Autophosphorylation-dependentinactivationofplant chimeric
calcium/calmodulin-dependent protein kinase
P. V. Sathyanarayanan and B. W. Poovaiah
1
Laboratory ofPlant Molecular Biology and Physiology, Department of Horticulture, Washington State University,
Pullman, WA, USA
Chimeric calcium/calmodulin dependent protein kinase
(CCaMK) is characterized by the presence of a visinin-like
Ca
2+
-binding domain unlike other known calmodulin-
dependent kinases. Ca
2+
-Binding to the visinin-like domain
leads to autophosphorylation and changes in the affinity
for calmodulin [Sathyanarayanan P.V., Cremo C.R. &
Poovaiah B.W. (2000) J. Biol. Chem. 275, 30417–30422].
Here, we report that the Ca
2+
-stimulated autophosphory-
lation of CCaMK results in time-dependent loss of enzyme
activity. This time-dependent loss of activity or self-inacti-
vation due to autophosphorylation is also dependent on
reaction pH and ATP concentration. Inactivationof the
enzyme resulted in the formation of a sedimentable enzyme
due to self-association. Specifically, autophosphorylation in
thepresenceof200l
M
ATP at pH 7.5 resulted in the for-
mation of a sedimentable enzyme with a 33% loss in enzyme
activity. Under similar conditions at pH 6.5, the enzyme lost
67% of its activity and at pH 8.5, 84% enzyme activity was
lost. Furthermore, autophosphorylation at either acidic or
alkaline reaction pH lead to the formation of a sedimentable
enzyme. Transmission electron microscopic studies on
autophosphorylated kinase revealed particles that clustered
into branched complexes. The autophosphorylation of wild-
type kinase in the presence of AMP-PNP (an unhydrolyz-
able ATP analog) or the autophosphorylation-site mutant,
T267A, did not show formation of branched complexes
under the electron microscope. Autophosphorylation-
dependent self-inactivation may be a mechanism of modu-
lating the signal transduction pathway mediated by
CCaMK.
Keywords: self-inactivation; self association; autophospho-
rylation; Ca
2+
; chimeric calcium/calmodulin dependent
protein kinase.
Ca
2+
regulates a large number of physiological and
developmental processes [1]. The effects of Ca
2+
are so
wide spread that it becomes difficult to pinpoint specific
mechanisms of Ca
2+
signal transduction. Ca
2+
signaling is
orchestrated through several calcium binding proteins such
as calmodulin, ion channels, Ca
2+
-dependent protein
kinases and Ca
2+
/calmodulin dependent protein kinases
[2,3]. A large number ofplant Ca
2+
-dependent protein
kinases (CDPK) have been reported [4–7]. These kinases
require Ca
2+
for autophosphorylation and substrate phos-
phorylation [4–7]. However, there is only limited informa-
tion available about the Ca
2+
/CaM-dependent protein
kinases in plants [7,8].
Chimeric calcium calmodulin dependent protein kinase
(CCaMK) has been cloned from lily anthers [9]. CCaMK is
stage-specifically expressed in tapetal cells and pollen
mother cells of anthers during male gametophyte develop-
ment [10]. CCaMK is characterized by a serine-threonine
kinase domain, an autoinhibitory domain overlapping with
calmodulin binding domain and a C-terminal visinin-like
domain with three calcium-binding sites [9]. Visinin-like
proteins are high affinity Ca
2+
-binding proteins and
function as Ca
2+
sensors in neurons [30–33]
2
. The calmod-
ulin-binding domain of CCaMK is very similar to CaM
kinase II [11,12]. Ca
2+
binding to the C-terminal visinin-like
domain leads to autophosphorylation of the kinase [8,12].
Unlike CDPKs, the substrate phosphorylation by CCaMK
requires both Ca
2+
and CaM [7,8,11]. The interaction
between the CCaMK and CaM is modulated by the Ca
2+
-
stimulated autophosphorylation [8]. CaM-dependent pro-
tein kinases reported from invertebrates and vertebrates
require Ca
2+
/CaM for autophosphorylation [13].
In the present study, we report a new property of CCaMK
associated with Ca
2+
-stimulated autophosphorylation.
Ca
2+
-stimulated autophosphorylation of CCaMK resulted
in a time-dependent loss ofkinase activity. This property,
described as self-inactivation, is sensitive to reaction pH and
ATP concentration. Furthermore, the autophosphorylation-
dependent inactivation leads to the formation of a sediment-
able enzyme. When observed under transmission electron
microscope, the autophosphorylated kinase appeared as
particles that are clustered into branched complexes.
EXPERIMENTAL PROCEDURES
Materials
AMP-PNP and ATP were purchased from Sigma Chemical
Co. and [c-
32
P]ATP (3000 CiÆmmol
)1
) from Dupont Corp.
Correspondence to B. W. Poovaiah, Laboratory ofPlant Molecular
Biology and Physiology, Department of Horticulture,
Washington State University, Pullman, WA 99164-6414, USA.
Fax: + 1 509 335 8690, Tel.: + 1 509 335 2487,
E-mail: poovaiah@wsu.edu
Abbreviations:CaM,calmodulin;CCaMK,chimericcalcium/
calmodulin-dependent protein kinase; TEM, transmission electron
microscopy; AMP-PNP, adenosine 5¢-(b,-imido)triphosphate;
CaMK II, calcium/calmodulin-dependentproteinkinase II.
(Received 24 October 2001, revised 20 March 2002,
accepted 22 March 2002)
Eur. J. Biochem. 269, 2457–2463 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02904.x
Tabbed-copper grids (400 mesh) and polystyrene sizing
beads (93 and 262 nm) were obtained from Ted Pella
(Redding, CA, USA)
3
.
Purification of CCaMK
CCaMK cDNA from Lily (Lilium longiflorum Thunb cv.
Nellie white) and phosphorylation mutant T267A were
cloned into the pET3b expression vector (Novagen. Inc.)
and expressed in E. coli. These proteins were purified as
described previously [11,12].
Autophosphorylation assays
The autophosphorylation assay of CCaMK and mutant
protein were carried out [11] for 10 min at 30 °Cinthe
presence of 50 m
M
Hepes, pH 7.5, containing 10 m
M
magnesium acetate, 1 m
M
dithiothreitol, 1 m
M
[c-
32
P]ATP and either 2.5 m
M
EGTA or 0.2 m
M
CaCl
2
.
For the autophosphorylation under different pH condi-
tions, 50 m
M
Hepes, at pH 6.5 and 8.5, were used under
the same conditions as described above. Aliquots (10 lL,
200 ng enzyme) were collected at 0, 2, 6 min and diluted
to 100 lLoficecold50m
M
Hepes, pH 7.5 with 10 m
M
EDTA. This terminated further autophosphorylation of
the enzyme. Samples were then centrifuged for 30 min at
16 000 g at 4 °C. The supernatant and pellet were
separated and suspended in SDS loading buffer (10%
glycerol, 15 m
M
dithiothreitol, 2.3% SDS and 62.5 m
M
Tris, pH 6.8) for SDS/PAGE analysis [14]. [
32
P]PO
4
incorporation was measured by excising the protein
bands from the gel and counting using a scintillation
counter.
Kinase assays
The autophosphorylation-induced changes in the kinase
activity was studied using a second stage assay. An
aliquot of autophosphorylated enzyme (2.5 lL, 100 ng of
enzyme) from the first stage reaction was added to the
second stage reaction mix (final volume, 20 lL) consisting
of 50 m
M
Hepes, pH 7.5, containing 10 m
M
magnesium
acetate, 1 m
M
dithiothreitol, 1 m
M
[c-
32
P]ATP, 0.2 m
M
CaCl
2
,0.5l
M
calmodulin, 100 l
M
Histone II AS. The
second stage reaction mix was preincubated at 30 °C
before the addition of enzyme. The substrate phosphory-
lation was allowed to proceed for 10 min. The reaction
was stopped by the addition of SDS loading buffer. The
samples were analyzed on a 12% SDS/polyacrylamide gel
and
32
PO
4
incorporation was measured by excising
protein bands from the gel and counting using a
scintillation counter.
Transmission electron microscopy
All the solutions used for transmission electron microscopy
were filtered using 0.2-lm filters to remove any impurities.
CCaMK was autophosphorylated as described above and
the reaction was terminated by the addition of EDTA (to a
final concentration of 50 m
M
) and the sample was kept in
ice. The autophosphorylated and unphosphorylated kinase
samples were deposited onto the carbon coated Formvar
grids by the floating method, as described previously [15].
Drops (50 lL) of the kinase sample and other solutions
were placed on Parafilm stretched over a top of a Petri
dish. A grid was placed on a drop of the autophospho-
rylated kinase for 45 s and then placed on a drop of uranyl
acetate stain (2% uranyl acetate in 25% methanol) for
1 min. The grid was subsequently placed on a drop of
distilled water for 45 s and air dried. The polystyrene sizing
beads (93 and 262 nm diameter) were applied to the grids
following the procedures outlined above for size reference.
Transmission electron microscopy was performed using
conventional procedures on a JOEL-100CX operating at
80 kV.
RESULTS
Autophosphorylation and kinase activity of CCaMK
Autophosphorylation of CCaMK was associated with a
time dependent loss of the Ca
2+
/CaM-dependent enzyme
activity at pH 6.5, 7.5 and 8.5 (Fig. 1). Activity meas-
urements of enzymes phosphorylated at these different
pH conditions indicated that the enzyme activity decrea-
sed overtime with either a decrease or increase in the
reaction pH (Fig. 1). Autophosphorylation of CCaMK
at pH 6.5 resulted in 67% loss of enzyme activity in
2 min. While at pH 7.5, autophosphorylation-dependent
loss was 33% and at pH 8.5, CCaMK lost about 84%
of enzyme activity. A time-course experiment was conduc-
ted to study the kinetics ofinactivation (Fig. 2, Table 1).
The results in Fig. 2 indicate that the loss of enzyme
activity follow an exponential decay curve (Fig. 2).
CCaMK lost 28% activity at pH 7.5, 54% activity at
pH 6.5 and 72% activity at pH 8.5 in 30 s after
autophosphorylation.
Fig. 1. Self-inactivation of CCaMK during autophosphorylation.
Autophosphorylation was initiated by the addition of 500 ng CCaMK
to the reaction mixture (see Experimental procedures) at different pH
conditions. Aliquots of the autophosphorylation mix were collected
at the indicated time points and Ca
2+
/CaM dependent activity was
determined as described. Each bar represents mean of four inde-
pendent measurements.
2458 P. V. Sathyanarayanan and B. W. Poovaiah (Eur. J. Biochem. 269) Ó FEBS 2002
Autophosphorylation results in the formation
of sedimentable enzyme
At 0, 2 and 6 min of autophosphorylation, an aliquot of the
reaction mixture at different pH and ATP concentration
were subjected to centrifugation for 30 min at 16 000 g,to
determine whether any sedimentable enzyme is formed
under these conditions. Figure 3 shows the formation of the
sedimentable enzyme after 2 min of autophosphorylation.
Autophosphorylation at different ATP concentrations
(200 l
M
and 1 m
M
; Fig. 3) shows that at higher concentra-
tions of ATP, less sedimentable enzyme is formed.
Transmission electron microscopy studies
of autophosphorylated CCaMK
The formation of sedimentable enzyme was visualized
using TEM. CCaMK phosphorylated at 200 l
M
ATP for
5 min produced distinct uranyl acetate staining structures
(Fig. 4A,B). These particles appeared to interconnect and
associate to form branched structures. Figure 4C shows the
electron micrograph of an autophosphorylation deficient
site directed mutant (T267A) of CCaMK that did not form
branched complexes in the presence of 200 l
M
ATP for
5 min. In addition, autophosphorylation of T267A for
10 min did not show any branched complexes under TEM.
Autophosphorylation in the presence of adenosine 5¢-(b-
imido) triphosphate (AMP-PNP)
4
, an unhydrolyzable ana-
logue of ATP also did not produce uranyl acetate staining
structures under TEM (Fig. 4D).
DISCUSSION
It was shown that unlike other reported CaM-dependent
kinases, CCaMK requires Ca
2+
for autophosphorylation
and Ca
2+
/CaM is required for substrate phosphorylation
[7–9,11,12]. The substrate phosphorylation depends on the
extent of Ca
2+
stimulated autophosphorylation [9,12].
Ca
2+
binding to the C-terminal visinin-like domain results
is phosphorylation of threonine 267 [12]. In this study, we
report autophosphorylation-dependentinactivation of
CCaMK. Autophosphorylation of CCaMK resulted in
time-dependent loss of enzyme activity (Figs 1 and 2;
Table 1) and formation of a sedimentable enzyme (Fig. 3).
Autophosphorylation-dependent loss of enzyme activity
was influenced by both acidic and alkaline reaction pH.
Autophosphorylation at pH 8.5 produced a robust inacti-
vation leading to a loss of 72% enzyme activity in 30 s
(Fig. 2, Table 1). Among the three different pH conditions
tested, loss of enzyme activity was lowest at pH 7.5 (Fig. 1).
Time-dependent decrease in the activity of autophosphor-
ylated CaMK II has also been reported [16–24]. Auto-
phosphorylation of CaMK II at pH 7.5 resulted in 15%
decrease in activity at 2 min [24]. CCaMK lost about 33%
of enzyme activity in 2 min, indicating that it is more
sensitive to autophosphorylation-dependent inactivation.
At pH 6.5, there was about 40% loss of enzyme activity in
2 min for CaMK II [24] and at this pH, CCaMK lost 67%
of enzyme activity. These results suggest that the enzyme
inactivation is dependent on the duration of autophospho-
rylation and reaction pH.
The fit to the time-dependent kinetics showed (Fig. 2)
that the inactivation followed exponential decay [R values:
Table 1. Time-dependent inactivationofkinase activity of CCaMK due to autophosphorylation. CCaMK was autophosphorylated as described in
Experimental procedures, and an aliquot (200 ng of CCaMK) was added at indicated time points to a second stage reaction mixture containing
200 l
M
Histone II AS, as substrate. The phosphorylation was allowed for 10 min and [
32
P]PO
4
incorporation into the Histone II AS was measured
by excising protein bands from the gel and counting using a scintillation counter. The phosphorylation (c.p.m.) represents mean of three
measurements.
Time
(min)
pH 6.5 pH 7.5 pH 8.5
Phosphorylation
(c.p.m.)
% Initial
activity
Phosphorylation
(c.p.m.)
% Initial
activity
Phosphorylation
(c.p.m.)
% Initial
activity
0 84010 100 110998 100 48267 100
0.5 38259 46 80140 72 13286 28
2 28315 34 74543 67 7789 16
4 24066 29 69208 62 4440 9
6 22410 27 59127 53 3997 8
8 22671 27 60190 54 4293 9
Fig. 2. Time course ofinactivationof CCaMK during autophosphory-
lation. Autophosphorylation was performed at different reaction pH
(data points m,pH7.5;d,pH6.5;j, pH 8.5) as described. Aliquots
of the autophosphorylation mix were collected at the indicated time
points and Ca
2+
/CaM dependent phosphorylation of Histone II AS
were determined as described. The data was analyzed using
SIGMAPLOT
and the fit to the data is represented. The data points are the mean of
three measurements.
Ó FEBS 2002 Chimeric calcium/calmodulin dependent proteinkinase (Eur. J. Biochem. 269) 2459
0.998 (pH 8.5), 0.999 (pH 6.5), 0.992 (pH 7.5)]. The largest
drop in enzyme activity occurred during the first 30 s of
autophosphorylation (Fig. 2, Table 1). The enzyme activity
continued to decrease, but the decrease was not as great as
compared to the loss in the first 30 s. This suggests that there
are two phases of inactivation. In the first phase (fast
inactivation), the enzyme loses activity very rapidly and in
the second phase (slow inactivation), loss ofinactivation is
significantly slower. Loss of enzyme activity showed these
two phases ofinactivation at all the pH conditions tried.
5
The autophosphorylation resulted in the formation of a
sedimentable enzyme (Fig. 3). After 2 min of autophospho-
rylation, CCaMK was detected in both the pellet and
supernatant fractions. However, at time zero, all the kinase
enzymes were seen in the supernatant, indicating that
autophosphorylation leads to the formation of sedimentable
CCaMK. The formation of a sedimentable enzyme was
observed after 2 min of autophosphorylation at all the
different reaction pH tested (Fig. 3). The role of the ATP
concentration in sedimentablity of the enzyme was tested by
conducting autophosphorylation at different ATP concen-
trations (200 l
M
and 1 m
M
). Figure 3 shows that a
sedimentable enzyme was formed at pH 6.5 and 8.5 at
both the ATP concentrations used for autophoshorylation.
At pH 7.5, a higher ATP concentration (1 m
M
)prevented
the formation of a sedimentable enzyme (Fig. 3). However,
under all the other conditions of pH and ATP concentra-
tions, kinase enzymes existed as both the sedimentable and
soluble form. This suggests that both pH and ATP
concentration determine sedimentability of CCaMK due
to autophosphorylation. It is not necessary that all of the
soluble enzyme remains unphosphorylated or all the
phosphorylated enzyme undergoes sedimentation. This
depends on pH and ATP concentration. At higher concen-
trations of ATP (1 m
M
) and at pH 7.5, all of the
phosphorylated enzyme remains in the supernatant (soluble
fraction). ATP concentration also influenced autophospho-
rylation-dependent formation of sedimentable CaMK II
[24]. Higher concentrations of ATP during autophospho-
rylation prevented sedimentation of CaMK II. Inactivation
was not accompanied by the formation of sedimentable
CaMK II at different ATP concentrations at pH 7.5 [24]. In
contrast, plantkinase showed the formation of a sediment-
able enzyme at low concentrations of ATP (200 l
M
),
though higher concentrations inhibited formation of sedi-
mentable enzyme as mentioned above. In conclusion, the
inactivation of CCaMK depends on the duration of
autophosphorylation, reaction pH and ATP concentration.
The autophosphorylation-dependentinactivation was
further studied by TEM. Figure 4A,B shows electron
micrographs that are representative fields through out the
entire grid and were observed only under the autophospho-
rylation conditions that produced sedimentable enzyme.
However, the percentage of sedimentable enzyme that
adopted this network-like structure is uncertain. The
autophosphorylated kinase after denaturation (by boiling
in the presence of detergents) was observed under TEM.
After denaturation, the autophosphorylated kinase did not
show the formation of the network-like structures (data not
shown) indicating that these complex structures are formed
due to self-association caused by autophosphorylation.
The role of ATP binding and ATP hydrolysis in the
formation of the complexes was investigated by replacing
the ATP with an unhydrolyzable ATP analogue, AMP-
PNP, in the autophosphorylation reaction mix. Figure 4C
shows that the network-like complexes are not formed in the
presence of AMP-PNP. This suggests that ATP hydrolysis
is required for the complex formation. The autophospho-
rylation mutant T267A [12] did not show the complex
formation (Fig. 4C) under similar conditions that produced
complex structures of wild-type CCaMK. These results
further suggest that the formation of the complexes seen
under TEM is phosphorylation-dependent.
Self-inactivation may be a mechanism of regulating
enzyme activity as a means of modulating metabolic
processes or signal transduction pathway. Self-inactivation
imposes an upper limit on bioactive prostanoid synthesis by
prostaglandin H synthase (PGHS) [25]. The cytochrome
Fig. 3. Formation of sedimentable enzyme
during autophosphorylation of CCaMK.
CCaMK was autophosphorylated at different
pH (6.5, 7.5 and 8.5) and at different ATP
concentrations (200 l
M
and 1 m
M
)at0,2and
6 min. At the indicated time points, auto-
phosphorylated enzyme was subjected to
centrifugation and processing as described in
Experimental procedures. The pellet (P) and
supernatant (S) fractions are indicated at each
time point.
2460 P. V. Sathyanarayanan and B. W. Poovaiah (Eur. J. Biochem. 269) Ó FEBS 2002
Fig. 4. Transmission electron micrographs of CCaMK after autophosphorylation showing the formation of complex structures. CCaMK was auto-
phosphorylated for 5 min at pH 7.5 in the presence of 200 l
M
ATP and processed for TEM as described. (A) A field of uranyl acetate staining
structures at X40 000 following autophosphorylation. (B) Complex structures due to autophosphorylation at ·100 000. (C) Autophosphorylation
inthepresenceofAMP-PNPat·30 000. (D) Autophosphorylation of T267A, ·30 000. Arrows indicate polystyrene sizing beads (93 nm in
diameter).
Ó FEBS 2002 Chimeric calcium/calmodulin dependent proteinkinase (Eur. J. Biochem. 269) 2461
P450 apoenzyme self-inactivation is accompanied by
oxidation of all-groups, carbonyl group formation, changes
in aggregate state and apoenzyme polymerization [26]. It is
possible that heme loss and oxidative modification of the
apoenzyme is an important step in the regulation of P450
decay in cell [26]. Redistribution of soluble CaM Kinase II
to particulate fractions due to autophosphorylation has
been demonstrated in both in vivo and in situ models of
ischemia [27–29]. We have documented that the autophos-
phorylation-dependent inactivationof CCaMK involves
self-association leading to the formation of a sedimentable
enzyme. Inactivation and subsequent formation of a
sedimentable enzyme depends on the duration of auto-
phosphorylation, reaction pH, and ATP concentration. To
our knowledge, no other proteinkinase reported from
plants shows such a phosphorylation-dependent loss of
activity. However, CCaMK-mediated protein phosphory-
lation is implicated in male gametophyte development in
plants [10]. Self-inactivation of CCaMK may be a mechan-
ism of modulating the Ca
2+
/CaM mediated signal trans-
duction pathway in anther. The elucidation of the molecular
mechanisms leading to the self-inactivation of CCaMK will
broaden our understanding of the regulation of Ca
2+
/
CaM-mediated signaling in plants.
ACKNOWLEDGEMENTS
We thank Dr Chris Davitt and Professor Vincent Franceschi of the
Electron Microscopy Center, WSU for their valuable help and
suggestions with electron microscopy, and Shima Nakanishi for help
with inactivation kinetics experiments. The support of the National
Science Foundation (Grant MCB 0082256) and the National Aero-
nautics and Space Administration (Grant NAG-10-0061) is gratefully
acknowledged.
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. Autophosphorylation-dependent inactivation of plant chimeric
calcium/calmodulin-dependent protein kinase
P. V. Sathyanarayanan and B. W study, we
report autophosphorylation-dependent inactivation of
CCaMK. Autophosphorylation of CCaMK resulted in
time-dependent loss of enzyme activity (Figs