Bundling of microtubules by motor and tail domains of a kinesin-like calmodulin-binding protein from Arabidopsis: regulation by Ca(2+)/Calmodulin

Kinesin-like calmodulin-binding protein (KCBP), a novel kinesin-like protein from plants, is unique among kinesins and kinesin-like proteins in having a calmodulin-binding domain adjacent to its motor domain. KCBP localizes to mitotic microtubule (MT) arrays including the preprophase band, the spindle apparatus, and the phragmoplast, suggesting a role for KCBP in establishing these MT arrays by bundling MTs. To determine if KCBP bundles MTs, we expressed C-terminal motor and N-terminal tail domains of KCBP, and used the purified proteins in MT bundling assays. The 1.5 C protein with the motor and calmodulin-binding domains induced MT bundling. The 1.5 C-induced bundles were dissociated in the presence of Ca(2+)/calmodulin. Similar results were obtained with a 1.4 C protein, which lacks much of the coiled-coil region present in 1.5 C protein and does not form dimers. The N-terminal tail of KCBP, which contains an ATP-independent MT binding site, is also capable of bundling MTs. These results, together with the KCBP localization data, suggest the involvement of KCBP in establishing mitotic MT arrays during different stages of cell division and that Ca(2+)/calmodulin regulates the formation of these MT arrays.     

Origin and evolution of Kinesin-like calmodulin-binding protein

Kinesin-like calmodulin-binding protein (KCBP), a member of the Kinesin-14 family, is a C-terminal microtubule motor with three unique domains including a myosin tail homology region 4 (MyTH4), a talin-like domain, and a calmodulin-binding domain (CBD). The MyTH4 and talin-like domains (found in some myosins) are not found in other reported kinesins. A calmodulin-binding kinesin called kinesin-C (SpKinC) isolated from sea urchin (Strongylocentrotus purpuratus) is the only reported kinesin with a CBD. Analysis of the completed genomes of Homo sapiens, Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, and a red alga (Cyanidioschyzon merolae 10D) did not reveal the presence of a KCBP. This prompted us to look at the origin of KCBP and its relationship to SpKinC. To address this, we isolated KCBP from a gymnosperm, Picea abies, and a green alga, Stichococcus bacillaris. In addition, database searches resulted in identification of KCBP in another green alga, Chlamydomonas reinhardtii, and several flowering plants. Gene tree analysis revealed that the motor domain of KCBPs belongs to a clade within the Kinesin-14 (C-terminal motors) family. Only land plants and green algae have a kinesin with the MyTH4 and talin-like domains of KCBP. Further, our analysis indicates that KCBP is highly conserved in green algae and land plants. SpKinC from sea urchin, which has the motor domain similar to KCBP and contains a CBD, lacks the MyTH4 and talin-like regions. Our analysis indicates that the KCBPs, SpKinC, and a subset of the kinesin-like proteins are all more closely related to one another than they are to any other kinesins, but that either KCBP gained the MyTH4 and talin-like domains or SpKinC lost them.     

CpKLP1: A CALMODULIN-BINDING KINESIN-LIKE PROTEIN FROM CYANOPHORA PARADOXA (GLAUCOPHYTA)

KCBP (kinesin-like calmodulin [CaM]-binding proteins), a member of the carboxy-terminal kinesin-like proteins (KLPs), is unique among KLPs in having a CaM-binding domain (CBD). CaM-binding KLPs have been identified from flowering plants and the sea urchin. To determine if CaM-binding KLP is present in phylogenetically divergent protists, we probed Cyanophora paradoxa protein extract with affinity-purified KCBP antibody. The KCBP antibody detected a polypeptide with a molecular mass of about 133 kDa in the crude extract. In a CaM–Sepharose column-purified fraction, the same band was detected with both KCBP antibody and biotinylated CaM. In a PCR reaction using degenerate primers corresponding to two conserved regions in the motor domain of kinesin, a 500-bp fragment (CpKLP1) was amplified from a cDNA library. The predicted amino acid sequence of CpKLP1 showed significant sequence similarity with KCBPs. In phylogenetic analysis, CpKLP1 fell into the KCBP group within the carboxy-terminal subfamily. These biochemical data, sequence, and phylogenetic analysis strongly suggest the presence of a calmodulin-binding KLP in C. paradoxa and that it is related to Ca²+/calmodulin regulated KLPs from plants. This is the first report on identification of any motor protein in C. paradoxa. Furthermore, our data suggest that CaM-binding KLPs may have evolved long before the divergence of plants and animals.     

Interaction of Arabidopsis kinesin-like calmodulin-binding protein with tubulin subunits: modulation by Ca2+-calmodulin

K inesin-like c almodulin-b inding p rotein (KCBP) is a recently identified novel kinesin-like protein that appears to be unique to and ubiquitous in plants. KCBP is distinct from all other known KLPs in having a calmodulin-binding domain adjacent to its motor domain. We have used different regions of KCBP to study its interaction with tubulin subunits and the regulation of this interaction by Ca²⁺-calmodulin. The results show that the carboxy-terminal part of the KCBP, with or without calmodulin-binding domain, binds to tubulin subunits and this binding is sensitive to nucleotides. In the presence of Ca²⁺-calmodulin the motor with calmodulin-binding domain does not bind to tubulin. This Ca²⁺-calmodulin modulation is abolished in the presence of antibodies specific to the calmodulin-binding domain of KCBP. Similar binding studies with the carboxy-terminal part of KCBP lacking the calmodulinbinding domain show no effect of Ca²⁺-calmodulin. These results indicate that Ca²⁺-calmodulin modulates the interaction of KCBP with tubulin subunits and this modulation is due to the calmodulin-binding domain in the KCBP. Calcium-dependent calmodulin modulation of KCBP interaction with tubulin suggests regulation of KCBP function by calcium, the first such regulation of a kinesin heavy chain among all the known kinesin-like proteins.     

Interaction of a kinesin-like calmodulin-binding protein with a protein kinase

Kinesin-like calmodulin-binding protein (KCBP) is a novel member of the kinesin superfamily that is involved in cell division and trichome morphogenesis. KCBP is unique among all known kinesins in having a myosin tail homology-4 region in the N-terminal tail and a calmodulin-binding region following the motor domain. Calcium, through calmodulin, has been shown to negatively regulate the interaction of KCBP with microtubules. Here we have used the yeast two-hybrid system to identify the proteins that interact with the tail region of KCBP. A protein kinase (KCBP-interacting protein kinase (KIPK)) was found to interact specifically with the tail region of KCBP. KIPK is related to a group of protein kinases specific to plants that has an additional sequence between subdomains VII and VIII of the conserved C-terminal catalytic domain and an extensive N-terminal region. The catalytic domain alone of KIPK interacted weakly with the N-terminal KCBP protein but strongly with full-length KCBP, whereas the noncatalytic region did not interact with either protein. The interaction of KCBP with KIPK was confirmed using coprecipitation assays. Using bacterially expressed full-length and truncated proteins, we have shown that the catalytic domain is capable of phosphorylating itself. The association of KIPK with KCBP suggests regulation of KCBP or KCBP-associated proteins by phosphorylation and/or that KCBP is involved in targeting KIPK to its proper cellular location.     

Characterization of microtubule binding domains in the Arabidopsis kinesin-like calmodulin binding protein.

The kinesin-like calmodulin binding protein (KCBP) is a new member of the kinesin superfamily that appears to be present only in plants. The KCBP is unique in its ability to interact with calmodulin in a Ca2+-dependent manner. To study the interaction of the KCBP with microtubules, we expressed different regions of the Arabidopsis KCBP and used the purified proteins in cosedimentation assays with microtubules. The motor domain with or without the calmodulin binding domain bound to microtubules. The binding of the motor domain containing the calmodulin binding region to microtubules was inhibited by Ca2+-calmodulin. This Ca2+-calmodulin regulation of motor domain interactions with microtubules was abolished in the presence of antibodies specific to the calmodulin binding region. In addition, the binding of the motor domain lacking the calmodulin binding region to microtubules was not inhibited in the presence of Ca2+-calmodulin, suggesting an essential role for the calmodulin binding region in Ca2+-calmodulin modulation. Results of the cosedimentation assays with the N-terminal tail suggest the presence of a second microtubule binding site on the KCBP. However, the interaction of the N-terminal tail region of the KCBP with microtubules was insensitive to ATP. These data on the interaction of the KCBP with microtubules provide new insights into the functioning of the KCBP in plants.     

Localization of a kinesin-like calmodulin-binding protein in dividing cells of Arabidopsis and tobacco

The cloning and characterization of a novel kinesin-like protein ( k inesin-like c almodulin- b inding p rotein, KCBP) from Arabidopsis and other plants has recently been described. Unlike all other known kinesin-like proteins, KCBP interacts with calmodulin in the presence of micromolar calcium. An antibody specific to KCBP was raised using a calmodulin-binding synthetic peptide that is unique to KCBP. The KCBP antibody detected a single protein of about 140 kDa in Arabidopsis and tobacco, the size predicted from cDNA sequences. In synchronized cell cultures, the amount of KCBP was abundant during M-phase and very low in interphase. To get some insight into the function of this novel motor protein, KCBP in Arabidopsis and tobacco cells was localized by indirect immunofluorescence microscopy using affinity-purified anti-KCBP antibody. The KCBP was localized to the pre-prophase band, the mitotic spindle and the phragmoplast. The association of KCBP with microtubule arrays in dividing cells suggests that this minus-end-directed microtubule motor protein is likely to be involved in the formation of these microtubule arrays and/or functions associated with these structures.     

A novel kinesin-like protein with a calmodulin-binding domain

Calcium regulates diverse developmental processes in plants through the action of calmodulin. A cDNA expression library from developing anthers of tobacco was screened with ³⁵S-labeled calmodulin to isolate cDNAs encoding calmodulin-binding proteins. Among several clones isolated, a kinesin-like gene (TCK1) that encodes a calmodulin-binding kinesin-like protein was obtained. The TCK1 cDNA encodes a protein with 1265 amino acid residues. Its structural features are very similar to those of known kinesin heavy chains and kinesin-like proteins from plants and animals, with one distinct exception. Unlike other known kinesin-like proteins, TCK1 contains a calmodulin-binding domain which distinguishes it from all other known kinesin genes. Escherichia coli-expressed TCK1 binds calmodulin in a Ca²⁺-dependent manner. In addition to the presence of a calmodulin-binding domain at the carboxyl terminal, it also has a leucine zipper motif in the stalk region. The amino acid sequence at the carboxyl terminal of TCK1 has striking homology with the mechanochemical motor domain of kinesins. The motor domain has ATPase activity that is stimulated by microtubules. Southern blot analysis revealed that TCK1 is coded by a single gene. Expression studies indicated that TCK1 is expressed in all of the tissues tested. Its expression is highest in the stigma and anther, especially during the early stages of anther development. Our results suggest that Ca²⁺/calmodulin may play an important role in the function of this microtubule-associated motor protein and may be involved in the regulation of microtubule-based intracellular transport.     

A plant kinesin heavy chain-like protein is a calmodulin-binding protein

Calmodulin, a calcium modulated protein, regulates the activity of several proteins that control cellular functions. A cDNA encoding a unique calmodulin-binding protein, PKCBP, was isolated from a potato expression library using protein-protein interaction based screening. The cDNA encoded protein bound to biotinylated calmodulin and ³⁵S-labeled calmodulin in the presence of calcium and failed to bind in the presence of EGTA, a calcium chelator. The deduced amino acid sequence of the PKCBP has a domain of about 340 amino acids in the C-terminus that showed significant sequence similarity with the kinesin heavy chain motor domain and contained conserved ATP- and microtubule-binding sites present in the motor domain of all known kinesin heavy chains. Outside the motor domain, the PKCBP showed no sequence similarity with any of the known kinesins, but contained a globular domain in the N-terminus and a putative coiled-coil region in the middle. The calmodulin-binding region was mapped to a stretch of 64 amino acid residues in the C-terminus region of the protein. The gene is differentially expressed with the highest expression in apical buds. A homolog of PKCBP from Arabidopsis (AKCBP) showed identical structural organization indicating that kinesin heavy chains that bind to calmodulin are likely to exist in other plants. This paper presents evidence that the motor domain has microtubule stimulated ATPase activity and binds to microtubules in a nucleotide-dependent manner. The kinesin heavy chain-like calmodulin-binding protein is a new member of the kinesin superfamily as none of the known kinesin heavy chains contain a calmodulin-binding domain. The presence of a calmodulin-binding motif and a motor domain in a single polypeptide suggests regulation of kinesin heavy chain driven motor function(s) by calcium and calmodulin.     

The cotton kinesin-like calmodulin-binding protein associates with cortical microtubules in cotton fibers

Microtubules in interphase plant cells form a cortical array, which is critical for plant cell morphogenesis. Genetic studies imply that the minus end-directed microtubule motor kinesin-like calmodulin-binding protein (KCBP) plays a role in trichome morphogenesis in Arabidopsis. However, it was not clear whether this motor interacted with interphase microtubules. In cotton (Gossypium hirsutum) fibers, cortical microtubules undergo dramatic reorganization during fiber development. In this study, cDNA clones of the cotton KCBP homolog GhKCBP were isolated from a cotton fiber-specific cDNA library. During cotton fiber development from 10 to 21 DPA, the GhKCBP protein level gradually decreases. By immunofluorescence, GhKCBP was detected as puncta along cortical microtubules in fiber cells of different developmental stages. Thus our results provide evidence that GhKCBP plays a role in interphase cell growth likely by interacting with cortical microtubules. In contrast to fibers, in dividing cells of cotton, GhKCBP localized to the nucleus, the microtubule preprophase band, mitotic spindle, and the phragmoplast. Therefore KCBP likely exerts multiple roles in cell division and cell growth in flowering plants.     

The calmodulin-binding domain from a plant kinesin functions as a modular domain in conferring Ca2+-calmodulin regulation to animal plus- and minus-end kinesins

Plant kinesin-like calmodulin-binding protein (KCBP) is a novel member of the kinesin superfamily that interacts with calmodulin (CaM) via its CaM-binding domain (CBD). Activated CaM (Ca(2+)-CaM) has been shown to inhibit KCBP interaction with microtubules (MTs) thereby abolishing its motor- and MT-dependent ATPase activities. To test whether the fusion of CBD to non-CaM-binding kinesins confers Ca(2+)-CaM regulation, we fused the CBD of KCBP to the N or C terminus of a minus-end (non-claret disjunction) or C terminus of a plus-end (Drosophila kinesin) motor. Purified chimeric kinesins bound CaM in a Ca(2+)-dependent manner whereas non-claret disjunction, Drosophila kinesin, and KCBP that lack a CBD did not. As in the case of KCBP with CBD, the interaction of chimeric motors with MTs, as well as their MT-stimulated ATPase activity, was inhibited by Ca(2+)-CaM. The presence of a spacer between the motor and CBD did not alter Ca(2+)-CaM regulation. However, KCBP interaction with MTs and its MT-stimulated ATPase activity were not inhibited when the motor domain and CBD were added separately, suggesting that Ca(2+)-CaM regulation of CaM-binding motors occurs only when the CBD is attached to the motor domain. These results show that the fusion of the CBD to animal motors confers Ca(2+)-CaM regulation and suggest that the CBD functions as a modular domain in disrupting motor-MT interaction. Our data also support the hypothesis that CaM-binding kinesins may have evolved by addition of a CBD to a kinesin motor domain.     

Different effects of intron nucleotide composition and secondary structure on pre-mRNA splicing in monocot and dicot plants.

We have found previously that the sequences important for recognition of pre-mRNA introns in dicot plants differ from those in the introns of vertebrates and yeast. Neither a conserved branch point nor a polypyrimidine tract, found in yeast and vertebrate introns respectively, are required. Instead, AU-rich sequences, a characteristic feature of dicot plant introns, are essential. Here we show that splicing in protoplasts of maize, a monocot, differs significantly from splicing in a dicot, Nicotiana plumbaginifolia. As in the case of dicots, a conserved branch point and a polypyrimidine tract are not required for intron processing in maize. However, unlike in dicots, AU-rich sequences are not essential, although their presence facilitates splicing if the splice site sequences are not optimal. The lack of an absolute requirement for AU-rich stretches in monocot introns in reflected in the occurrence of GC-rich introns in monocots but not in dicots. We also show that maize protoplasts are able to process a mammalian intron and short introns containing stem--loops, neither of which are spliced in N.plumbaginifolia protoplasts. The ability of maize, but not of N.plumbaginifolia to process stem--loop-containing or GC-rich introns suggests that one of the functions of AU-rich sequences during splicing of dicot plant pre-mRNAs may be to minimize secondary structure within the intron.     

Crystal structure of kinesin regulated by Ca(2+)-calmodulin

Kinesins orchestrate cell division by controlling placement of chromosomes. Kinesins must be precisely regulated or else cell division fails. Calcium, a universal second messenger in eukaryotes, and calmodulin regulate some kinesins by causing the motor to dissociate from its biological track, the microtubule. Our focus was the mechanism of calcium regulation of kinesin at atomic resolution. Here we report the crystal structure of kinesin-like calmodulin-binding protein (KCBP) from potato, which was resolved to 2.3 A. The structure reveals three subdomains of the regulatory machinery located at the C terminus extension of the kinesin motor. Calmodulin that is activated by Ca2+ ions binds to an alpha-helix positioned on the microtubule-binding face of kinesin. A negatively charged segment following this helix competes with microtubules. A mimic of the conventional kinesin neck, connecting the calmodulin-binding helix to the KCBP motor core, links the regulatory machine to the kinesin catalytic cycle. Together with biochemical data, the crystal structure suggests that Ca(2+)-calmodulin inhibits the binding of KCBP to microtubules by blocking the microtubule-binding sites on KCBP.     

Plant Kinesin-Like Calmodulin Binding Protein Employs Its Regulatory Domain for Dimerization

Kinesin-like calmodulin binding protein (KCBP), a Kinesin-14 family motor protein, is involved in the structural organization of microtubules during mitosis and trichome morphogenesis in plants. The molecular mechanism of microtubule bundling by KCBP remains unknown. KCBP binding to microtubules is regulated by Ca²⁺-binding proteins that recognize its C-terminal regulatory domain. In this work, we have discovered a new function of the regulatory domain. We present a crystal structure of an Arabidopsis KCBP fragment showing that the C-terminal regulatory domain forms a dimerization interface for KCBP. This dimerization site is distinct from the dimerization interface within the N-terminal domain. Side chains of hydrophobic residues of the calmodulin binding helix of the regulatory domain form the C-terminal dimerization interface. Biochemical experiments show that another segment of the regulatory domain located beyond the dimerization interface, its negatively charged coil, is unexpectedly and absolutely required to stabilize the dimers. The strong microtubule bundling properties of KCBP are unaffected by deletion of the C-terminal regulatory domain. The slow minus-end directed motility of KCBP is also unchanged in vitro. Although the C-terminal domain is not essential for microtubule bundling, we suggest that KCBP may use its two independent dimerization interfaces to support different types of bundled microtubule structures in cells. Two distinct dimerization sites may provide a mechanism for microtubule rearrangement in response to Ca²⁺ signaling since Ca²⁺- binding proteins can disengage KCBP dimers dependent on its C-terminal dimerization interface.     

Sequence of a genomic DNA clone for the small subunit of ribulose bis-phosphate carboxylase-oxygenase from tobacco.

We have cloned and sequenced a gene for the small subunit (SS) of ribulose bis-phosphate carboxylase-oxygenase from Nicotiana tabacum. The tobacco gene is most closely related to the SS genes from the dicots soybean and pea, and less so to the monocots wheat and Lemna; the deduced amino acid sequence of the mature protein is in all cases more closely conserved than is its chloroplast transit sequence. Unlike the genomic sequences of the two monocots, which have one intron, and the two other dicots, which have two introns, the tobacco gene has three introns. The third tobacco intron lies within a highly conserved region of the protein. Its position coincides with the boundary of a 12 amino acid insertion in the SS genes of higher plants, relative to those of blue green algae. The 5' flanking end of the gene carries 67 bp inverted repeats, which flank a series of eight direct repeats; the direct repeats themselves each carry inverted repeats. The 3' untranslated end of this gene differs by only 2 bp from that of an N. sylvestris SS gene.     

Intron loss and gain during evolution of the catalase gene family in angiosperms.

Angiosperms (flowering plants), including both monocots and dicots, contain small catalase gene families. In the dicot, Arabidopsis thaliana, two catalase (CAT) genes, CAT1 and CAT3, are tightly linked on chromosome 1 and a third, CAT2, which is more similar to CAT1 than to CAT3, is unlinked on chromosome 4. Comparison of positions and numbers of introns among 13 angiosperm catalase genomic sequences indicates that intron positions are conserved, and suggests that an ancestral catalase gene common to monocots and dicots contained seven introns. Arabidopsis CAT2 has seven introns; both CAT1 and CAT3 have six introns in positions conserved with CAT2, but each has lost a different intron. We suggest the following sequence of events during the evolution of the Arabidopsis catalase gene family. An initial duplication of an ancestral catalase gene gave rise to CAT3 and CAT1. CAT1 then served as the template for a second duplication, yielding CAT2. Intron losses from CAT1 and CAT3 followed these duplications. One subclade of monocot catalases has lost all but the 5''-most and 3''-most introns, which is consistent with a mechanism of intron loss by replacement of an ancestral intron-containing gene with a reverse-transcribed DNA copy of a fully spliced mRNA. Following this event of concerted intron loss, the Oryza sativa (rice, a monocot) CAT1 lineage acquired an intron in a novel position, consistent with a mechanism of intron gain at proto-splice sites.     

The kinesin-like calmodulin binding protein is differentially involved in cell division

The kinesin-like calmodulin (CaM) binding protein (KCBP), a minus end-directed microtubule motor protein unique to plants, has been implicated in cell division. KCBP is negatively regulated by Ca(2)+ and CaM, and antibodies raised against the CaM binding region inhibit CaM binding to KCBP in vitro; therefore, these antibodies can be used to activate KCBP constitutively. Injection of these antibodies into Tradescantia virginiana stamen hair cells during late prophase induces breakdown of the nuclear envelope within 2 to 10 min and leads the cell into prometaphase. However, mitosis is arrested, and the cell does not progress into anaphase. Injection of antibodies later during cell division has no effect on anaphase transition but causes aberrant phragmoplast formation and delays the completion of cytokinesis by approximately 15 min. These effects are achieved without any apparent degradation of the microtubule cytoskeleton. We propose that during nuclear envelope breakdown and anaphase, activated KCBP promotes the formation of a converging bipolar spindle by sliding and bundling microtubules. During metaphase and telophase, we suggest that its activity is downregulated.     

Information contents and dinucleotide compositions of plant intron sequences vary with evolutionary origin

The DNA sequence composition of 526 dicot and 345 monocot intron sequences have been characterized using computational methods. Splice site information content and bulk intron and exon dinucleotide composition were determined. Positions 4 and 5 of 5 splice sites contain different statistically significant levels of information in the two groups. Basal levels of information in introns are higher in dicots than in monocots. Two dinucleotide groups, WW (AA, AU, UA, UU) and SS (CC, CG, GC, GG) have significantly different frequencies in exons and introns of the two plant groups. These results suggest that the mechanisms of splice-site recognition and binding may differ between dicot and monocot plants.     

The question of cotyledon homology in angiosperms

The flowering plants (Magnoliophyta) are separated into two large classes distinguished by the morphology of their embryos. The embryos of monocots (class Liliopsida) have a single terminal cotyledon, while the embryos of dicots (class Magnoliopsida) usually have two lateral cotyledons. The cotyledons of monocots and dicots also differ in form, and there are no true intermediates. In addition, the third leaf of Nymphaealean seedlings appears to be identical to the single cotyledon of monocots. From this it is concluded that the cotyledons of monocots and dicots are not homologous. In addition, dissimilarity of cotyledons and succeeding leaves in dicots, together with recent genetic studies, suggests that the two cotyledons of dicots are not homologous with the succeeding leaves of the same plant. This interpretation is consistent with the view that the Nymphaealean embryo’s third leaf is homologous to the first leaf (cotyledon) of monocots. Because dicotyledonous embryos are common among seed plants and are present in the Gnetopsids, the most likely scenario is that the dicots share a widespread seed plant symplesiomorphy and that the monocots have lost this character state. A less parsimonious hypothesis of monocotyledonous embryos as plesiomorphic for angiosperms is also discussed. Genetic analysis of early embryo development in a variety of vascular plants may be the only way to conclusively determine the evolutionary origin of the distinctive difference between monocot and dicot embryos.