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Plant Physiol. (1999) 120: 859-866
Autophosphorylation-Dependent Activation of a
Calcium-Dependent Protein Kinase from Groundnut1
Subho Chaudhuri,
Anindita Seal, and
Maitrayee Das Gupta*
Department of Biochemistry, Ballygunge Science College, Calcutta
University, 35 Ballygunge Circular Road, Calcutta 700019, India
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ABSTRACT |
Ca2+-dependent protein
kinases (CDPKs) containing a calmodulin-like domain integrated in
their primary sequence are present primarily in plants. A member of
this family was characterized from the groundnut (Arachis
hypogea) plant and called GnCDPK (M. DasGupta [1994]
Plant Physiol 104: 961-969). GnCDPK specifically uses the
myosin light chain synthetic peptide (MLCpep), which is the
phosphate-accepting domain of smooth muscle myosin light chains
(KKRPQRATSNVFS), as an exogenous substrate under in vitro experimental
conditions. In this report we show that GnCDPK undergoes intramolecular
autophosphorylation. This self-phosphorylation occurs in threonine
residues in a Ca2+-dependent
(K0.5 = 0.5 µM) and
calmodulin-independent manner. The kinase activity toward MLCpep and
its sensitivity to Ca2+ were unaffected by prior
autophosphorylation when measured under saturating ATP concentrations.
The role of autophosphorylation in the exogenous substrate MLCpep
phosphorylation reaction was reinvestigated at low ATP concentrations.
A pronounced lag time of 1 to 2 min, followed by a linear increase of
activity for 7.5 min, was seen in the initial rate of MLCpep
phosphorylation under such suboptimal conditions. Prior
autophosphorylation completely abolished this lag phase, and a sharp
rise of exogenous substrate phosphorylation was seen from the 1st min.
Our results suggest that autophosphorylation is a prerequisite for the
activation of GnCDPK.
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INTRODUCTION |
CDPKs act as sensors for intracellular Ca2+
fluxes and translate them into physiological responses by reversibly
phosphorylating Ser and/or Thr residues of relevant essential enzymes
(Roberts and Harmon, 1992 ). Plants were shown to contain a novel group of CDPKs that are regulated by direct binding of
Ca2+ and are independent of CaM or phospholipids
(Harmon et al., 1987; Battey and Venis, 1988). Sequence analysis
revealed that the unique biochemistry of these kinases results from a
novel structural arrangement within a single polypeptide, wherein the
catalytic domain is joined to a CaM-like regulatory domain containing
four Ca2+-binding EF hands through a junction
domain (Harper et al., 1991). Almost 40 different CDPK-like sequences
are now available in the database, and some have significantly
degenerate sequences in the CaM-like domain. Some of these CDPK-related
protein kinases do not require Ca2+ as an
activator (Furumoto et al., 1996). These kinases, possibly with
variable Ca2+-binding properties, are thus
capable of translating quantitative Ca2+ signals
to qualitative messages. Because Ca2+ is involved
in many of the cellular functions of higher plants (Hepler and Wayne,
1985; Trewavas, 1986), it is reasonable to believe that novel
Ca2+-mediated regulatory pathways operate in
plant signal transduction through these kinases. The upstream or
downstream events of the CDPKs in such pathways and the details of
their mechanisms of regulation are not yet known.
Autophosphorylation, a common regulatory property of protein kinases,
leads to changes in their activity and/or changes in dependence on
their activators. The kinases become independent of their respective
effectors by autophosphorylation of a pseudosubstrate sequence, whereby
the inhibitory interaction of the domain is prevented (Schworer et al.,
1988; DasGupta and Blumenthal, 1995). Two independent studies
(Harmon et al., 1994; Harper et al., 1994) also demonstrated
pseudosubstrate-type autoinhibitory properties of the junction domain
of CDPKs. Because the autophosphorylation properties of CDPKs are not
known in detail, studies in the direction of understanding the
relationship between pseudosubstrate-type autoinhibition and
autophosphorylation of CDPK-type kinases are yet to be
undertaken.
The CDPK from groundnut (Arachis hypogea), a
unique member of the CDPK family, was identified and characterized from
dry groundnut seeds and was named GnCDPK (DasGupta, 1994). This enzyme
specifically phosphorylates the phosphate-accepting domain of chicken
smooth-muscle myosin light chains. GnCDPK has been purified to
homogeneity and is composed of a single subunit with a native
Mr of 53,000. As with several other CDPKs
(Roberts and Harmon, 1992), GnCDPK was found to autophosphorylate in a
Ca2+-dependent manner. The purpose of this
investigation was to study the autophosphorylation phenomenon of GnCDPK
in detail and to determine the role (if any) of
autophosphorylation in modulating the activity of GnCDPK toward its
exogenous substrate, MLCpep. Our results show that prior
autophosphorylation of GnCDPK completely abolishes the characteristic
lag observed during its exogenous substrate phosphorylation reaction.
We believe that autophosphorylation is an activation step for GnCDPK.
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MATERIALS AND METHODS |
Materials
Groundnut (Arachis hypogea var JL24) seeds were
obtained from the National Research Centre for Groundnuts (Gujrat,
India). [ -32P]ATP (specific activity 3000 Ci/mmol) was obtained from the Bhaba Atomic Research Centre (Bombay,
India). Q Sepharose and Sephacryl 300 were purchased from Pharmacia.
Blue Sepharose was prepared according to the method of Botime et al.
(1972). W7, W5, calmidazolium, and cellulose plates were obtained from
Sigma. All other reagents of analytical grade were obtained from Sisco
Research Laboratories (Bombay, India).
Protein Purification
GnCDPK was purified as described previously (DasGupta, 1994). The
final enzyme preparation was further clarified through a preparative
polyacrylamide gel (Laemmli, 1970) in a slab-gel apparatus in the
absence of SDS (nondenaturing conditions). Two-millimeter sections were
cut from the gel, crushed, and left in 1 mL of 20 mM
Tris-HCl, pH 7.4, at 4°C overnight. The centrifuged supernatants were
concentrated using an ultrafiltration unit (Centricon 30, Amicon,
Beverly, MA). The highest active fraction showed a single band in 10%
SDS-PAGE and was used for all of the experiments described. Protein
estimation was according to the method of Bradford (1976).
Kinase Assays
GnCDPK activity was determined by phosphate incorporation into
MLCpep using the P81 filter-binding method as described by DasGupta et
al. (1989) with the modifications described by DasGupta (1994). The
final reaction mixture (25 µL) contained 50 mM Tris-HCl, pH 7.4, 150 µM substrate peptide, 10 mM
MgOAc, 200 µM CaCl2, 250 µM [32-P]ATP (300 cpm/pmol),
and 0.25 µg of enzyme. The reaction was carried out for 10 min at
25°C. Autophosphorylation was performed under the same conditions
except that the [32-P]ATP concentration was
1 µM (30,000 cpm/pmol) and MLCpep was omitted. Reactions
were terminated at the indicated times by the addition of SDS-PAGE
sample buffer. Samples were subjected to 10% or 17.5% SDS-PAGE, and
the gels were stained for 30 min and destained for 1 h before
autoradiography. The phosphorylated protein was excised and the
incorporated 32P was determined by liquid
scintillation counting. For Ca2+-dependence
curves, free Ca2+ levels were set using
Ca2+/EGTA buffers with the stability constants
described by Martell and Smith (1974).
The Effect of Autophosphorylation of GnCDPK on MLCpep
Phosphorylation
The effect of autophosphorylation on MLCpep phosphorylation
activity was measured by a two-step assay. In the first step
(preincubation) the autophosphorylation reaction was carried out with
GnCDPK (0.25 µg/assay volume) in a 15-µL reaction mixture as
described above except that nonradioactive 1 µM ATP was
used. After 10 min the entire reaction mixture was added to a cocktail
for MLCpep phosphorylation, so that the final reaction mixture (25 µL) contained 50 mM Tris-HCl, pH 7.4, 150 µM substrate peptide, 10 mM MgOAc, 200 µM CaCl2, and 250 µM
or 5 µM [32-P]ATP (300 or
30,000 cpm/pmol). The reaction was monitored for 7.5 min at 25°C. In
control experiments GnCDPK was preincubated under identical conditions
but without 1 µM nonradioactive ATP.
Phosphoamino Acid Analysis
Purified GnCDPK was autophosphorylated under standard conditions.
Free ATP was removed by gel filtration through Sephadex G25
(Pharmacia). The eluted protein was extensively dialyzed, and the
filtrate was evaporated in a freeze drier (Speed-Vac, Savant
Instruments, Holbrook, NY) and subjected to hydrolysis in 6 N HCl for 2 h at 110°C. Hydrolysates were mixed with
standard phosphoserine, phosphothreonine, and phosphotyrosine and
analyzed by thin-layer electrophoresis on cellulose plates at pH 3.5 (acetic acid:pyridine:water, 50:5:945 [v,v]) at 800 V for 45 min
(Cooper et al., 1983). Radiolabeled amino acids were identified by
autoradiography, and the standards were identified by ninhydrin
staining.
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RESULTS |
Autophosphorylation of GnCDPK
Pure GnCDPK was found to autophosphorylate its own sequence (Fig.
1A, lane a). This self-catalyzed
phosphate incorporation was inhibited in the presence of 1 mM EGTA (Fig. 1A, lane b), indicating that the reaction
occurs in a Ca2+-dependent manner. The
concentration of Ca2+ needed for half-maximal
autophosphorylation of GnCDPK was 0.5 µM (Fig. 1B), which
is similar to the Ca2+ requirement for
exogenous substrate phosphorylation by GnCDPK (Table
I). The Mg2+ ion
requirement of 5 to 10 mM was the same for both catalytic activities of GnCDPK (Table I). However, a wide difference was noted in
the affinity of GnCDPK toward ATP, depending on whether it used its own
structure or an exogenous substrate as its target. There was about a
100-fold difference in the enzyme's Km for
ATP in autophosphorylation (100 nM) in the
absence of substrate compared with the Km
for ATP in the exogenous substrate phosphorylation (10 µM) reaction (Table I). Such differences were
also noted in phosphorylase kinase, in which the
Km value of ATP for autophosphorylation was
found to be 27 µM compared with 240 µM for kinase-dependent phosphorylase
phosphorylation (Hallenbeck and Walsh, 1983).
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| Figure 1.
Ca2+-dependent autophosphorylation of
GnCDPK. A, Purified GnCDPK was autophosphorylated as described in
``Materials and Methods''. The standard reaction mixture (25 µL)
contained 50 mM Tris-HCl, pH 7.4, 10 mM MgOAc,
200 µM CaCl2, 1 µM
[32-P]ATP (30,000 cpm/pmol), and 0.25 µg of enzyme.
Reactions were carried out for 10 min at 25°C and terminated by the
addition of SDS-PAGE sample buffer. Samples were analyzed in 10%
polyacrylamide gels and autoradiographed. Lane a, Standard reaction
mixture; lane b, plus 1 mM EGTA. B, Autophosphorylation of
GnCDPK was monitored in different concentrations of free
Ca2+ by using Ca2+/EGTA buffers in otherwise
standard reaction mixtures. Catalytic rates are expressed relative to
the reaction rate under standard assay conditions.
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Effect of CaM and CaM Antagonists on GnCDPK Activity
The nature of the Ca2+/CaM dependence in the
autophosphorylation reaction and in the exogenous substrate
phosphorylation reaction of GnCDPK was found to
be the same. In a previous report (DasGupta, 1994) it was shown that
exogenous CaM did not have an effect on GnCDPK activity, but that
CaM-binding drugs such as W7 and R2457 (calmidazolium) inhibited GnCDPK
activity with an IC50 of 30 and 10 µM, respectively. Here we show that the addition of
exogenous CaM (10 µM) did not have any effect on
autophosphorylation or on GnCDPK-catalyzed MLCpep phosphorylation (Fig.
2, lane b). However, in the presence of
50 µM R2457 (calmidazolium) or W7, both
autophosphorylation and exogenous substrate phosphorylation by GnCDPK
were completely inhibited (Fig. 2, lanes c-d). Under the same
conditions W5, a much less potent analog of W7, had no inhibitory
effect on GnCDPK activity (Fig. 2, lane e). Considering the proper
order of potency of these drugs and the identical sensitivity of both
of the catalytic functions of GnCDPK toward these drugs, the mechanism
of Ca2+ dependence of both autophosphorylation
and MLCpep phosphorylation reactions catalyzed by GnCDPK appears to
be mediated through a CaM-like sequence integrated into its primary
structure.
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| Figure 2.
Effect of CaM and CaM antagonists on
autophosphorylation of GnCDPK and phosphorylation of MLCpep. Purified
GnCDPK was incubated in standard reaction mixtures as described in
``Materials and Methods''. The final reaction mixture (25 µL)
contained 50 mM Tris-HCl, pH 7.4, 150 µM
substrate peptide, 10 mM MgOAc, 200 µM
CaCl2, 5 µM [32-P]ATP
(30,000 cpm/pmol), 0.25 µg of enzyme, and the indicated effectors.
Reactions were carried out for 10 min at 25°C and terminated by the
addition of SDS-PAGE sample buffer. Samples were analyzed in 17.5%
polyacrylamide gels and autoradiographed. Lane a, Standard reaction
mixture; lane b, plus 10 µM CaM; lane c, plus 50 µM R2457; lane d, plus 50 µM W7; and lane
e, plus 50 µM W5.
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Mechanism of Autophosphorylation
Autocatalysis of GnCDPK could occur by two potential reaction
mechanisms, intermolecular and intramoleular. To determine by which
mechanism autophosphorylation occurs, the dependency of the rate of
autophosphorylation was examined as a function of the GnCDPK
concentration. The reaction rate was linearly dependent on the GnCDPK
concentration over a 20-fold (25-500 nM) range of GnCDPK
concentrations, which is diagnostic of an intramolecular reaction (Fig.
3A, lanes a-d). Support for this model
was also provided by analysis of the relationship between the reaction rate and the enzyme concentration using Van't Hoff's plot, which had
a slope of 1.042 (Fig. 3B).
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| Figure 3.
Effect of enzyme concentration on
autophosphorylation. A, Purified GnCDPK was successively diluted to the
indicated concentrations and autophosphorylated as described in
``Materials and Methods'' for 1 min. The standard reaction mixture
(25 µL) contained 50 mM Tris-HCl, pH 7.4, 10 mM MgOAc, 200 µM CaCl2, 1 µM [32-P]ATP (30,000 cpm/pmol), and
enzyme in varying amounts. Lane a, 25 nM (0.03 µg/25
µL); lane b, 50 nM (0.06 µg/25 µL); lane c, 250 nM (0.3 µg/25 µL); and lane d, 500 nM (0.6 µg/25 µL). Reactions were terminated by the addition of SDS-PAGE
sample buffer, and samples were analyzed in 10% polyacrylamide gels
and autoradiographed. The radioactivity associated with GnCDPK bands
was quantitated by scintillation counting and the specific activity was
plotted against the enzyme concentration. B, Van't Hoff plot of log
velocity versus log enzyme concentration using the data in A.
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The time course of the autophosphorylation reaction is shown in Figure
4. Saturation of this self-catalyzed
phosphate incorporation was attained within 2 min under our
experimental conditions. The level of phosphorylation remained the same
thereafter (Fig. 4, inset). This level of phosphorylation was found not
to change even after 24 h (data not shown). To determine the
stoichiometry of phosphate incorporation, the gel bands shown in the
inset of Figure 4 were counted for radioactivity, as described in
``Materials and Methods''. The stoichiometry of phosphate
incorporation in GnCDPK was 0.2 mol/mol, which remained unchanged even
after prolonged incubation with ATP (Fig. 4). Therefore, under the
prescribed experimental conditions, at most only about 20% of the
kinase could have been phosphorylated to the extent of 1 mol/mol at the
point of saturation.
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| Figure 4.
Time course and stoichiometry of
autophosphorylation of GnCDPK. Purified GnCDPK (0.25 µg) was
autophosphorylated as described in ``Materials and Methods'' for the
indicated time periods. The standard reaction mixture (25 µL)
contained 50 mM Tris-HCl, pH 7.4, 10 mM MgOAc,
200 µM CaCl2, and 1 µM
[32-P]ATP (30,000 cpm/pmol). Samples were analyzed in
10% SDS PAGE. The phosphorylated enzyme was visualized by
autoradiography and the radioactivity was quantitated by scintillation
counting. Inset, Autoradiogram of the analyzed samples.
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The stoichiometry of phosphate incorporation into the
autophosphorylation reaction was determined in four of our enzyme
preparations and was consistently found to be in low order, indicating
that low phosphate incorporation is not due to inactivation of the enzyme during purification. In another well-characterized CDPK from
soybean, the stoichiometry of phosphate incorporation in the
autophosphorylation reaction was also found to be approximately 0.2 mol/mol (Roberts and Harmon, 1992). The low stoichiometry of
autophosphorylation in in vitro conditions could have been due to the
absence of unknown factors that may determine optimum autophosphorylation in vivo. For example, in the case of phosphorylase kinase phosphorylation by cAMP-dependent protein kinase, the
stoichiometry of phosphorylation was found to increase 28- to 36-fold
by increasing the Mg2+ concentration (Hallenbeck
and Walsh, 1983), indicating that reaction conditions have a profound
influence on phosphorylation reactions.
An important difference was noted in the choice of the amino acid as
the site of self-catalyzed phosphorylation compared with exogenous
substrate phosphorylation. GnCDPK was found to be predominantly autophosphorylated in Thr residues, as shown in Figure
5. This is in contrast to what was
observed in its exogenous substrate, MLCpep, in which the site of
phosphorylation was preferably Ser (DasGupta, 1994).
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| Figure 5.
Identification of the phosphoamino acid residue in
autophosphorylated GnCDPK. Autophosphorylation of GnCDPK was carried
out as described in ``Materials and Methods''. Phosphorylated GnCDPK
was hydrolyzed in 6 N HCl, and the hydrolysate was dried,
dissolved in water, and subjected to thin-layer electrophoresis at pH
3.5. The positions of the standards visualized by ninhydrin staining
are indicated.
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Effect of Autophosphorylation on GnCDPK Activity
For prior autophosphorylation, GnCDPK was preincubated in the
presence of 1 µM ATP for 10 min, and this
autophosphorylated enzyme was then used for MLCpep phosphorylation.
Under saturating concentrations of ATP (250 µM) in a
MLCpep phosphorylation reaction, autophosphorylation of GnCDPK was
found to have no effect on the rate of its exogenous substrate
phosphorylation activity. As shown in Figure
6A, the progress in MLCpep
phosphorylation was identical in previously autophosphorylated and
nonautophosphorylated GnCDPK. However, for Ca2+-dependent
protein kinases, autophosphorylation is known to affect not only the
enzymatic activity but also the response of the kinases to the metal
ion. Such changes in the Ca2+ dependence of
CaMKII are reported to range from a decrease in affinity for the metal
ion to a formation of a Ca2+-independent form
(Lai et al., 1986; Schworer et al., 1986). Autophosphorylation of
GnCDPK was found to have no such effect on the
Ca2+ dependence of the enzyme. As shown in Figure
6A, phosphorylation of MLCpep by both autophosphorylated GnCDPK and
nonautophosphorylated GnCDPK was inhibited in the presence of EGTA,
indicating that both reactions are Ca2+
dependent.
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| Figure 6.
Effect of autophosphorylation on MLCpep
phosphorylation by GnCDPK. GnCDPK (0.25 µg/assay volume) was
autophosphorylated in the presence of 50 mM Tris-HCl, pH
7.4, 10 mM MgOAc, 200 µM CaCl2,
and 1 µM nonradioactive ATP for 10 min at 25°C as
described in ``Materials and Methods''. Control experiments were
performed in which GnCDPK was preincubated under identical conditions
except that water was substituted for ATP. GnCDPK-dependent MLCpep
phosphorylation was then tested according to the procedure described in
detail in ``Materials and Methods''. The specific reaction conditions
are indicated below. Maximal activity is defined as total counts per
minute incorporated in the substrate under the indicated experimental
conditions of assay at the 7.5-min time point. A, MLCpep
phosphorylation assayed in the presence of 250 µM
[32-P]ATP (300 cpm/pmol) for the indicated time
periods with nonautophosphorylated GnCDPK ( ), nonautophosphorylated
GnCDPK and 1 mM EGTA ( ), autophosphorylated GnCDPK
( ), and autophosphorylated GnCDPK and 1 mM EGTA ( ).
Values represent the means of triplicate assays. B, MLCpep
phosphorylation assayed in the presence of 5 µM
[32-P]ATP (300 cpm/pmol) in otherwise standard
conditions for the indicated time periods with nonautophosphorylated
GnCDPK () or with autophosphorylated GnCDPK (). Values represent
the means of triplicate assays. C and D, MLCpep phosphorylation assayed
in the presence of 5 µM [32-P]ATP
(30,000 cpm/pmol) in otherwise standard conditions for 15 s (lanes
a), 30 s (lanes b), 45 s (lanes c), 1 min (lanes d), 2.5 min
(lanes e), 5 min (lanes f), or 7.5 min (lanes g) with
nonautophosphorylated (C) or autophosphorylated (D) GnCDPK. The
reaction mixtures were analyzed in 17.5% SDS PAGE and
autoradiographed. The positions of GnCDPK and MLCpep are indicated.
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These experiments for studying the effect of autophosphorylation of
GnCDPK were then repeated in the presence of low ATP concentration (5 µM) during substrate phosphorylation to slow the initial
rate of reaction. The reaction temperature (25°C) was kept unchanged. Interestingly, under such suboptimal conditions, without prior autophosphorylation of GnCDPK, there was a significant lag in the
progress curve of MLCpep phosphorylation (Fig. 6B). When GnCDPK was
incubated with 1 µM ATP for 10 min, this lag in MLCpep
phosphorylation was completely abolished and there was a sharp increase
in the reaction rate in the 1st min. The time of preincubation with ATP for autophosphorylation of GnCDPK in this experiment could be reduced
to 2 min without significant loss of the stimulatory effect on the
initial rates of MLCpep phosphorylation (data not shown).
The reaction mixtures described in Figure 6B were also analyzed in
SDS-PAGE to monitor the specific labeling kinetics of the enzyme
autophosphorylation and MLCpep phosphorylation, both with nonautophosphorylated enzyme (Fig. 6C) and with autophosphorylated enzyme (Fig. 6D). In the nonautophosphorylated enzyme the lag in
MLCpep phosphorylation by GnCDPK could be seen clearly (Fig. 6C).
Autophosphorylation under these conditions had no lag and progressed
linearly until saturation was obtained after 2 min (Fig. 6C). The
observed lag in MLCpep phosphorylation was completely abolished by
prior autophosphorylation of the enzyme, as shown in Figure 6D. In this
case, however, radiolabeled autophosphorylated GnCDPK bands were absent
because of saturation of the autophosphorylation reaction in the
preincubation period (Fig. 6D). This also indicates that the
autophosphorylation reaction reaches a plateau by 2 min and therefore
fulfills the structural change required to show optimum kinase
activity. Preincubation of GnCDPK with a nonhydrolyzable ATP analog
failed to show a similar effect on GnCDPK activity, indicating that it
is not an ATP-dependent conformational change but autophosphorylation
that is responsible for the modulation of the activity of GnCDPK toward
its exogenous substrate (data not shown).
The next question concerns which aspect of GnCDPK that is changed by
autophosphorylation leads to an increase in its activity toward
MLCpep, as demonstrated under limiting conditions and shown in Figure
6B. For this answer we examined the affinities of
pre-autophosphorylated and nonautophosphorylated GnCDPK for
Ca2+ and MLCpep under the same limiting assay
conditions (5 µM ATP), combined with short assay times (2 min). As shown in Table II, the
K0.5 values for Ca2+
were the same in both pre-autophosphorylated and nonautophosphorylated GnCDPK, indicating that autophosphorylation does not change the sensitivity of the enzyme toward this divalent ion. Experiments performed under saturating conditions also indicated that
autophosphorylation does not affect the Ca2+
dependence of the GnCDPK enzyme (Fig. 6A).
Under the same set of limiting conditions, the
pre-autophosphorylated and the nonautophosphorylated GnCDPK
differed widely in their affinities toward MLCpep, as reflected in the
estimated Km values of 50 and 250 µM, respectively (Table II). This observation indicates that a conformational change in GnCDPK after
autophosphorylation enables the enzyme to phosphorylate its exogenous
substrate by increasing its affinity toward the exogenous substrate. We
also examined the ATP dependence of GnCDPK using short assay times for both its autophosphorylated and nonautophosphorylated forms. The
estimated Km values under such conditions
were found to be 10 µM in both cases,
indicating that autophosphorylation does not change the affinity of the
enzyme for the nucleotide.
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DISCUSSION |
Autophosphorylation of several protein kinases was found to lead
to changes in activity and/or changes in the dependence of the enzymes
on their activators. For example, autophosphorylation of phosphorylase
kinase caused an increase in enzymatic activity (King et al., 1983),
whereas autophosphorylation of CaMKII rendered it autonomous of
Ca2+ and CaM (Lai et al., 1986). However,
autophosphorylation is not always necessarily linked to a modulation of
kinase activity. It also regulates the subcellular distribution of
protein kinases, promoting rapid and preferential modulation
of specific targets within a defined microenvironment in response to
diffusible second messengers. For example, CaMKII was released
from the cytoskeleton after autophosphorylation (Saitoh and Schwartz,
1985). The phenomenon of autophosphorylation was demonstrated in many
CDPKs (Roberts and Harmon, 1992) and shown to have an up- or
down-regulatory effect on the associated kinase activity (Bogre et al.,
1988; Saha and Singh, 1995). For wingbean CDPK, the autophosphorylation reaction was found to be independent of Ca2+,
although the exogenous substrate phosphorylation was strictly dependent
on Ca2+ (Saha and Singh, 1995). The present
studies were undertaken to investigate autophosphorylation and its
regulatory effect on GnCDPK.
Autophosphorylation-dependent up-regulation of CDPK activity was
previously demonstrated in a heterogeneous preparation of CDPK from
alfalfa, in which it was shown that incubation with unlabeled ATP
before the addition of labeled ATP and exogenous substrate resulted in
a 4-fold increase in rate but no difference in
Ca2+ sensitivity (Bogre et al., 1988). However,
for another well-characterized, pure CDPK from soybean, no such
regulatory effect of autophosphorylation could be detected under
comparable conditions (Roberts and Harmon, 1992). Similar experiments
with pure GnCDPK also indicated that the rate of phosphorylation of
MLCpep by GnCDPK, as well as its Ca2+
sensitivity, remained unchanged by prior autophosphorylation of the
pure enzyme (Fig. 6A).
In a similar set of experiments in which the MLCpep phosphorylation
reaction by GnCDPK was performed under suboptimal
concentrations of ATP, interesting deviations were
observed. With nonautophosphorylated GnCDPK under such
conditions, a significant lag in MLCpep phosphorylation was
noted (Fig. 6B), suggesting that autophosphorylation and the substrate
phosphorylation of GnCDPK may not be a concomitant phenomenon. It is
possible that autophosphorylation precedes substrate phosphorylation and is a prerequisite needed by GnCDPK to show its exogenous substrate phosphorylation. This proposition is supported by the experiments described in Figure 6, B and D, where prior autophosphorylation completely abolished the lag in MLCpep phosphorylation by GnCDPK.
As shown in Figure 6, B and D, there was a sharp increase in the
initial rate of MLCpep phosphorylation with autophosphorylated GnCDPK.
The lag period in the nonautophosphorylated GnCDPK is due to the
autophosphorylation reaction and the subsequent change of conformation
that enables the enzyme to interact with its exogenous substrate,
MLCpep, with increasing affinity (Table II) and to phosphorylate it.
However, the maximal rate of MLCpep phosphorylation attained by the
nonautophosphorylated enzyme after overcoming the lag period was only
one-fourth of the maximal rate of MLCpep phosphorylation by the
preautophosphorylated enzyme (Fig. 6B), even though the
autophosphorylation reaction in the presence of MLCpep is saturated
within 2.5 min (Fig. 6C). A possible explanation may be that the prior
presence of MLCpep in the vicinity of the enzyme is inhibitory toward
the requisite structural change that follows in the enzyme after it is
autophosphorylated.
In an earlier report on CDPK from alfalfa (Bogre et al., 1988), the
difference in the labeling kinetics of the enzyme protein and its
substrate, histone, was discussed without any presented data.
Autophosphorylation of this alfalfa CDPK was reported to be saturated
within 30 s, with an increased rate of histone phosphorylation thereafter. This is worth mentioning because it is similar to what was
observed with GnCDPK. Another possibility that arises from these data
is that autophosphorylation may be a manifestation of the formation of
an enzyme-bound covalent intermediate during turnover of the phosphate
to MLCpep. The large difference between the catalytic constants of the
autophosphorylation reaction (0.9 nmol min 1
mg1) and the exogenous substrate
phosphorylation reaction (600 nmol min1
mg1) is a strong argument against such a
proposition, however.
We propose a working model based on our observations, shown in Figure
7, of GnCDPK-dependent phosphorylation
that occurs in two steps. In step I, the enzyme is autophosphorylated
in a Thr residue in the presence of Ca2+ and
undergoes a structural change. This structural change enables the
enzyme to recognize and phosphorylate MLCpep in step II. Identity of
the phosphorylation site associated with the transition of GnCDPK from
step I to step II is not yet known. The present set of data cannot
explain how the low extent of autophosphorylation of GnCDPK (0.2 mol/mol) could be responsible for the observed effects.
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| Figure 7.
Possible model for the role of autophosphorylation
in the activation of GnCDPK. The shaded structure represents GnCDPK and
the line structure represents MLCpep. Details of the model are
discussed in the text.
|
|
One explanation, based on our preliminary observations (S. Chaudhuri
and M. DasGupta, unpublished data), can be suggested. In all of our
efforts to purify GnCDPK, 10% to 30% GnCDPK activity was found to be
eluted in high-Mr fractions, which do not
autophosphorylate. It is possible that a low extent of
autophosphorylation of GnCDPK initiates cooperative interaction between
the phosphorylated and nonphosphorylated enzyme, resulting in the
formation of a multimeric active enzyme complex that is resistant
to further autophosphorylation. This explanation accounts for and
explains how 0.2 mol/mol self-catalyzed phosphate incorporation can be
responsible for the activation of GnCDPK. Such a cooperative model
for phosphorylation and activation was suggested to take place in CaMKs
by Kwiatkowski et al. (1988). Autophosphorylation of a recently
characterized atypical Ca2+/CaM-dependent protein
kinase was found to occur in a stoichiometry of 0.339 mol phosphate
mol1 kinase (Patil et al., 1995; Takezawa et
al., 1996). The low level of autophosphorylation of this kinase also
leads to stimulation of its kinase activity. Why certain kinases tend
to have a very low level of self-catalyzed phosphorylation and how such
low levels of phosphorylation enable them to effect their activity
toward exogenous substrates needs more investigation.
| |
FOOTNOTES |
1
This work was supported by the Council of
Scientific and Industrial Research (grant no. 38-0894-95-EMR II),
Government of India.
*
Corresponding author; e-mail maitrayee_d{at}hotmail.com; fax
91-33-476-4419.
Received December 4, 1998;
accepted March 18, 1999.
| |
ABBREVIATIONS |
Abbreviations:
CaM, calmodulin.
CaMKII, type II CaM-dependent
protein kinase.
CDPK, Ca2+-dependent protein kinase.
MLCpep, myosin light-chain synthetic peptide.
R2457, (1-[bis-(4-chlorophenyl)methyl]-3-[2-(2,4-dichlorophenyl)-2-[(2,4-dichloro-phenyl)methoxy]-ethyl]-1H-imidazolium
chloride) .
W5, N-(6-aminohexyl)-1-naphthalene
sulfonamide.
W7, N-(6-aminohexyl)-5-chloro-1-naphthalene
sulfonamide.
| |
ACKNOWLEDGMENT |
We thank Dr. D.K. Blumenthal for generously providing the MLCpep.
| |
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Abstract
Introduction