The crucial enzyme in diacylglycerol-mediated
signaling is protein kinase C (PKC). In this paper we provide evidence
for the existence and role of PKC in maize. A protein of an apparent
molecular mass of 70 kDa was purified. The protein showed kinase
activity that was stimulated by phosphatidylserine and oleyl acetyl
glycerol (OAG) in the presence of Ca2+. Phorbol
12-myristate 13-acetate (PMA) replaced the requirement of OAG.
[3H]PMA binding to the 70-kDa protein was competed by
unlabeled PMA and OAG but not by 4
-PMA, an inactive analog. The
kinase phosphorylates histone H1 at serine residue(s), and this
activity was inhibited by H-7 and staurosporine. These properties
suggest that the 70-kDa protein is a conventional serine/threonine
protein kinase C (cPKC). Polyclonal antibodies raised against the
polypeptide precipitate the enzyme activity and immunostained the
protein on Western blots. The antibodies also cross-reacted with a
protein of expected size from sorghum, rice, and tobacco. A rapid
increase in the protein level was observed in maize following PMA
treatments. In order to assign a possible role of PKC in gene
regulation, the nitrate reductase transcript level was investigated.
The transcript level increased by PMA, not by 4
-PMA treatments, and
the increase was inhibited by H-7 but not by okadaic acid. The data
show the existence and possible function of PKC in higher plants.
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INTRODUCTION |
Protein phosphorylation/dephosphorylation is an important
signaling mechanism in animals (1, 2). Although this appears to be the
same in plants, much less is known about these processes (3, 4). The
regulation of phosphorylation is controlled by a large number of
protein kinases which are grouped into different categories depending
on the activating signals, the substrates which become phosphorylated
by these kinases, and the specific amino acid residues that become
phosphorylated (5, 6). The discovery of a novel class of protein
kinases, the Ca2+-dependent protein kinases (7,
8), has provided substantial evidence for a central role of
Ca2+ in a variety of signal transduction processes (9, 10).
The activity of this class of kinases is dependent on Ca2+,
but independent of calmodulin, and the enzyme has been purified from a
number of plant species (11-14). In addition several genes for these
enzymes have been cloned from plants (15-19). Besides Ca2+-dependent protein kinases, other classes
of kinases have also been reported; however, an unequivocal
demonstration of the existence of cAMP-dependent protein
kinase and PKC1 equivalent in
plants is still missing (20, 21). Besides Ca2+, the
phosphatidylinositol cycle has been shown to be involved in signal
transduction (22). The receptor-mediated hydrolysis of membrane-bound
phosphatidylinositol bisphosphate by phospholipase C (PLC) results in
the generation of inositol trisphosphate (IP3) and
diacylglycerol (DAG). Inositol trisphosphate releases Ca2+
from internal stores, whereas DAG transduces signals via the activation
of PKC, an enzyme belonging to the class of serine/threonine kinases
(23, 24). Protein kinase C is also activated by the tumor-promoting
phorbol esters (25, 26). The PKC gene family contains at least 10 different isozymes with different activation requirements, subcellular
distributions, and substrate specificities (27).
In recent years, the involvement of the phosphatidylinositol cycle in a
variety of plant signal responses has been demonstrated (19). However,
the characterization of PKC is still lacking. A number of reports
confirms the existence of such kinases in plants. The presence of a
Ca2+- and phospholipid-dependent kinase was
first shown in cytosolic fractions of zucchini (28). Later such kinases
were reported from oat (29) and the chloroplast envelope (30). The
presence of PKC was also shown by using partially purified fractions
from Amarnthus tricolor (31, 32) and rice (33) by using
antibodies raised against the animal enzyme. In rice an enzyme fraction
was found to phosphorylate a specific PKC substrate, MARCKS, and this enzyme activity was inhibited by staurosporine and calphostine (33). In
the same system, Komatsu and Hirano (34) found a staurosporine-inhibited kinase activity. They further characterized a
Ca2+-, PS-, and PMA-stimulated kinase activity, and the
phosphorylation of 45- and 43-kDa polypeptides was increased by PMA.
Earlier, in wheat, a lipid- and PMA-stimulated kinase was reported
(35). A partially purified kinase activity was also obtained from
Brassica compestris and maize, and both of them were
stimulated by Ca2+, PS, and OAG or PMA (36, 37). The
presence of a PKC equivalent enzyme is also supported by the presence
of PKC inhibitors (38, 39). The isolation of a DAG kinase gene and the
identification of PLC further suggested the existence of PKC in plants
(40). The presence of PKC in higher plants has been indicated either by
demonstrating the stimulation of partially purified enzyme fractions by
Ca2+ and phospholipids or by immunoblotting with antibodies
raised against animal PKC. However, the purification and detailed
biochemical analyses of the lipid-stimulated kinases in the plants are
still lacking.
We have shown earlier that serotonin, an activator of
phosphatidylinositol cycle and PMA could replace the requirement of light for the induction of nitrate reductase (NR) transcripts and
enzyme activities in maize. A PKC equivalent enzyme activity was
partially purified (41-45). In this paper we report the purification of a PKC-type kinase to homogeneity, its partial characterization, and
the production of polyclonal antibodies against the first plant PKC. We
further demonstrate that the PKC level is regulated by PMA, a chemical
which also stimulated the nitrate reductase transcript level.
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EXPERIMENTAL PROCEDURES |
Plant Material and Light Sources--
Seeds of Zea
mays var. Ganga-5 were obtained from National Seed Corp., New
Delhi, India and were grown on germination paper at 25 ± 1 °C
in complete darkness. PMA treatments were given to excised etiolated
leaves as described earlier (41).
PS, OAG, HEPES, histone, PMA, 4
-PMA, Tris, MES, BCIP, HRP conjugate,
and o-phenylenediamine were purchased from Sigma.
[3H]PMA was purchased from NEN Life Science Products,
[
-32P]ATP and [
-32P]ATP from Bhabha
Atomic (BARC), Bombay, India. All other reagents were of analytical
grade.
Purification of Protein Kinase--
Unless mentioned otherwise
all steps of protein purification were carried out at 4 °C.
Etiolated leaves were frozen in liquid nitrogen and homogenized in a
buffer containing 20 mM HEPES, 2 mM EDTA, 5 mM EGTA, 5 mM DTT, 2 mM PMSF, 10%
glycerol. The homogenate was first centrifuged at 15,000 rpm in RPR
20-2 rotor using a Hitachi centrifuge for 30 min and then at
100,000 × g for 1 h. The final supernatant was
loaded onto DEAE-Sephacel (80-100 ml) in a Bio-Rad column
pre-equilibrated with equilibration buffer (10 mM HEPES (pH
7.5), 2 mM EDTA, 5 mM EGTA, 5 mM
DTT, 2 mM PMSF, and 10% glycerol) as reported earlier
(37). After loading the extract, the column was washed with 10 mM HEPES (pH 7.5), 2 mM EDTA, 5 mM
EGTA, 10 mM mercaptoethanol, 10% glycerol, and the proteins were eluted with a linear salt gradient of 0-0.4
M KCl in 300 ml of equilibration buffer at a flow rate of
30 ml/h, and 8-ml fractions were collected. The peak fractions showing
kinase activity were pooled and reverse-dialysed to 10-15 ml with PEG 20,000 at 4 °C. The concentrated DEAE-Sephacel-pooled fractions were
dialysed against equilibration buffer (without EGTA and EDTA) for
2 h at 4 °C, and leupeptin (100 µM) was added
before loading onto the PS affinity column, which was prepared
according to the procedure of Uchida and Filburn (46). The protein was
eluted first with 15 ml of column buffer containing 14 mM
CaCl2 followed by 45 ml of 1 mM
CaCl2, followed by CaCl2, and the final elution was done with 2 mM EGTA and additional 3-ml fractions were
collected. All fractions were collected in plastic tubes and
immediately assayed for kinase activity and protein estimations.
Purified protein was passed through gel filtration in a Sephacryl 300 (90 × 2 cm) column equilibrated with equilibration buffer, and
the elution was carried out at a flow rate of 20 ml/h using the same
buffer. Fractions containing kinase activity were pooled, reverse-dialysed, and stored at
20 °C.
Protein Electrophoresis--
SDS-PAGE was done according to
Laemmli (47). A 10% gel was used, and proteins were visualized by
Coomassie Brilliant Blue or silver staining (48).
Enzyme Renaturation from Gel Slices--
The protein was eluted
from gel slices using a modified method of Satiel et al.
(49). Briefly, after SDS-PAGE, one lane was stained, destained, and
aligned with the rest of the gel. The protein band of interest was cut
and minced in buffer containing 10 mM HEPES, 5 mM DTT, 2 mM PMSF, 10% glycerol and incubated
for 2 h at 4 °C.
Protein Kinase Assay--
The kinase C-type activity was assayed
using histone H-1 (Type III) as a substrate. The reaction volume (100 µl) contained 30 mM HEPES (pH 7.5), 5 mM
MgCl2, 40 µg of histone, 10 µg of PS, 100 µM CaCl2, 4 µg of OAG or 15 ng of PMA, and
50 µl of enzyme solution. The enzymatic reaction was started by the
addition of 100 µM [
-32P]ATP (200,000 cpm, specific activity of 3000 Ci/mmol; obtained from BARC). The
reaction was carried out at 30 °C for 5 min and stopped by ice-cold
trichloroacetic acid (10%). Precipitates were collected on GF/C
Whatman filter discs, washed 4-5 times with cold 10% trichloroacetic
acid, dried, and counted in a LKB scintillation counter. Enzyme assays
were performed by using P81 paper (50). The specific activity is
expressed as pmol of 32P incorporated min
1
mg
1 protein. Protein concentrations were estimated by the
method of Bradford (51) using bovine serum albumin as standard.
Phosphoamino Acid Analysis--
For identifying amino acids that
are phosphorylated in histone, samples were digested in the presence of
5.7 N HCl at 110 °C for 2 h. The acid hydrolysate
was lyophilized, resolubilized in water (100 µl), and applied on
Whatman chromatography paper along with standards. Phosphoamino acids
were separated by using a solvent consisting of propionic acid:1
M NH4OH:isopropyl alcohol (45:17.5:17.5) as
used by Neufeld et al. (52). After the run, paper was dried and sprayed with ninhydrin to develop the spots. The paper was exposed
to x-ray films.
Binding Studies with Labeled PMA--
Binding studies with
[3H]PMA were performed as described earlier (53). The
conditions used for binding were similar to those used for kinase
assays. Binding reactions were carried out at 4 °C for 2 h
followed either by PEG 8000 precipitation (54) or collected on GF/C
(46) or GF/F filters as described (53). To analyze PMA binding on gel
the precipitates were collected, washed with buffer (30 mM
HEPES, 5 mM DTT, 10% glycerol, 0.1 mM PMSF,
and 100 µM leupeptin), dissolved in native gel buffer,
and analyzed by electrophoresis. The gel was cut into different
segments and counted in a scintillation counter.
Raising Polyclonal Antibodies against ZmcPKC70--
New Zealand
White rabbit was used for raising antibodies. A homogenous preparation
of the enzyme eluted from the gel after SDS-PAGE was used for
immunization (55). Four intradermal injections (0.5 ml) were given to
the rabbits at a gap of 1 month at multiple sites. The test bleeding
was done from the ear vein, and the debris was cleared by
centrifugation at 12,000 rpm.
ELISA and Immunotitration--
For ELISA, wells of the
microtiter plate were coated with purified kinase of different
dilutions (1:500, 1:1000, 1:2000). The enzyme was immunoprecipitated by
adding different amounts of antiserum to a fixed concentration (1 µg)
of purified kinase. The mixture was incubated at 4 °C for 7-8 h,
centrifuged at 2000 × g for 5 min, and the enzyme
activity was checked in the supernatant. 1:4000 and 1:5000 dilutions of
the antisera were used. To detect the antibodies, HRP conjugate was
used with o-phenylenediamine as the substrate, and
absorbance at 492 nm was read on an ELISA reader (Labsystems Multiscan
Biochromatic by Biological Diagnostic Supplies Ltd., Aryashire,
UK).
Western Blot Analysis--
Proteins were separated on SDS-PAGE
and transferred to nitrocellulose paper according to the procedure of
Towbin et al. (56). For developing immunoblots, the
antibodies raised for Z. mays kinase were used as primary
antibodies at 1:200 dilution. Blocking was done with bovine serum
albumin (3% w/v in phosphate-buffered saline) for 2 h at 37 °C
with constant shaking. The blots were incubated in the primary
antibodies for 2-3 h and then for 30 min with the secondary
antibodies, horseradish peroxidase-linked antibodies (HRP) or alkaline
phosphatase-linked antibodies (1:30,000). Three washes of 5 min each
were given with PBST (140 mM NaCl, 2.7 mM KCl,
10 mM Na2HPO4, 1.8 mM
KH2PO4 (pH 7.3), Tween 20 (0.1%)) after each
incubation. The protein-antibody complex was detected by using
4-chloronaphthol as a substrate for the HRP-linked assay and with BCIP
and NBT for the alkaline phosphatase-linked assay.
RNA Analysis--
RNA was isolated according to the methods of
Logemann et al. (57) and as described earlier (44). Thirty
µg of RNA samples were denatured in the presence of 6% formaldehyde
and 50% formamide at 50 °C for 1 h and blotted on a GeneScreen
Plus membrane. The RNAs were cross-linked to the membrane utilizing the
stratalinker (Stratagene). The filters were baked at 80 °C. A
32P-labeled probe was generated by the random primer
extension method using a NR cDNA of maize (58) or a chicken actin
cDNA. The labeling was performed by a commercial labeling kit (New
England Biolabs) according to the manufacturer's instructions. The
specific activity of the probe was 9 × 108 dpm/µg.
Pre-hybridization was performed for 15-30 min at 65 °C in 0.5 M NaCl, 0.1 M NaH2PO4, 0.1 M Tris base, 2 mM EDTA, 1% SDS, and 100 µg/ml denatured salmon sperm DNA. Denatured probe (l × 106 dpm/ml) was added, and the hybridization was continued
for 24 h at 65 °C. Washings were done using 10 mM
sodium phosphate buffer (pH 7.0), 2 mM EDTA, and 1% SDS,
initially at room temperature and then at hybridization
temperature.
For chicken actin probe the specific activity obtained was 5 × 106 dpm/µg. The hybridization was carried out at 55 °C
in 6 × SSC, 1% SDS, and 100 µg/ml denatured salmon sperm DNA.
Washings were done in 2 × SSC, 1% SDS at hybridization
temperature followed by 0.5 × SSC at room temperature. The washed
filters were exposed for autoradiography.
Statistics--
Each experiment was repeated at least three
times and in addition the kinase assays were done in triplicate. For
Northern blot, data from a representative experiment are given.
 |
RESULTS |
Purification of ZmcPKC from Maize--
Seven-day-old etiolated
seedlings of Z. mays (var. Ganga-5) were used for the
extraction of the enzyme as given under "Experimental Procedures."
The supernatant obtained after centrifugation at 100,000 × g was loaded onto a DEAE-Sephacel column. The protein was
eluted with a linear salt gradient (0-0.4 M), and the
fractions were assayed for kinase activity in the presence of PS, PMA,
Ca2+ using histone H1 as a substrate. Fig.
1A shows the protein elution and kinase activity profiles of DEAE-Sephacel fractions. The fractions showing maximum kinase activities were pooled, leupeptin was added to
avoid proteolytic degradation of PKC, and the fractions were loaded
onto a PS affinity column. The elution was done first with 1 mM Ca2+ followed by a buffer containing 0.1 mM Ca2+ and finally by EGTA (2 mM).
All fractions were assayed for kinase activity. Fig. 1B
shows the protein and kinase activity profiles of the fractions
obtained after PS affinity column chromatography. Most of the protein
was eluted with 1 mM Ca2+. An analysis of the
protein composition of fraction 18 on SDS-PAGE, which showed the
maximum enzyme activity and eluted with 0.1 mM Ca2+, showed two polypeptides of 70 and 55 kDa. Both
polypeptides could also be detected on native gels.

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Fig. 1.
Elution profile following DEAE-Sephacel
column and PS affinity chromatography. Every alternate fraction
from DEAE-Sephacel (A) was tested for kinase activity in the
presence of PS, PMA, and Ca2+. *------*, activity profile;
+, protein profile. Inset of A shows the
separation of proteins of peak fraction (17) on 10% SDS-PAGE. The
elution from affinity column (B) was done with l
mM CaCl2 until the 10th fraction followed by
elution with 0.1 mM CaCl2. Last elution was
performed with EGTA (2 mM). The kinase activity was
measured in the presence of PS, PMA, and Ca2+.
Inset of B shows the separation of proteins of
peak fraction (18) on SDS-PAGE and on native PAGE. The gel
was stained with Coomassie Blue.
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To separate these two proteins, the peak kinase activity fractions were
pooled, reverse-dialysed, and loaded onto a gel filtration column. Fig.
2A shows the protein profile
and kinase activity profile. The analysis of the protein composition
from three different fractions showed that the PS, PMA, and
Ca2+ kinase activity was associated with a 70-kDa protein
(Fig. 2A, inset).

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Fig. 2.
Elution profile after gel filtration on
Sephacryl S-300 before (A) and after purification through
SDS-PAGE (B). The eluted fractions were assayed for
the kinase activity in presence of PS, OAG, and Ca2+.
Protein absorbance was taken at 280 nm. The markers used for
calibrating the gel filtration column were phosphorylase (97 kDa),
bovine serum albumin (67 kDa), ovalbumin (43 kDa), and carbonic
anhydrase (31 kDa). Insets show the separation of proteins
on SDS-PAGE of different fractions (A) and the final
purified protein (B). M, marker; A,
peak fraction showing kinase activity. Gel was silver-stained according
to Merril et al. (48).
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To further purify the protein and to improve its recovery, the side
fractions, which also contained the 55-kDa polypeptide, were combined
and loaded onto a preparative SDS-PAGE, and the 70-kDa protein was
gel-eluted and again loaded on to a SDS gel. The eluted protein was
finally loaded onto a gel filtration column. Fig. 2B shows
the elution profile of the kinase activity, and the inset
(SDS-PAGE) shows the homogeneity of the protein. The kinase was
enriched 329-fold with a yield of approximately 0.07% (Table
I). The molecular mass of the
gel-purified protein (70 kDa) corresponds to that found in gel
filtration assays (69.8 kDa). This suggests that the protein functions
as a monomer. It was named Z. mays conventional protein
kinase C (ZmcPKC).
Characterization of ZmcPKC--
Fig.
3 shows that the optimum pH for the
purified kinase activity was 7.5. Fig. 4
shows that the optimal Mg2+ concentration was 5 mM in the presence of optimal PS and OAG at optimum free
Ca2+ concentrations. The effects of varying concentrations
of different lipids (PS, OAG, and PMA) were checked in the presence of
optimum Ca2+ concentration (100 µM). The free
Ca2+ was estimated to be 1.48 × 10
8
M as calculated by Sillen and Martell (59). Fig. 4 also
shows that the PS optimum for kinase activity was 10 µM,
that of OAG was 4 µM, and that of PMA was 15 nM in the presence of optimal PS and Ca2+
concentrations. Fig. 5 shows that the
kinase activity in the presence of PS and PMA is dependent on
Ca2+. In the absence of lipids, Ca2+ alone
stimulated the kinase activity 20-30% and even with increasing concentrations of Ca2+ the activity of the kinase was not
further stimulated. However, to activate the kinase maximally by
lipids, Ca2+ was essential. At 100 µM
Ca2+, a 12-fold stimulation in the activity was achieved by
the addition of PS and PMA. When calmodulin was added in place of
lipids, the kinase activity was not stimulated more than 35% (data not
given). These results indicated that the enzyme is a lipid-stimulated and Ca2+-requiring kinase.

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Fig. 3.
Effect of pH on the kinase activity. The
pH optimum for the kinase activity was determined by using different
buffers (30 mM), sodium acetate (3.5-5.5), MES (5.5-7.5),
Tris (7.5-9.5), and Tris-MES (6.0-9.0). The kinase activity was
checked in the presence of PS, OAG, and Ca2+
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Fig. 4.
Effect of various effectors on ZmcPKC70
activity. ZmcPKC70 activity was monitored in standard protein
kinase assay mixtures at different concentrations of Mg2+
(A), PS (B), OAG (C), and PMA
(D).
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Fig. 5.
Effect of Ca2+ concentration on
ZmcPKC70. Effect of Ca2+ on histone phosphorylation
was monitored in the presence (PS and PMA) ( ------ ) and in
absence of lipids (|------|).
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To confirm the nature of the kinase each component was tested at its
optimal level either individually or together. Fig.
6A shows that the PKC
equivalent was maximally stimulated in the presence of PS, PMA, and
Ca2+. The extent of stimulation in the presence of either
PMA or OAG were comparable. To check this further, the substrate
histone, H1, was separated, loaded onto a SDS-PAGE following
phosphorylation by ZmcPKC70, and autoradiographed. Fig. 6B
shows that PMA or OAG along with Ca2+ and PS maximally
stimulated the phosphorylation of histone H1. To determine the amino
acid residue at which histone H1 is phosphorylated, the phosphorylated
histone band from SDS-PAGE was cut and digested with acid.
Phosphoamino acid analysis was performed on paper chromatography as
described under "Experimental Procedures." As is seen in Fig. 6C the histone was phosphorylated at serine residues.

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Fig. 6.
Effect of lipids and Ca2+ on
histone phosphorylation by ZmcPKC70 and phosphoamino acid analysis of
phosphorylated histone. Assays were done in different conditions
(presence and absence of Ca2+/lipids (PMA, OAG, PS)), and
activity was measured using histone type IIIS as substrate
(A). The activity was maximally stimulated in presence of
PMA or OAG together with Ca2+ + PS in assay mixture using
histone as a substrate analyzed on SDS-gel followed by autoradiography
(B). Phosphorylated histone was digested and separated by
paper chromatography using propionic acid: (1 M)
NH4OH:isopropyl alcohol (45:17.5:17.5) solvent as described
under "Experimental Procedures." The paper was sprayed with
ninhydrin to detect the position of phosphoamino acid standards, and
the paper was exposed to films for autoradiography for the detection of
labeled phosphoamino acid (C).
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Kinetic Properties of ZmcPKC70--
Using histone H1 as a
substrate the kinetic behavior of ZmcPKC in the presence and absence of
lipids was studied. Fig. 7 shows that the
Km was high (9.7 µM) in the absence of
PS, PMA, and Ca2+. The Km value was
lowered to 7.8 µM on the addition of PS and PMA and was
further lowered to 3.12 µM by the addition of
Ca2+ suggesting that the enzyme activity is dependent on
lipid and Ca2+. Decrease in Vmax in
the presence of PS, PMA, and Ca2+ suggest the
noncompetitive inhibition (Table II). In
addition the Ca2+- and lipid-dependent kinase
activity of ZmcPKC was inhibited in the presence of the kinase
inhibitors H-7 and staurosporine. As shown in Fig.
8 the IC50 value was 6 nM for H-7 and 4 nM for staurosporine.

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Fig. 7.
Determination of Km and
Vmax for ZmcPKC70 using
histone as substrate. Protein kinase assays were performed with
purified preparations of ZmcPKC70 (150 ng) and a different
concentration of histone was used as substrate in presence of PS, PMA,
and Ca2+. A plot of 1/v versus
1/s was made. The inset shows a
"Michaelis-Menten" curve for the same data.
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Fig. 8.
Effect of kinase inhibitors on ZmcPKC70
activity. Effect of H-7 ( ) and staurosporine ( ) was tested
at different concentrations in standard kinase assay conditions using a
purified preparation of ZmcPKC70. The enzyme was incubated for 5 min
with the inhibitors before the addition of the substrate.
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ZmcPKC70 Binds to Labeled PMA--
One of the important properties
of ZmcPKC represents its binding site for PMA. ZmcPKC70 purified from
maize not only showed stimulation of kinase activity by PMA but also
binds to labeled PMA. The purified enzyme was used to monitor PMA
binding by four different methods following the protocols of Bazzi and
Nelsestuen (53). Irrespective of the method used, 39 × 103 to 42 × 103 counts were incorporated
into the protein. Fig. 9, A
and B, shows that the binding of labeled PMA to ZmcPKC70 was
competed by cold PMA and also by OAG. The Kd value
estimated from the binding analysis using Scatchard plot was 0.6 nM, and the Ki for OAG was 1.25 nM. An inactive analog of PMA, 4
-PMA, did not show any
competition suggesting that the binding is specific. The binding of PMA
to ZmcPKC was also confirmed by autoradiography of the purified enzyme
labeled with PMA following gel electrophoresis as shown in Fig.
9C. When the gel was sliced and the pieces were counted, the
label was exclusively found in the stained band.

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Fig. 9.
Binding of [3H]PMA to
ZmcPKC70. Binding competition of labeled PMA to purified ZmcPKC70
was performed with cold PMA and an inactive analog of PMA (4 -PMA)
(++) and with OAG (A). After binding labeled PMA, purified
ZmcPKC70 protein was precipitated with PEG and separated on PAGE
followed by fluorography and autoradiography (A,
autoradiogram; S, stained). Gel was sliced and counted in a
scintillation counter (B).
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ELISA Immunotitration of ZmcPKC70 and Western Blot
Analysis--
Fig. 10A
demonstrates that the kinase activity was decreased with increasing
amounts of antiserum. Preimmune serum did not show any inhibition.
Immunocross-reactivity was checked on Western blots by using a
partially purified DEAE-Sephacel fraction and the purified enzyme. Fig.
10B shows that a single band reacted with the purified
protein and with the DEAE-Sephacel fraction initially used for loading
the PS affinity column. However, in crude extracts an additional faint
band of approximately 82 kDa was noticed (data not shown).

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Fig. 10.
Immunotitration curve of ZmcPKC70 and
Western blot analysis. Immunotitration was done by using
polyclonal antiserum ( ------ ) and preimmune serum (+------+). 1 µg of purified protein was taken for microtitration reactions, and
immunotitration (A) was performed as given under
"Experimental Procedures" (A). For Western analysis the
original DEAE-fractions that were loaded on an affinity column and the
purified protein were run on SDS-PAGE, and the proteins were
transferred onto nitrocellulose membrane. The blot was incubated with
primary antibodies (1:200) and alkaline phosphatase-linked secondary
antibodies (1:30,000), and the color was developed using BCIP/NBT
(B) (D, DEAE-Sephacel fraction; P,
purified).
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Tissue Specificity and Cross-reactivity--
Immunoblots with
protein extracts from different parts of dark-grown seedlings revealed
that the protein was present in both roots and shoots; however, the
level in roots was significantly lower. ZmcPKC70 was not detected in
the hypocotyls whereas in the epicotyls, the protein content was almost
the same as in the shoots (data not shown). The immunoresponse was
checked with extracts from different plant species. A protein with an
expected molecular mass of 70 kDa immunostained in crude protein
fractions from shoots of sorghum (Sorghum bicolor), tobacco
(Nicotiana tabaccam), and rice (Oryza sativa var)
(Fig. 11). This suggests that ZmcPKC70 is present in both monocots and dicots.

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Fig. 11.
Cross-reactivity of antibodies against
ZmcPKC70 with different plants. Proteins from monocots (sorghum,
maize, rice) and a dicot (tobacco) were run on SDS-PAGE and transferred
to nitrocellulose. The blots were probed with ZmcPKC70 antibodies as
described in the legend to Fig. 9.
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Effect of H-7 and Okadaic Acid on PMA-mediated NR Gene
Expression--
Earlier we have shown that PMA stimulated the nitrate
reductase (NR) transcript level similar to red light irradiation (44). Fig. 12 shows that the effect of PMA on
the stimulation of NR transcripts in dark-grown seedlings is specific
as the inactive analog 4
-PMA could not induce NR transcripts in
dark-grown leaves. When PMA was applied along with H-7, an increase in
the NR transcript level was inhibited. Under the same conditions,
okadaic acid had no effect, again confirming specificity (Fig. 12).

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Fig. 12.
Effect of PMA, H-7, and OKA on NR transcript
level. 8- to 9-day-old etiolated primary leaves were treated with
PMA, 4 -PMA, PMA + H-7, and PMA + OKA followed by incubation in
KNO3 (60 mM) for 4 h. Total RNA was
isolated and blotted as described under "Experimental Procedures."
The blot was probed with labeled genes encoding for NR and actin.
|
|
 |
DISCUSSION |
A number of reports have indicated the presence of PKC-type
activities in plants; however, to our knowledge, this is the first report on the purification and characterization of a PKC homolog from a
higher plant. The present study suggests a role of protein phosphorylation events mediated by such kinases in regulating gene
expression.
Presence of a PKC Homolog in Plants--
Much information is
available regarding the amino acid sequences and biochemical properties
of PKC in animal systems (27, 60). Based on sequence homologies and
biochemical characterizations, the various isoforms of PKC can be
classified as conventional PKCs (cPKC), novel PKCs (nPKC), and atypical
PKCs (aPKC) (24, 27, 60). Generally aPKC-type kinases are not
stimulated by DAG and not activated by PMA. The nPKC kinases do not
show Ca2+ dependence. Further these kinases are unable to
phosphorylate a commonly used PKC substrate, histone III, except after
partial proteolytic digestion (61). Although there are reports on the presence of lipid-stimulated kinases in plants (introduction to the
text), their classification type is still a matter of discussion (62).
In plants, none of the lipid-stimulated protein kinases have been
purified to homogeneity.
Earlier we had shown the presence of a protein kinase activity that was
stimulated in the presence of OAG or PMA (41). Although the existence
of such kinases has been confirmed (36, 37, 63), none of the enzymes
have been purified to homogeneity and characterized in detail. Based on
our biochemical data, ZmcPKC70 belong to the cPKC category (26, 64). We
were successful in purifying this enzyme by utilizing affinity
chromatography followed by gel purification. The 70-kDa protein
ZmcPKC70 can be activated by either OAG or PMA and showed
Ca2+ concentration dependence for its activation. The
lowering of Km by the addition of Ca2+
in the presence of PS and PMA also suggests that this protein belongs
to the cPKC category. ZmcPKC can phosphorylate histone H1 as a
substrate without proteolytic degradation, and the phosphorylation occurs at serine residue(s) indicating that this protein is a serine/threonine kinase.
The binding studies with labeled PMA and its competition with unlabeled
PMA and OAG suggest that there may be only a single binding site for
PMA and OAG (54). Since there was no competition in the presence of the
inactive analog 4
-PMA, the binding site appears to be specific.
These results, together with the inhibition of the kinase activity by
general PKC inhibitors (65, 66), strongly suggest that ZmcPKC70 belongs
to the C-type PKC. Since no further stimulation was observed when DAG
and PMA were added together, it is unlikely that ZmcPKC belongs to the
PKD type (67). Thus ZmcPKC appears to be a homolog of cPKC and differs
from kinases from other sources. This is supported by the observation
that there are differences with respect to concentrations of various activators required to obtain optimal kinase activities (68-72). More
work is required to find out the catalytic and regulatory domains of
ZmcPKC70.
ZmcPKC70 Homologs Are Present in Other Plants--
We have been
successful in obtaining antibodies against ZmcPKC70. The antibodies
cross-reacted with the enzyme on Western blots (Fig. 11) and could also
precipitate the kinase activity (Fig. 10). Recently a PKC homolog shown
to be present in potato also used
-peptide as a substrate (63). The
potato protein was recognized by animal PKC antibodies. In our studies
we also found that the plant enzyme activity can be precipitated by
antibodies raised against animal PKC (data not shown).
The maize PKC antibodies also cross-reacted with a 70-kDa protein in
sorghum, rice, and tobacco indicating that PKC homologs may be present
in both monocot and dicot. Earlier the presence of PKC-type activities
was indicated in rice, Brassica, wheat, oat, and soybean
(29, 30, 32, 33, 35, 36, 66), and a PKC homolog partial cDNA clone
has been reported from rice callus (73). Physiological processes such
as swelling of protoplasts and activation of enzymes by activators of
PKC in monocots and dicots indicated the presence of such kinases in
different systems (41, 64, 74-76).
PMA Stimulates NR Transcript Level--
We have earlier
demonstrated that PMA affects the NR activity and transcript level (41,
44). This effect was similar to that obtained after irradiation of
dark-grown leaves with red light. It was further shown that the
red-light effect was mediated via a protein phosphorylation event (43).
The present data confirm our earlier observation. We further show that
the effect of PMA in stimulating the nitrate-induced NR transcripts is
specific since 4
-PMA was inactive. From the present data we further
infer that PMA-mediated stimulation of NR transcript is related to a phosphorylation event since H-7, a known kinase inhibitor which inhibits cPKC in plants, inhibited the PMA effect. An inhibitor of
protein phosphatases I and IIA, okadaic acid, had no effect. These
results suggests that PMA kinase-mediated protein phosphorylation is
involved in plant cell signal transduction pathway. In addition we have
evidence to show that PMA can increase the level of the kinase (data
not shown), thus it is possible that the expression of the kinase
gene(s) may be an early event in PMA-mediated physiological processes.
NR cDNA was provided by Prof. W. Campbell, and the chicken actin clone was from Dr. R. Bamezai. We are
thankful to Dr. Robert Fluhr, Weizmann Institute of Science (Israel),
for preliminary discussion on the manuscript and Dr. Ralf
Oelmüller, Botanisches Institut der
Ludwig-Maximilians-Universität (Germany) for his valuable
suggestions and cooperation in preparation of the manuscript.