From the Institut für Biologie II/Botanik, Universität Freiburg, Schänzlestrasse 1, 79104 Freiburg, Germany
Received for publication, August 31, 2000, and in revised form, December 4, 2000
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ABSTRACT |
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The common plant regulatory factors (CPRFs) from
parsley are transcription factors with a basic leucine zipper motif
that bind to cis-regulatory elements frequently found in
promoters of light-regulated genes. Recent studies have revealed that
certain CPRF proteins are regulated in response to light by changes in their expression level and in their intracellular localization. Here, we describe an additional mechanism contributing to the light-dependent regulation of CPRF proteins. We show that
the DNA binding activity of the factor CPRF4a is modulated in a
phosphorylation-dependent manner and that cytosolic
components are involved in the regulation of this process. Moreover, we
have identified a cytosolic kinase responsible for CPRF4a
phosphorylation. Modification of recombinant CPRF4a by this kinase,
however, is insufficient to cause a full activation of the factor,
suggesting that additional modifications are required. Furthermore, we
demonstrate that the DNA binding activity of the factor is modified
upon light treatment. The results of additional irradiation experiments
suggest that this photoresponse is controlled by different
photoreceptor systems. We discuss the possible role of CPRF4a in
light signal transduction as well as the emerging regulatory network
controlling CPRF activities in parsley.
Light is probably the most important environmental stimulus for
plants, controlling central developmental processes such as germination, deetiolation, and the transition from the vegetative to
the reproductive phase (1). To monitor differences in the quality,
intensity, and direction of light, plants have evolved different
photoreceptor systems that control the expression of an enormous number
of genes (2). The analysis of some of these light-controlled genes has
revealed several cis-acting elements that are involved in
mediating light responsiveness. One of these elements, the so-called
G-box, is a hexameric DNA-motif (CACGTG) that is frequently found not
only in the promoters of light-regulated genes but also
in promoters that respond to other environmental or endogenous stimuli,
such as hormones, stress, or cell cycle-related signals (3). Screening
of expression libraries with G-box probes has led to the identification
of several G-box binding proteins that belong to the family of basic
leucine zipper motif
(bZIP)1 transcription
factors, including the common plant regulatory factors (CPRFs) from
parsley and the G-box binding factors (GBFs) from
Arabidopsis (5-7). The bZIP motif is frequently found in eukaryotic transcription factors and mediates sequence specific DNA
binding, as well the formation of either homo- or heterodimers (8).
The results of previous studies suggest that several of the identified
G-box binding proteins are involved in light signal transduction. For
example, we recently reported that one member of the CPRF family,
CPRF2, is exclusively localized in the cytosol in the dark and that
light treatment causes an almost complete import of the factor into the
nucleus (9). In contrast, the factor CPRF1 is constitutively localized
in the nucleus, whereas CPRF4a is found in the nucleus as well as in
the cytosol under all conditions tested (9). The molecular mechanisms
leading to the regulation of the intracellular distribution of CPRF2
are not fully understood. However, we recently described that CPRF2 is
phosphorylated in vivo in a light-dependent
manner (10). Because phosphorylation reactions are frequently involved
in the regulation of the intracellular distribution of transcription factors of yeast and animals (11, 12), the phosphorylation of CPRF2
might play a role in triggering the nuclear import of the factor. This
idea is supported by the fact that the import and the phosphorylation
of CPRF2 are both strongly influenced by red light treatment, pointing
to an involvement of the red light sensing phytochrome photoreceptors
(10).
In this report, we describe the role of phosphorylation in the
regulation of the parsley transcription factor CPRF4a. We demonstrate that the DNA binding activity of the factor is modulated in a phosphorylation-dependent manner and that cytosolic
components are involved in this process. In agreement with this result,
we have identified a cytosolic CPRF4a-kinase. Phosphorylation of recombinant CPRF4a by this kinase, however, is not sufficient to cause
a full activation of the factor, suggesting that additional modifications are required. Moreover, we found that light treatment of
dark-grown parsley cells leads to an increased DNA binding activity of
CPRF4a. This effect is accompanied by a change of the CPRF4a-specific
DNA binding pattern observed in an electrophoretic mobility shift assay
(EMSA). The results of further irradiation experiments point to an
involvement of different photoreceptor systems in the regulation of
this process. We discuss the possible role of CPRF4a in light signal
transduction, as well as the different mechanisms that contribute to
the regulation of CPRF transcription factors.
Isolation of Cytosolic and Nuclear Extracts from Cultured Parsley
Cells--
Cytosolic and nuclear extracts were isolated from a
dark-grown parsley cell culture 6 days after subcultivation as
described previously (9, 13-15). For irradiation experiments,
evacuolated protoplasts were irradiated for 20 min with white light,
red light, or far-red light or kept in darkness (13). Preparation of
cytosolic extracts from irradiated evacuolated protoplasts was done in
green safety light (13).
Expression and Purification of Recombinant
CPRFs--
Restriction fragments encoding full-length CPRF1, CPRF2,
and CPRF4a were subcloned into the BamHI sites of the
vectors pQE70 (CPRF2) or pQE30 (CPRF1 and CPRF4a) to produce fusion
proteins with C-terminal (CPRF2) or N-terminal (CPRF1 and CPRF4a)
histidine tags. Transformation of the vectors in Escherichia
coli and expression and purification of the proteins on nickel
nitrilotriacetic acid-agarose were performed under denaturing
conditions, as described in the manufacturer's protocol (Qiagen). The
purified proteins were refolded by removing urea by gel filtration
through NAP 5 columns (Amersham Pharmacia Biotech) against 25 mM Tris/HCl, pH 7.8, 100 mM NaCl, and 1 mM dithiothreitol. The protein content of the eluate was determined after centrifugation (1 h at 100,000 × g
and 4 °C) using a method that is based on Coomassie Blue G-250 (16).
2 µg of the recombinant proteins were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using a 12% (w/v) acrylamide gel (17).
Subsequently, the proteins were stained with Coomassie Blue R-250.
In Vitro Phosphorylation of Recombinant CPRFs--
2 µg of the
recombinant CPRFs were mixed in a total volume of 20 µl with 50 µg
of cytosolic protein or 20 µg of nuclear protein and
For kinase inhibitor experiments, the reaction mixtures containing
recombinant CPRF4a and cytosolic protein were supplemented with one of
the following substances (final concentrations are indicated):
Me2SO (0.1% (v/v)), K252a (100 nM),
H-89 (50 µM), staurosporine (200 nM),
chelerythrine chloride (10 µM), genistein (50 µM), KN62 (10 µM), and hypericin (5 µM). All kinase inhibitors were purchased from Calbiochem
and diluted in Me2SO. The total concentration of
Me2SO did not exceed 0.1% (v/v). For activation of
hypericin, the samples were irradiated for 10 min with white light
prior to addition of [ Electrophoretic Mobility Shift Assays, Supershift Assays, and
Phosphatase Treatment--
For EMSA, either a monomeric G-box probe
(5'-AATTCTCCCTTATTCCACGTGGCCATCCGG-3') or a tetrameric
G-box probe (5'-(ACCACGTGGC)4-3') was used
(G-box core sequences are underlined). For the mutated tetrameric G-box
probe, the core sequence of the second G-box repeat was changed from
CACGTG to CACTGT. Preparation of the radioactively labeled probes and
experimental conditions for EMSA were described previously (9, 13). For
supershift assays the binding reaction mixtures containing either
cytosolic or nuclear protein in a total volume of 15 µl were
incubated for 10 min on ice with 1 µl of the CPRF antisera or the
corresponding preimmunosera prior to EMSA. The CPRF antisera used in
this study have been described previously (9). Treatment of protein
extracts with alkaline phosphatase was performed as follows: 0.5 µl
of 4.2 units/µl alkaline phosphatase (Sigma, P-7915) was added to 50 µg of cytosolic or 20 µg of nuclear protein in a total volume of 10 µl and incubated for 10 min at room temperature. Identical samples
were supplemented with 0.5 µl of the phosphatase storage buffer (50%
(v/v) glycerol, 1 mM MgCl2, 0.1 mM
ZnCl2, and 30 mM triethanolamine, pH 7.6) for control reactions. Subsequently, the DNA probe was added, and the
samples were subjected to EMSA. The experimental conditions for the
dephosphorylation of cytosolic extracts with immobilized alkaline
phosphatase and for the rephosphorylation of dephosphorylated cytosolic
protein have been described previously (13).
DNA Binding Activity of Recombinant CPRF4a after
Phosphorylation--
The phosphorylation kinetics of recombinant
CPRF4a was performed as described previously (10).
Expression and Characterization of Recombinant CPRF
Proteins--
To allow for a comparative analysis of different CPRF
transcription factors, three members of the CPRF family
(e.g. CPRF1, CPRF2, and CPRF4a) were expressed in E. coli as fusion proteins with terminal histidine tags. The use of
histidine tags allowed a purification of the recombinant proteins by
affinity chromatography. Under all conditions tested, overexpression of
the CPRFs resulted in an almost complete incorporation of the
recombinant proteins into inclusion bodies. Therefore, inclusion bodies
were isolated, and the recombinant proteins were solubilized by
treatment with a solution containing 6 M guanidinium
hydrochloride. Subsequently, purification of the proteins was performed
in the presence of 8 M urea using nickel nitrilotriacetic
acid-agarose. Fig. 1A shows the purified proteins after SDS-PAGE and Coomassie staining. Whereas the CPRF1 and CPRF4a preparations were highly pure (Fig. 1A,
lanes 1 and 3), the CPRF2 preparation contained a minor
contaminant of a lower apparent molecular weight (Fig. 1A, lane
2). Western blotting analysis using specific CPRF2 and histidine
tag antibodies revealed that this protein is a C-terminal CPRF2
fragment (data not shown). Folding of the purified CPRF proteins was
performed by removing urea by gel filtration. For all CPRFs, the
protein yield after the folding procedure was about 50%. The factors
were subjected to EMSA to test their DNA binding activities. As shown in Fig. 1B, all CPRFs were capable of binding a G-box
containing probe. This result is in agreement with previous reports
showing G-box binding activity to be a characteristic feature of native CPRF proteins (4, 5, 19).
Cytosolic and Nuclear Kinase Activities for CPRF Proteins--
In
yeast and in animals, the important role of phosphorylation in the
regulation of transcription factor activities has been described in
detail (12). Because we were recently able to report a
light-dependent phosphorylation of the factor CPRF2 (10), we addressed the question of whether phosphorylation events are involved in the regulation of other CPRF proteins as well. A
prerequisite for such a regulatory mechanism is the existence of
specific kinases for the individual factors. Therefore, we initiated
our study by analyzing compartment specific CPRF phosphorylation
activities. For this purpose, we mixed the recombinant bZIP factors
with cytosolic and nuclear extracts, which were obtained from
evacuolated parsley protoplasts. These subcellular extracts were chosen
due to our previous results showing that CPRFs are localized in the
nucleus as well as in the cytosol (9). After [ Characterization of the Cytosolic CPRF4a Kinase Activity by
Inhibitor Studies--
In a previous report, we studied the
phosphorylation activity for CPRF2 in detail, showing that this factor
is modified in its C-terminal half by a 40-kDa serine kinase in a
light-dependent reaction (10). To gain a better
understanding of the mechanisms regulating CPRF activities, we focused
our analysis on the compartment-specific kinase activity for CPRF4a. We
performed in vitro phosphorylation experiments with
cytosolic extracts as described above in the presence of different
kinase inhibitors to characterize the CPRF4a specific kinase in more
detail. The results of these experiments (Fig.
3) revealed that the CPRF4a kinase is
strongly inhibited by staurosporine (lane 3) and H-89
(lane 8), whereas other substances caused no or only very
weak effects. Staurosporine is a general inhibitor of serine/threonine
kinases (20), suggesting that CPRF4a is modified on either serine or
threonine residues, or both. H-89 was originally designed to
specifically inhibit protein kinase A in nanomolar concentrations (20).
However, higher concentrations of H-89 (50 µM was used in
Fig. 3) affect other types of kinases as well (20). Interestingly, H-89
in micromolar concentrations strongly inhibits the G-box binding
activity of bZIP-like proteins that are localized in the cytosol of
evacuolated parsley protoplasts (13). Because a pool of CPRF4a factors
is found in the cytosol (9) and the cytosolic phosphorylation of CPRF4a
is strongly decreased by H-89 treatment, we next tested whether
phosphorylation may contribute to the regulation of its DNA binding
activity.
The DNA Binding Activity of CPRF4a Is Controlled by
Phosphorylation--
To study the possible effect of phosphorylation
on the activity of CPRF4a, we isolated nuclear and cytosolic extracts
to perform DNA binding studies with endogenous CPRF4a. Fig.
4 shows the results of EMSA experiments
in which the extracts were tested using a monomeric G-box probe. The
signals deriving from CPRF4a were identified by addition of a specific
CPRF4a antiserum to the binding reactions, resulting in the reduction
of the CPRF4a-signals in cytosolic as well as nuclear extracts, with a
concomitant appearance of supershifted DNA-CPRF4a antibody complexes
(Fig. 4, A and B, lane 5). No effects were
observed when the corresponding preimmunoserum was added (Fig. 4,
A and B, lane 6). To remove peptide-bound
phosphate residues, we added alkaline phosphatase to cytosolic as well
as nuclear extracts prior to EMSA (Fig. 4, A and B,
lane 3). This treatment resulted in a strong decrease of the
CPRF4a-specific signals. In contrast, mock treatment caused no effects,
indicating that the reduction of the DNA binding activity of CPRF4a is
not due to a modification of the reaction conditions (Fig. 4,
A and B, lane 4). Taken together, these results
suggest that the DNA binding activity of CPRF4a is controlled in a
phosphorylation-dependent manner.
We next tested whether the cytosolic kinase activity for CPRF4a is
sufficient for an activation of the factor. For this purpose, we mixed
recombinant CPRF4a with ATP-containing or ATP-free cytosol and
incubated the reaction mixtures over a time period of 30 min. After
different incubation times, aliquots of the reaction mixtures were
removed, and the samples were subjected to EMSA using a monomeric G-box
probe (Fig. 5). Whereas the DNA binding
activity of CPRF4 incubated in ATP-free cytosol remained unchanged
(Fig. 5, -ATP), we observed a gradual up-shift as well as a
weak increase of the signals deriving from CPRF4a incubated in
ATP-containing cytosol during the course of the experiment (Fig. 5,
+ATP). Interestingly, under the identical experimental
conditions, no change in the DNA binding activity of recombinant CPRF2
was found (10). Controls displaying the weak endogenous DNA binding
activity of the cytosolic extracts indicate that the signals described
above derived mainly from recombinant proteins (Fig. 5,
cytosol).
In contrast to the strong decrease of the DNA binding activity of
endogenous CPRF4a after phosphatase treatment (Fig. 4), the effects
shown in Fig. 5 were relatively weak. We have demonstrated that
recombinant CPRF4a is phosphorylated in the presence of ATP-containing cytosol (Fig. 2). Therefore, the results shown in Fig. 5 suggest that
phosphorylation of recombinant CPRF4a by the cytosolic kinase activity
is insufficient to cause a full activation of the factor (see under
"Discussion" for further details).
CPRF4a Is Identical to the Previously Described Cytosolic bZIP-like
Factors--
As mentioned above, cytosolic bZIP-like proteins from
parsley have been described in a previous report in which they were identified by their ability to bind to a probe containing four G-box
elements (tetrameric G-box probe). The results of several experiments
presented in this study and elsewhere strongly suggested that these
factors are identical with CPRF4a: (i) CPRF4a and the unknown factors
contribute very strongly to the overall G-box binding activity. (ii)
Both CPRF4a and the unknown factors are localized in the cytosol
and in the nucleus, displaying a comparable intracellular distribution
(9, 13). (iii) The unknown factors were classified as bZIP-like by
their interaction with antibodies raised against the
Arabidopsis bZIP factor GBF1 (13). Likewise, GBF1 antiserum
strongly cross-reacts with endogenous
CPRF4a.2 To test whether
these factors are, in fact, identical with CPRF4a, we performed
supershift experiments with cytosolic extracts using the tetrameric
G-box probe (Fig. 6A). In
agreement with previous results (13), we observed the formation of
three distinct protein-DNA complexes (Fig. 6A, lane 2). The
addition of the specific CPRF4a antiserum to the binding reaction
mixtures caused a strong decrease of all three signals as well as a
supershifted band (Fig. 6A, lane 3). In contrast, neither
addition of a specific CPRF2 serum nor the corresponding preimmunosera
caused any effects (Fig. 6A, lanes 4-6). These results
indicate that CPRF4a is involved in the formation of all three
bands.
The appearance of three distinct signals deriving from CPRF4a activity
can be readily explained by different amounts of the factor that are
bound to the tetrameric G-box probe. This would lead to DNA-protein
complexes of different sizes that would be differentially retarded in
the gel. To test this possibility, we used a tetrameric probe for EMSA
in which one of the binding-sites was disrupted by nucleotide changes
within the G-box core sequence (Fig. 6B). In accordance with
our hypothesis, we observed a decrease in the number of signals
compared with the nonmutated probe (Fig. 6A), suggesting
that the number of factors that can bind to the probe is reduced.
As shown in Fig. 4, we have demonstrated that the DNA binding activity
of CPRF4a is reduced after phosphatase treatment. To confirm that the
three signals observed in Fig. 6A are indeed derived from
CPRF4a, we tested whether these signals are affected by
dephosphorylation as well. For this purpose, cytosol was treated with
alkaline phosphatase that was immobilized on Sepharose beads. Prior to
EMSA, the immobilized phosphatase was removed by centrifugation. As
shown in Fig. 6C, lane 3, the formation of all three signals was abolished by phosphatase treatment. This result supports our conclusion that all three signals derive from CPRF4a-activity.
After Dephosphorylation, the DNA Binding Activity of CPRF4a Can Be
Restored in an ATP-dependent Reaction--
As shown in
Figs. 4 and 6C, the DNA binding activity of CPRF4a is
reduced after phosphatase treatment suggesting that phosphorylation is
crucial for the regulation of the factor. This idea was confirmed by an
experiment, in which ATP was added to alkaline phosphatase-treated cytosol, from which the immobilized enzyme had been removed by centrifugation. This treatment caused an almost complete restoration of
the DNA binding activity of CPRF4a reflected by the reappearance of all
three signals in EMSA (Fig. 6C, lane 4; compare with
lane 2), whereas in an ATP-free control reaction, no effects
were observed (Fig. 6C, lane 3). This result indicates that
ATP-dependent processes are involved in the regulation of
the DNA binding activity of endogenous CPRF4a. Furthermore, it
demonstrates that cytosolic components are involved in the regulation
of the factor.
The DNA Binding Properties of CPRF4a Are Influenced by
Light--
Interestingly, the uppermost of the three signals that
derive from CPRF4a activity was never detected in cytosolic extracts that were isolated from cells kept in darkness (13). Further experiments revealed that light induces the formation of this signal in
an ATP-dependent reaction (13). To determine the
photoreceptor systems that are involved in the regulation of this
process, we irradiated dark-kept evacuolated protoplasts with different
light qualities and subsequently isolated cytosolic extracts. These extracts were tested in EMSA using the tetrameric G-box probe (Fig.
7). Whereas white light strongly induced
the formation of the third, uppermost signal (Fig. 7, lane
4), such a DNA-CPRF4a complex was not observed in extracts
isolated from dark-kept protoplasts (Fig. 7, lane 2).
Additionally, the signal of the middle band was enhanced. In comparison
to white light treatment, irradiation with either red or far-red light
caused similar but significantly weaker effects (Fig. 7, lanes
3 and 5). In total, light treatment resulted in an
enhancement of the overall G-box binding activity of cytosolic CPRF4a
and in the formation of an additional signal that might reflect a
different modification state of the factor (see under
"Discussion").
The effects of red light treatment and of far-red light treatment
indicate an involvement of the red light sensing phytochrome photoreceptors in the control of CPRF4a activity (1). However, white
light had a stronger effect on the DNA binding activity of the factor
than red light alone, suggesting that photoreceptor systems in
addition to phytochromes contribute to the regulation of CPRF4a activity.
In a previous report, we have studied the regulation of bZIP-like
factors from parsley that are localized in the cytosol as well as in
the nucleus (13). Here, we were able to show that these factors are
identical with CPRF4a (Fig. 6). This finding allows us to integrate the
previously reported data (13) with the results presented in this study.
We have demonstrated that the DNA binding activity of endogenous CPRF4a
is affected by the kinase inhibitor H-89 as well as by two different
phosphatase inhibitors (13). These results implied that a
kinase/phosphatase system contributes to the regulation of the factor.
In agreement with this idea, the DNA binding activity of endogenous
CPRF4a is strongly reduced by dephosphorylation (Figs. 4 and
6C) and the G-box binding activity of cytosolic CPRF4a after
dephosphorylation can be restored in an ATP-dependent
reaction (Fig. 6C). Taken together, the results presented in this study and elsewhere (13) indicate the important role of phosphorylation in
the regulation of CPRF4a.
As mentioned above, we were able to show that the DNA binding activity
of cytosolic CPRF4a after dephosphorylation can be restored in an
ATP-dependent reaction (Fig. 6C). Interestingly, in a
similar experiment, the G-box binding activity of nuclear localized
CPRF4a could not be restored (13). These results imply that cytosolic
components are necessary for the activation of the factor. The
identification of a cytosolic CPRF4a-specific kinase (Fig. 2), as well
as the phosphorylation-dependent regulation of the DNA
binding activity of CPRF4a, suggested that the kinase might directly
activate the factor by changing its phosphorylation state. In
accordance with this idea, we found similar effects of the kinase
inhibitor H-89 on the activity of the CPRF4a-kinase (Fig. 3) and on the
DNA binding activity of the factor (see above). However, compared with
the nonphosphorylated factor, the DNA binding activity of recombinant
CPRF4a changes only weakly after phosphorylation (Fig. 5). We conclude,
therefore, that the phosphorylation of CPRF4a by the cytosolic kinase
is not sufficient for a full activation of the factor and that
additional modifications are therefore required. In this scenario,
recombinant CPRF4a was not fully activated by ATP-containing cytosol
because these additional modifications require either noncytosolic
components (e.g. membrane proteins) and/or an intact cell
structure. Accordingly, the ATP-dependent restoration of
the DNA binding activity of cytosolic CPRF4a after dephosphorylation
(Fig. 6C) would indicate that the dephosphorylation process
does not affect the additional modifications that are required for a
full activation of the factor.
CPRF4a contains a conserved nuclear localization sequence (5). However,
a large pool of CPRF4a was found in the cytosol under all conditions
tested (9). Therefore, it has been proposed that CPRF4a is partly
retained in the cytosol by an unknown mechanism (9). Intracellular
distributions similar to those observed for CPRF4a have been described
for the bZIP factors GBF1 from Arabidopsis and for
G/HBF1 from soybean (21, 22). Interestingly, the DNA binding
activities of both factors are regulated by phosphorylation. Whereas
GBF1 is phosphorylated by a nuclear casein kinase II (23, 24), G/HBF1
is rapidly phosphorylated in elicited soybean cells by a cytosolic
kinase, leading to an enhancement of its DNA binding activity in
vitro (22). Dephosphorylation of G/HBF1 leads to a changed
immunoreactivity of the factor, pointing to a major conformational
change within the protein (22). It has been proposed that this
conformational change leads to an unmasking of the nuclear localization
signal of G/HBF1 allowing its nuclear import (22). An altered
conformation might also explain the additional CPRF4a signal that
appeared upon irradiation. Our results suggest that the appearance of
this band is due to an increased number of factors that are bound to
the tetrameric G-box probe (Fig. 6). This effect cannot be solely
explained by an increase of the overall DNA binding activity of the
cytosolic CPRF4a pool after irradiation because even high amounts of
protein from dark-kept cytosolic extracts do not cause the formation of
this band (13). An altered conformation, however, could facilitate
binding to the different G-boxes of the tetrameric probe that are in
close proximity by reducing steric hindrance between the factors.
The appearance of the additional CPRF4a signal in cytosolic extracts is
not only light-dependent but also ATP-dependent
(13). Furthermore, this effect is accompanied by an increase of the DNA
binding activity of CPRF4a (Fig. 7). As a working hypothesis, we
therefore suggest that the kinase/phosphatase system that is involved
in controlling the DNA binding activity of CPRF4a is also involved in
its light-dependent modification.
Interestingly, a G-box binding pattern that is similar to that of
cytosolic CPRF4a after irradiation has been observed for nuclear
localized CPRF4a (13). Therefore, a modification of CPRF4a might be a
prerequisite for nuclear localization. Moreover, it has been
demonstrated by cotranslocation assays using GBF1 antibodies that white
light treatment resulted in an enhanced translocation of cytosolic
bZIP-like factors to the nucleus (13). Because GBF1 antibodies strongly
cross-react with CPRF4a,2 this result could indicate a
light-induced nuclear import of CPRF4a. However, we cannot rule out the
possibility that this effect is due to cross-reactions of the
GBF1 antibodies with cytosolic bZIP factors others than CPRF4a. In
supershift experiments, we failed to detect a major change of the
cytosolic CPRF4a pool after light treatment (9). However, CPRF4a
accumulates very rapidly upon irradiation (5). This newly synthesized
protein could replace those factors that are transported to the
nucleus, leading to a relatively constant number of proteins in the
cytosolic CPRF4a pool.
The results of our irradiation experiments suggest that different
photoreceptor systems contribute to the regulation of CPRF4a. The
effect of red and far-red light on the DNA binding properties of CPRF4a
implies the involvement of the red light sensing phytochrome photoreceptors (1, 2). However, compared with irradiation with white
light, red light caused only minor effects. This result can be readily
explained by the involvement of a second photoreceptor system that acts
with phytochromes in an additive manner. Good candidates for such
receptors are the UV-A/blue light sensing cryptochromes, which, like
the phytochromes, play an important role in the development of plants
(25). This idea is supported by the fact that irradiation with UV and
blue light causes a rapid accumulation of CPRF4a protein (5). However,
further experiments have to be performed to confirm a role of
cryptochromes in the regulation of CPRF4a.
The important role of G-boxes in light-dependent gene
regulation has been confirmed recently by the identification of the G-box binding factors HY5 and PIF3 from Arabidopsis
(26-28). Reduced levels of these factors cause a decrease in the
photoresponsiveness of their putative target genes (29). Interestingly,
it has been shown that these factors are not capable of inducing all
light-regulated genes that contain functional G-boxes in their
promoters, indicating that additional factors are required (29). The
results of this study suggest that CPRF4a could be such an additional
G-box binding factor. A putative target gene for CPRF4a is the chalcone
synthase gene from parsley that is induced by UV light. It has been
recently suggested that a CPRF1-containing bZIP heterodimer is involved in the regulation of this gene (30). Because CPRF4a can form heterodimers with CPRF1 (5), it is a likely candidate for being the
partner of CPRF1 in chalcone synthase regulation. In addition to its
contribution in the formation of heterodimer complexes, CPRF4a might
also compete with other G-box binding proteins (e.g. HY5 or
PIF3) for binding sites and thereby may fine-tune the activities of
these factors. It is remarkable that other CPRF factors that are
regulated in response to light are controlled by mechanisms that are
different to that of CPRF4a. For example, light treatment does not
alter the DNA binding properties of CPRF2 (10). However, although CPRF2
is absent from the nucleus in the dark (9), light treatment leads to a
nuclear import of the factor. These results imply that an activation of
CPRF2 is achieved by its intracellular redistribution (9). Furthermore,
in contrast to CPRF4a, CPRF2 is efficiently modified in response to red
light, whereas other light qualities have only minor effects (9, 10).
These differences in light responsiveness and in the mechanisms
regulating their activities suggest that CPRF2 and CPRF4a contribute to
different aspects of light signal transduction and thereby act in a
nonredundant manner.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP, the reactions were
stopped with 500 µl of 6 M guanidine hydrochloride, 0.1 M NaH2PO4, 0.01 M Tris,
pH 8.0. The histidine-tagged proteins were isolated on nickel
nitrilotriacetic acid-agarose and eluted with 50 µl of 8 M urea, 0.1 M NaH2PO4,
0.01 M Tris, pH 6.3, and 100 mM EDTA. The
eluates were subjected to SDS-PAGE using a 12% (w/v) acrylamide gel
(17). Subsequently, the gels were silver-stained (18), dried, and
analyzed by autoradiography.
-32P]ATP. The labeling
reactions, as well as the purification of the recombinant proteins and
their analysis, were performed as described above.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Purification and characterization of the
recombinant CPRFs. A, after purification, 2 µg of
recombinant CPRFs (as indicated) were analyzed by SDS-PAGE and
Coomassie Blue staining. The position of protein molecular mass markers
in kilodaltons are indicated on the right. B,
after the folding procedure, the recombinant CPRF proteins were
subjected to EMSA to test their G-box binding activities. A monomeric
G-box probe was used for this assay. In lanes 2-4, 200 ng
of the individual factors were tested. In lane 1, no protein
was added (free probe).
-32P]ATP
was added, the reaction mixtures were incubated for 1 min at room
temperature. Subsequently, the recombinant factors were purified on
nickel nitrilotriacetic acid-agarose under denaturing conditions and
subjected to SDS-PAGE. Silver staining of the gels showed that the
recombinant proteins were recovered in similar amounts (Fig.
2, A and B, panel
II). Thus, the signals obtained after autoradiography (Fig. 2,
A and B, panel I) were compared directly. CPRF1
was only marginally phosphorylated in the presence of cytosolic
extracts (Fig. 2A, lane 1) and nuclear extracts (Fig. 2B, lane 1), whereas CPRF2 was strongly labeled in the
presence of both extracts (Fig. 2, A and B, lane
3). In contrast, CPRF4a was strongly phosphorylated by cytosolic
extracts (Fig. 2A, lane 5) but very weakly by nuclear
extracts (Fig. 2B, lane 5). Neither cytosolic nor nuclear
extracts showed detectable signals in the autoradiogram in the absence
of purified recombinant CPRFs (Fig. 2, A and B, lanes
2, 4, and 6). Taken together, our results show different and, in the case of CPRF4a, compartment-specific kinase activities for the individual CPRF proteins.
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Fig. 2.
Cytosolic and nuclear phosphorylation
activities for the recombinant CPRFs. 2 µg of the recombinant
proteins were mixed with cytosolic (A) or nuclear
(B) extracts. The phosphorylation reaction and the
purification of the factors were performed as outlined in the text.
After SDS-PAGE, the purified proteins were analyzed by autoradiography
(panel I) and silver staining (panel II).
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Fig. 3.
Inhibition of the cytosolic CPRF4a kinase
activity. 2 µg of recombinant CPRF4a were mixed with 50 µg of
cytosolic protein and 1 µl of Me2SO
(DMSO)-diluted kinase inhibitors as indicated (lanes
3-9). As controls, identical samples were supplemented with
either 1 µl of H2O (lane 1) or
Me2SO (DMSO) (lane 2) to a final
concentration of 0.1% (v/v). The phosphorylation reaction and the
purification of the factors were performed as outlined in the text.
After SDS-PAGE, the purified proteins were analyzed by autoradiography
(panel I) and silver staining (panel II).
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Fig. 4.
The DNA binding activity of CPRF4a is reduced
after phosphatase treatment. 50 µg of cytosolic protein
(A) and 20 µg of protein from a nuclear extract
(B) were subjected to EMSA using a monomeric G-box probe.
The extracts were either treated with alkaline phosphatase prior to
EMSA (lane 3) or mock-treated (lane 4).
CPRF4a-specific signals were identified by addition of a specific
CPRF4a polyclonal serum (lane 5) or the corresponding
preimmunoserum (lane 6). The signals deriving from CPRF4a
are indicated (C4). Supershifts are marked with an
arrow. In lane 1, no protein was added
(free probe), and in lane 2, the extracts were
tested without further treatment.
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Fig. 5.
Analysis of the DNA binding activity of
recombinant CPRF4a after phosphorylation. Recombinant CPRF4a was
mixed with ATP-containing cytosol (+ATP) or ATP-free cytosol
(-ATP). Reactions were stopped after 1, 2, 5, 10, 20, or 30 min as indicated. As a control, endogenous DNA binding activities of
ATP-free and ATP-containing cytosol are also shown
(cytosol). The samples were tested by EMSA using a monomeric
G-box probe.
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Fig. 6.
Identification of CPRF4a in cytosolic
extracts. A, 20 µg of cytosolic protein were tested
in EMSA using a tetrameric G-box probe. The three signals deriving from
untreated cytosolic extracts (lane 2) are numbered
(1-3) on the right. To identify CPRF-specific signals, 1 µl of either CPRF4a antiserum (lane 3) or CPRF2 antiserum
(lane 5), as well as of the corresponding preimmunosera
(lanes 4 and 6), was added to the binding
mixtures prior to EMSA. An arrow indicates the supershifted
CPRF4a-DNA complex in lane 3. In lane 1, no protein was
added (free probe). B, 20 µg of cytosolic
protein were tested in EMSA using a tetrameric probe in which the
second of the G-box repeats was disrupted by nucleotide exchanges
within the core sequence. No protein was added in lane 1 (free probe). C, 20 µg of cytosolic protein
were treated with immobilized alkaline phosphatase (lanes
3-4) or mock-treated (lane 2). Subsequently, the
immobilized phosphatase was removed by centrifugation. The samples were
then either supplemented with ATP (lane 4) or mock-treated
(lanes 2 and 3) and, after a 30-min incubation
step, subjected to EMSA. The three signals deriving from cytosolic
extracts are numbered (1-3) on the right. In
lane 1, no protein was added (free probe).
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Fig. 7.
Light-dependent modification of
the DNA binding properties of CPRF4a. Dark-grown evacuolated
protoplasts were either irradiated for 20 min with red light
(lane 3), white light (lane 4), or far-red light
(lane 5) or kept in darkness (lane 2).
Subsequently, cytosolic extracts were isolated, and 10 µg of protein
were analyzed by EMSA using a tetrameric G-box probe. In lane
1, no protein was added (free probe). The three signals
deriving from cytosolic extracts after irradiation are numbered
(1-3) on the left.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* This work was supported in part by Deutsche Forschungsgemeinschaft Grant SFB 388 (to E. S. and K. H.) and Human Frontier Science Program Grant RG-43/97 (to K. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: California Institute of Technology, Division of
Biology 156-29, Pasadena, CA 91125. Supported by the Graduiertenkolleg Molekulare Mechanismen pflanzlicher Differenzierung.
§ To whom correspondence should be addressed. Tel.: 49-761-203-2686; Fax: 49-761-203-2612; E-mail harterkl@uni-freiburg.de.
Published, JBC Papers in Press, December 5, 2000, DOI 10.1074/jbc.M007971200
2 F. Wellmer, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: bZIP, basic leucine zipper motif; CPRF, common plant regulatory factor; GBF, G-box binding factor; PAGE, polyacrylamide gel electrophoresis; EMSA, electrophoretic mobility shift assay.
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REFERENCES |
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