(Received for publication, July 26, 1995; and in revised form, December 4, 1995)
From the
Calmodulin, a ubiquitous calcium-binding protein, regulates many
diverse cellular functions by modulating the activity of the proteins
that interact with it. Here, we report isolation of a cDNA encoding a
novel kinesin-like calmodulin-binding protein (KCBP) from Arabidopsis using biotinylated calmodulin as a probe.
Calcium-dependent binding of the cDNA-encoded protein to calmodulin is
confirmed by S-labeled calmodulin. Sequence analysis of a
full-length cDNA indicates that it codes for a protein of 1261 amino
acids. The predicted amino acid sequence of the KCBP has a domain of
about 340 amino acids in the COOH terminus that shows significant
sequence similarity with the motor domain of kinesin heavy chains and
kinesin-like proteins and contains ATP and microtubule binding sites
typical of these proteins. Outside the motor domain, the KCBP has no
sequence similarity with any of the known kinesins, but contains a
globular domain in the NH
terminus and a putative
coiled-coil region in the middle. By analyzing the calmodulin binding
activity of truncated proteins expressed in Escherichia coli,
the calmodulin binding region is mapped to a stretch of about 50 amino
acid residues in the COOH terminus region of the protein. Using a
synthetic peptide, the calmodulin binding domain is further narrowed
down to a 23-amino acid stretch. The synthetic peptide binds to
calmodulin with high affinity in a calcium-dependent manner as judged
by electrophoretic mobility shift assay of calmodulin-peptide complex.
The KCBP is coded by a single gene and is highly expressed in
developing flowers and suspension cultured cells. Although many kinesin
heavy chains and kinesin-like proteins have been extensively
characterized at the biochemical and molecular level in evolutionarily
distant organisms, none of them is known to bind calmodulin. The plant
kinesin-like protein with a calmodulin binding domain and a unique
amino-terminal region is a new member of the kinesin superfamily. The
presence of a calmodulin-binding motif in a kinesin heavy chain-like
protein suggests a role for calcium and calmodulin in kinesin-driven
motor function(s) in plants.
Calcium is a key messenger in transducing many hormonal and environmental signals in plants(1, 2, 3, 4) . Several recent studies demonstrated elevation of cytosolic calcium by various hormonal and physical signals(3) . Increased cytosolic calcium is believed to control biochemical and molecular processes by modulating the activity of specific proteins either directly or through calmodulin(1, 2) . Calmodulin, a highly conserved multifunctional calcium-binding protein, is implicated in many calcium-dependent cellular processes in plant and animal cells(2, 5) . However, unlike animal calmodulin genes, plant calmodulin genes are highly responsive to various signals. The expression of calmodulin is found to be induced by many signals in plants(2, 6, 7) . Furthermore, the amount of calmodulin varies in different tissues and is high in actively dividing tissues(8, 9, 10, 11) . In plants, calmodulin is implicated in controlling a wide variety of cellular functions and physiological processes. These include phytochrome-regulated gene expression and chloroplast development(4) , cell division(12, 13) , thigmomorphogenesis(7) , gravitropism(6, 14) , and microtubule organization(15) .
Calmodulin action in
regulating biochemical and molecular events and ultimately
physiological processes involves its interaction with other proteins
called calmodulin-binding proteins. The effect of this interaction
usually results in regulation of enzymatic activity of the binding
protein. In animal systems over 20 calmodulin-binding proteins such as
protein kinases, a protein phosphatase, a plasma membrane calcium
ATPase, an inositol trisphosphate kinase, a nitric oxide synthase,
transcription factors, enzymes involved in cyclic nucleotide
metabolism, and several cytoskeletal proteins have been characterized (2, 16, 17) . In plants, little is known
about the identity and function of calmodulin-binding proteins. Using
gel overlay assays, it has been shown that there are a number of
proteins in plants that bind to calmodulin in a calcium-dependent
manner(18, 19, 20) . Some of these proteins
are detected only in specific tissues or cells suggesting their tissue
specific role. To understand calmodulin-regulated processes, various
strategies have been used to isolate, identify, and characterize the
proteins that interact with calmodulin. These studies have resulted in
identification of a few calmodulin-modulated proteins such as NAD
kinase, calcium ATPase, nuclear nucleoside triphosphatase, a vacuolar
ion channel, glutamate decarboxylase, elongation factor-1, and
protein kinases in plants (1, 21, 22, 23, 24, 25, 26) .
Complementary DNAs that encode calmodulin-binding proteins of unknown
function have also been isolated from maize and
tobacco(27, 28) .
The kinesin superfamily of
microtubule motor proteins is comprised of conventional kinesin heavy
chains and other related proteins called kinesin-like
proteins(29, 30) . The common feature among the
members of the kinesin superfamily is a highly conserved motor domain
of about 350 amino acid residues that contains ATP and microtubule
binding sites. These motor proteins hydrolyze ATP and use the derived
energy to translocate unidirectionally on microtubules. Kinesins and
kinesin-like proteins are implicated in controlling diverse functions
including spindle formation, chromosome segregation during cell
division, and organellar and vesicular
transport(30, 31, 32, 33, 34) .
The conventional kinesin is a tetramer consisting of two heavy chains
and two light chains, and the motor activity is associated with the
heavy chain(35, 36) . The heavy chain has three
structural domains: a motor domain that is located in the
amino-terminal region, and contains conserved ATP and microtubule
binding sites, a central stalk region that forms an -helical
coiled-coil region involved in dimerization, and a globular tail that
binds to two light
chains(37, 38, 39, 40) . However,
kinesin-like proteins are either dimeric or
monomeric(41, 42, 43) . The motor domain in
kinesin-like proteins is located either in the NH
terminus,
the COOH terminus, or in the middle region of the protein (33, 39, 41, 44, 45) .
Outside the motor region kinesin-like proteins show limited or no
sequence homology. Because of the presence of a superfamily of
kinesins, it is suggested that the motor domain performs many diverse
microtubule-based transport functions by being fused to unique domains
that are specific to the cargo that they transport. Little is known
about kinesins and their role in cellular functions in plants. Using a
monoclonal antibody to the calf brain kinesin, an immunoreactive
homolog of kinesin was identified in the tobacco pollen
tube(46) . More recently, Mitsui et al.(47, 48) used primers corresponding to conserved
regions in the motor domain of kinesin heavy chains to isolate three
cDNAs (KatA, KatB, and KatC) encoding
kinesin-like proteins from Arabidopsis. The predicted amino
acid sequence of KatA, KatB, and KatC showed
significant sequence similarity to motor domains of kinesins and
kinesin-like proteins. This report describes the isolation and
characterization of a cDNA which encodes a novel kinesin-like protein
with a calmodulin binding domain from Arabidopsis. The
calmodulin-binding motif, which is absent in all the previously
reported kinesins and kinesin-like proteins, is mapped to a short
stretch of 23 amino acids in the carboxyl-terminal region of the
protein. The predicted amino acid sequence shows significant sequence
similarity with the motor domain of kinesin heavy chain and contains
structural features associated with kinesins and kinesin-like proteins.
However, the calmodulin binding region is unique to this protein,
suggesting that it is a new member of the kinesin superfamily. To our
knowledge, this is first report to show that a calmodulin-binding
protein is a kinesin heavy chain-like protein. The presence of a
calmodulin binding domain and a motor domain in a single protein
implies a role for calcium and calmodulin in kinesin heavy chain-driven
motor functions in plant cells.
Fluorescence spectra of free
and calmodulin-bound synthetic peptide was recorded with a Hitachi
F-3010/4010 spectrofluorometer. The concentration of peptide and
calmodulin were 166 pmol in a buffer containing 5 mM Tris-HCl,
pH 7.3, 0.5 mM CaCl. The excitation wavelength was
290 nm and the bandwidth for excitation and emission was 5 nm.
Corrections were made for the protein and solvent blanks.
Figure 1:
Calcium-dependent binding
of the fusion protein from isolated clones to S-labeled
calmodulin. Left, line diagram showing the length of the
different cDNAs that are used for binding studies. Open bars represent
the coding region in the cDNA and solid lines represent 3` untranslated
region. Right, plaque purified recombinants isolated using
biotinylated calmodulin as a probe and a positive control
(
ICM-1), which encodes a calmodulin-binding protein from
mouse (53) were probed with
S-labeled
calmodulin(50) . Recombinant phages were plated with
appropriate bacteria and incubated at 42 °C for 3 h. The fusion
protein was induced by applying an IPTG-soaked nitrocellulose filter.
The filter containing the fusion protein was incubated with the binding
buffer containing 18 nM
S-labeled calmodulin and
1 mM calcium chloride for overnight(50) . One-half of
each filter was then washed with binding buffer containing 1 mM CaCl
(1) and the other half with binding
buffer containing 5 mM EGTA (2). The filters were
dried and exposed to x-ray film.
Figure 9:
Expression of KCBP in different tissues
and suspension culture. Total RNA (80 µg) was electrophoresed on a
formaldehyde-containing agarose gel, transferred to a Hybond
N membrane and sequentially hybridized with
P-labeled KCBP cDNA (a 1.4-kb fragment) (A) and
ubiquitin cDNA (B). C, ethidium bromide-stained gel. Lane 1, flowers; lane 2, leaves; lane 3,
roots; lane 4, suspension of cultured cells. Numbers at left indicate the size of the RNA markers in kilobase pairs for
blot A.
Figure 2: Nucleotide and deduced amino acid sequences of KCBP. The amino acid sequence is presented below the nucleotide sequence. Numbers at right correspond to nucleotides and deduced amino acids. The underscored bases surrounding the translation initiation codon represent the Kozak's (57) consensus nucleotides. Amino acid sequences in bold denote putative PEST sequences(60) . The underscored amino acids (623-641, 755-774, and 774-793) represent potential nuclear localization signals. Bold and underscored amino acid sequence corresponds to a synthetic peptide that binds calmodulin.
Figure 3:
Alignment of predicted amino acid
sequences (1) of KCBP of Arabidopsis(79) with the motor domain of kinesin-like proteins from Chlamydomonas (KHP1) (58) , Saccharomyces
cerevisiae (kar3)(80) , Arabidopsis (Kata)(47) , and Drosophila ncd (Clar)(81) . The conserved ATP binding site is shown with
asterisks. Four highly conserved sequences in the microtubule binding
region are underlined. Dashes indicate amino acid residues that are
identical to KCBP. Upper case letters denote aligned
nonidentical amino acids, and lower case letters denote
unaligned amino acids. Gaps in alignment are denoted by dots. The amino
acid sequence that showed calcium dependent binding to calmodulin is
boxed.
In the region outside the motor
domain, KCBP showed no significant sequence similarity with other
kinesin heavy chains or heavy chain-like proteins. Analysis of the
sequence amino-terminal to motor domain indicates that it is likely to
be composed of two domains. First, amino acids from 610 to 890 form an
-helix (Fig. 4A). Analysis of the predicted amino
acid sequence using a computer program that predicts coiled-coil
structure (59) has revealed that a region (from amino acid
residues 610 to 890) has extremely high probability to form coiled-coil
structure (Fig. 4B). The presence of the coiled-coil
region implies that the native KCBP may form a dimer. The second domain
is a globular domain that extends from the beginning of the protein to
the coiled coil region. This region showed some sequence similarity
with myosins. It has been shown that ``PEST'' motifs that are
rich in proline, glutamate, serine, and threonine are usually
associated with proteins that have a short
half-life(60, 61) . Using PESTFIND program, we found
two potential PEST sequences in the amino-terminal region (residues
5-70, 334-350 with PESTFIND scores of +1 and +4,
respectively) of KCBP (Fig. 4C). Hence, it is likely
that the KCBP is a rapidly turned over protein. Proteins that move into
the nucleus contain nuclear targeting sequences such as a bipartite
signal motif in which two regions of basic amino acids are separated by
a spacer of 10 or more amino acids(62) . The deduced amino acid
sequence of KCBP has three likely bipartite signal motifs
(623-641, 755-774, and 774-793), suggesting that it
may be a nuclear protein (Fig. 2).
Figure 4:
Predicted structural features of KCBP
based on primary sequence. A, the secondary structure of KCBP
as predicted by Robson-Garnier and Chou-Fasman methods. Regions
predicted to be helices (Hlx),
sheets (Sht), or
turns (Trn) are indicated by solid boxes. B, location of putative coiled-coil region in
KCBP. The probability that each residue of KCBP will participate in a
coiled-coil region is calculated according to Lupas et al.(59) and represented in a bar graph. C, schematic
diagram of predicted protein of KCBP showing different structural
features.
Figure 5: Localization of calmodulin binding domain using different fusion proteins. Fusion protein from the 1.4-kb cDNA (amino acids 860-1261, Fus.1), 1-kb (amino acids 860-1210, Fus.2), and 0.4-kb cDNA (amino acids 1210-1261, Fus.3) were tested for their ability to bind calmodulin. A, diagrammatic representation of the cDNA parts that were expressed in E. coli. Open bars represent the coding region in the cDNA and solid lines represent the 3`-untranslated region. B, Coomassie-stained SDS-polyacrylamide gel showing insoluble protein fraction from uninduced (U) and induced (I) cultures containing the fusion constructs. The arrowheads indicate the fusion protein. C, binding of calmodulin to fusion protein. Insoluble protein fractions from uninduced and induced cultures of the three constructs were electrophoresed as in B, transferred to a nitrocellulose membrane, and probed with 60 nM biotinylated calmodulin. Calmodulin binding to fusion protein was detected with Vectastain ABC-horseradish peroxidase as described by Fordham-Skelton et al.(50) . Calcineurin (P), a known calmodulin-binding protein, was used as a positive control. D, binding of the fusion protein from the 1.4-kb cDNA (Fus.1) to calmodulin-Sepharose. The E. coli expressed protein was solubilized and passed through a calmodulin-Sepharose column, and the bound fraction was eluted as described under ``Experimental Procedures.'' The eluted fraction was separated on duplicate denaturing gels. One gel (lane 1) was stained with Coomassie Blue, and the second gel was blotted onto a nitrocellulose membrane and probed with biotinylated calmodulin (lane 3) as described above. Lane 2 represents calcineurin probed with biotinylated calmodulin.
More than 34
kinesin heavy chain proteins have been characterized at the molecular
and biochemical level from phylogenetically divergent
organisms(41) . However, none of them was shown to be the
target of calmodulin. Matthies et al.(63) tested
bovine brain light and heavy chains of kinesin for their ability to
bind calmodulin and found that the heavy chain does not bind to
calmodulin, whereas the light chain showed calmodulin binding. These
results raise the possibility that kinesin heavy chains that bind to
calmodulin are unique to plants, or such proteins are yet to be
discovered in animals. Recently, we have isolated a calmodulin-binding
protein cDNA from developing potato tubers that showed significant
sequence similarity with kinesin heavy chain motor domain. ()Furthermore, the calmodulin binding domain in Arabidopsis is highly conserved in potato kinesin-like
calmodulin-binding protein, whereas this region is not present in any
of the animal kinesin heavy chains or kinesin-like proteins. Hence, it
is likely that calmodulin-binding proteins that share homology with the
kinesin heavy chain motor domain are widely distributed in plants and
are involved in kinesin heavy chain driven motor functions.
Figure 6:
Binding of a 23-amino acid-long synthetic
peptide (amino acids corresponding to 1218-1240) to calmodulin. A, One hundred ng (lane 1), 500 ng (lane 2),
and 1 µg (lane 3) of the 0.4-kb fusion protein (T), synthetic peptide (M), or bovine serum albumin (B) was applied to a nitrocellulose membrane and probed with
biotinylated calmodulin. B, predicted -helical wheel
diagram of amino acids 1218-1231. Positively charged and
negatively charged amino acids are denoted with + and -
superscripts, respectively. Hydrophobic amino acids are circled.
Figure 7:
Analysis of calmodulin binding to a
synthetic peptide by electrophoretic mobility shift in polyacrylamide
gel containing 4 M urea. A and B, calmodulin
(221 pmol) was incubated with increasing concentrations of synthetic
peptide in the presence of 4 M urea, 100 mM Tris-HCl,
pH 8.0, and 1 mM CaCl (A) or 5 mM EGTA (B), and the reaction mixtures were analyzed on urea
containing gels. Lane 1, calmodulin alone; lane 2,
calmodulin plus synthetic peptide (1:1); lane 3, calmodulin
plus synthetic peptide (1:2); lane 4, calmodulin plus
synthetic peptide (1:3). Numbers in parentheses indicate calmodulin to
peptide molar ratios. C and D, calmodulin (166 pmol)
was incubated with increasing concentrations of synthetic peptide as
above in the presence of either 1 µM CaCl
(C) or 5 mM EGTA (D) and analyzed on
urea containing gels. Lane 1, calmodulin alone; lanes
2-8, different molar ratios of synthetic peptide to
calmodulin; lane 2, 0.1:1; lane 3, 0.2:1; lane
4, 0.4:1; lane 5, 0.6:1; lane 6, 0.8:1; lane
7, 1:1; lane 8, 2:1.
The binding of a peptide to calmodulin can also be monitored by fluorescence spectroscopy if the peptide contains a tryptophan residue which is not present in calmodulin. The binding of a tryptophan-containing peptide to calmodulin has been shown to shift the fluorescence spectrum and often change the intensity of fluorescence (55, 67, 68, 69) . Since the synthetic peptide used in our studies contains a tryptophan residue, we tested if the fluorescence properties are altered in the presence of calmodulin. As shown in Fig. 8, the synthetic peptide showed a shift in fluorescence spectra and an increase in the fluorescence intensity in the presence of calmodulin. The wavelength of the emission maximum decreased from 342 to 330 nm.
Figure 8:
Fluorescence emission spectrum of
synthetic peptide in the presence(- - - - -) or absence
(-) of calmodulin. The concentration of peptide and
calmodulin was 166 pmol in a buffer containing 5 mM Tris-HCl,
pH 7.3, 0.5 mM CaCl. The excitation wavelength was
290 nm, and the bandwidth for excitation and emission was 5
nm.
Figure 10:
Southern blot analysis of genomic DNA.
Five µg of genomic DNA were digested with different restriction
enzymes: B, BamHI; E, EcoRI; H, HindIII; E-H, EcoRI and HindIII; E-B, EcoRI and BamHI; H-B, HindIII and BamHI. The digested DNA was
electrophoresed through a 0.8% agarose gel, transferred onto a Hybond N
membrane, and probed with P-labeled, 1.4-kb cDNA which
contains the coding region for the motor domain and the calmodulin
binding domain. Molecular mass markers are shown on the left in
kilobase pairs.
We isolated a full-length cDNA encoding a calmodulin-binding
protein (KCBP) using a protein-protein interaction-based screening.
Several approaches have been used to demonstrate that the cDNA-encoded
protein binds to calmodulin with high affinity in a calcium-dependent
manner. Studies with S-labeled and biotinylated calmodulin
show that KCBP binds to calmodulin in a calcium-dependent manner ( Fig. 1and Fig. 5). This was further confirmed by
calmodulin column chromatography (Fig. 5). Calmodulin-binding
studies with truncated proteins and a synthetic peptide have shown that
the calmodulin binding domain is located in the carboxyl-terminal end
next to the motor domain. Binding of calmodulin to synthetic peptide in
solution and analysis of calmodulin-peptide complex by mobility shift
assay on urea containing gels indicate that the peptide has high
affinity to calmodulin (Fig. 7). The binding of synthetic
peptide to calmodulin occurred at 1 µM calcium (Fig. 7C). We used 59 nM of biotinylated
calmodulin for screening the libraries and 18 nM for
S-labeled calmodulin to confirm the isolated clones. This
concentration of calmodulin is well within the physiological levels of
calmodulin in plant cells(70) . Furthermore, the concentration
of calmodulin used in our studies is similar or lower as compared to
other calmodulin-binding
studies(22, 24, 25, 71, 72, 73) .
The fact that the screening of about 800,000 plaques resulted in
isolation of only two different cDNAs also suggests the specificity of
the probe. These results clearly show that the binding of KCBP to
calmodulin occurs at physiological levels of calmodulin and calcium and
raise an interesting possibility that calcium and calmodulin may be
involved in regulating the function of KCBP.
The sequence similarity
between the motor domain of kinesin heavy chain and KCBP suggests that
the KCBP is a member of the kinesin superfamily of proteins. Structural
analysis indicates that it, like most other kinesins and kinesin-like
proteins, contains a coiled-coil region and a globular tail. However,
the KCBP is a new member of kinesin-like proteins since none of the
known kinesin heavy chains contains a calmodulin binding domain.
Several lines of evidence suggest that the presence of a motor domain
and a calmodulin binding domain on the isolated clone is not due to
cloning artifact, but is derived from a single gene. Polymerase chain
reaction amplification of first strand cDNA with one primer
corresponding to the motor domain and the other primer to the
calmodulin binding domain produced a single amplified product of
expected size (data not shown). Southern blotting with a cDNA probe
containing the coding region for both motor and calmodulin binding
domains showed a single band (Fig. 10). We isolated genomic
clones in which motor and calmodulin binding domains are contiguous. ()Screening of two different cDNA libraries has yielded the
cDNAs that are identical and contained both the kinesin motor domain
and calmodulin binding domain. Finally, a homolog of KCBP has been
isolated from potato, which, like KCBP, contains both motor and
calmodulin binding domains.
Phylogenetic analysis of motor regions of all the known kinesins indicates that they fall into five distinct groups that are likely to play different roles in basic cellular processes(41) . Only one of these five groups contains the motor region in the carboxyl-terminal region, whereas the remaining four have their motor regions located in the amino-terminal regions(41) . All of the kinesins that have motor domain in the amino-terminal region perform plus end-directed movement along microtubules, whereas the kinesins with the motor domain in the carboxyl-terminal end perform minus end-directed movement(41, 74, 75) . The presence of the motor domain in the carboxyl-terminal end of the KCBP suggests that it may be involved in minus end-directed translocation processes. In vitro motility assays with purified E. coli-expressed protein should help determine its motor activity as well as the direction of translocation.
Three cDNAs (KatA, KatB, and KatC) encoding kinesin-like proteins have been isolated from Arabidopsis(47, 48) . The predicted amino acid sequence from these cDNAs is less than 800 amino acids, and the COOH-terminal half of the protein showed strong sequence similarity to the motor domain of kinesins and kinesin-like proteins. So far, these are the only kinesin-like proteins that have been characterized from plants. The cDNA that we isolated from Arabidopsis encodes a much longer protein (KCBP, 1261 amino acids) and contains a calmodulin binding region that is absent in KatA, KatB, and KatC, suggesting that the KCBP is distinct from the previously reported kinesin-like proteins from the same system. Virtually nothing is known about the kinesin-driven motor functions in plants. A number of events during cell division in plants involve movement of a variety of subcellular structures. These include reorientation of microtubules, distribution of chromosomes, and targeted deposition of vesicles containing the cell wall material during cytokinesis(76) . Calcium and calmodulin are implicated in some of these aspects of cell division that involve subcellular movement. The high level of calmodulin in meristematic tissues and growing regions of plants (8, 9, 19) suggests its involvement in some events in cell division. Furthermore, immunofluorescence and immunogold staining studies have shown that calmodulin is localized to mitotic apparatus, especially microtubule converging centers and kinetochore microtubules (12) . The distribution of calcium is also found to be the same as that of calmodulin(12) . Based on these results it was suggested that calcium and calmodulin are involved in chromosome movement. Movement of vesicles during phragmoplast formation, a process unique to plants, may also be carried out by calcium-regulated microtubule motor proteins, since calcium and microtubules are implicated in the transport of vesicles to the cell plate during cytokinesis in plants(77, 78) . It is likely that microtubule motor proteins such as kinesins that are regulated by calcium and calmodulin are involved in the movement of subcellular structures in plants. The finding that KCBP, a putative microtubule motor protein, is a calmodulin-binding protein and is highly expressed in dividing cells suggests that it may be involved in the movement of subcellular structures that is regulated by calcium and calmodulin. In summary, we have isolated a novel gene encoding a calmodulin-binding protein that has significant sequence similarity with the motor-domain of kinesin heavy chain and kinesin heavy chain-like proteins. The presence of a calmodulin binding domain and a motor domain in KCBP suggests a role for calcium and calmodulin in at least some of the microtubule-based motor functions. The availability of the KCBP cDNA will allow us to express the protein to study its motor activity and the role of calcium and calmodulin in kinesin driven motor functions.