(Received for publication, September 6, 1995; and in revised form, January 17, 1996)
From the
A chimeric Ca/calmodulin-dependent protein
kinase (CCaMK) gene characterized by a catalytic domain, a
calmodulin-binding domain, and a neural visinin-like
Ca
-binding domain was recently cloned from plants
(Patil, S., Takezawa, D., and Poovaiah, B. W.(1995) Proc. Natl.
Acad. Sci. U. S. A. 92, 4797-4801). The Escherichia
coli-expressed CCaMK phosphorylates various protein and peptide
substrates in a Ca
/calmodulin-dependent manner. The
calmodulin-binding region of CCaMK has similarity to the
calmodulin-binding region of the
-subunit of multifunctional
Ca
/calmodulin-dependent protein kinase (CaMKII).
CCaMK exhibits basal autophosphorylation at the threonine residue(s)
(0.098 mol of
P/mol) that is stimulated 3.4-fold by
Ca
(0.339 mol of
P/mol), while
calmodulin inhibits Ca
-stimulated autophosphorylation
to the basal level. A deletion mutant lacking the visinin-like domain
did not show Ca
-stimulated autophosphorylation
activity but retained Ca
/calmodulin-dependent protein
kinase activity at a reduced level. Ca
-dependent
mobility shift assays using E. coli-expressed protein from
residues 358-520 revealed that Ca
binds to the
visinin-like domain. Studies with site-directed mutants of the
visinin-like domain indicated that EF-hands II and III are crucial for
Ca
-induced conformational changes in the visinin-like
domain. Autophosphorylation of CCaMK increases
Ca
/calmodulin-dependent protein kinase activity by
about 5-fold, whereas it did not affect its
Ca
-independent activity. This report provides
evidence for the existence of a protein kinase in plants that is
modulated by Ca
and Ca
/calmodulin.
The presence of a visinin-like Ca
-binding domain in
CCaMK adds an additional Ca
-sensing mechanism not
previously known to exist in the
Ca
/calmodulin-mediated signaling cascade in plants.
The signal-induced change in free Ca concentration in the cytoplasm has been portrayed as a switch
that turns on various cellular processes in plants and
animals(1, 2, 3) .
Ca
-mediated protein phosphorylation is one of the
major mechanisms by which eukaryotic cells transduce extracellular
signals into intracellular
responses(4, 5, 6) .
Ca
/calmodulin-dependent protein kinases are involved
in amplifying and diversifying the action of
Ca
-mediated signals(7, 8) . In
animals, several types of Ca
/calmodulin-dependent
protein kinases have been identified, including myosin light chain
kinases, phosphorylase kinase, and EF-2 kinase, as well as
Ca
/calmodulin-dependent protein kinase I, II, and
IV(9, 10) . The multifunctional
Ca
/calmodulin-dependent protein kinase (CaMKII) (
)is one of the well characterized kinases, and it is known
to play a pivotal role in cellular regulation because of its ability to
phosphorylate a large number of proteins (11) .
Although
Ca-dependent protein kinases are found in many plant
species(12, 13) , little is known about
Ca
/calmodulin-dependent protein kinases in plants.
Ca
and Ca
/calmodulin-dependent
protein phosphorylation has been demonstrated in a number of plant
extracts (14, 15, 16) . However, convincing
biochemical evidence for the presence of calmodulin-dependent protein
kinase in plants has not been reported previously. Most of the evidence
of calmodulin dependence has been indirect, based on the use of
calmodulin antagonists and on activation studies with exogenous
calmodulin(1, 14, 16) . Watillon et al.(17) reported a homolog of mammalian CaMKII from plants,
but the biochemical properties of this kinase are not known.
CCaMK
is a novel Ca/calmodulin-dependent protein kinase
characterized by two distinct regulatory domains; a visinin-like domain
is regulated by Ca
, while the other is regulated by
Ca
/calmodulin. The visinin-like domain of CCaMK
contains three conserved Ca
-binding EF-hand motifs,
similar to neural visinin-like proteins(18, 19) ,
which are members of a family of Ca
-sensitive
regulators. The chimeric feature of CCaMK with three distinct domains
in a single polypeptide suggests that it has evolved from a fusion of
two genes that are functionally different in origin. The CCaMK gene is
preferentially expressed during anther development, and it is regulated
in a stage-specific manner during microsporogenesis, which implies that
it may play a central role in the development of the male
gametophyte(20) .
Here we report the biochemical properties
of CCaMK, which has structural features resembling both mammalian
Ca/calmodulin-dependent protein kinases and plant
Ca
-dependent protein kinases. The results presented
here show a dual mode of regulation of CCaMK by Ca
and Ca
/calmodulin.
To study the Ca/calmodulin-dependent kinase
activity of CCaMK, the E. coli-expressed protein was purified.
The protein was essentially pure as revealed by SDS-PAGE and was stable
at 4 °C for a few days. The purified protein was used to
phosphorylate different substrates such as casein, histones, myelin
basic protein, and synthetic peptides. Histone IIAS was found to be the
most reactive protein substrate for CCaMK and was used for studying
calmodulin concentration-dependent protein kinase activity. The
addition of increasing amounts of calmodulin in the presence of 0.5
mM Ca
stimulated CCaMK activity (Fig. 1A). Kinase activity was saturated at calmodulin
concentrations around 1.0 µM. The concentration of
calmodulin required for half-maximal activity (K
)
of CCaMK was approximately 0.2 µM. The time course studies
revealed that histone IIAS phosphorylation was saturated after 10 min
in the presence of Ca
/calmodulin (Fig. 1B). In the presence of 2.5 mM EGTA or
0.5 mM Ca
alone, the enzyme has basal
activity that is 10-15-fold lower than the maximal activity
achieved with Ca
/calmodulin. Among other protein
substrates tested, CCaMK phosphorylated histone IIIS and myelin basic
protein, but it did not phosphorylate phosvitin, phosphoenolpyruvate
carboxylase, synapsin I, and casein. CCaMK also phosphorylated
synthetic peptides such as GS peptide, myelin basic protein peptide,
and syntide-2. Among these peptides, GS peptide was most efficiently
phosphorylated by CCaMK in the presence of
Ca
/calmodulin.
Figure 1:
Ca/calmodulin-dependent
protein kinase activity of CCaMK. A, histone IIAS was
phosphorylated with CCaMK in the presence of 0.5 mM CaCl
and increasing amounts of calmodulin (µM) at 30
°C for 2 min. CCaMK activity is presented as nmol of
phosphate/min/mg of CCaMK. B, time course of phosphorylation
of histone IIAS by CCaMK in the presence of 2.5 mM EGTA
(
) or 0.5 mM CaCl
(
) or 0.5 mM CaCl
and 1 µM calmodulin (
). CCaMK
activity is represented as nmol of phosphate/mg of
CCaMK.
Calmodulin-binding affinity of the
CCaMK was studied by using different concentrations of S-labeled calmodulin. Binding of calmodulin to CCaMK
saturated at concentrations above 300 nM (Fig. 2). From
the saturation curve, the dissociation constant (K
) of calmodulin for CCaMK was estimated to be
around 55 nM. The binding of calmodulin to CCaMK was
completely blocked in the presence of 5 mM EGTA. The Scatchard
analysis indicated that CCaMK has a single calmodulin binding site. (Fig. 2, inset).
Figure 2:
Saturation curve of S-calmodulin binding to purified CCaMK. E.
coli-expressed CCaMK protein (4 pmol) was separated by SDS-PAGE
and electrophoretically transferred onto a nitrocellulose filter and
incubated with different amounts of
S-labeled calmodulin.
After washing in the buffer without
S-calmodulin,
radioactivity of the filter was measured by using a liquid
scintillation counter. The amount of bound calmodulin at each point was
represented as percent of the maximal binding. The inset shows
a Scatchard plot of data indicating that the binding ratio of
calmodulin to CCaMK is 1:1. Bound/free and bound calmodulin are
expressed as B/F and B,
respectively.
To identify the calmodulin-binding
region of CCaMK, truncated mutant constructs were prepared (Fig. 3A). The CCaMK mutant 1-356 lacks the
COOH-terminal domain, which has high homology to visinin-like proteins.
Another CCaMK mutant, 1-322, is further truncated, but it has all
11 domains conserved in serine/threonine protein kinases(28) .
Wild-type CCaMK(1-520), and truncated mutants 1-356 and
1-322 were expressed in E. coli and purified as
described under ``Experimental Procedures.'' These proteins
were used for S-calmodulin binding assays in the presence
of Ca
. The binding of calmodulin to wild-type and
mutant 1-356 CCaMKs were similar; whereas, calmodulin did not
bind to the mutant CCaMK 1-322 (Fig. 3A, boxed region), indicating that amino acid residues
322-356 (Fig. 3A) are essential for
calmodulin-binding to CCaMK. Another mutant CCaMK 1-341 also
binds to calmodulin in the presence of Ca
(data not
shown). Similar results were obtained when biotinylated calmodulin was
used instead of
S-calmodulin. Calmodulin binding to
wild-type and mutant CCaMKs was prevented by the addition of 5 mM EGTA, indicating the requirement of Ca
for
calmodulin binding. Comparison of amino acid residues of this region of
CCaMK corresponding to regions of animal CaMKII
revealed high
homology (Fig. 3B).
Figure 3:
Identification of calmodulin binding site
of CCaMK. A, schematic diagram of wild-type and truncation
mutants of CCaMK, which were used for S-calmodulin binding
assays are shown on the left. The mutants 1-356 and
1-322 represent CCaMK lacking the visinin-like domain and both
visinin-like and calmodulin-binding domains. E. coli-expressed
wild-type and mutant CCaMKs were electrophoresed on SDS-polyacrylamide
gel and transferred onto nitrocellulose filter. The excised bands
containing the expressed proteins were subjected to
S-calmodulin binding assay. The autoradiogram is shown on
the right of each diagram (boxed area). The
radioactivity (cpm) of bound
S-calmodulin was 11,600 for
wild-type, 12,500 for the mutant 1-356, and 99 for the mutant
1-322, respectively. B, comparison of amino acid
sequences surrounding the putative calmodulin-binding sites of CCaMK
and
subunit of CaMK II. C, calmodulin binding to the
synthetic peptides in a gel mobility-shift assay. Nondenaturing gel
electrophoresis was performed in the presence of 0.5 mM CaCl
. Top, lane 1, calmodulin alone (100
pmol); lanes 2-5, a mixture of calmodulin (100 pmol) and
each of the following peptides (1 nmol): lane 2, CCaMK
328-340; lane 3, CCaMK 322-340; lane 4,
317-340; and lane 5, 311-340. Bottom, mixtures of calmodulin and CCaMK peptide 322-340 at
different molar ratios. Lane 1, calmodulin alone (100 pmol); lanes 2-8, calmodulin (100 pmol) and 40, 80, 160, 240,
320, 480, and 640 pmol of the peptide 322-340. The bands of
calmodulin and calmodulin-peptide complex were visualized by staining
with Coomassie Brilliant Blue. D, helical wheel projection of
calmodulin-binding sequences in CCaMK (left) and CaMKII
(right). Hydrophobic amino acid residues are boxed.
Basic amino acid residues are marked with
(+).
Different lengths of synthetic
peptides from the calmodulin-binding region (amino acid residues
311-340) were used to identify amino acid residues necessary for
calmodulin binding. Calmodulin binding to these peptides was studied by
gel mobility shift assay using nondenaturing polyacrylamide gel.
Calmodulin mixed with peptides 311-340, 317-340, and
322-340 migrated above the position of calmodulin alone; whereas,
peptide 328-340 did not affect the mobility of calmodulin (Fig. 3C, top), suggesting that the
calmodulin-binding site exists between amino acid residues
322-340. The addition of these peptides to calmodulin in the
presence of 2.5 mM EGTA did not affect the mobility of
calmodulin, suggesting that peptide binding to calmodulin is
Ca-dependent. Increasing amounts of the peptide
322-340 facilitates the gel mobility shift toward the upper
position (Fig. 3C, bottom). Similar results
were obtained when the peptides 317-340 and 311-340 were
used, suggesting that the amino acid residues 322-340 have a
pivotal role in calmodulin binding of CCaMK. The helical wheel
projection revealed that amino acid residues 325-338 of CCaMK
form a basic amphiphilic
-helix (29) similar to
CaMKII
(Fig. 3D).
To study autophosphorylation,
CCaMK was incubated at 30 °C with 10 mM magnesium acetate,
1 mM [-
P]ATP, and 2.5 mM EGTA. In 30 min, approximately 0.098 mol of
P/mol of
CCaMK was incorporated. This basal autophosphorylation was induced to
approximately 3.4-fold in the presence of 0.5 mM CaCl
(0.339 mol of
P/mol of CCaMK) (Fig. 4A). Increasing the incubation time to 60 min did
not improve the stoichiometry of Ca
-dependent
autophosphorylation. Ca
-dependent autophosphorylation
was inhibited to the basal level (0.061 mol of
P/mol of
CCaMK) by the addition of 1 µM calmodulin (Fig. 4A). Calmodulin inhibits
Ca
-stimulated autophosphorylation in a
concentration-dependent manner (Fig. 4B). These results
indicate that Ca
and calmodulin have opposing effects
on autophosphorylation of CCaMK. Phosphoamino acid analysis revealed
that CCaMK autophosphorylates at the threonine residue(s) (Fig. 4C), which was stimulated by Ca
and inhibited by Ca
/calmodulin.
Figure 4:
Effects of Ca and
Ca
/calmodulin on autophosphorylation of CCaMK. A, time course of autophosphorylation of CCaMK in the presence of 2.5
mM EGTA (
) or 0.5 mM CaCl
(
)
or 0.5 mM CaCl
and 1 µM calmodulin
(
). The autophosphorylation is presented as pmol of
P
incorporated per 21.4 pmol of CCaMK. B, effect of calmodulin
on Ca
-dependent autophosphorylation of CCaMK. CCaMK
was autophosphorylated in the presence of CaCl
(0.5
mM) and increasing concentrations of calmodulin. Lane
1, +CaCl
, (0.5 mM); lanes
2-6, +CaCl
(0.5 mM) and 60, 120,
240, 360, and 480 nM calmodulin respectively. C, phosphoamino acid analysis of autophosphorylated CCaMK. CCaMK (200
ng) was autophosphorylated either in the presence of 2.5 mM EGTA (-Ca), 0.5 mM CaCl
(+Ca) or 0.5 mM CaCl
plus 1
µM calmodulin (+Ca/CaM). Autophosphorylated
CCaMK was subjected to phosphoamino acid analysis. The positions of
phosphoserine (S) and phosphothreonine (T) are
marked.
Apart from
the calmodulin-binding domain, CCaMK has another regulatory domain
toward the COOH terminus, which has high homology to animal
visinin-like proteins. The visinin-like domain of CCaMK contains three
EF-hand motifs with conserved Ca-ligating amino acid
residues (Fig. 5A). To study
Ca
-binding properties of the visinin-like domain of
CCaMK, recombinant visinin-like domain protein was expressed in E.
coli, using the pET14b expression vector. The visinin-like domain
protein was expressed to high levels upon induction with 0.5 mM isopropyl-1-thio-
-D-galactopyranoside, and most of
the protein was present in the soluble fraction. The expressed protein
was purified using the Ni
resin column. The protein
eluted from the column with 1 M imidazole buffer was dialyzed
in 50 mM Tris-Cl, pH 7.5, and used for
Ca
-dependent mobility shift assay. Electrophoretic
mobility of the recombinant visinin-like domain protein was just above
the 20.1-kDa molecular weight marker in the presence of 2.5 mM EGTA; whereas, the addition of Ca
shifted the
electrophoretic mobility toward the lower molecular weight (Fig. 5B). This suggests that Ca
binding to the recombinant visinin-like domain protein induces a
conformational change. To verify that the EF-hand motifs in the
visinin-like domain are responsible for the
Ca
-dependent mobility shift, site-directed mutants of
the visinin-like domain protein were created. Each of the EF-hands (I,
II, and III) were mutated by replacing the amino acid residue at the
-x position (D417A, S453A, and T495A) in the EF-hands (Fig. 5A), which are known to be primary determinants
of the Ca
dissociation rate (30) . The mutant
in which all three EF-hands are mutated was expressed in E. coli and purified, and the protein was also analyzed by SDS-PAGE in the
presence of Ca
. The visinin-like protein mutated in
the EF-hand I migrated at a similar position to the wild-type protein,
suggesting that this site may not be functional. However, mutations in
EF-hands II and III shifted the mobility of the protein toward the
higher molecular weight. The mutant of the EF-hand III migrated to a
similar position to the protein in which all three EF-hands are mutated (Fig. 5B). The migration of EF-hand III mutant in the
presence of Ca
was also similar to the wild-type
protein in the absence of Ca
. These results suggest
that Ca
binding to the EF-hands II and III contribute
to the Ca
-dependent mobility shift of the
visinin-like domain protein. Removal of Ca
by EGTA
shifts the mobility of all the mutant proteins to similar positions
toward the higher molecular weight (data not shown).
Figure 5:
Binding of Ca to
visinin-like domain of CCaMK. A, amino acid sequences of the
three EF-hand motifs in the visinin-like domain of CCaMK. Six
Ca
ligating residues denoted as x, y, z, -y, -x,
-z are marked. Site-directed mutants were prepared by
substituting the amino acid residues at -x position with
alanine (A). B, Ca
-dependent
mobility shift of wild-type and site-directed mutants of visinin-like
domain protein. E. coli-expressed recombinant visinin-like
domain proteins were electrophoresed on 14% SDS-polyacrylamide gel. In
the presence of 2.5 mM EGTA (lane 1) or 0.5 mM CaCl
(lanes 2-6). Wild-type protein (lanes 1 and 2), proteins mutated in the EF-hand I (lane 3), EF-hand II (lane 4), EF-hand III (lane
5), and all three EF-hands (lane 6) are
shown.
To study the
role of the visinin-like domain in Ca-stimulated
autophosphorylation, the CCaMK mutant 1-356 lacking the
visinin-like domain was used for autophosphorylation and substrate
phosphorylation. Autophosphorylation of mutant 1-356 was not
stimulated by Ca
(Fig. 6, A and C); however, it retained
Ca
/calmodulin-dependent kinase activity at a
substantially reduced level (Fig. 6, B and D).
This indicates that the visinin-like domain is required for
Ca
-stimulated autophosphorylation as well as for
maximal substrate phosphorylation.
Figure 6:
Comparison of enzyme activity of wild-type (A and B) and the truncated mutant(1-356) (C and D) of CCaMK. Ca-dependent
autophosphorylation (A and C) and
Ca
/calmodulin-dependent histone IIAS phosphorylation (B and D). The assays were carried out in the
presence of 2.5 mM EGTA (-Ca), 0.5 mM CaCl
(+Ca), or 0.5 mM CaCl
and 1 µM calmodulin
(+Ca/CaM).
In order to understand the
significance of Ca-stimulated autophosphorylation,
the autophosphorylated CCaMK was used to study its effect on substrate
phosphorylation. First we attempted to study the activity of the
autophosphorylated CCaMK using histone IIAS as a substrate. However, in
the presence of histone IIAS, calmodulin did not suppress the
Ca
-dependent autophosphorylation of CCaMK. It is
probable that histone IIAS was interacting with acidic proteins such as
calmodulin and the visinin-like domain of CCaMK. Therefore, we used GS
peptide as a substrate for studying the activity of autophosphorylated
CCaMK. The rate of phosphorylation of the GS peptide by
unphosphorylated CCaMK was stimulated by increasing concentrations of
calmodulin, but the maximal stimulation was only 3-4-fold higher
as compared with the basal activity. However, when autophosphorylated
CCaMK was used, calmodulin stimulated the rate of phosphorylation of
the GS peptide with similar kinetics as histone IIAS (Fig. 7A). To study the effect of autophosphorylation
on kinase activity using GS peptide as substrate, we compared
Ca
/calmodulin-dependent and
Ca
/calmodulin-independent activity of
autophosphorylated to unphosphorylated CCaMKs. Autophosphorylated CCaMK
exhibits approximately 5-fold increased
Ca
/calmodulin-dependent kinase activity as compared
with the unphosphorylated enzyme. The maximal stimulation of
autophosphorylated CCaMK by Ca
/calmodulin was
20-25-fold as compared with the EGTA control (Fig. 7B). Ca
/calmodulin-independent
activity was not significantly affected by autophosphorylation. These
results suggest that Ca
-induced autophosphorylation
stimulates Ca
/calmodulin-dependent activity of CCaMK.
Figure 7:
The effect of autophosphorylation of CCaMK
on GS peptide phosphorylation. A, effect of increasing
concentrations of calmodulin on the GS peptide phosphorylation by
autophosphorylated CCaMK. B, effect of CCaMK
autophosphorylation on Ca/calmodulin-dependent and
-independent activity. Autophosphorylated and unphosphorylated enzymes
were used for studying phosphorylation of the GS peptide. Column
1, CCaMK autophosphorylated in the presence of 0.5 mM CaCl
at 30 °C for 20 min and was used for
Ca
/calmodulin-dependent GS peptide phosphorylation (hatched bar). Column 2, unphosphorylated enzyme
incubated at 30 °C for 20 min and used for
Ca
/calmodulin-dependent GS peptide phosphorylation (hatched bar). Solid bars represent the activity of
autophosphorylated CCaMK (column 1) and unphosphorylated CCaMK (column 2) in the presence of 2.5 mM EGTA.
This report provides the biochemical evidence for a
Ca/calmodulin-dependent protein kinase in plants.
Although several Ca
/calmodulin-dependent kinases have
been characterized from animal systems(10) , CCaMK is the only
plant kinase whose activity is regulated by both Ca
and Ca
/calmodulin. Among the substrates tested,
histone IIAS and synthetic GS peptide are the most efficient phosphate
acceptors. CCaMK exhibits a higher K
value
(150-200 nM) for calmodulin (Fig. 1A and Fig. 7A) compared with CaMKII (20-100
nM) (31) and CaMKIV (26-150
nM)(32, 33) , indicating that plant kinase
requires a higher concentration of calmodulin for its activity. This is
probably due to a higher dissociation constant of calmodulin for CCaMK
(55 nM) than for animal
Ca
/calmodulin-dependent protein kinases (1-10
nM)(34) .
S-Labeled calmodulin binding
and peptide binding assays revealed that calmodulin binding site of
CCaMK is present between amino acid residues 322 and 340 (Fig. 3). This region has homology to animal CaMKII, with
conserved basic (Arg-326, Arg-327, and Lys-328) as well as hydrophobic
(Phe-323, Ala-325, Ala-332, and Leu-338) amino acid residues.
The
visinin-like Ca-binding domain, a novel feature of
CCaMK, is not known to exist in other protein kinases. The visinin-like
domain contains three EF-hand motifs (Fig. 5A) similar
to animal visinin-like proteins. Frequenin, neurocalcin, and
visinin-like proteins are known to be members of
Ca
-sensitive guanylyl cyclase activators that are
involved in cation channel regulation in neuronal tissues(35) .
Visinin-like proteins typically contain three conserved EF-hand motifs,
each with different affinities to
Ca
(36, 37) . The
Ca
-dependent mobility shift assay suggests that
binding of Ca
to the EF-hands II and III is important
for inducing conformational changes in the visinin-like domain of CCaMK (Fig. 5B). Ca
-induced conformational
change in the visinin-like domain may be critical for regulation of
CCaMK activity. The CCaMK mutant 1-356 lacking this domain did
not show Ca
-dependent autophosphorylation. The mutant
1-356 also exhibited reduced activity as compared with the
wild-type enzyme, suggesting that the visinin-like domain is required
for the maximal activation of CCaMK. It is unlikely that this reduced
activity is due to lowered affinity of mutant 1-356 to
calmodulin, since the saturation curve of
S-calmodulin
binding for mutant 1-356 indicated that it has a similar K
(60 nM) for calmodulin (data not
shown). However, it is possible that the visinin-like domain may
stabilize the conformation of CCaMK, which is indispensable for its
maximal activity.
The suppression of Ca-dependent
autophosphorylation of CCaMK by Ca
/calmodulin is
intriguing. Phosphoamino acid analysis revealed that CCaMK
autophosphorylation is due to the phosphorylation of the threonine
residue(s) (Fig. 4C). Autophosphorylation of CCaMK
increased its Ca
/calmodulin-dependent kinase activity
by 5-fold (Fig. 7B).
Ca
/calmodulin-dependent autophosphorylation of animal
CaMKII at Thr-286 NH
-terminal to the calmodulin binding
site is known to stimulate Ca
-independent
activity(11, 38, 39) . In contrast,
Ca
/calmodulin-independent basal autophosphorylation
at Thr-305 and -306 within the calmodulin-binding site inactivates
CaMKII by inhibiting its ability to bind
calmodulin(40, 41) . Although the calmodulin binding
region of CCaMK has similarity to the calmodulin-binding region of
CaMKII, there are no threonine residues around this area (Fig. 3A). The inhibition of the
Ca
-stimulated CCaMK autophosphorylation by calmodulin
may be due to the conformational change induced by the calmodulin
binding to CCaMK(42) . Inhibition of autophosphorylation by
calmodulin is also reported in smooth muscle myosin light chain
kinases(43) , where all three phosphorylated residues are
present in proximity to the calmodulin-binding site. The absence of
threonine residues around the calmodulin-binding region of CCaMK
suggests that the mechanism of CCaMK regulation by autophosphorylation
is different from myosin light chain kinases and CaMKII.
Signal-induced changes in cytosolic Ca concentration are believed to be important for many cellular
processes in plants(2, 44, 45) . Our results
indicate that Ca
has a dual effect on the stimulation
of CCaMK activity. In the presence of calmodulin, Ca
binds to calmodulin and stimulates CCaMK activity. In the absence
of calmodulin, Ca
alone stimulates
autophosphorylation of CCaMK, which further increases
Ca
/calmodulin-dependent kinase activity (Fig. 7B).
Plants have multiple isoforms of
calmodulin, and their expression is developmentally regulated and
responsive to environmental
signals(22, 46, 47) . Plant calmodulin mRNA
and protein are also reported to have a relatively rapid turnover rate
in the cell(48) . Signal-induced expression and rapid turnover
suggest that there is a dynamic regulation of calmodulin in
vivo. Therefore, it is likely that CCaMK activity is
differentially controlled by signal-induced transient changes in free
Ca concentration and calmodulin. In plant cells, the
Ca
concentration required for
Ca
-dependent autophosphorylation and the
Ca
concentration required for
Ca
/calmodulin-dependent substrate phosphorylation may
be different. In order to determine how the two regulatory domains
control kinase activity, the site(s) of autophosphorylation and the
critical concentrations of Ca
required for substrate
phosphorylation and autophosphorylation need to be determined. These
experiments are currently being carried out.
A unique feature of
CCaMK is its stage-specific expression in developing
anthers(20) . We recently cloned a tobacco cDNA encoding a
protein kinase with structural features similar to CCaMK, including
calmodulin-binding and visinin-like Ca-binding
domains. Transgenic tobacco plants expressing the antisense RNA of this
cDNA clone showed impaired pollen development, (
)indicating
a crucial role for CCaMK in male gametophyte development.
The
Ca signaling pathway in plants is receiving
considerable attention and is beginning to be unraveled at the
molecular and biochemical levels(1, 2) . The
Ca
-signaling pathway mediated by
Ca
/calmodulin-dependent kinases is well established
in animals. Unfortunately, calmodulin-binding proteins, especially the
kinases, have not been well characterized in plants. Therefore, the
discovery of Ca
/calmodulin-dependent protein kinase
and the elucidation of its biochemical properties will impact future
studies on the role of calmodulin in Ca
-mediated
signaling in plants.