  ; Hasezawa and Kumagai, 2002). These microtubule (MT) arrays are the basis for scaffolds along which chromosomes are aligned and separated into daughter nuclei, and cell wall material is transported to the site where the new cell plate emerges. The exact order of events demands for a perfect orchestration of the action of many proteins. Phosphorylation is an important regulatory mechanism in the control of MT organization during the mitotic processes.
Plant A-type cyclin-dependent kinases (CDKs), such as CDKA;1 in Arabidopsis, are the principal regulators of the orderly progression of cell cycle. CDKA;1 is associated with MTs in dividing and interphase cells (Stals et al., 1997
In yeast and animal cells, CDK regulates MT organization and function by controlling the activity or distribution of multiple proteins that are involved in MT arrangement and transport activities. CDK phosphorylates MT-associated proteins, which are important for MT dynamics and stability (Cassimeris, 1999
Several kinesins carry one or more putative CDK consensus Ser/Thr phosphorylation sites. For example, the bimC kinesin subfamily contains a phosphorylation site around a conserved sequence motif, called the bimC box. Phosphorylation of the embedded Thr in the human bimC kinesin Eg5 is a prerequisite for Eg5 to localize to the mitotic spindle and to ensure the formation of a bipolar organization of the spindle (Blangy et al., 1995 We report the isolation and characterization of two Arabidopsis kinesin-like proteins that interact with CDKA;1, designated KCA1 and KCA2 (acronym for kinesin CDKA;1 associated). KCA1 and KCA2 are unusual kinesins in that they are unique to plants and possess an N-terminal motor domain that is most similar to that of the C-terminal subfamily of kinesins. We show that KCA1 and KCA2 dimerize through a coiled-coil region in the center and fold intramolecularly through interactions within the tail domain. These conformational properties were regulated by a phosphorylation-dependent control mechanism that involves a putative CDKA consensus phosphorylation site.
Cloning of KCA1 and KCA2
We screened a cDNA
Because KCA1 is potentially phosphorylated by CDKA;1, the presence of consensus CDK phosphorylation sites (S/T-P-X-K/R) was searched for. The tail contained putative A-type CDK phosphorylation sites at positions 698, 849, and 853 in KCA1 and at positions 827 and 831 in KCA2 (Fig. 1B). A single SPGR site in the N-terminal part of the tail domain was fully conserved in KCA-like sequences of other species (Fig. 1C).
The presence of KCA mRNA transcripts was analyzed in different plant organs and developmental stages by reverse transcription (RT)-PCR with gene-specific primers. DNA fragments of the expected size corresponding to KCA1 or KCA2 transcripts were visualized after hybridization (Fig. 2A ). KCA1 and KCA2 mRNA were detected in young seedlings and in log-phase cell suspension cultures. KCA1 transcripts were more abundant and required a shorter film exposure to be visualized. Expression of KCA1 and KCA2 transcripts was found in all the organs tested (roots, leaves, stems, and flowers) and was more elevated in roots and flowers. Steady-state expression levels were observed for the ACT2 gene, which served as a loading control.
To determine the transcriptional activity of KCA1 and KCA2 during the course of the cell cycle, an Arabidopsis cell suspension culture was synchronized at G1/S by applying aphidicolin (Menges and Murray, 2002 ). Samples were prepared from 1-h intervals after drug release and RNA subjected to RT-PCR analysis. The transcriptional levels of KCA1 and KCA2 were constant throughout the cell cycle (Fig. 2B). This is consistent with the ubiquitous expression pattern revealed by immunoblot and immunolocalization by Kong and Hanley-Bowdoin (2002).
The CDKA;1-binding sites of KCA1 and KCA2 were determined by two-hybrid analysis whereby positive interactions were defined by the ability to grow on medium without His. The different KCA1 and KCA2 fragments used for the analysis are presented in Figure 3A . The CDKA;1-binding sites were mapped to the head domain (KCA11-497 and KCA236-507), the centrally located stalk region (KCA1395-926, KCA1473-866, KCA2425-617, and KCA2425-864), and the N-terminal part of the tail domain (KCA1660-862 and KCA2655-864). In addition, the full-length KCA1 protein showed CDKA;1-binding affinity (Fig. 3A). The interaction strength of the individual peptide fragments and full length was estimated by including the HIS3 competitive inhibitor 3-aminotriazole (3-AT) in the growth medium. The strongest interactions were observed with KCA fragments that included the conserved CDK phosphorylation site SPGR, with the exception of the complete tail domain (KCA1660-1,273) that did not interact with CDKA;1. These data indicate that the three separate KCA domains contribute to the interaction with CDKA;1 and that the C-terminal part of the tail has an inhibitory effect on the binding with CDKA;1.
The KCA-CDKA;1 interaction was also analyzed by coimmunoprecipitation experiments using a coupled transcription-translation system in which the KCA1 and KCA2 fragments were tagged with c-Myc and CDKA;1 with hemagglutinin epitope (HA; Fig. 3B). In the control experiment, the c-Myc-tagged peptides precipitated with a c-Myc monoclonal antibody, confirming the correct synthesis of the KCA peptide fragments. Next, the c-Myc- and HA-tagged translation products were mixed and pulled down with monoclonal anti-HA antibodies. The KCA1 peptide fragments containing either the N-terminal tail (KCA1660-862) or the head (KCA11-497) cosedimented with HA-CDKA;1, whereas the fragment containing the C-terminal tail domain (KCA1875-1,273) did not (Fig. 3B, left panel). Similar results were obtained with peptide fragments of KCA2. The stalk (KCA2425-617) and the N-terminal part of the tail (KCA2655-864) were pulled down together with HA-CDKA;1, whereas the C-terminal tail (KCA2855-1,267) was not (Fig. 3B, right panel). The results confirmed the KCA and CDKA;1 interactions that had been revealed by the two-hybrid analysis. None of the KCA fragments was able to bind with the anti-HA antibody in the absence of HA-CDKA;1 (data not shown).
To investigate the association of KCA1 with CDKA;1 in vivo, CDK-protein complexes were purified from Bright Yellow-2 (BY-2) transgenic cell cultures that produced green fluorescent protein (GFP)-tagged versions of the full-length and fragments containing the motor (KCA11-497) or the tail domain (KCA1660-1,273; Fig. 3C). Extracts of the transgenic cultures and control wild-type BY-2 cells were mixed with p10CKS1AtCDKA;1 affinity beads. Crude extract, pellet, and supernatant were analyzed by western blot and developed with polyclonal GFP antibody. As shown in Figure 3C, GFP-fusion products corresponding to the predicted molecular mass were detected in the separated crude BY-2 extracts. Compared to the noninduced protein extract (–), three kinesin-related protein products were present in the preparations from dexamethasone-induced cells producing the full-length kinesin GFP-fusion protein. The 160-kD band corresponded to the intact GFP-KCA1 protein, whereas the smallest 40-kD protein resulted from degradation or prematurely arrested translation. The high molecular mass band at approximately 250 kD may represent a GFP-KCA1 dimer. The presence of kinesin dimers in denaturing polyacrylamide gels has been reported before (Fontijn et al., 2001
An important property of kinesin molecules is their ability to attach to MTs either for transport or control of MT organization (Walczak, 2003
The full-length GFP-KCA1 fusion protein was followed during cell division (Fig. 4J). Throughout mitosis, the fusion protein remained in the cortical cytoplasm and the cytoplasmic strands, and it invaded the unrestricted space of the spindle in metaphase and anaphase cells (Fig. 4J). Fluorescence was diffuse and did not reveal fibrous structures, indicating that the fusion protein did not attach to MTs (Fig. 4J). Once the daughter chromosomes were separated, GFP-KCA1 fluorescence accumulated at the midline of the emerging phragmoplast where Golgi-derived vesicles accumulate to form the cell plate (Fig. 4J). In a second stage of cell plate development, concomitant with expansion of the phragmoplast, fluorescence was most bright at the leading edges (Fig. 4J). Reduced fluorescence was observed at the center of the centrifugally expanding phragmoplast, where the cell plate starts to mature and MTs are depolymerized.
In contrast to the findings of Kong and Hanley-Bowdoin, GFP-KCA1 did not concentrate in the nucleus, nor did it associate with condensed chromosomes in metaphase cells. As the N-terminal domain in front of the motor domain may be implicated in nuclear targeting or chromosome binding, we analyzed the subcellular localization of a C-terminal fusion of KCA1 in BY-2 cells. KCA1-GFP was excluded from the nucleus and vacuoles (Fig. 4K). In the cytoplasm, it was associated with a reticulated network resembling the endoplasmic reticulum (ER; Fig. 4K). During division (Fig. 4L), KCA1-GFP was distributed to the polar sides of the spindle and the midline of the phragmoplast reminiscent to the subcellular localization of an ER-targeted marker in BY-2 cells (Saint-Jore et al., 2002
Several observations suggested that the KCA kinesins adopt different folding configurations with distinct properties in terms of interaction with CDKA;1 and in relation to their subcellular localization. Firstly, the N-terminal part of the tail domain could bind CDKA;1 only when the C-terminal part was not included. The inhibitory activity of the C-terminal part of the tail was not evident when the full-length proteins were tested, indicating that these had taken on an alternative configuration immune to control by the tail domain. Secondly, the full-length KCA1 GFP-fusion product was excluded from the nucleus while the head and tail as separate GFP fusion fragments entered the nucleus. Thirdly, MTs were not labeled with GFP fused to full-length proteins but with the GFP-head fusion product. Therefore, we examined the intra- and intermolecular interactions of KCA1 and KCA2 peptides by two-hybrid and immunoprecipitation assays. Full-length KCA and peptide fragments containing the complete or part of the central coiled-coil region resulted in yeast growth (combinations pGBT-KCA1395-926 with pGAD-KCA1473-866 and pGBT-KCA2425-617 with pGAD-KCA2425-864), indicating that both KCA1 and KCA2 could form homodimers (Fig. 5A ). Evidence for heterodimerization through the central coiled-coil region followed from yeast growth when the stalk domains of both kinesins were tested against each other (combinations of both pGBT-KCA2425-617 and pGBT-KCA2425-864 with pGAD-KCA1473-866).
Two-hybrid interactions were also observed between the N-terminal and the C-terminal halves of the tail of KCA1 (combination pGBT-KCA1660-862 with pGAD-KCA1875-1,273) and KCA2 (combination pGBT-KCA2425-864 with pGAD-KCA2855-1,267), pointing out that the tail domains had a tendency to fold onto themselves. A similar type of interaction also occurred between the N- and C-terminal tail domains of KCA1 and KCA2 (combinations pGBT-KCA1660-862 with pGAD-KCA2855-1,267 and pGBT-KCA1875-1,273 with pGAD-KCA2425-864). The folding of the KCA tails probably occurred via bending of two predicted hinge regions that were present in the tail domain (Fig. 1B). The tail fragment upstream of the first hinge region was essential for the interaction (Fig. 5), whereas that downstream of the second hinge did not interact in two-hybrid tests, indicating that the tail fragment between the two hinges was responsible for the interactions observed (combination pGBT-KCA11,067-1,273 and pGAD-KCA1473-866, and pGBT-KCA21,052-1,267 with pGAD-KCA2425-864, and the reciprocal combinations). The tail interactions of KCA1 and KCA2 were confirmed by coimmunoprecipitation assays (Fig. 5B). Protein fragments containing the N-terminal part of the tail of KCA1 or KCA2 pulled down a KCA1 fragment containing sequences downstream of the first hinge region (Fig. 5B).
We showed that KCA1 and KCA2 formed homo- or heterodimers and that both proteins can occur in a folded conformation. In addition, both proteins bind to CDKA;1 and contain CDKA;1 phosphorylation sites in the tail domain. Hence, we investigated whether CDKA;1 phosphorylation was implicated in KCA dimerization and folding.
Western blot of BY-2 cells transformed with GFP fused to the full-length KCA1 protein (Fig. 3C) revealed two high molecular mass bands, one of which probably represents a dimeric form of GFP-KCA1. To test the role of phosphorylation in dimer formation, a phosphatase inhibitor was applied to the cells. BY-2 cells transformed with GFP-KCA1 were treated with vanadate (10 mM; Brown et al., 1999 To investigate whether CDKA;1 phosphorylation could have implications for CDKA;1 binding and KCA folding, we introduced mutations in the putative phosphorylation sites at positions 698 to 701 (TPNK) and 841 to 848 (SPGR/SPVR) in the pGBT-KCA1660-862 sequence that contains the N-terminal tail of KCA1. Thr (T698) and Ser (S841 and S845) were replaced by either an Ala (A) as a nonphosphorylable residue or by a Glu (E) that mimics the phosphorylated residue (Table I). We assessed the effects these changes had on the ability to interact with either CDKA;1 or with the KCA tail by means of two-hybrid analysis. Replacement of T698 by either an A or E had no consequences on the interaction with CDKA;1. On the contrary, substitution of the consensus sequences further downstream, S841 or S841/845 (S841 and S845 double substitution) with E residues, disallowed the yeast strain to grow on selective medium. Replacement of these residues by an A had little or no effect. These results indicated that CDKA;1 binding was sensitive to the phosphorylation status of residues S841 and S845.
The same mutagenized KCA1660-862 fragments were tested against the C-terminal tail fragments KCA1875-1,273 and KCA2855-1,267 in the pGAD vector. Alteration of T698 in either A or E had no effect on the tail interactions. However, substitution of S841 into an E residue strongly reduced the interaction with the C-terminal tail regions of both KCA1 and KCA2, while replacement of both S841 and S845 completely abolished the interaction (Table I). Changing these residues by an A did not alter the growth of yeast. The results suggest that phosphorylation at the consensus sequences 841-844 and 845-848 in KCA1 and 827-830 in KCA2 influences the protein conformation that has important consequences concerning their activity.
CDKA governs control over the progression of the cell division processes through the interaction with several partners and selective phosphorylation of target proteins. Despite considerable efforts, only few potential targets phosphorylated by CDKA complexes have been identified in plants so far (Reindl et al., 1997 ; Nakagami et al., 1999; Boniotti and Gutierrez, 2001). The kinesin KCA1 is a novel candidate target that is phosphorylated in a cell cycle kinase-dependent manner in insect cells (Kong and Hanley-Bowdoin, 2002). We report on the interaction of KCA1, and a highly homologous protein KCA2, with CDKA;1 and the role of a conserved CDKA phosphorylation site in dimerization and folding.
KCA1 and KCA2 share high sequence identity and have a conserved structural organization that is reminiscent to classic kinesin molecules. The N-terminal region contains an ATP-loop and an MT-binding site and is most similar to the motor domain of the C-terminal subfamily of kinesins (Kim and Endow, 2000
The calmodulin-binding protein KCBP is a minus-end kinesin that belongs to the C-terminal clad, which is nearest to that containing KCA1 and KCA2 (Dagenbach and Endow, 2004
Binding of MT with KCBP is controlled by calcium through the interaction with calcium calmodulin (Narasimhulu et al., 1997
KCBP carries an MT-binding site in the N-terminally located tail domain that is independent from calmodulin (Narasimhulu and Reddy, 1998
As certain folding configurations may no longer have been possible because of the presence of the GFP moiety, a different subcellular localization of KCA was revealed. C-terminally tagged KCA1-GFP appeared to associate with the ER. It concentrated at the polar sides of the metaphase spindle where ER and Golgi derived organelles are known to congregate (Nebenführ et al., 2000
The KCA proteins have been shown to interact with CDKA;1 in two-hybrid assays (De Veylder et al., 1997
The coiled coils in the stalk region have been implicated in kinesin oligomerization that is necessary for proper control of motility and cargo binding (Vale and Fletterick, 1997 The KCA tail domain carries a CDKA;1 phosphorylation site that is conserved in all KCA-like kinesins found in the publicly available databases. Therefore, this site is the best candidate for a general role in the functioning of the KCA kinesins. Two-hybrid and immunoprecipitation experiments revealed that the N-terminal part of the tail domain interacted with CDKA;1 as well as intramolecularly between the N-terminal part of the tail and a downstream region flanked by the two hinges. These interactions are probably mutually exclusive because the tail, when tested in its entirety, did not bind CDKA;1. The folding of the tail fragment would have prevented an interaction with CDKA;1. How could CDKA;1 affect the conformational changes and functioning of KCA? The phosphorylation, dimerization, and the internal tail interactions are probably interdependent and may be implicated in phosphorylation-controlled activation and/or binding of cargo. Point mutations in the putative CDKA phosphorylation sites of the KCA1 tail abolished the intramolecular tail interaction. Thus, KCA molecules not phosphorylated at the Ser residues in the tail would have a compact folding conformation. This conformational stage might keep the KCA inactive until modulated by the cell cycle-controlled CDKA;1 kinase. The opening up of the tail would prepare the single KCA molecules to bind the cargo they need to transport. Alternatively to the stimulation of cargo binding upon phosphorylation, it is also possible that the opened-up kinesin tail no longer prevents the homo- or heterodimerization that is driven by the stalk domain.
Isolation and cDNA Characterization of KCA1 and KCA2
A cDNA
The Arabidopsis KCA proteins and KCA homologs were aligned by the Clustal method (PileUp) from the GCG Wisconsin package version 10.1 program (Accelrys, San Diego) without penalizing gaps. A set of analysis tools was applied for a compressive sequence interpretation. As BLAST browsers, the programs AtBlast (http://www.arabidopsis.org/blast/) (Huala et al., 2001
RNA was prepared from 200 mg of 3-d-old Arabidopsis cell suspensions, 3-week-old plants (roots, rosette leaves, stems, and flowers), and 1-week-old seedlings. Total RNA was isolated with the RNeasy plant mini kit (Qiagen, Hilden, Germany) and used as template for semiquantitative RT-PCR with Superscript RT II reverse transcriptase (Invitrogen, Carlsbad, CA) and oligo(dT)18. From the 50-µL PCR reaction, 10 µL was separated on a 1% Tris-acetate EDTA agarose gel and transferred onto Hybond N+ membranes (Amersham Bioscience, Little Chalfont, UK). The membranes were hybridized at 65°C with fluorescein-labeled probes (Gene Images random prime module; Amersham Biosciences) and detected with the CDP Star detection module (Amersham Biosciences). For RT-PCR, the following primers were used: 5'-GTGCCGGTTTTATCCTCGTTGACATCC-3' and 5'-CGTATCAAGATATCGAACAGGGG-3' for the KCA1 gene (position 1,185–2,556 bp); 5'-CCGATGATCGTCAACATTTGTCCAAGTGC-3' and 5'-ACGGATTCTTGAAACTACAGATACC-3' for the KCA2 gene (position 1,275–2,592 bp); and 5'-CTAAGCTCTCAAGATCAAAGGCTTA-3' and 5'-TTAACATTGCAAAGAGTTTCAAGGT-3' for Arath;ACT2 (U41998).
For cell cycle-dependent expression analysis, a suspension culture of the Arabidopsis cell line MM2d (Menges and Murray, 2002
RNA was prepared following the method described by Leyman et al. (2000)
The CDKA;1 interaction site was mapped by constructing deletion fragments of the KCA genes. The DNA fragments were created by PCR with the Pfu polymerase (Stratagene, La Jolla, CA) and subcloned in the pGBT9 vector. The CDKA;1 was inserted into the pGAD424 vector (CLONTECH). A series of deletion fragments of KCA1 and KCA2 cDNA were amplified with primers containing EcoRI and BamHI enzyme restriction sites. The amplified DNA fragments were inserted into the EcoRI and BamHI sites of the pGBT9 and pGAD424 vectors. The original pGAD-TH65 clone (residues 473–866) was also included as a positive control for CDKA;1 binding. Yeast strain HF7c reporter strain (MATa ura3-52 his3-200 ade2-101 lys2-801 trp1-901 leu2-3112 gal4-542 gal80-538 LYS2::GAL1UAS-GAL1TATA-HIS3, URA3::GAL417-mers(x3)-CYC-1TATA_Lacz; CLONTECH) was cotransformed with pGAD-KCAx (x is deletion fragment) and pGAD-CDKA;1 or the empty vector as described previously (De Veylder et al., 2001
The same KCA deletion fragments generated by PCR for the two-hybrid assays were used to generate the pBSK-c-Myc and pBSK-HA vectors (Stratagene). The Arabidopsis CDKA;1 was recloned from the pGADCDKA;1 vector in the pBSK-HA vector with EcoRI and BamHI restriction sites. Plasmids were sequenced to verify in-frame cloning with the c-Myc tag and HA tag. In vitro transcription and translation experiments were performed separately for each construct with the TNT T7-coupled wheat germ extract kit (Promega, Madison, WI) primed with the appropriate template for 90 min at 30°C. For immunoprecipitation assays, 10 µL of the c-Myc-KCA total in vitro translated extract (50 µL) was mixed with 5 µL of the HA-CDKA;1 total in vitro translated extract, diluted at 1:5 in Nonidet P-40 buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 10 µg/mL leupeptin/aprotinin/pepstatin), and incubated for 2 h at 4°C with anti-HA antibodies (9E10; BabCo-Covance, Berkeley, CA). Protein-A-Sepharose beads (40 µL, 25% [v/v]) were added and incubated for 1 h at 4°C. The beads were washed four times with 1 mL Nonidet P-40 buffer. Immune complexes were eluted with 10 µL of 2x SDS sample buffer, analyzed by a 13% SDS-PAGE, and autoradiographed. The same procedure was followed using the c-Myc-KCAs and HA-KCAs constructs to test for dimerization.
The KCA1 full-length open reading frame and fragments were cloned behind the open reading frame of enhanced GFP by using the GATEWAY system (Invitrogen). GATEWAY-compatible vectors were designed by inserting the EGFP-coding region and the GATEWAY rfA cassette into the pBin19 backbone. The expression of the fusion was under the control of the cauliflower mosaic virus 35S promoter and 35S polyadenylation signal. For the construction of the inducible vector, a similar strategy was followed. The EGFP-rfA cassette was cloned into the pTA7002 vector allowing dexamethasone (Sigma-Aldrich)-inducible expression of the fusion protein (Aoyama and Chua, 1997 The KCA1 full-length and fragments were amplified with GATEWAY attB-flanked primers with the Pfx polymerase (Invitrogen). Via BP and subsequent LR reactions, the fragments were introduced into the destination vectors described above. The borders of the inserted fragments were sequenced prior to further analysis.
Growth of BY-2 cells was according to Nagata et al. (1992) BY-2 cultures were transformed with pBin19GFP carrying the KCA1 motor (KCA11-497), and the KCA1 tail (KCA1660-1,273), leading to constitutive expression of fusion protein. For subcellular localization of the full-length KCA1 protein, the dexamethasone-inducible vector pTA7002GFP carrying the KCA11-1,273 fragment was used. Calli producing GFP-fusion proteins were identified by fluorescence microscopy. BY-2 cells transformed with the inducible expression vector were induced overnight on BY-2 agar containing 10 µM dexamethasone. Callus material was transferred on a slide with a coverslip, and observed with an epifluorescence microscope (Axioplan 2; Zeiss, Jena, Germany) equipped with a fluorescein isothiocyanate filter set. GFP-positive calli were transferred to liquid BY-2 medium with selection and grown as cell suspensions. Confocal images were taken with a scanning confocal microscope (LSM 510; Zeiss) with argon laser illumination at 488 nm and a fluorescein isothiocyanate filter set. For transmission light images, differential interference contrast optics was used. Images were taken with 25% laser power to reduce photobleaching.
Cells were incubated in liquid BY-2 medium containing 0.1% Triton X-100 (Sigma-Aldrich) for 15 min with gentle agitation. Cells were washed twice in BY-2 medium, transferred to a slide, and covered with a coverslip. The detergent-extracted cells were observed directly with the confocal microscope.
Three-day-old liquid cultures of BY-2 transgenic lines, also used for GFP-localization experiments, and wild-type BY-2 cells were ground in liquid nitrogen with a mortar and pestle and homogenized in ice-cold P10 buffer (25 mM Tris-HCl, pH 7.6, 15 mM ethyleneglycol-bis( For drug analysis, 10 mM Na3VO4 was added to a 2-d-old BY-2 culture, transformed with the inducible GFP-KCA1 construct. As control, a nontreated culture was cultured simultaneously. After 24 h of growth at 28°C, crude protein extracts were prepared in the P10 homogenization buffer. Thirty micrograms of proteins was loaded on a 12% gel and processed as described above.
Point mutations were introduced by PCR site-directed mutagenesis in the pGTB-KCA1660-862 plasmid with the Advantage polymerase mix (CLONTECH). The linear PCR product was circularized by ligation. The nucleotide changes were verified by sequencing the KCA1 inserts in two directions.
Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes. Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession numbers AX449336 and AX449307.
We thank Martine De Cock for help preparing the manuscript. Received April 19, 2004; returned for revision May 19, 2004; accepted May 19, 2004.
1 This work was supported by grants from Interuniversity Poles of Attraction Programme-Belgian Science Policy (P5/13), by the Instituut voor de aanmoediging van Innovatie door Wetenschap en Technologie in Vlaanderen and the Consejo Nacional de Ciencia y Tecnologia, Mexico (CONACYT; grant no. 120198; predoctoral fellowships to M.V. and J.A.T.A., respectively), and by the Fund for Scientific Research-Flanders (postdoctoral fellowships to L.D.V. and D.G.). 
2 Present address: Zentrum für Angewandte Genetik Universität für Bodenkultur, Muthgasse 18, A–1190 Wien, Austria. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.044818. * Corresponding author; e-mail dirk.inze{at}psb.ugent.be; fax 32 9 3313809.
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ABSTRACT
RESULTS
phage library with a partial kinesin fragment TH65 that had been isolated previously in a two-hybrid screen with CDKA;1 (De Veylder et al., 1997
-aminoethyl)tetra-acetate, 1 mM dithiothreitol, 15 mM MgCl2, 85 mM NaCl, 15 mM pNO2PhePO4, 60 mM glycerol phosphate, 0.1% Nonidet P-40, 1 mM NaF, 0.1 mM Na3VO4, and 100 µL protease inhibitor cocktail; Sigma-Aldrich). The homogenate was centrifuged at 10,000g for 10 min in an Eppendorf centrifuge 5417 at 4°C to remove cell debris. The supernatant was then centrifuged at 14,000g for 10 min. A sample was taken and kept on ice as crude extract. Then, 50 µL of 50% (v/v) p10CKS1At Sepharose beads was added to 300 µg of proteins and incubated at 4°C for 1 h on a rotating wheel. The beads were collected by centrifugation; the supernatant was removed and kept on ice. Beads were washed three times with bead buffer (50 mM Tris-HCl, pH 7.5, 5 mM NaF, 250 mM NaCl, 5 mM EGTA, 5 mM EDTA, 10 µg/mL leupeptin, 10 µg/mL aprotinin, 0.1 mM benzamidine, and 0.1 mM Na3VO4). Loading buffer (Laemmli, 1970
