Báo cáo Y học: A Ca2+/CaM-dependent kinase from pea is stress regulated and in vitro phosphorylates a protein that binds to AtCaM5 promoter ppt

ACa
2+
/CaM-dependent kinase from pea is stress regulated
and
in vitro
phosphorylates a protein that binds to
AtCaM5
promoter
Sona Pandey*, Shiv B. Tiwari
†
, Wricha Tyagi, Mali K. Reddy, Kailash C. Upadhyaya and Sudhir K. Sopory
School of Life Sciences, Jawaharlal Nehru University, New Delhi, India and International Center for Genetic Engineering and
Biotechnology, New Delhi, India
An immuno-homologue of maize Ca
2+
/calmodulin (CaM)-
dependent protein kinase with a molecular mass of 72 kDa
was identified in pea. The pea kinase (PsCCaMK) was
upregulated in roots in response to low temperature and
increased salinity. Exogenous Ca
2+
application increased
the kinase level and the response was faster than that
obtained following stress application. Low temperature-
mediated, but not salinity-mediated stress kinase increase
was inhibited by the application of EGTA and W7, a CaM
inhibitor. The purification of PsCCaMK using immuno-
affinity chromatography resulted in coelution of the kinase
with another polypeptide of molecular mass 40 kDa (p40).
Western blot revealed the presence of PsCCaMK in nuclear
protein extracts and was found to phosphorylate p40 in vitro.

Gel mobility shift and South-Western analysis showed that
p40 is a DNA-binding protein and it interacted specifically
with one of the cis acting elements of the Arabidopsis CaM5
gene (AtCaM5) promoter. The binding of p40 to the specific
elements in the AtCaM5 promoter was dependent of its
dephosphorylated state. Our results suggest that p40 could
be an upstream signal component of the stress responses.
Keywords: calmodulin; DNA–protein interaction; plant
protein kinase; protein phosphorylation; stress signaling.
Plants perceive a variety of signals from the external
environment as well as from the internal cellular milieu
generated during various developmental processes. Signals,
such as light, nutrients and various environmental stresses,
etc. are perceived by specific receptors. Following percep-
tion, a number of second messengers are generated that
regulate the activity of other proteins such as kinases and
phosphatases to transduce the signal downstream. Towards
the end of the signal transduction pathway, these second
messengers and/or other accessory protein(s) affected by
them modulate the activity of the transcription factors,
which regulate the expression of specific gene(s) leading to
the final response. The events related to perception of a
signal and corresponding changes in the activity and
concentration of various second messengers have been
studied in great detail. However, the downstream pathways
leading to the final control of gene expression have been
elucidated in few cases only [1–3].
Calcium ions are the most important second messengers,
controlling a variety of cellular and physiological responses
[4,5]. Cytosolic concentration of calcium ([Ca

2+
]
cyt
) chan-
ges in response to a number of external stimuli and
internal physiological developments [6,7]. Besides, a num-
ber of other second messenger such as cyclic ADP-ribose,
inositol-3-phosphate and various proteins such as calmo-
dulin (CaM) and calcium-dependent protein kinases
(CDPKs) are also affected by changes in ([Ca
2+
]
cyt
) [6,8].
The information through ([Ca
2+
]
cyt
) is transduced by two
main pathways, one involving CaM and CaM-related
proteins, and the other involving CDPKs. Both of these
pathways cross-talk at various points in the signaling
cascade [9]. The presence of CaM has been detected in
different plant cell compartments especially in the nuclei
[10]. In animal systems, it has been shown conclusively that
nuclear CaM, in combination with nuclear Ca
2+
changes,
regulates the expression of various genes either by inter-
acting with the transcription factors directly [11] or via

specific calmodulin kinases (CaMKs) [12–14]. In plant
systems too, recent studies have shown that CaM is
involved in the regulation of gene expression. Szymanski
et al. [15] have shown that CaM affects the binding of
TGA3 to the Arabidopsis CaM3 promoter. A study by van
der Luit et al. [16] showed that distinct calcium signaling
pathways that operate during cold (predominantly cyto-
plasmic) and wind (predominantly nucleus) signaling are
regulated via CaM gene expression. They have identified
two different CaM isoforms, having different nucleotide
sequences, but coding for the same polypeptide, and only
one of these (NpCaM-1) is affected by cold and wind
signaling.
CaMKs are important junctions in signal transduction
where Ca
2+
-dependent and CaM-dependent signaling
pathways converge and these kinases are potential candi-
dates in regulating gene expression. Reports on the existence
Correspondence to S. Pandey, 208 Mueller Laboratory,
Biology Department, Pennsylvania State University,
University Park, PA 16802, USA.
Fax: + 1 814 865 9131, E-mail:
Abbreviations: CaM, calmodulin; CaMK, calmodulin kinase;
CDPK, calcium-dependent protein kinase; GMSA, gel mobility shift
analysis; W7, N-(6-aminohexyl)-5-chloro-1-napthalenesulphonamide
hydrochloride; HMG, high mobility group.
Note: S. Pandey and S. B. Tiwari contributed equally to this work.
*Present address: 208 Mueller Laboratory, Biology Department, The
Pennsylvania State University, University Park, PA 16802, USA.

Present address: Biochemistry Department, 117 Schweitzer Hall,
University of Missouri, Columbia, MO 65211, USA.
(Received 15 February 2002, revised 8 May 2002,
accepted 13 May 2002)
Eur. J. Biochem. 269, 3193–3204 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02994.x
of this class of kinase are limited in plants [17–21]. The
Ca
2+
/CaM kinase isolated from maize roots has been
shown to be involved in gravitropism [19] and the Ca
2+
/
CaM kinase isolated from lily anthers binds with the
translational elongation factor II [22].
We reported previously the purification and characteri-
zation of a novel Ca
2+
/CaM-dependent protein kinase,
ZmCCaMK from etiolated maize coleoptiles [21,23]. We
have now identified an immuno-homologue of this kinase in
pea (PsCCaMK) and in this study we show that PsCCaMK
is involved in Ca
2+
/CaM-regulated stress signaling in
plants. PsCCaMK is tightly associated with its substrate,
a 40-kDa protein (p40) that binds to specific sequence
elements in the promoter of the Arabidopsis CaM5 gene
(AtCaM5) gene in a phosphorylation/dephosphorylation-
dependent manner.
EXPERIMENTAL PROCEDURES

Plant material, growth conditions and stress
treatments
Pea (Pisum sativum) plants were grown in moist vermiculite
(16-h light/8-h dark) at 25 °C for 5 days. For salinity stress,
5-day-old plants were treated in 50, 100, 200, 300 or 500 m
M
NaCl solution for specified time periods. For osmotic stress,
plants were similarly treated with 300 m
M
mannitol for 24,
48 or 96 h. For temperature stress, 5-day-old plants were
transferred either to 4 °C (cold stress) or 40 °C (heat stress)
for specified time periods. Calcium treatment (10, 25, 50 or
100 m
M
) was given to the 5-day-old plants for 3, 6, 12 or
24 h. Plants grown under normal conditions for the same
time period served as controls.
For pharmacological experiments, 5-day-old plants were
treated with EGTA (10 m
M
), lanthanum (10 m
M
)orW7
(60 l
M
) for 12 h. These plants were further subjected to
different stresses as described earlier. Plants treated with
these compounds but without stress conditions served as
control for these experiments. After the treatments roots

and shoots of the treated as well as untreated (control)
plants were harvested separately, frozen in liquid N
2
and
stored at )80 °C until use.
Protein extraction, SDS/PAGE and immunoblotting
Frozen tissue was ground to a fine powder in liquid N
2
and
extracted with 3 vols extraction buffer [20 m
M
Hepes
pH 7.5, 2 m
M
EDTA, 5 m
M
EGTA, 2 m
M
phenyl-
methanesulfonyl fluoride, 5 m
M
dithiothreitol and 10%
glycerol (v/v)] on ice. The slurry obtained was centrifuged at
12 000 g for 30 min and the supernatant was used for SDS/
PAGE and immunoblotting studies. Protein estimation was
carried out according to Bradford [24] and equal amounts
of proteins were resolved on SDS/PAGE according to
Laemmli [25]. Gels were run in duplicate; one part was used
for staining with Coomassie brilliant blue R250 to ascertain
equal loading of proteins, and the second part was

electrophoretically transferred to nitrocellulose membrane.
Equal amount of the proteins on blots was further
confirmed by staining with Ponceau S (Sigma Chemical
Co.). The blots were probed with antibodies raised in rabbit
against purified ZmCCaMK [21] at 1 : 25000 dilution. Goat
anti-(rabbit IgG) Ig conjugated with alkaline phosphatase
(Sigma Chemical Co.) was used as secondary antibody and
the antigen–antibody complex was visualized by the reac-
tion of 5-bromo-4-chloro-3-indocyl phosphate/nitroblue
tetrazolium (Sigma Chemical Co.) as described by Harlow
& Lane [26].
Protein purification using immuno-affinity
chromatography
For purification of the pea immuno-homologue of ZmC-
CaMK, 5-day-old pea plants were treated overnight with
CaCl
2
(100 m
M
); roots were harvested and washed exten-
sively with water. Further steps were performed at 4 °C
unless stated otherwise. Tissue was ground in presence of
liquid N
2
, extracted with 3 vol. extraction buffer as
described earlier and centrifuged at 15000 g for 45 min.
The crude protein extract obtained was precipitated with
0–40%, 40–50% and 50–80% ammonium sulfate and
dialyzed extensively against 50 vols extraction buffer. To
detect the presence of the ZmCCaMK homologue, all the

three fractions were immunoblotted with anti-ZmCCaMK
Ig. The 40–50% ammonium sulfate precipitated proteins
containing the kinase homologue were utilized for immuno-
affinity chromatography.
Protein A sepharose purified anti-ZmCCaMK Ig were
linked to CNBr-activated Sepharose 6B (Pharmacia Bio-
tech) according to the manufacturer’s instructions and the
matrix was packed in a 4-mL column. The column was
equilibrated with buffer containing 50 m
M
Tris pH 7.5,
10 m
M
MgSO
4
,1m
M
phenylmethanesulfonyl fluoride,
1m
M
dithiothreitol, 0.1% Triton-X 100, 10% glycerol (v/v).
The proteins were bound to the column by recirculating
twice through it. The bound proteins were washed exten-
sively with the same buffer containing 50 m
M
NaCl until the
A
280
of the column flow-through reached zero. Specifically
bound proteins were eluted either with a salt gradient of

0.05–1
M
in the presence of 50 m
M
Tris pH 7.5, 10 m
M
MgSO
4,
1m
M
phenylmethanesulfonyl fluoride, 1 m
M
dithiothreitol, 10 m
M
2-mercaptoethanol, 0.5% Triton-X
100 and 10% (v/v) glycerol or with 2.9 pH glycine buffer
[26]. When low pH glycine buffer was used for elution, the
samples were neutralized immediately using a calibrated
amount of Tris pH 7.5.
In vitro
phosphorylation assays
In vitro phosphorylation assays were performed as described
earlier [21]. Briefly, protein eluted from the affinity column
(1 lg) was incubated in phosphorylation buffer (30 m
M
Hepes pH 7.5, 5 m
M
MgCl
2
,0.5m

M
dithiothreitol, 25 m
M
NaCl) in the absence (control) or presence of 100 l
M
calcium and 110 n
M
CaM. The reaction was started by
the addition of 100 l
M
[c-
32
P]ATP (Amersham Biosciences
Corp.) to the reaction mix in a total volume of 50 lLand
incubated at 30 °C for 5 min. An equal volume of SDS
sample buffer was added to stop the reaction. The reaction
mix was boiled at 100 °C for 5 min and resolved by SDS/
PAGE. The gel was dried and exposed for autoradiography.
To test the effect of KN-62, 50 l
M
of the compound was
also included in the reaction mix. To determine if anti-
ZmCCaMK Ig blocked the phosphorylation reaction, the
protein fraction was first incubated with 1 lg of antibodies
at room temperature for 1 h with shaking. The antigen–
3194 S. Pandey et al. (Eur. J. Biochem. 269) Ó FEBS 2002
antibody complex was then precipitated out using protein A
sepharose beads and the resulting supernatant was used for
phosphorylation assay.
RT-PCR

Approximately 0.5 g plant tissue (control and stress treated)
was ground in liquid nitrogen and total RNA was extracted
with TRIzol reagent (Life Technologies) according to the
manufacturer’s protocol. Total RNA concentration was
determined by UV absorbance at 260 nm. For each sample
5 lg total RNA was reverse transcribed with an oligo dT
primer and Superscript II (Life Technologies) according to
manufacturer’s instructions. One ll of reaction product was
used as template in PCR reaction with AtCaM5 primers
(forward primer: 5¢-GATGTTGATGGTGATGGTCA-3¢;
reverse primer: 5¢-AAACCAGCCATGAATGAAAT-3¢)
and with actin primers (forward primer: 5¢-GTTGGGAT
GAACCAGAAGGA-3¢; reverse primer: 5¢-GAACCA
CCGATCCAGACACT-3¢) as a control. Reactions with
no DNA added served as a negative control. The PCR
cycling profile was: denaturation at 92 °C for 30 s, annealing
at 58 °C for 1 min and extension at 72 °Cfor1.5minfor25
cycles. PCR products were analyzed on 1% agarose gels.
Preparation of nuclear protein extract, heparin–agarose
chromatography and gel mobility shift analysis
Pea nuclei were isolated as described previously [27] and
purified on a discontinuous percoll gradient. For prepar-
ation of nuclear protein extract, the nuclei were washed with
buffer containing 50 m
M
Tris pH 7.8, 5 m
M
MgCl
2
,1m

M
dithiothreitol, 20% glycerol (v/v) and collected by gentle
centrifugation. Nuclei were resuspended in washing buffer
containing 110 m
M
KCl, 10 m
M
phenylmethanesulfonyl
fluoride, and 5 lgÆmL
)1
each of antipain and leupeptin and
the suspension was brought to 40% ammonium sulfate
saturation. The suspension was centrifuged at 100 000 g for
1 h in a Beckman Ti 75 rotor. The supernatant obtained
was brought to 70% saturation and the nuclear proteins
were obtained by centrifugation at 100 000 g for 1 h. The
proteins were finally resuspended in the same buffer without
MgCl
2
and stored frozen in small aliquots at )80 °C.
A pre-packed 2.5-mL heparin–agarose column (Sigma
Chemical Co.) was equilibrated with 10 column vols of
binding buffer (50 m
M
Tris pH 7.2, 10 m
M
MgSO
4
,1m
M

dithiothreitol, 1 m
M
phenylmethanesulfonyl fluoride and
10% glycerol, v/v). Ten to 20 mg proteins were loaded on to
the column and washed with binding buffer containing
50 m
M
NaCl until the A
260
of the flow through reached
zero. The proteins specifically bound to the column were
eluted with a 0.05 to 1
M
NaCl gradient in binding buffer
and eluted proteins were tested for binding with the
AtCaM5 promoter fragment. Active fractions were pooled,
dialyzed and stored in small aliquots at )80 °C.
Gel mobility shift analysis (GMSA) were performed
according to Ausubel et al. [28] either with the labeled
AtCaM5 promoter fragment ()588 to )339) or with specific
oligonucleotides designed from the same promoter frag-
ment. The sequences of the oligonucleotides used for these
experiments are: Oligo I, 5¢-CAAGGACGTTCGATGCA
CTTCCAAAAAACATATAAT-3¢; Oligo II, 5¢-CAAT
GTAGTATTAAAAAGTAGTAGTTAAAAGC-3¢; Oligo
III, 5¢-GTTTTTATCCGATGCAAATTTTTGCTTTGT
GATTG-3¢.
The reaction was performed in 20 lL DNA-binding
buffer containing (50 m
M

Tris pH 7.4, 50 m
M
KCl, 1 m
M
dithiothreitol, 6% glycerol, v/v) supplemented with
1 lg sonicated calf thymus DNA. Labeled probe
( 10 000 c.p.m.) was incubated with the required amount
of protein at room temperature for 10 min. DNA–protein
complexes formed were fractionated by 5% nondenaturing
PAGE and autoradiographed. To abolish any protein–
protein interaction, 0.5% deoxycholate (Sigma Chemical
Co) was incubated in the reaction mixture. For supershift
analysis, the required dilution of anti-ZmCCaMK Ig was
included in the reaction mixture with or without deoxy-
cholate. Competition analyses were performed by including
1000 · concentration of self or nonself oligonucleotides in
the reaction mix. To test the affect of phosphorylation on
binding of p40 with DNA, p40 was phosphorylated using
cold ATP as described above and used for assays. An
identical experiment performed with labeled ATP was run
on a gel to verify the phosphorylation and to confirm the
integrity of protein (data not shown). For dephosphoryla-
tion 10 lg of phosphorylated protein was incubated in
buffer containing 50 m
M
Hepes pH 7.5, 1 m
M
MgCl
2
,

0.5 m
M
dithiothreitol and calf intestinal phosphatase (10 U)
in a total volume of 50 lL, at 30 °C for 10 min. Reactions
were stopped by the addition 5 lL100m
M
sodium
pyrophosphate. Protein was precipitated using ice-cold
acetone and resuspended in DNA-binding buffer to study
DNA–protein interaction.
For South-Western analysis, proteins separated on 10%
SDS/PAGE were electro-blotted on nitrocellulose mem-
brane. The membrane was blocked with the binding buffer
(described above) containing 3% BSA at room temperature
with gentle shaking. The membrane was washed twice with
same buffer containing 0.25% BSA. Hybridization was
carried out in the presence of 50 lgÆmL
)1
sonicated calf
thymus DNA and labeled probe at room temperature for
1 h. The membrane was washed, dried and exposed for
autoradiography.
RESULTS
Immuno-homologue of maize ZmCCaMK is upregulated
by low temperature and salinity stresses in pea roots
Western blot analysis using anti-ZmCCaMK Ig of total
protein extracts from pea shoots and roots showed the
presence immuno-homologue of maize kinase in pea
(PsCCaMK). PsCCaMK showed a development-depend-
ent and tissue-specific expression (S. Pandey & S. K.

Sopory, unpublished data). The level of PsCCaMK was
very low in roots as compared to the shoots (Fig. 1,
control lanes). As some protein kinases are involved in
stress signaling pathways [29] and recent work points
towards the involvement of Ca
2+
/CaM-dependent protein
kinases in stress signaling [16], changes in the level of
PsCCaMK was evaluated under various stress conditions.
Five-day-old pea seedlings were subjected to temperature,
salinity and osmotic stress (as described in Experimental
procedures) and the level of PsCCaMK was monitored by
Western blotting in both roots and shoots using anti-
ZmCCaMK Ig. It was seen that PsCCaMK level remained
Ó FEBS 2002 Role of PsCCaMK in stress signaling (Eur. J. Biochem. 269) 3195
unchanged under all the conditions tested in shoots
(Fig. 1). However, a strong upregulation of the protein
level was observed in roots when salt (0.3
M
NaCl) or low
temperature (4 °C) treatment was given to the plants.
Osmotic stress or heat shock had no effect on the level of
PsCCaMK suggesting that this kinase is not a general
stress-regulated kinase but may specifically be involved in a
signaling pathway associated with salinity and low tem-
perature stress.
As both NaCl and low temperature upregulated the
kinase level, time-kinetics experiments were performed for
these stress treatments. The optimum concentration of
NaCl required to upregulate the kinase level was also

determined. As shown in Fig. 2, the level of PsCCaMK
started increasing in response to 50 m
M
NaCl, reached
maxima at 300 m
M
and then remained constant up to
500 m
M
NaCl. The kinase level started increasing after 6 h
of treatment of plants with NaCl as well as low temperature
and the maximum level was observed following 24 h of
treatment.
Calcium upregulates the PsCCaMK level in a time-
and concentration-dependent manner
Because PsCCaMK showed a strong upregulation in
response to low temperature and salinity stress in pea roots
under in vivo conditions, and the signaling pathways for
both of these stresses are often mediated by Ca
2+
,theeffect
of exogenous Ca
2+
was analyzed on the protein level of
PsCCaMK. As shown in Fig. 3, the level of the kinase was
strongly upregulated by Ca
2+
. The kinase level started
increasing at 10–25 m
M

exogenous Ca
2+
and the maximum
level was observed at 100 m
M
. The time-kinetics data
showed that the appearance of PsCCaMK after Ca
2+
Fig. 2. Western blots showing the kinetics of induction of PsCCaMK
following NaCl and cold treatment. Five-day-old pea plants were given
NaCl or cold treatment for indicated time periods and concentrations.
Roots were harvested and immediately frozen. Twenty-five lgtotal
protein extracts were separated by SDS/PAGE, and Western blotting
was performed using anti-ZmCCaMK Ig. Equal loading of proteins
per lane was confirmed by Ponceau S staining of the Western blot.
Lane C denotes protein isolated from control plants that were not
given any stress treatment.
Fig. 1. Western blot analysis of level of PsCCaMK in response to dif-
ferent stresses in roots and shoots of pea. Five-day-old pea plants were
given heat stress (42 °C), low temperature stress (4 °C), salt stress
(0.3
M
NaCl) and osmotic stress (0.3
M
mannitol) for 24 h and roots
and shoots were harvested separately. Extracted proteins were separ-
ated by SDS/PAGE (25 lg per lane) and probed with anti-ZmC-
CaMK Ig. Roots and shoots from normal vermiculite-grown plants
served as controls. Numbers on the left indicate molecular weight
markers in kDa.

Fig. 3. Effect of exogenous calcium on the level of PsCCaMK. Calcium
treatment was given to the 5-day-old pea plants for the indicated time
periods and concentration and roots were harvested. Total protein
extracted (25 lg per lane) was Western blotted and probed with anti-
ZmCCaMK Ig. Mg denotes plants treated with 50 m
M
MgCl
2
instead
of CaCl
2
. Lane C denotes protein isolated from control plants not
given any calcium treatment.
3196 S. Pandey et al. (Eur. J. Biochem. 269) Ó FEBS 2002
treatment was earlier (at 3 h) than that obtained following
NaCl and low temperature stress treatment (at 6 h). To
exclude the possibility of this upregulation being mediated
via a divalent cation in general, the effect of Mg
2+
was also
tested. Plants treated with 50 m
M
Mg
2+
for 24 h did not
show any upregulation of the PsCCaMK level.
Low temperature, but not salinity-stimulated kinase
level is mediated via a Ca
2+
/CaM pathway

To further confirm that the upregulation of PsCCaMK by
NaCl and low temperature is mediated via a Ca
2+
/CaM
signaling pathway, the plants were pretreated with EGTA
(10 m
M
), lanthanum (10 m
M
)orW7(60l
M
), before
exposure to low temperature, NaCl or Ca
2+
.Plants
treated with these compounds but not given any further
stress treatments served as controls along with normal
vermiculite-grown plants. The Western blot analysis
showed that EGTA as well as W7 almost completely
blocked low temperature- and Ca
2+
-induced upregulation
of the kinase but that the NaCl-stimulated kinase level
was unaffected by these treatments (Fig. 4). These results
suggest that the low temperature-induced response may be
mediated by Ca
2+
/CaM whereas the salt-induced upreg-
ulation might be mediated via some other pathway.
Lanthanum had no effect on the expression level of the

kinase.
Purification of PsCCaMK by immuno-affinity
chromatography: a 40-kDa protein always coelutes
To purify PsCCaMK, 5-day-old pea plants were treated
overnight with 100 m
M
Ca
2+
and then roots were excised
for purification of protein. The total soluble protein extract
was fractionated with 40–50% ammonium sulfate and
loaded on to an immuno-affinity column prepared using
anti-ZmCCaMK Ig. The protein was bound in the
presence of 50 m
M
NaCl and eluted either using a salt
gradient of 0.05–1
M
containing 0.5% TritonX-100 and
10 m
M
2-mercaptoethanol or with low pH glycine buffer
(pH 2.9). Under both of these sets of conditions a 40-kDa
protein eluted first from the column followed by elution of
the 72-kDa protein corresponding to the PsCCaMK
(Fig. 5). Under all elution conditions tested, the 72-kDa
protein could not be eluted independently of the 40-kDa
protein. Western blot analysis of the eluted fractions with
the same antibodies that were used for making the
immuno-affinity column showed cross-reactivity with

the 72-kDa PsCCaMK protein only. This suggests that
the 40-kDa protein does not bind to the antibodies on the
column directly but possibly it is very tightly associated
with PsCCaMK.
PsCCaMK is present in nuclear protein extracts
and possibly interacts with DNA
A number of CaM kinases from animal systems have been
reported to be present in nuclei and to regulate gene
expression [30]. Our studies with the AtCaM5 promoter,
which is induced under different stress conditions (S. B.
Tiwari & K. C. Upadhyaya, unpublished data), gave
indications that the anti-ZmCCaMK Ig affected binding of
the AtCaM5 promoter with specific proteins eluted from
Fig. 4. Effect of various pharmacological compounds on the expression
level of PsCCaMK. Five-day-old pea plants were pretreated with
EGTA (10 m
M
), lanthanum (La, 10 m
M
)orW7(60l
M
). Pre-treated
plants were given low temperature (4 °C),NaCl(0.3
M
)orcalcium
(100 m
M
) treatment for 24 h. Pre-treated plants, not given further
treatment, as well as normal vermiculite-grown plants (C) served as
different controls. Twenty-five lg total proteins extracted from roots

were separated by SDS/PAGE, Western blotted and probed with anti-
ZmCCaMK Ig.
Fig. 5. Purification of PsCCaMK from immuno-affinity columns.
Roots of 5-day-old plants treated with calcium (100 m
M
)for24 hwere
used for the purification of kinase using the immuno-affinity column
prepared using anti-ZmCCaMK Ig. Elution was with a 0.05–1
M
NaCl
gradient. The left panel shows the SDS/PAGE profile of proteins from
fractions 7, 9 and 11 after silver staining. The right panel shows the
Western blot of the same fractions using anti-ZmCCaMK Ig.
Ó FEBS 2002 Role of PsCCaMK in stress signaling (Eur. J. Biochem. 269) 3197
the heparin–agarose column. Moreover, expression of
AtCaM5 gene is strongly upregulated in response to
identical conditions of low temperature and salinity stress
as analyzed by RT-PCR (Fig. 6A). Taking clues from these
observations we tested for the presence of PsCCaMK in
nuclear protein fractions as well as in the proteins
fractionated on the heparin–agarose column. As shown in
Fig. 6B, the antibodies cross-reacted with a 72-kDa protein
in the total nuclear protein fractions indicating its possible
nuclear localization. The antibodies also cross-reacted with
the nuclear proteins fractionated on the heparin–agarose
column. The 72-kDa kinase band could be detected
specifically in the 0.2–0.4
M
salt-eluted protein fractions.
The same fractions also showed binding to the AtCaM5

promoter under different physiological conditions (unpub-
lished data). As the salt concentration used was high
enough not to let nonspecific proteins interact with the
heparin column, it was predicted that this protein might be
interacting with DNA directly. To confirm this observa-
tion, we performed GMSA of total pea nuclear protein
extract with a 249-bp labeled AtCaM5 promoter region
()588 to )339) in the presence and absence of anti-
ZmCCaMK Ig. This particular region was selected as it
showed maximum protection and structural changes when
foot-printing analysis was performed (S. B. Tiwari & K. C.
Upadhyaya, unpublished data). As shown in Fig. 6B, a
strong DNA–protein complex was formed with the
AtCaM5 promoter fragment and total pea nuclear protein
extract, which showed a supershift with the anti-ZmC-
CaMK Ig. Addition of 0.5% deoxycholate to abolish any
ionic protein–protein interactions showed the presence of
two loose complexes. Addition of antibodies along with
deoxycholate also showed a supershift, giving further
indications that the kinase interacts with DNA either
directly or it is strongly associated with some DNA binding
protein. Antibodies alone showed no interaction with
DNA. We could not determine the direct binding of the
purified kinase with DNA as we could not obtain the
kinase preparation without p40 under any conditions and
attempts to purify the kinase by gel elution gave a very low
yield and the eluted protein was highly labile.
The 40-kDa protein is phosphorylated by PsCCaMK
and binds directly with the
AtCaM5

promoter
As p40 remains tightly bound to the PsCCaMK during
the purification process, the possibility of it being a
substrate for PsCCaMK was tested. Further it was also
examined if p40 binds with the specific regions of AtCaM5
promoter. To ascertain these facts, proteins eluted from
the immuno-affinity column were pooled in two different
fractions, one containing pure p40 (fraction A) and other
containing both the p72 and p40 (fraction B). Both of
these fractions were used for in vitro phosphorylation
experiments. As shown in Fig. 7A, no phosphorylation
could be detected in fraction A under any of the
Fig. 6. RT-PCR, Western blot and supershift analyses. (A) RT-PCR of AtCaM5 gene. RNA isolated from roots of control, low temperature (4 °C)
and salinity (0.3
M
NaCl) stressed plants was reverse transcribed and amplified with AtCaM5 gene-specific primers. PCR with actin primers is
included as control. (B) Western blot analysis of pea nuclear extracts. Five lg pea nuclear proteins (NP) and 2 lg pea nuclear proteins fractionated
on heparin–agarose column (HAFr) were separated by SDS/PAGE in duplicate. One set was silver stained while the other set was Western blotted
and probed with anti-ZmCCaMK Ig. (C) Interaction of pea nuclear proteins with AtCaM5 promoter and supershift analysis. The nuclear proteins
were used in GMSAs to analyze their interaction with AtCaM5 promoter fragment. Analysis was also performed in the presence or absence of 0.5%
deoxycholate and anti-ZmCCaMK Ig to determine supershift. Antibodies (Abs) alone were included as control.
3198 S. Pandey et al. (Eur. J. Biochem. 269) Ó FEBS 2002
conditions tested, but a Ca
2+
-dependent, CaM-stimulated
phosphorylation of p40 could be seen in fraction B. This
phosphorylation could be specifically blocked by KN-62, a
Ca
2+
/CaM kinase inhibitor as well as with anti-ZmC-

CaMK Ig. Addition of 50 ng fraction B proteins into
fraction A led to phosphorylation of p40 in fraction A
also. On longer exposure of the blots a faint signal could
be detected at the 72-kDa position, corresponding to the
autophosphorylated PsCCaMK in fraction B, but no such
signal was observed with fraction A (data not shown),
confirming that p40 has no phosphorylation activity of its
own. These results clearly established that the PsCCaMK
has Ca
2+
/CaM kinase activity and uses p40 as its in vitro
substrate.
To ensure the DNA-binding property of p40, both
fractions A and B were used for South-Western analysis
with the AtCaM5 promoter fragment ()588 to )339) that
was used earlier for GMSA. A strong signal was
observed at the 40-kDa position with both the fractions,
showing that p40 binds to this promoter fragment
(Fig. 7B). To confirm this observation GMSA was
performed with the AtCaM5 promoter fragment and
fraction B, in the presence of excess of calf thymus DNA.
As shown in Fig. 7C two specific DNA–protein com-
plexes were formed. These data showed that p40 binds
directly to the AtCaM5 promoter, but whether PsC-
CaMK binds with DNA directly or through p40 remains
inconclusive.
p40 binds to the specific
cis
-elements in the AtCaM5
promoter region and the binding is affected

by phosphorylation
To further analyze the specific binding of p40 to DNA,
GMSAs were performed with fraction A (pure p40) and
three specific oligonucleotides (see Experimental proce-
dures). These oligonucleotides were designed based on the
protected regions of the AtCaM5 promoter fragment ()588
to )339) in the foot-printing experiments (data not shown).
p40 showed binding with two of the sequences, Oligo I and
Oligo III but not with Oligo II (data for Oligos I and II are
shown in Fig. 8). The specificity of the binding was further
confirmed by competition assays where excess concentra-
tions (1000 ·) of the self-oligonucleotides as well as nonself
oligonucleotides were used. As shown in the autoradiogram
(Fig. 8), the DNA–protein complex could not be detected
when the self-oligonucleotides were used at higher concen-
tration,whereasnosucheffectcouldbeseenwiththe
nonself oligonucletides.
As p40 was found to be an in vitro substrate for the
PsCCaMK, the effect of phosphorylation on its DNA-
binding property was tested. The protein was in vitro
phosphorylated using cold ATP and used for DNA binding
studies. No binding was detected when prephosphorylated
protein was used for the DNA binding reaction. To confirm
the reversibility of the phosphorylation reaction and the
associated DNA-binding activity, phosphorylated p40 was
Fig. 7. In vitro protein phosphorylation, South-Western and DNA GMSA with pea nuclear proteins fractionated by immuno-affinity chromatography.
(A) Protein fractions eluted from kinase antibody affinity column containing 1 lg purified p40 (fraction A) and those containing 1 lgofbothp40
and p72 PsCCaMK kinase (fraction B) were used for in vitro phosphorylation in the absence or presence of Ca
2+
, CaM, kinase antibodies and KN-

62. In one set 50 ng fraction B was added to fraction A. (B) Proteins from fraction A and B were separated by PAGE and probed with labeled
AtCaM5 promoter fragment in South-Western analysis. (C) GMSA was performed in the absence (control) or presence of fraction B proteins with
labeled AtCaM5 promoter fragment.
Ó FEBS 2002 Role of PsCCaMK in stress signaling (Eur. J. Biochem. 269) 3199
treated with calf intestinal phosphatase. A DNA–protein
complex could be detected using the dephosphorylated
protein, confirming that p40 could bind to the AtCaM5
promoter only in the dephosphorylated form.
DISCUSSION
Role of PsCCaMK in stress signaling
Protein kinases and phosphatases are important compo-
nents of signaling cascades, which by changing the
phosphorylation status of the target proteins transduce
the signal to elicit the final response [31]. A number of
studies in recent years have linked the stress signaling with
changes in the calcium level and the upregulation of
various protein kinase transcripts [1–3,32–36]. A receptor
protein kinase RPK1 is regulated in response to multiple
stresses and probably has a role at the very beginning of
multiple stress signaling pathways [37]. Involvement
of CDPKs in stress signaling has also been shown by
using chimeric gene constructs containing abscisic-acid-
responsive elements and the GFP reporter gene [29]. In this
system, transient expression of CDPKs could be observed
in response to various stresses as well as exogenous
calcium. A number of kinases of the mitogen activated
protein signaling cascade have also been reported to be
involved in stress signaling pathways [38].
Upregulation of protein kinase transcripts is a complex
process, as multiple signals (stress, light as well as phyto-

hormones) affect the level of same kinase and in turn
different kinases are modulated by the same signal,
depending on the specificity of calcium signal and position
of the kinase in the signaling cascade. Though the role of
some protein kinases is established in the stress signaling
pathways, the exact sequence of events that leads to the final
gene expression in response to a particular signal is not very
well elucidated.
We have reported earlier the purification and character-
ization of ZmCCaMK and the presence of its immuno-
homologues in a variety of other plants [21] including
Arabidopsis (data not shown). We now show that the kinase
homologue from pea is involved in stress signaling. Con-
ditions such as 0.3
M
NaCl and 4 °C are widely reported to
cause severe stress to plants. Under these conditions, pea
plants showed visible effect of stress in both roots and
shoots. However, the upregulation of kinase level was
observed in roots only. Shoots had higher kinase levels
compared to roots to begin with, and it was not altered by
stress treatment. This is similar to the Nicotiana tobacum
CDPKl transcript that could not be detected in leaves of the
normal plants, but shows a strong upregulation in response
to different stresses [39].
The kinase level also increases in response to exogenous
Ca
2+
and even though a maximum increase in the kinase
Fig. 8. GMSA with p40 with specific cis elements (oligonucleotides) of AtCaM5 promoter. Oligonucleotides I and II representing specific cis-regions

of the AtCaM5 promoter were used to study the specific interaction of p40. Proteins from fraction A (see Fig. 7) interacted specifically with Oligo I
(A) but not with Oligo II (B). For competition a 1000 · excess of self or nonself oligonucleotides were included in the reaction mix. Addition of ATP
abolished the DNA–protein complex formation with Oligo I. The binding with Oligo I was also studied following phosphorylation/dephospho-
rylation of p40 (C).
3200 S. Pandey et al. (Eur. J. Biochem. 269) Ó FEBS 2002
level was obtained with 100 m
M
Ca
2+
, stimulatory effects
are seen at much lower concentrations (10–25 m
M
) as well.
As these treatments were given to whole plants (not to
isolated protoplasts or proteins) for very short duration, the
actual Ca
2+
uptake would be much lower. Moreover, the
low temperature- and Ca
2+
-induced increase in kinase level
could be blocked by EGTA and W7 (a CaM inhibitor) and
the high Mg
2+
treatment given for 24 h has no effect on the
kinase level; it is therefore very likely that Ca
2+
is acting as a
signal molecule. However, the possibility that at such high
concentrations Ca

2+
might be causing some pleiotropic
effects to cell physiology cannot be ruled out totally.
It has been shown earlier that both salt- and low
temperature stress-mediated signaling pathways are modu-
lated via Ca
2+
[2,40–42]. In most of the cases the same
protein is affected in response to both of these stresses, but
proteins affected by low temperature but not by salt or
dehydration and vice versa are also known [34,43,44].
Recently, the role of a novel kinase SOS2 in salt tolerance
has been suggested [36,45]. Specific sequence elements have
been identified (e.g. DRE) that are important for cold and/
or dehydration responses [46]. In some cases different
proteins bind to the same sequence under different physio-
logical conditions to give specific responses [43,47,48].
PsCCaMK does not appear to be a general stress-
responsive kinase, as its expression does not change in
response to mannitol or heat shock. As dehydration or
osmotic shock are known to affect the level of proteins
regulated by salt stress, this kinase falls into a different
category. The response of PsCCaMK to NaCl is similar to
that of the SOS3 gene [42] and Ca
2+
appears to be involved
in its regulation by upregulating the protein level; also the
response is faster than that to either NaCl or to low
temperature treatment. The differential effects of EGTA
and W7 on low temperature- and salinity-mediated expres-

sion of PsCCaMK indicate that their responses are medi-
ated by different signaling pathways and that Ca
2+
/CaM
signaling is involved only in the upregulation of PsCCaMK
in response to low temperature. To ascertain whether this
kinase acts as a junction point of two different signaling
pathways needs further work.
Purification of PsCCaMK and its substrate protein
and their functional characterization
PsCCaMK is present in the nuclear protein fractions as well
as in the protein fractions eluted from the heparin–agarose
column and possibly interacts with the AtCaM5 promoter.
In animal systems, a number of CaM kinases have been
identified in the nuclei that are shown to affect the
expression of specific genes by changing the phosphoryla-
tion status of transcription factors [30,49]. It has been shown
that plant nuclear extracts could be phosphorylated [50],
showing the presence of kinase(s) in the nuclei. In addition,
different signaling pathways point towards the possibility of
Ca
2+
and CaM acting through the nucleus via specific CaM
kinases [16]. However, our study provides strong evidence
of the presence of a CaM kinase and its possible function in
plant nuclei.
It has been observed that during purification of PsC-
CaMK using an immuno-affinity column, p40 always
coelutes with the PsCCaMK, even under very stringent
conditions. Moreover, p40 does not cross-react with the

kinase antibodies used to make the column, showing that it
is not interacting directly with the antibodies linked with the
column but is possibly very tightly associated with the
PsCCaMK. There are other examples where substrate or
interacting protein has been eluted from the affinity column
along with the relevant protein [45]. In vitro phosphoryla-
tion experiments show that p40 could not be phosphoryl-
ated on its own, but only in the presence of PsCCaMK, in a
Ca
2+
-dependent, CaM-stimulated manner. The phos-
phorylation of p40 could be blocked by KN-62 (a specific
CaM kinase inhibitor) as well as the anti-ZmCCaMK Ig
(Fig. 7). This shows that PsCCaMK properties are similar
to those of ZmCCaMK and p40 is one of its in vitro
substrates.
To check the interaction of these proteins with DNA, we
selected the promoter of the Arabidopsis CaM5 gene
(AtCaM5). The reason to use this promoter was that the
AtCaM5 gene was strongly upregulated in response to
identical conditions of salinity and low temperature stress.
Besides, PsCCaMK was specifically present in the same
fractions of heparin–agarose eluted proteins that show
binding with the AtCaM5 gene promoter. We also had
preliminary data for this promoter, using GMSA and
DNase I foot-printing, about the regions of the promoter
showing structural changes under different physiological
conditions. Based on these studies we used the )588 to )399
fragment of this promoter (that showed maximum protec-
tion and changes in footprinting analysis) for our studies.

Detection of DNA–protein complexes by South-Western
analysis, using the labeled fragment and the affinity purified
protein fraction, shows the binding of the promoter with the
p40 protein and not with PsCCaMK (Fig. 7). The absence
of binding with PsCCaMK could be due either to the fact
that it does not bind to DNA at all, to a weak interaction, or
to a very much lower amount of protein that could not be
detected. However, GMSAs with the nuclear protein extract
and anti-ZmCCaMK Ig showed a supershift though weak,
even in presence of high concentration of deoxycholate,
indicating the possible interaction of this kinase directly with
the DNA (Fig. 6B). On the other hand, pure p40 protein
showed direct binding with the AtCaM5 gene promoter
fragment by GMSA. Binding experiments with specific
oligonucleotides confirm that the p40 interacts with defined
sequence elements of the promoter, and that the binding is
highly specific. The same oligonucleotides, which do not
show binding with p40 when tested for binding with total
pea nuclear extract, show a strong binding (data not shown)
which further confirms that p40 binds to some specific cis
sequences only. The question of whether PsCCaMK binds
directly or via its association with p40 has not been fully
resolved. During purification of the protein the association
of the protein kinase and its substrate was not affected even
though stringent conditions were used. It is possible
therefore, that these two proteins physically interact and
even in the presence of 0.5% deoxycholate this interaction
remains intact. In this case the supershift could be a result of
antibody interacting with the PsCCaMK bound to p40,
whichinturninteractsdirectlywithDNA.

Of the several strategies that modulate the binding of a
protein with its target DNA, to effect transcription,
phosphorylation is regarded as one of the major mecha-
nisms [51,52]. In plants too, a number of studies have shown
that phosphorylating the transcription factors affects their
Ó FEBS 2002 Role of PsCCaMK in stress signaling (Eur. J. Biochem. 269) 3201
binding with DNA [27,53–55]. We have found that the
binding of p40 is dependent on the dephosphorylation
status of the protein and the binding is fully abolished once
the protein is phosphorylated.
Analysis of the specific sequence elements, with which
the p40 interacts, shows that these elements are not yet
reported in any of the stress responsive genes. On
computational analysis, both of the sequences with which
p40 interacts show the presence of a binding site for high
mobility group proteins (HMG boxes). HMG box
proteins have been identified from many plant species
and it has been shown that their binding with DNA is
affected by the phosphorylation status of the proteins [56–
58]. In animal systems it has been shown conclusively that
these proteins usually interact with the AT-rich sequences
that show high structural changes [59–62], which is the
case with the oligonucleotides that we used. It could
therefore be speculated that p40 is a protein similar to
HMG box proteins; this, however, requires further char-
acterization.
Proposed mechanism of action
Based on all these results we propose that p40 is a negative
regulator of the CaM5 gene promoter that may act in a
similar way to the DREAM protein, a negative transcrip-

tional regulator that acts in a calcium-dependent manner
[63]. During normal growth and development, p40 is
bound to the promoter. When the plant is under salinity or
low temperature stress, the calcium level inside the cell
increases. This elevated calcium could then affect the level
and activity of the Ca
2+
/CaM-dependent protein kinase
homologue, which is either always present in the nuclei or
it gets translocated to the nuclei [55,64]. Once activated,
the kinase phosphorylates p40 and as a result its binding
to DNA is abolished and the protein is released from the
DNA. We also have preliminary data (not shown) which
show that some other protein of unknown identity binds
to the same sequence elements, after p40 dissociates from
it following phosphorylation. There are earlier reports
which show that different proteins, though unrelated, bind
to the same sequence elements, as in case of DREB1A and
DREB2A [48]. The present study thus proposes that
protein kinases that are upregulated in response to specific
stress function in the nuclei, might use DNA binding
proteins as substrate(s) and affect their binding property
thereby regulating the expression of stress-induced genes.
To be able to apply this statement in a broader perspec-
tive, it would be essential to look for genes for both
CCaMK and p40 to be able to functionally dissect the
underlying mechanistic pathways. It would also be
important to look for true Ôfunctional orthologuesÕ of the
kinase and p40 in other plant species, especially in
Arabidopsis where powerful genetic tools are available

for further studies.
ACKNOWLEDGEMENTS
This work was supported partly by internal grants from the
International Center for Genetic Engineering and Biotechnology and
funds from Department of Science and Technology, Government of
India. We thank Prof. S. Assmann for critical reading of the manuscript
and constructive suggestions and B. Yadav for technical help.
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