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INTRODUCTION |
Ca2+ is a universal second messenger that regulates
diverse developmental and physiological processes in plants (1). One of
the major mechanisms decoding the change of intracellular
Ca2+ and transducing Ca2+ signal is the action
of Ca2+-modulated proteins. Calmodulin
(CaM)1 is a highly conserved
and the most widely distributed Ca2+-binding protein (2).
CaM is believed to be a primary receptor for intracellular
Ca2+ and functions as a regulatory element for its target
proteins when activated by Ca2+. One group of CaM-modulated
proteins is composed of Ca2+/CaM-dependent
protein kinases.
Extensive investigation of protein phosphorylation and
dephosphorylation has revealed that protein kinases play key roles in
various signal transduction pathways leading to cellular and physiological responses (3). Ca2+/CaM-dependent
protein phosphorylation has been implicated in regulating a broad array
of biological functions and is believed to play a pivotal role in
amplifying and diversifying the action of Ca2+/CaM-mediated
signals in animals. A number of different types of
Ca2+/CaM-dependent protein kinases, including
CaM kinases (CaMKs) I-IV, phosphorylase kinase, and myosin light chain
kinase, have been cloned and demonstrated to regulate various cellular
processes (4, 5). Some indirect evidence for the existence of
Ca2+/CaM-dependent protein phosphorylation has
been reported in plants (1). However, until recently, no direct and
convincing evidence has been presented. In recent years, three
Ca2+/CaM-dependent protein kinase genes having
some features similar to the mammalian multifunctional
Ca2+/CaM-dependent protein kinase (CaMKII) were
cloned from plants (6-8). The apple
Ca2+/CaM-dependent protein kinase was isolated
using an interaction screening method with labeled CaM as a ligand
probe (6). CCaMK from lily and MCK1 from maize were cloned using the
polymerase chain reaction screening method with oligonucleotide primers
designed based on the conserved regions of mammalian CaMKs (7, 8). However, the sequence similarity between lily CCaMK, maize MCK1, and
apple Ca2+/CaM-dependent protein kinase is only
observed in their conserved kinase domains. No obvious sequence
similarity exists outside their kinase domains. Although these three
kinases were directly or indirectly demonstrated to bind CaM in a
Ca2+-dependent manner, only biochemical
properties for CCaMK have been reported to show that its kinase
activity is regulated by Ca2+/CaM (9, 10).
CCaMK has a unique structural feature characterized by the presence of
a kinase domain, a CaM-binding domain, and a neural visinin-like
Ca2+-binding domain within a single polypeptide (7). Its
kinase domain and CaM-binding domain are highly similar to those of
mammalian CaMKII. The sequence of its C-terminal domain does not show
significant homology to any known protein kinase. This domain has high
similarity with a family of neural visinin-like
Ca2+-binding proteins (7), and its Ca2+-binding
property was confirmed by biochemical characterization (9). In plants,
a predominant and widely expressed form of protein kinase is the
Ca2+-dependent protein kinase or CaM-like
domain protein kinase (CDPK) (11, 12). CDPK contains a kinase domain
similar to that of mammalian CaMKII, a junction domain, and a CaM-like
domain (11). Although both CCaMK and CDPK contain a kinase domain and a
Ca2+-binding domain and have similarity in their overall
structures, they distinctly differ in terms of their sequences as well
as regulation of their kinase activities. There is no striking sequence similarity shared between them except their kinase domains. Unlike the
CaM-like domain of CDPK, the Ca2+-binding domain of CCaMK
contains three EF-hand Ca2+-binding sites and has higher
similarity to visinin-like proteins than CaM (7). Unlike CCaMK, the
full-length CDPK does not bind to CaM, and its kinase activity is
Ca2+-dependent but does not require exogenous
CaM (13). Another difference between these kinases is that CDPK is
encoded by a large family of genes and is ubiquitously distributed
among plant tissues, while CCaMK is a single copy gene in lily and its
expression is developmentally and spatially regulated (7).
Study of protein-protein interactions between a kinase and its target
proteins is crucial to illustrate intracellular signal transduction
that eventually leads to physiological responses. Isolation and
characterization of interacting proteins/substrates of CCaMK is a major
step in understanding its in vivo regulation, cellular
compartmentalization, and biological functions. In this study, the
yeast two-hybrid interaction cloning system was used to isolate
cDNAs encoding proteins that interact with CCaMK. Here we report
that one of the cDNA clones (LlEF-1
1) isolated from the screening is homologous to the eukaryotic elongation factor-1
(EF-1
), and CCaMK phosphorylates LlEF-1
1 in a
Ca2+/CaM-dependent manner, but their binding
does not require Ca2+.
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EXPERIMENTAL PROCEDURES |
Plant Material--
Lily (Lilium longiflorum Thunb
cv. Nellie White) plants were grown in the greenhouse. Various organs
and anthers of different stages were collected and immediately frozen
in liquid nitrogen for isolation of RNA and proteins.
Cloning of LlEF-1
1 Using the Yeast Two-hybrid System--
The
two-hybrid cDNA cloning kit was purchased from Stratagene, and the
screening procedure was followed as recommended by the manufacturer.
The mRNA was isolated from immature lily anthers (flower bud size
0.5-2.5 cm) using standard protocols (14). A lily immature anther
cDNA library was constructed in HybriZAP two-hybrid
vector and
expressed as fusion proteins with the activation domain of GAL4 in
pAD-GAL4. The full coding region of lily CCaMK was cloned in frame into
pBD-GAL4 and expressed as a fusion protein with the binding domain of
GAL4. The yeast strain YRG-2 carrying the pBD-GAL4/CCaMK plasmid was
transformed with the plasmids excised from the library. Approximately
5 × 105 transformants were screened for growth on
medium lacking leucine, tryptophan, and histidine. Growing yeast
colonies were screened for expression of
-galactosidase using the
chromogenic substrate, 5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside. The
positive clones from the His+ and LacZ+
colonies were retransformed into YRG-2 alone, with pBD-GAL4/CCaMK, or
with the negative control plasmids (pGD-GAL4, p53, and pLamin C). Only
clones that did not activate lacZ alone and in the presence of the negative control plasmids were characterized further. In order
to clone the cDNA of LlEF-1
1 containing the complete
coding region, the partial clone obtained from the screening was used as a probe to screen a cDNA library derived from developing anthers of lily (7).
Preparation of the Anti-CCaMK Antibody and Western
Analysis--
A rabbit polyclonal antibody was raised against the
visinin-like domain (amino acids 358-520) of lily CCaMK. The
anti-CCaMK antibody was further purified from antiserum by blot
affinity purification essentially as described (15). Proteins (60 µg) extracted from different tissues of lily and anthers at different stages were separated on a 10% SDS-polyacrylamide gel and transferred onto polyvinylidene difluoride membrane (Millipore Corp.). The membrane
was immunoblotted with either the affinity-purified anti-CCaMK antibody
or a polyclonal anti-EF-1
antibody, followed by incubation with the
goat anti-rabbit IgG conjugated with horseradish peroxidase (Bio-Rad).
The immunoblot was developed using an enhanced chemiluminescent substrate (Pierce) and exposed to autoradiography film.
Preparation of CaM-binding Proteins--
Lily anthers (flower
bud size 0.9-2.0 cm) were powdered and homogenized in the extraction
buffer (40 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.1% Triton X-100)
containing 1 mM CaCl2, 1 mM
phenylmethylsulfonyl fluoride, 5 µg/ml antipain, 5 µg/ml aprotinin,
5 µg/ml leupeptin, and 5 µg/ml pepstatin. The homogenate was
centrifuged at 21,000 × g for 15 min and at
100,000 × g for 1 h at 4 °C. The supernatant
was applied onto a calmodulin-Sepharose 4B (Amersham Pharmacia Biotech)
column pre-equilibrated with the extraction buffer containing 1 mM CaCl2. The column was then washed extensively with the extraction buffer containing 1 mM
CaCl2. The bound proteins were eluted with 50 mM Tris-HCl (pH 7.5) containing 2.5 mM EGTA,
0.05% Tween 20, 200 mM NaCl, 1 mM DTT, and 1 mM phenylmethylsulfonyl fluoride and concentrated with a
Centricon-30 concentrator (Amicon). Aliquots of eluted proteins were
separated on a 12% SDS-polyacrylamide gel and immunoblotted with
either the anti-EF-1
antibody or the anti-CCaMK antibody.
Plasmid Construction and Site-directed Mutagenesis--
The
original cDNA clone isolated from the yeast two-hybrid screening
encodes the C-terminal portion (amino acids 204-447) of LlEF-1
1.
This cDNA clone was amplified, and BamHI and
EcoRI cloning sites were introduced by polymerase chain
reaction. The amplified fragment was subcloned in frame into the
expression vector pGEX-3X (Amersham Pharmacia Biotech), resulting in
pmC. The cDNA fragment with engineered BamHI and
EcoRI restriction sites coding for the N-terminal portion
(amino acids 1-221) of LlEF-1
1 was amplified and inserted in frame
into pGEX-3X to generate pmN. The full coding region of
CRPK2, a Ca2+-dependent protein
kinase gene from corn root tips (16), was amplified, and
NdeI and EcoRI restriction sites were introduced and subcloned in frame into pET-14b (Novagen) to generate the CRPK2
expression construct pCRPK2. All of these constructs were sequenced to
confirm that no mutation was introduced. The site-directed mutagenesis
was performed using a kit purchased from Stratagene. The
oligonucleotide primers used for generating the mutant
pmC257A (Thr was mutated into Ala at the amino acid residue
257 in pmC) were 5'-CAGTCGGCCGTGTGGAGGCTGGTATTGTGAAG-3' and
5'-CTTCACAATACCAGCCTCCACACGGCCGACTG-3'. The sequence of the mutant was
confirmed by DNA sequencing, and no additional mutation was introduced.
Expression and Purification of CCaMK, CRPK2, and LlEF-1
1
Deletion Mutants--
CCaMK was expressed and purified as described
previously (9). For expression and purification of CRPK2,
Escherichia coli BL21 (DE3) cells transformed with pCRPK2
were grown at 37 °C in M9 minimal medium supplemented with 0.2%
casein enzymatic hydrolysate, 0.4% glucose, 10 mM
magnesium sulfate, and 100 mg/liter ampicillin. The expression of CRPK2
was induced for 3 h by adding 0.5 mM isopropyl
-D-thiogalactoside after A600
reached 0.6 units. CRPK2 was purified according to the protocol
provided by the manufacturer (Novagen). The glutathione
S-transferase (GST) fusion proteins (mN, mC, and mC257A) were purified on glutathione-Sepharose 4B columns
using standard procedures, except that the columns were washed
extensively with phosphate buffer containing 1 mM DTT,
0.1% Tween 20, 2 mM ATP, 10 mM
MgSO4, and 1.2 M NaCl to further eliminate
contamination. Eluted proteins were thoroughly dialyzed with
Centricon-30 against Tris-HCl (pH 7.5) buffer containing 1 mM DTT and 10% ethylene glycol. Protein concentrations
were determined by Bradford's method or by SDS-PAGE using bovine serum
albumin as a standard.
Protein Kinase Assay--
Phosphorylation assays were carried
out as previously reported (9). The indicated amounts of CCaMK, CRPK2,
and LlEF-1
1 deletion or site-directed mutants were added into the
reaction mixtures. Proteins were resolved by SDS-PAGE and stained with Coomassie Brilliant Blue. The gels were dried and subjected to autoradiography. 32P incorporation was determined by
counting the excised protein bands in a liquid scintillation counter.
In Vitro Binding Assay--
In vitro binding
experiments were performed as described by Watanabe et al.
(17). The assays were carried out in 500 µl of 40 mM
Tris-HCl (pH 7.5) containing 1 mM DTT and 30 µl of
glutathione-Sepharose 4B. CCaMK and the deletion mutant of LlEF-1
1
(mN or mC) were added into the mixture in the presence of 0.5 mM CaCl2, 0.5 mM CaCl2
plus 1 µM CaM, or 2.5 mM EGTA. The reaction
was incubated at 4 °C for 1 h. The Sepharose beads were
collected and washed three times in 1 ml of 40 mM Tris-HCl
buffer containing 300 mM NaCl and either 0.5 mM
CaCl2 or 2.5 mM EGTA. After the final wash, the
beads were resuspended in 50 µl of 2.5× SDS-PAGE sample buffer and
boiled for 5 min. Eluted proteins were subjected to SDS-PAGE and
visualized by staining with Coomassie Brilliant Blue or transferred onto polyvinylidene difluoride membrane for immunoblot analysis.
Immunoprecipitation--
Lily anthers (flower bud size 0.9-2.0
cm) were powdered and homogenized in the extraction buffer containing 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml antipain, 5 µg/ml aprotinin, 5 µg/ml leupeptin, and 5 µg/ml pepstatin in the
presence of either 1 mM CaCl2 or 2.5 mM EGTA. The homogenate was centrifuged at 21,000 × g for 30 min at 4 °C. The supernatant was used as the
crude protein extract. 60 µl of protein G-agarose beads (Life
Technologies, Inc.) were incubated with 4 µg of the anti-CCaMK
antibody in the extraction buffer for 4 h at 4 °C with gentle
agitation. The beads were collected and washed three times in 1 ml of
the extraction buffer. An aliquot of the crude extract containing 800 µg of protein was added to the conjugate of the anti-CCaMK antibody
and protein G-agarose beads and incubated with gentle agitation for
4 h at 4 °C. The immunoprecipitate was washed three times with
1 ml of the extraction buffer in the presence of either 1 mM CaCl2 or 2.5 mM EGTA and
subjected to immunoblot analysis with a mouse monoclonal antibody
against urchin EF-1
. Control experiments were carried out by
incubation of protein G-agarose beads with either the anti-CCaMK
antibody alone or the crude protein extract alone.
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RESULTS |
Cloning of LlEF-1
1 cDNA--
The yeast two-hybrid
interaction cloning method was used to isolate genes encoding
interacting proteins and/or substrates of CCaMK. Since lily CCaMK is
expressed in a stage-specific manner during anther development (7), a
cDNA library was constructed using mRNA isolated from lily
anthers at the corresponding stages and expressed as fusion proteins
with the GAL4 activation domain. The fusion protein of the GAL4
DNA-binding domain/CCaMK was expressed in pBD-GAL4 and utilized as the
bait protein to screen the cDNA library. Among the positive clones
identified from several rounds of screening, sequence comparison showed
that one of these clones has high homology with the eukaryotic
elongation factor-1
. The clone is 1016 base pairs long and encodes
the C-terminal portion (amino acids 204-447) of LlEF-1
1 (Fig.
1). This cDNA clone was used as a
probe to obtain its clone containing the full coding region by
screening a cDNA library from which CCaMK was originally isolated
(7). The clone coding for the full-length polypeptide was designated as
LlEF-1
1 (Fig. 1).

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Fig. 1.
Nucleotide and deduced amino acid sequences
of LlEF-1 1. The nucleotides are numbered on
the left, and the corresponding amino acids are indicated on
the right. All serine and threonine residues located in the
motifs of RXX(S/T) and KXX(S/T) are
underlined. The site (Thr-257) phosphorylated by CCaMK is
italicized and underlined.
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LlEF-1
1 encodes a polypeptide of 447 amino acid residues
(Fig. 1), which shares high homology with EF-1
s from other species (Fig. 2). It has very high homology
(97.8% similarity and 95.7% identity) with a maize EF-1
and also
significant homology with a protozoan parasite (Trypanosoma
brucei) EF-1
(82.6% similarity and 76.1% identity) and a
rabbit EF-1
(82.2% similarity and 75.9% identity). LlEF-1
1
contains the putative GTP-binding and tRNA-binding regions, and the
sequences in these regions are highly conserved among different
EF-1
s (Fig. 2).

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Fig. 2.
Comparison of the predicted amino acid
sequence of LlEF-1 1 with those of
EF-1 s from other organisms and their putative
functional domains. The deduced amino acid sequences of
LlEF-1 1, maize EF-1 (GenBankTM accession number
D45408), T. brucei EF-1 (U10562), and rabbit EF-1
(X62245) are aligned in this comparison. Gaps are introduced to
maximize the homology among the sequences. The putative functional
domains are underlined involving tRNA binding (region J) and
GTP binding and hydrolysis (regions A, C, G, and E). The site (Thr-257)
phosphorylated by CCaMK is marked by an asterisk. Identical
amino acids and introduced gaps are indicated by dashes and
dots, respectively.
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Expression of CCaMK and EF-1
--
Since the visinin-like domain
is unique to CCaMK (7), the fusion protein of the visinin-like domain
was expressed and purified from E. coli for producing a
polyclonal antibody to analyze the expression pattern of CCaMK. CCaMK
is specifically expressed only in root tips and anthers, and its
expression is undetectable among other organs examined (Fig.
3). The expression of CCaMK is
developmentally regulated in anthers. At the early stages, the protein
level is very low. CCaMK expression reaches the highest level at the
stage when the flower bud size is between 0.9 and 2.0 cm, coinciding with microsporogenesis (18). Its expression then decreases until it
becomes undetectable at the later stages of anther development. On the
other hand, EF-1
is a ubiquitous protein and expressed in all of the
organs examined. However, its expression level varies dramatically.
EF-1
is highly expressed in root tips and anthers at the early
stages of development, coinciding with the expression pattern of CCaMK.
It appears that both CCaMK and EF-1
are highly expressed in tissues
with high mitotic and meiotic activities.

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Fig. 3.
Expression of CCaMK and
EF-1 in different organs and at different
stages of anther development. Immunoblot analysis showing CCaMK
and EF-1 protein levels in different organs of lily. The
numbers in parentheses indicate the length of the
flower buds when anthers were collected.
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CCaMK binds CaM in a Ca2+-dependent manner (7,
9). Endogenous CCaMK was obtained from lily anthers by passing the
protein extract through a calmodulin-Sepharose column. Interestingly, EF-1
was copurified with CCaMK on the calmodulin-Sepharose column (Fig. 4). However, this result does not
demonstrate a direct interaction between CCaMK and EF-1
, since
EF-1
was reported to bind to CaM in the presence of Ca2+
(19, 20).

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Fig. 4.
Copurification of CCaMK and
EF-1 from lily anthers on a
calmodulin-Sepharose column. CaM-binding proteins were purified
from lily anthers (flower bud size 0.9-2.0 cm) on a
calmodulin-Sepharose column in the presence of Ca2+ and
eluted with EGTA. The eluted proteins were separated on an
SDS-polyacrylamide gel and immunoblotted with the polyclonal
anti-EF-1 antibody and the anti-CCaMK antibody, respectively.
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Ca2+/CaM-dependent Phosphorylation of
LlEF-1
1 by CCaMK--
The fact that LlEF-1
1 was
cloned using the yeast two-hybrid system indicates that it interacts
with CCaMK. There may be different ways in which CCaMK interacts with
LlEF-1
1: (a) LlEF-1
1 may physically bind to CCaMK;
(b) it may be one of the substrates of CCaMK; and
(c) it may serve as an effector of CCaMK. To determine the
nature of interaction between CCaMK and LlEF-1
1, we first examined
whether CCaMK can phosphorylate LlEF-1
1 because of the identity of
CCaMK as a protein kinase. The N-terminal and C-terminal regions of
LlEF-1
1 were expressed and purified as GST fusion proteins (mN and
mC, Fig. 5A). In the presence
of EGTA or Ca2+, CCaMK phosphorylated the mC at the basal
level (Fig. 5B). The phosphorylation level was stimulated up
to around 25-fold by adding Ca2+ and CaM, indicating that
CCaMK phosphorylates the mC in a
Ca2+/CaM-dependent manner. The stimulation of
the kinase activity by Ca2+/CaM is comparable with previous
results where other substrates were used (9). The mN was not
phosphorylated by CCaMK even in the presence of Ca2+ and
CaM (Fig. 5C). The mC was phosphorylated by CCaMK, which agrees with the result of the yeast two-hybrid screening, since the
original cDNA isolated from the screening codes for the C-terminal region (204-447) of LlEF-1
1.

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Fig. 5.
Phosphorylation of
LlEF-1 1 by CCaMK in a
Ca2+/CaM-dependent manner. A,
schematic diagrams of the N-terminal and C-terminal deletion mutants
(mN and mC) of LlEF-1 1. Both were expressed as GST fusion proteins.
B, Ca2+/CaM-dependent
phosphorylation of the mC by CCaMK. The mC (0.2 µg) was incubated in
20 µl of phosphorylation reaction buffer with 0.2 µg of CCaMK in
the presence of 2.5 mM EGTA, 0.5 mM
Ca2+, or 0.5 mM Ca2+ plus 1 µM CaM. The reaction was carried out at 30 °C for 2 min and terminated by adding 20 µl of 2.5 × sample buffer and
boiling for 5 min. C, left, immunoblot showing
the positions of GST, the mN, and the mC. GST, the mN, and the mC were
separated by SDS-PAGE and immunoblotted with the goat anti-GST antibody
(Amersham Pharmacia Biotech) and visualized with the rabbit anti-goat
IgG conjugated with horseradish peroxidase (Sigma). The positions of
protein size markers in kDa are shown on the left.
Right, the phosphorylation assays were performed as
described except that 0.1 µg of the mC was used.
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Identification of the Phosphorylation Site for CCaMK in
LlEF-1
1--
CCaMK is similar to the mammalian multifunctional
CaMKII in terms of the sequences in their kinase and CaM-binding
domains and the regulation of their substrate phosphorylation (7, 9). The phosphorylation sites of CCaMK in LlEF-1
1 were predicted based
on the consensus sequence of the minimal motif, RXX(S/T), which is present in majority of the phosphorylation site sequences of
the CaMKII protein substrates (5, 21). There are only two putative
phosphorylation sites in LlEF-1
1 (Fig. 1). One is Thr-72 in the N
terminus. Since the mN containing Thr-72 was not phosphorylated by
CCaMK (Fig. 5C), obviously this site is not the
phosphorylation site for CCaMK. The other phosphorylation site is
Thr-257 in the putative tRNA-binding region (Figs. 1 and 2). Therefore,
Thr-257 in the mC was mutated into Ala to investigate whether Thr-257
is a real phosphorylation site for CCaMK. The result revealed that the
mC257A was no longer phosphorylated by CCaMK (Fig.
6), indicating that Thr-257 is indeed the
phosphorylation site for CCaMK. Taken together, it is clear that
Thr-257 is the only phosphorylation site for CCaMK, which is located in
the putative tRNA-binding domain of LlEF-1
1.

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Fig. 6.
Phosphorylation of the mC and its
site-directed mutant by CCaMK. Left panel, immunoblot
showing approximately equal amounts of the mC and the
mC257A were used for the phosphorylation assays.
Right panel, phosphorylation of the mC and the
mC257A by CCaMK. The mC and the mC257A (1 µg)
were phosphorylated by CCaMK at 30 °C for 10 min.
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Ca2+-dependent and CaM-independent
Phosphorylation of LlEF-1
1 by CRPK2--
In plants, it has been
reported that EF-1
can be phosphorylated by CDPK (22). To
investigate whether LlEF-1
1 can also be phosphorylated by CDPK, one
of CDPK isoforms (CRPK2) isolated from corn root tips was
expressed and purified from E. coli. Although CCaMK and
CRPK2 show overall structural resemblance (Fig.
7A), there is no significant
sequence similarity outside of their kinase domains. CRPK2 is a typical
form of CDPK and has extensive and significant homology to other CDPKs
(16). The amino acid sequence of CRPK2 shares 75% similarity and 65%
identity with the prototype of CDPK, soybean CDPK
, along their three
functional domains (kinase domain, junction domain, and CaM-like
domain) (11).

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Fig. 7.
Phosphorylation of
LlEF-1 1 by CRPK2 in a
Ca2+-dependent and CaM-independent manner.
A, schematic diagrams showing the functional domain
organization of CCaMK and CRPK2 for their structural comparison.
Domains of CCaMK were as follows: kinase domain (KD);
CaM-binding domain (CD); and neural visinin-like domain
(VLD). The three EF-hand Ca2+-binding sites in
the neural visinin-like domain are marked. Amino acids are numbered
below. Domains of CRPK2 were as follows: N-terminal domain
(ND); kinase domain (KD); junction domain
(JD); and CaM-like domain (CLD). The four EF-hand
Ca2+-binding sites in the CaM-like domain are marked. Amino
acids are numbered based on comparison of CRPK2 with soybean CDPK .
B, phosphorylation of LlEF-1 1 by CRPK2. 0.2 µg each of
GST, the mN, and the mC was used in the phosphorylation assays in the
presence of 2.5 mM EGTA, 0.5 mM
Ca2+, or 0.5 mM Ca2+ plus 1 µM CaM. CRPK2 (0.1 µg) was added into each of the
reaction mixtures and incubated at 30 °C for 2 min. The positions of
protein size markers are indicated on the left.
C, phosphorylation of the mC and the mC257A by
CRPK2. Left, immunoblot showing that relative equal amounts
of the mC and the mC257A were used for the phosphorylation
assays. Right, the mC and the mC257A (0.2 µg)
were phosphorylated by CRPK2 in the presence of 0.5 mM
Ca2+.
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Like other CDPKs, CRPK2 showed Ca2+-dependent
and CaM-independent kinase activity (Fig. 7B). There was no
kinase activity in the absence of Ca2+, and its kinase
activity was not stimulated by CaM. CRPK2 phosphorylated both the mN
and the mC in a Ca2+-dependent and
CaM-independent manner. In fact, CaM slightly decreased the
phosphorylation of the mN by CRPK2. The reason may be that CaM masked
some of CRPK2 phosphorylation sites by binding to the mN, since CaM
binds to EF-1
in the presence of Ca2+ (Refs. 19 and 20;
Fig. 4). In an attempt to determine whether CCaMK and CRPK2
phosphorylate the same site, the mC257A, which was not
phosphorylated by CCaMK (Fig. 6), was tested for CRPK2 phosphorylation.
Fig. 7C showed that CRPK2 phosphorylated both the mC and the
mC257A to a similar extent, suggesting that Thr-257 may not
be the phosphorylation site for CRPK2. CDPKs were reported to
preferentially phosphorylate serine and threonine residues in the
four-residue motifs of both RXX(S/T) and KXX(S/T)
(12). Besides the two RXXT motifs, there are also a number
of KXX(S/T) motifs in LlEF-1
1 (Fig. 1). The mN and the mC
cover the full-length LlEF-1
1 and share a stretch of overlapping
sequence (amino acids 204-221; Fig. 5A). Since CRPK2
phosphorylated both the mN and the mC (Fig. 7B), we studied
whether CRPK2 phosphorylated only sites in this overlapping region.
There is indeed a threonine (Thr-215) residue located in the motif
KXXT in this region. Thr-215 was mutated to Ala in both the
mN and the mC. However, both of the site-directed mutants were still
phosphorylated by CRPK2 (data not shown). These results indicate that
CRPK2 phosphorylates multiple sites of LlEF-1
1 in a
Ca2+-dependent and CaM-independent manner, and
its phosphorylation sites are different from that for CCaMK.
Binding of CCaMK to LlEF-1
1--
To test whether there is
direct association between CCaMK and LlEF-1
1, in vitro
binding assays were carried out, and the results are shown in Fig.
8. CCaMK did not bind to
glutathione-Sepharose beads when incubated with GST in the presence of
EGTA, Ca2+, or Ca2+ plus CaM, suggesting that
CCaMK did not bind to glutathione-Sepharose beads and GST. The protein
that migrated at a similar molecular mass as CCaMK (~58 kDa) was only
observed when CCaMK was incubated with the mN or the mC in the absence
of Ca2+. In the presence of Ca2+ or
Ca2+ plus CaM, this protein band disappeared, indicating
that its association with the mN and the mC was disrupted. To examine
whether the protein bands were CCaMK, a similar experiment was carried out, and the proteins were blotted onto polyvinylidene difluoride membrane for an immunoblot assay with the anti-CCaMK antibody. The
result showed that the proteins immunologically reacted with the
anti-CCaMK antibody and were indeed CCaMK. These results suggest that
CCaMK binds to LlEF-1
1 in a Ca2+-independent manner, and
Ca2+ disrupts their direct and physical interaction.

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Fig. 8.
In vitro binding assay showing
Ca2+ inhibition of binding of CCaMK to
LlEF-1 1. Left panel, in
vitro binding of CCaMK to LlEF-1 1. GST, the mN, and the mC
(2-8 µg) were incubated with CCaMK (1 µg) in the presence of EGTA,
Ca2+, or Ca2+ plus CaM as described under
"Experimental Procedures." Proteins bound to glutathione-Sepharose
4B beads were analyzed by SDS-PAGE and visualized by staining with
Coomassie Brilliant Blue. The position of CCaMK is marked with a
solid arrow on the left. Right
panel, immunoblot showing bound CCaMK in the in vitro
binding assays. A binding experiment was performed as described for the
left panel. Bound proteins were separated by SDS-PAGE,
transferred to polyvinylidene difluoride membrane, and immunoblotted
with the anti-CCaMK antibody.
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Association between CCaMK and EF-1
in Lily Anthers--
All of
the in vitro binding assays mentioned above were carried out
using the N-terminal and C-terminal deletion mutants of LlEF-1
1. To
examine whether CCaMK binds to intact EF-1
in lily anthers, we
performed immunoprecipitation assays using the protein extract from
lily anthers. The affinity-purified anti-CCaMK antibody was used for
immunoprecipitation to investigate whether a protein complex containing
CCaMK and EF-1
exists in lily anthers. The result showed that
EF-1
was coimmunoprecipitated with CCaMK in the presence of EGTA in
the extraction buffer (Fig. 9). In the presence of Ca2+, no EF-1
was detected in the
immunoprecipitates. When the anti-CCaMK antibody alone or the protein
extract alone was incubated with protein G-agarose beads in the control
assays, there was no signal detected by the monoclonal anti-EF-1
antibody. This experiment demonstrates that CCaMK binds to EF-1
in
lily anthers in a Ca2+-independent manner.

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Fig. 9.
Coimmunoprecipitation of CCaMK and
EF-1 from lily anthers. The crude protein
extract of lily anthers was used for immunoprecipitation with the
anti-CCaMK antibody in the presence of EGTA or Ca2+.
Immunoprecipitates were immunoblotted with the mouse monoclonal
anti-EF-1 antibody. Ab and CE represent the
anti-CCaMK antibody and the crude protein extract of lily anthers,
respectively. Treatments of the antibody alone (Ab) without
the crude protein extract and the crude protein extract alone
(CE) without the antibody were used as controls. The
positions of protein size markers are indicated on the left.
The position of EF-1 is marked on the right.
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DISCUSSION |
In this study, we identified a CCaMK-interacting protein using the
yeast two-hybrid interaction screening method. Sequence comparison
revealed that this protein (LlEF-1
1) is a homolog of EF-1
. It is
well known that the eukaryotic EF-1
is an essential component of
polypeptide synthesis complex and promotes the
GTP-dependent binding of aminoacyl-tRNA to the A-site on
ribosomes during the elongation phase of translation in the cytoplasm
(23). In eukaryotes, EF-1
is a ubiquitous and highly conserved
protein. It is one of the most abundant proteins and is normally
present in excessive molar ratios compared with other essential
components of the translational machinery. Growing evidence indicates
that EF-1
is a multifunctional protein and that it is involved in
other cellular processes besides protein synthesis (23-25). An EF-1
from tobacco cells was reported to have similar properties to
vitronectin, an adhesion protein in animals (26). In carrot cells, an
EF-1
homolog was identified as an activator of a plasma membrane
phosphatidylinositol 4-kinase (22). EF-1
has been demonstrated to
bind to actin filaments and microtubules and affect their dynamics both
in vitro and in vivo (19, 24, 27-30). These
lines of evidence indicate that EF-1
possesses other biological
functions beyond its activity in protein synthesis.
In both plants and protozoan parasites, the interaction between CaM and
EF-1
has been demonstrated, and their association depends on the
presence of Ca2+ (19, 20). Since CaM is an important
receptor for intracellular Ca2+ in regulating cytoskeletal
functions (2), the finding of the Ca2+-dependent association of CaM and EF-1
led to speculation that this might be one of the mechanisms mediating
the dynamics and functions of cytoskeleton by Ca2+/CaM. The
significance of interaction between Ca2+/CaM and EF-1
has been implicated in several studies. It has been reported that the
ability of EF-1
to bundle microtubules and F-actin was regulated by
CaM in the addition of Ca2+ (24, 31, 32). Considering the
multipotentiality of EF-1
, the connection between Ca2+,
CaM, and EF-1
may involve not only the dynamics of cytoskeleton but
also other cellular and biochemical processes.
EF-1
undergoes three different post-translational modifications,
i.e. methylation, formation of
glycerylphosphoryl-ethanolamine, and phosphorylation (23, 33). However,
the effects of these post-translational modifications on its
translational activity remain unclear. Protein phosphorylation is one
of the major mechanisms in regulating cellular and biochemical events.
It is also highly involved in protein synthesis through phosphorylation
of various components of the translational machinery including EF-1
(33). EF-1
was reported to be phosphorylated by protein kinase C, S6 kinase, and plant CDPK (22, 34, 35). Phosphorylation of EF-1
would
facilitate its interaction with aminoacyl-tRNA and decrease its binding
to ribosomes, and dephosphorylation is required for the transfer of
aminoacyl-tRNA to 80 S ribosomes (33). Phosphorylation of EF-1 by
multipotential S6 kinase resulted in stimulation of EF-1 activity (35).
However, an in vitro assay indicated that phosphorylation of
isolated EF-1
by protein kinase C did not affect its function as an
elongation factor (34). Instead of its effect on protein synthesis,
phosphorylation of a carrot EF-1
was shown to be pivotal to its
function as an activator of phosphatidylinositol 4-kinase (22). These
studies indicate that phosphorylation of EF-1
may not necessarily
regulate its activity only in protein synthesis and may be involved in
mediating its other functions also.
In this report, we demonstrated that a homolog of EF-1
was
phosphorylated by a plant Ca2+/CaM-dependent
protein kinase in a Ca2+/CaM-dependent manner.
The only phosphorylation site for CCaMK is located in the putative
tRNA-binding region. This threonine residue is conserved among all
EF-1
sequences available in eukaryotes. The functional significance
of the phosphorylation and the localization of the phosphorylation site
in the tRNA-binding region are currently not known. It is unclear
whether the same site can be phosphorylated by the mammalian CaMKII or
other protein kinases. We also showed that LlEF-1
1 was
phosphorylated by a CDPK in a Ca2+-dependent
but CaM-independent manner, which is consistent with the finding of a
previous study (22). The CDPK phosphorylates multiple sites in
LlEF-1
1, and these sites differ from the phosphorylation site of
CCaMK. This suggests that these two kinases have some structural
similarities, yet they differ in their regulation of kinase activity.
Phosphorylation of EF-1
by CDPK was reported to be crucial for its
function as an activator of phosphatidylinositol 4-kinase (22). Since
CCaMK and CDPK phosphorylate different sites in EF-1
, it is
reasonable to speculate that their phosphorylation may regulate
different aspects of its biological functions.
In vitro binding experiments revealed that CCaMK binds to
EF-1
in a Ca2+-independent manner, and the addition of
Ca2+ disrupts their association. In eukaryotic cells, a
large family of protein kinases regulate a variety of cellular
processes. How these kinases coordinate their functions is a very
challenging and fundamental area in signal transduction. In recent
years, progress in this area has revealed that one of the mechanisms for accomplishing the coordination is regulation of their subcellular localization through association with their anchoring proteins (36-38). Association between CCaMK and EF-1
may provide a mechanism in the regulation of the kinase activity of CCaMK by localizing it into
specific cellular compartments. This hypothesis is supported by the
fact that EF-1
is associated with many proteins, including the
components of the translational machinery, microtubules, and actin
filaments (19, 23, 27, 28, 39, 40). Both CCaMK and EF-1
are highly
expressed in similar tissues in lily (Fig. 3). It is very intriguing
that targeting CCaMK into cytoskeleton or translational machinery
through EF-1
may be one of the mechanisms to localize CCaMK into the
neighborhood of its substrates.
The results revealed that CCaMK phosphorylates EF-1
in a
Ca2+/CaM-dependent manner but binds to it in a
Ca2+-independent manner. We propose that CCaMK may be
localized in cells through direct association with EF-1
in a
Ca2+-independent manner. The transient kinase/substrate
interaction of CCaMK with EF-1
requires Ca2+/CaM, while
EF-1
serves as a substrate for CCaMK. It can be envisioned that
CCaMK is compartmentalized into the subcellular domains, where its
substrates exist, by association with EF-1
at the resting level of
Ca2+ in cells. At this stage in the process, CCaMK is
inactive. When Ca2+ concentration increases in cells due to
external or internal signals, CCaMK dissociates from EF-1
and
subsequently, upon binding Ca2+/CaM, becomes active to
phosphorylate its substrates such as EF-1
or proteins near EF-1
.
To some extent, this possible mechanism is analogous to the regulation
of another second messenger-regulated protein kinase,
cAMP-dependent protein kinase, where activation of the
kinase is achieved through the release of the catalytic subunits from
the inactive holoenzyme by cAMP (3). Various binding proteins for
several protein kinases have been identified. Their interactions are
mediated by either kinase domains or other regions and are subject to
different types of regulation (41-45). It remains to be investigated
how CCaMK mediates its direct association with EF-1
and how
Ca2+ disrupts their binding. Future studies will aim at
illustrating the functional significance of the phosphorylation of
EF-1
by CCaMK and its possible roles in tRNA binding, protein
synthesis, and/or other cellular processes.