Phosphatidic acid binds to and inhibits the activity of Arabidopsis CTR1.
ABSTRACT Phosphatidic acid (PA) has only recently been identified as an important eukaryotic lipid-signalling molecule. In plants, PA formation is triggered by various biotic and abiotic stresses, including wounding, pathogen attack, drought, salinity, cold, and freezing. However, few molecular targets of PA have been identified so far. One of the best characterized is Raf-1, a mammalian MAPKKK. Arabidopsis thaliana CTR1 (constitutive triple response 1) is one of the plant homologues of Raf-1 and functions as a negative regulator of the ethylene signalling pathway. Here, it is shown that PA binds CTR1 and inhibits its kinase activity. Using different PA-binding assays, the kinase domain of CTR1 (CTR1-K) was found to bind PA directly. Addition of PA resulted in almost complete inhibition of CTR1 kinase activity and disrupted the intramolecular interaction between CTR1-K and the CTR1 N-terminal regulatory domain. Additionally, PA blocked the interaction of CTR1 with ETR1, one of the ethylene receptors. The basic amino acid motif shown to be required for PA binding in Raf-1 is conserved in CTR1-K. However, mutations in this motif did not affect either PA-binding or PA-dependent inhibition of CTR1 activity. Subsequent deletion analysis of CTR1's kinase domain revealed a novel PA-binding region at the C-terminus of the kinase.
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ABSTRACT: Recent evidence has demonstrated that both copper amine oxidase (CuAO; EC 220.127.116.11) and phospholipase D (PLD; EC 18.104.22.168) are involved in abscisic acid (ABA)-induced stomatal closure. In this study, we investigated the interaction between CuAO and PLD in the ABA response. Pretreatment with either CuAO or PLD inhibitors alone or that with both additively led to impairment of ABA-induced H2O2 production and stomatal closure in Vicia faba. ABA-stimulated PLD activation could not be inhibited by the CuAO inhibitor, and CuAO activity was not affected by the PLD inhibitor. These data suggest that CuAO and PLD act independently in the ABA response. To further examine PLD and CuAO activities in ABA responses, we used the Arabidopsis mutants cuaoζ and pldα1. Ablation of guard cell-expressed CuAOζ or PLDα1 gene retarded ABA-induced H2O2 generation and stomatal closure. As a product of PLD, phosphatidic acid (PA) substantially enhanced H2O2 production and stomatal closure in wide type, pldα1, and cuaoζ. Moreover, putrescine (Put), a substrate of CuAO as well as an activator of PLD, induced H2O2 production and stomatal closure in WT but not in both mutants. These results suggest that CuAO and PLD act independently in ABA-induced stomatal closure.Journal of Plant Research 05/2014; · 2.06 Impact Factor
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ABSTRACT: Phospholipase D (PLD) and its product phosphatidic acid (PA) are incorporated in a complex metabolic network in which the individual PLD isoforms are suggested to regulate specific developmental and stress responses, including plant programmed cell death (PCD). Despite the accumulating knowledge, the mechanisms through which PLD/PA operate during PCD are still poorly understood. In this work, the role of PLDα1 in PCD and the associated caspase-like proteolysis, ethylene and hydrogen peroxide (H2O2) synthesis in tomato suspension cells was studied. Wild-type (WT) and PLDα1-silenced cell lines were exposed to the cell death-inducing chemicals camptothecin (CPT), fumonisin B1 (FB1) and CdSO4. A range of caspase inhibitors effectively suppressed CPT-induced PCD in WT cells, but failed to alleviate cell death in PLDα1-deficient cells. Compared to WT, in CPT-treated PLDα1 mutant cells, reduced cell death and decreased production of H2O2 were observed. Application of ethylene significantly enhanced CPT-induced cell death both in WT and PLDα1 mutants. Treatments with the PA derivative lyso-phosphatidic acid and mastoparan (agonist of PLD/PLC signalling downstream of G proteins) caused severe cell death. Inhibitors, specific to PLD and PLC, remarkably decreased the chemical-induced cell death. Taken together with our previous findings, the results suggest that PLDα1 contributes to caspase-like-dependent cell death possibly communicated through PA, reactive oxygen species and ethylene. The dead cells expressed morphological features of PCD such as protoplast shrinkage and nucleus compaction. The presented findings reveal novel elements of PLD/PA-mediated cell death response and suggest that PLDα1 is an important factor in chemical-induced PCD signal transduction.Protoplasma 04/2013; · 2.86 Impact Factor
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ABSTRACT: We previously identified a gene related to the SEC14-gene phospholipid transfer protein superfamily that is induced in Nicotiana benthamiana (NbSEC14) in response to infection with Ralstonia solanacearum. We here report that NbSEC14 plays a role in plant immune responses via phospholipid-turnover. NbSEC14-silencing compromised expression of defense-related PR-4 and accumulation of jasmonic acid (JA) and its derivative JA-Ile. Transient expression of NbSEC14 induced PR-4 gene expression. Activities of diacylglycerol kinase, phospholipase C and D, and the synthesis of diacylglycerol and phosphatidic acid elicited by avirulent R. solanacearum were reduced in NbSEC14-silenced plants. Accumulation of signaling lipids and activation of diacylglycerol kinase and phospholipases were enhanced by transient expression of NbSEC14. These results suggest that the NbSEC14 protein plays a role at the interface between lipid signaling-metabolism and plant innate immune responses.PLoS ONE 01/2014; 9(5):e98150. · 3.53 Impact Factor
Journal of Experimental Botany, Vol. 58, No. 14, pp. 3905–3914, 2007
doi:10.1093/jxb/erm243Advance Access publication 13 November, 2007
This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
Phosphatidic acid binds to and inhibits the activity
of Arabidopsis CTR1
Christa Testerink1,*,†, Paul B. Larsen2,†, Dieuwertje van der Does1, John A. J. van Himbergen1and
1Section of Plant Physiology, Swammerdam Institute for Life Sciences, University of Amsterdam, Kruislaan 318,
NL-1098 SM Amsterdam, The Netherlands
2Department of Biochemistry, University of California-Riverside, Riverside, California, CA 92521, USA
Received 3 August 2007; Revised 13 September 2007; Accepted 14 September 2007
Phosphatidic acid (PA) has only recently been identified
as an important eukaryotic lipid-signalling molecule. In
plants, PA formation is triggered by various biotic and
abiotic stresses, including wounding, pathogen attack,
drought, salinity, cold, and freezing. However, few
molecular targets of PA have been identified so far. One
of the best characterized is Raf-1, a mammalian
MAPKKK. Arabidopsis thaliana CTR1 (constitutive triple
response 1) is one of the plant homologues of Raf-1
and functions as a negative regulator of the ethylene
signalling pathway. Here, it is shown that PA binds
CTR1 and inhibits its kinase activity. Using different
PA-binding assays, the kinase domain of CTR1 (CTR1-K)
was found to bind PA directly. Addition of PA resulted
in almost complete inhibition of CTR1 kinase activity
and disrupted the intramolecular interaction between
CTR1-K and the CTR1 N-terminal regulatory domain.
Additionally, PA blocked the interaction of CTR1 with
ETR1, one of the ethylene receptors. The basic amino
acid motif shown to be required for PA binding in Raf-1
is conserved in CTR1-K. However, mutations in this
motif did not affect either PA-binding or PA-dependent
analysis of CTR1’s kinase domain revealed a novel
PA-binding region at the C-terminus of the kinase.
Key words: Constitutive triple response 1, ethylene, lipid
signalling, phosphatidic acid, plant stress signalling, protein
Phosphatidic acid (PA) has been shown to represent an
important lipid second messenger in several eukaryotic
systems, including plants. It is produced via the phospho-
lipase D (PLD) or the phospholipase C/diacylglycerol
kinase-mediated pathways (Meijer and Munnik, 2003;
Testerink and Munnik, 2004; Wang, 2004, 2005).
In plants, PA levels increase rapidly and transiently
in response to several environmental stress conditions
including drought, wounding, high salinity, pathogen attack,
chilling, and freezing (Testerink and Munnik, 2005).
Analyses of Arabidopsis thaliana PLD mutants with
reduced PA accumulation have further established the
significance of stress-induced PA formation. Knockout or
knockdown mutations of the PLDa1 isoform display altered
responses to ABA, drought, wounding, and freezing (Wang
et al., 2000; Sang et al., 2001; Welti et al., 2002; Zhang
et al., 2004; Mishra et al., 2006). Similarly, PLDd knockout
mutants have reduced drought, UV, and freezing resistance
(Zhang et al., 2003; Li et al., 2004), which may result from
an increased sensitivity to reactive oxygen species that are
generated from these stresses. PLDf isoforms seem to play
a role in coping with phosphate starvation as well as in
normal root development and auxin responses (Ohashi et al.,
2003; Cruz-Ramirez et al., 2006; Li et al., 2006; Li and
Xue, 2007). By contrast, the early PA responses induced
by pathogens and cold seem to be generated by the
phospholipase C/diacylglycerol kinase pathway (Van der
Luit et al., 2000; Ruelland et al., 2002; de Jong et al., 2004;
Gomez-Merino et al., 2004).
* To whom correspondence should be addressed. E-mail: firstname.lastname@example.org
yThese authors contributed equally to this work.
Abbreviations: ER, endoplasmic reticulum; GST, glutathione S-transferase; MBP, myelin basic protein or maltose binding protein (as indicated); PA,
phosphatidic acid; PC, phosphatidylcholine; PI4P, phosphatidylinositol 4-phosphate; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PLD, phospholipase
D; PS, phosphatidylserine.
ª 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which
permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
While these genetic analyses have shown that various
plant stress responses require a PA signal. However, little
is known about the mechanisms underlying plant PA
signalling because few in vivo PA targets have been
identified. One reported PA target in Arabidopsis is
phosphoinositide-dependent protein kinase 1 (AtPDK1),
which binds PA through its pleckstrin homology domain
(Deak et al., 1999). Moreover, PA was found to stimulate
AtPDK1 activity as well as the in vivo activity of its
substrate AGC2-1 in a PDK1-dependent manner (Anthony
et al., 2004, 2006). AGC2-1 is identical to OXI1, a protein
kinase that mediates oxidative stress responses (Rentel
et al., 2004). Another demonstrated Arabidopsis PA target
is ABI1, which is a protein phosphatase 2C that is
a negative regulator of ABA signalling and is specifically
inhibited by PA (Zhang et al., 2004; Mishra et al., 2006).
Whereas functional ABI1 translocates to the plasma
membrane in response to ABA, a non-PA-binding mutant
did not, suggesting a role for PA in membrane targeting.
Several other putative plant PA targets, including the
PP2a regulatory subunit RCN1, were identified in a screen
using PA affinity beads coupled with mass spectrometry-
based identification of the isolated proteins (Testerink et al.,
2004). Moreover, PA was shown to play a role in actin
organization via binding of heterodimeric capping protein
(Lee et al., 2003; Huang et al., 2006).
PA targets have also been identified in other eukaryotic
systems. In yeast, the inositol-regulated transcriptional
repressor Opi1 has been identified as a PA target that
translocates from the nucleus to the endoplasmic re-
ticulum (ER) when PA levels in the ER increase (Loewen
et al., 2004). Mammalian PA targets comprise proteins
involved in mitogenic signalling, vesicular trafficking, and
the oxidative burst (Jenkins and Frohman, 2005; Testerink
and Munnik, 2005; Stace and Ktistakis, 2006). One of the
best characterized of these is the MAPKKK Raf-1, which
binds PA specifically at a concise region of basic amino
acids in its kinase domain (Ghosh and Bell, 1997).
Disruption of these amino acids severely reduced the PA-
binding capacity (Rizzo et al., 2000) and resulted in
abnormal development of zebrafish embryos (Ghosh
et al., 2003).
A plant homologue of Raf-1 is constitutive triple
response 1 (CTR1). It functions to negatively regulate
ethylene responses in Arabidopsis (Kieber et al., 1993).
Ethylene is a classic plant hormone that is responsible for
a variety of developmental phenomena such as fruit
ripening and senescence, together with responses to
pathogen attack and wounding. Ethylene signal trans-
duction starts with a family of five receptors that bind
ethylene (Chang and Bleecker, 2004; Guo and Ecker,
2004; Chen et al., 2005). Immediately downstream of
these receptors lays CTR1, which has been shown to
directly interact with several of the ethylene receptors at
the ER (Gao et al., 2003). In the absence of ethylene, this
interaction is thought to maintain CTR1 in an active state,
thus repressing ethylene responses. Mutational loss of the
ethylene receptors results in a constitutive ethylene re-
sponse phenotype (Hua and Meyerowitz, 1998; Huang
et al., 2003). Little is known about the biochemical
regulation of CTR1 activity. The CTR1 protein has been
expressed in insect cells and characterized. The full-length
protein as well as the kinase domain alone was shown to
have ser/thr kinase activity (Huang et al., 2003).
Interestingly, CTR1’s kinase domain has an amino acid
motif composed of several basic amino acids that is
similar to the PA-binding site found in Raf-1. This
prompted us to investigate whether CTR1 is a putative
PA target. Here, the specific binding of PA to CTR1 is
reported, along with its effects on CTR1 kinase activity
and the intra- and intermolecular interactions in which
Materials and methods
Generation of recombinant glutathione S-transferase (GST)
fusion proteins and35S-labelled proteins
The GST–CTR11–821and GST–CTR1551–821fusion proteins were
produced by transfection of Sf9 insect cells by recombinant
baculovirus using the Baculovirus Expression Vector System (BD-
Pharmingen). Mutant versions of the GST–CTR1 fusion proteins
were generated by using the Stratagene Quikchange site-directed
mutagenesis kit (La Jolla, CA, USA).
The GST, GST–PDK1, GST–CTR1654–821, CTR1654–742, and
CTR1739–821proteins were produced in Escherichia coli. GST–
CTR1 fusion constructs were obtained by amplifying the CTR1
fragments by PCR and cloning them into the destination vector
pDest15, using the Gateway recombination system. The primers
used were: 5#-AAAAAGCAGGCTGTATG-GCTTATGATGTGGC-
T-3# and 5#-AGAAAGCTGGGTTTTACAAATCCGAGCGGTT-3#
for CTR1654–821, 5#-AAAAAGCAGGC-TGTATGGCTTATGATG-
TGGCT-3# and 5#-AGAAAGCTGGGTCAAGCTACCACAAGAT-
GAC-3# for CTR1654–742, and 5#-AAAAAGCAGGCTGGGTCATC-
TTGTGGGAGCTT-3# and 5#-AGAAAGCTGGGTTTTACAAAT-
CCGAGCGGTT-3# for CTR1739–821. The constructs were trans-
formed to E. coli strain BL21-AI, and expression was induced by
arabinose according to the manufacturer’s instructions (Invitrogen).
Total soluble protein was isolated and GST-tagged proteins were
purified using glutathione–Sepharose.
GFP–SnRK2.10 protein was expressed in tobacco BY-2 cells (C
Testerink et al., unpublished data). In short, cells were treated with
250 mM NaCl for 15 min to induce activity of the kinase. GFP–
SnRK2.10 protein was immunoprecipitated using an anti-GFP
Radiolabelled test proteins were synthesized using the TnT T7
Coupled Transcription/Translation System (Promega, Madison, WI,
USA) using Redivue [35S] Pro-mix containing L-[35S]methionine
and L-[35S]cysteine (Amersham Biosciences, Piscataway, NJ, USA).
In vitro protein kinase assays
Approximately 50 ng of the various GST–CTR1 fusion proteins or
GFP–SnRK2.10 protein was incubated with 5 lg of myelin basic
protein (MBP) (Sigma Chemical, St Louis, MO, USA) for 30 min
at 22 ?C in a total volume of 30 ll. Reaction conditions consisted
of 50 mM TRIS (pH 7.5), 2 mM DTT, 10 mM MgCl2, 10 lM
3906 Testerink et al.
non-radioactive ATP, and 5 lCi c-[32P]ATP in the absence or
presence of synthetic lipids (Avanti Polar Lipids). Following
incubation, reactions were terminated by addition of 63 SDS
sample loading buffer after which samples were separated by SDS–
PAGE, and results were visualized by autoradiography.
In vitro protein binding assays
Maltose binding protein (MBP)-fusion proteins were produced as
previously described (Larsen and Cancel, 2003). For this assay,
5 lg of each fusion protein was used in a binding assay with 25 ll
of CTR1551–821-radiolabelled protein either in the presence or ab-
sence of 100 nmol of PC or PA in a total volume of 400 ll. Assays
were performed as previously described (Clark et al., 1998), except
that samples were incubated for 16 h at 4 ?C.
PA bead assays were performed according to Testerink et al.
(2004). For this assay, 300 ng of purified protein was added to 3 ll
of beads (containing 2.6 lmol of dipalmitoyl PA ml?1). Samples
were separated on SDS–PAGE and subjected to western analysis
using an anti-GST antibody (Santa Cruz).
Liposome assays were performed as described before (Levine and
Munro, 2002; Loewen et al., 2004) with some modifications. Per
sample, 640 nmol of total lipids were used. Synthetic dioleoyl
phosphatidylcholine (PC) and phosphatidylserine (PS) and natural
PA (from egg yolk, mainly consisting of C16:0, C18:1 PA),
phosphatidylinositol 4-phosphate (PI4P), and phosphatidylinositol
4,5-bisphosphate [PI(4,5)P2] (from brain tissue) were used (all from
Avanti Polar Lipids, Alabaster, AL, USA). Lipids were mixed in the
right molar ratios in chloroform, or chloroform:MeOH 20:9 for
phosphoinositides, dried, and rehydrated in extrusion buffer (250
mM raffinose, 25 mM TRIS pH 7.5, 1 mM DTT) for 1 h.
Unilamellar vesicles were produced using a lipid extruder (0.2 lm
filters; Avanti Polar Lipids) according to the manufacturer’s
instructions. Liposomes were diluted in three volumes of binding
buffer (125 mM KCl, 25 mM TRIS pH 7.5, 1 mM DTT, 0.5 mM
EDTA) and pelleted by centrifugation at 50 000 g for 15 min.
Liposomes were resuspended in binding buffer, added to 1250 ng
purified GST-tagged protein, and incubated for 30–45 min in a total
volume of 50 ll at room temperature. Liposomes were harvested by
centrifugation at 16 000 g for 30 min, washed once in binding
buffer, and resuspended in Laemmli sample buffer. Samples were
run on SDS–PAGE and gels were stained with colloidal Coomassie
staining (Sigma), scanned, and quantified with ImageQuant
(Molecular Dynamics, Sunnyvale, CA, USA). For in vitro translated
35S-labelled proteins, 5 ll of the total volume was used in a standard
liposome assay, and run on SDS–PAGE.35S-Labelled proteins were
visualized by autoradiography and quantified by phosphoimaging
The kinase domain of CTR1 binds PA
In order to test whether CTR1 is able to bind PA, both
full-length protein (CTR1–FL; CTR11–821) and the kinase
domain alone (CTR1-K; CTR1551–821) were produced as
GST-fusion proteins in Sf9 insect cells transfected with
recombinant baculovirus. Binding to pure PA was tested
by using PA beads (previously described in Manifava
et al., 2001; Lim et al., 2002; Testerink et al., 2004). Both
bound PA (Fig. 1), with GST–CTR1551–821
a higher affinity than GST–CTR11–821. GST-tagged
AtPDK1, which has previously been shown to bind PA
and several phosphoinositides (Deak et al., 1999; Anthony
et al., 2004), was included as a positive control. In
contrast, GST alone did not bind the PA beads, indicating
that the observed interaction between GST–CTR1 and PA
was not dependent on the GST domain (Fig. 1).
To determine whether CTR1 specifically bound PA,
lipid-binding specificity of GST–CTR11–821was tested in
a liposome assay. In this assay, vesicles consisting of PC
mixed with low concentrations of different charged
phospholipids, including PA, were loaded with raffinose.
Liposomes were pelleted and protein from the supernatant
and pellet fractions was analysed by SDS–PAGE. GST–
PDK1 and GST alone were used as controls. GST–
CTR11–821specifically bound liposomes containing 5%
PA, with negligible binding to liposomes that contained
the same amount of PI4P or PI(4,5)P2. Some binding was
observed for liposomes containing 20% PS, but this was
less than for PA liposomes (7% binding versus 19%
binding, respectively; Fig. 2). By contrast to CTR1,
AtPDK1 associated equally well with liposomes contain-
ing 5% PA, PI4P, or PI(4,5)P2, as has been found before
using a lipid overlay assay (Deak et al., 1999). GST alone
did not bind to any of the liposomes tested. From these
results, it appears that CTR1 preferentially binds PA.
Fig. 1. CTR1 binds PA beads. To determine whether CTR1 was able to
bind PA, GST fusions of full-length CTR1 (GST–CTR1–FL, CTR11–821)
and the CTR1 kinase domain (GST–CTR1–K, CTR1551–821) as well as
the positive control protein GST–PDK1 and the negative control GST
were incubated with 3 ll control beads (C) or PA beads (PA). Start
material (7%), supernatant (non-bound; 7%), and pellet (bound; 100%)
fractions were separated by SDS–PAGE and visualized by western
analysis using an anti-GST antibody.
Phosphatidic acid regulates CTR1 activity 3907
CTR1 kinase activity is inhibited by PA
To test whether the observed association of PA with
CTR1 has an effect on CTR1 activity, an in vitro kinase
assay was used in which the activity of GST–CTR11–821
was tested in the presence or absence of 1-palmitoyl-2-
oleoyl-PC, PS, or PA. For this assay, approximately
50 ng of GST–CTR11–821was incubated with myelin
basic protein (MBP), which served as the substrate for
CTR1, c-[32P]ATP, and the appropriate buffer. Follow-
ing incubation, the samples were separated electropho-
retically using an SDS–PAGE system and visualized by
GST–CTR11–821in the presence of MBP gave the
previously described phosphorylation pattern (Huang
et al., 2003; Larsen and Cancel, 2003), including
phosphorylation of MBP and autophosphorylation of
CTR1 (Fig. 3A). Addition of 1 nmol of PC had no
detectable effect on either substrate phosphorylation or
autophosphorylation. In contrast, addition of 1 nmol of PS
had a moderately inhibitory effect on both CTR1
activities, while addition of 1 nmol of PA resulted in
almost complete inhibition of both substrate phosphory-
(Fig. 3A), suggesting that PA is a negative regulator of
It was tested whether acyl chain length is a determi-
nant for the effectiveness of PA as a negative regulator
of CTR1 function. For this analysis, the inhibitory
effect of long-chain 1-palmitoyl-2-oleoyl-PA (C16:0,
C18:1) was compared with short-chain dioctanoyl-PA
(di-C8:0) using the aforementioned kinase assay. While
1 nmol of long-chain PA resulted in almost complete
inhibition, 1 nmol of short-chain PA had only a moder-
ate effect on CTR1 activity (Fig. 3B). Thus, long-chain
PA was significantly more effective in inhibition of CTR1
activity compared with short-chain PA, indicating that
both the lipid head group and chain length are critical
Next, it was investigated whether PA could inhibit
activity of the isolated CTR1 kinase domain, GST–
CTR1551–821. The kinase domain alone appeared to be
less active than the full-length protein. Again, PA was
found to cause profound inhibition of kinase activity, both
in terms of autophosphorylation and MBP phosphory-
lation (Fig. 3C).
In order to ascertain whether PA inhibition of CTR1
activity could represent a non-specific detergent effect, it
was determined whether PA also had an impact on
SnRK2.10 activity, which is a protein kinase that has
been identified previously as a PA-binding protein
(Testerink et al., 2004). SnRK2.10 was generated in
tobacco BY-2 cells as a GFP fusion with immunoprecipi-
tated GFP–SnRK2.10 being tested with the in vitro kinase
assay using MBP as the substrate either in the presence or
absence of 1-palmitoyl-2-oleoyl-PC, PS, or PA. Neither of
these lipids at 0.1 nmol or 1 nmol had a measurable effect
on GFP–SnRK2.10 activity (Fig. 3D).
Association of PA with CTR-K and subsequent
inhibition of kinase activity is not dependent on the
conserved Raf-1 PA-binding motif
In mammalian Raf-1 kinase, a short, highly basic amino
acid motif has been demonstrated to bind PA. Since
a similar motif is found in the kinase domain of CTR1, we
hypothesized that this motif might also be responsible for
PA binding. Since mutation of two or three of the basic
amino acid residues to alanines reduced or abolished PA-
binding in Raf-1 (Ghosh et al., 2003), the corresponding
mutations were introduced into the GST–CTR11–821
baculovirus construct (Fig. 4A). However, as shown in
Fig. 4B, the triple mutant (CTR1K601A,R602A,R604A) bound
to PA liposomes just as well as the wt GST–CTR11–821
To determine whether the mutations had an effect on
PA’s ability to inhibit CTR1 activity, GST–CTR11–821,
GST–CTR1K601A,R604A, and GST–CTR1K601A,R602A,R604A
proteins were tested in the in vitro kinase assay using
MBP as a substrate. Consistent with the findings on PA
(CTR1K601A,R602A,R604A) mutations had no effect on the
strongly inhibitory effect of PA (Fig. 4C). Surprisingly,
the CTR1K601A,R604Aand CTR1K601A,R602A,R604Amutant
proteins showed a progressive reduction in kinase activity
compared with the CTR1 wt protein, indicating that
these amino acid residues are somehow required for
CTR1 activity (Fig. 4D). Based on the present binding
and activity assays, it is unlikely that this basic amino
acid motif is responsible for the PA binding of CTR1,
Fig. 2. CTR1 preferentially binds liposomes containing PA. Lipid-
binding specificity of CTR1 was assessed in a liposome-binding assay.
Using a lipid extruder, large unilamellar vesicles of different phospho-
lipid composition were produced. GST-tagged protein (1250 ng) was
incubated with 640 nmol of liposomes. Bound (p; 100%) and unbound
(s; 50%) protein was separated on SDS–PAGE and visualized by
colloidal Coomassie brilliant blue staining. Lipid mixtures: PS, PC/PS
80:20; PA, PC/PS/PA 80/15/5; PIP2, PC/PS/PIP280/15/5; PIP, PC/PS/
PIP 80:15:5. The concentration of PA that would be available for
binding was 640 lM.
3908 Testerink et al.
suggesting that a novel undefined motif in CTR1’s kinase
associates with PA.
The PA binding capacity of CTR1 resides in the
C-terminal region of its kinase
Since binding of PA to CTR1’s kinase domain does not
follow the Raf-1 paradigm, another approach was used.
To find the PA-binding region, [35S]methionine-labelled
in vitro-translated truncations of CTR1’s kinase were
analysed for their ability to bind PA liposomes. The
CTR1551–821, CTR1600–821, and CTR1654–821fragments
(Fig. 5A) bound 5% PA-containing liposomes with similar
high affinity (Fig. 5B), indicating that the PA-binding
capacity resides in the extreme C-terminus of the protein.
Based on the above results, three new constructs were
designed (Fig. 5A). The CTR1654–821fragment, and two
smaller fragments, CTR1654–742and CTR1739–821, were
produced as GST fusion proteins in E. coli and sub-
sequently tested for their ability to bind liposomes
containing increasing concentrations of PA. The GST–
CTR1654–821fragment was found to have affinity for PA
(Fig. 5A), confirming the binding data of the in vitro-
translated fragments. When GST–CTR1654–821was sub-
divided into smaller fragments, mainly GST–CTR1654–742
and, to a lesser extent, GST–CTR1739–821were found to
contribute to PA binding.
CTR1739–821had some affinity for PS- (0% PA in Fig.
5C) or PA-containing liposomes, but showed no specific-
ity for PA, since binding did not increase upon increasing
the concentration of PA in the liposomes. On the other
hand, GST–CTR1654–742bound PA specifically, although
with lower affinity than the GST–CTR1654–821fragment
(Fig. 5C). The lower affinity can be accounted for by
reduction in non-selective basal binding to 0% PA-
liposomes. Thus the PA-binding region seems to be
primarily determined by the amino acid sequence of the
CTR1654–742fragment, although residues in the CTR1739–821
fragment may serve to increase the affinity for PA.
Examination of the CTR1654–742region did not reveal any
highly basic amino acid motifs that could be responsible
PA inhibits intra- and intermolecular interactions of
CTR1’s kinase domain
Previous work using an in vitro binding assay revealed
that the kinase domain and amino-terminal regulatory
domain of CTR1 interact, suggesting that this association
may control CTR1 activity (Larsen and Cancel, 2003).
Fig. 3. PA inhibits CTR1 kinase activity in vitro. (A) To determine whether PA had an effect on CTR1 kinase activity, 50 ng of the full-length
protein, GST–CTR11–821were incubated for 30 min with 5 lg of myelin basic protein (MBP) and 5 lCi c-[32P]ATP in the presence or absence of
1 nmol PC, PS, or PA (all C16:0, C18:1). Proteins were separated by SDS–PAGE and phosphorylation was visualized by autoradiography.
Arrowheads indicate MBP phosphorylation and GST–CTR1 auto-phosphorylation. (B) The in vitro kinase assay was repeated for GST–CTR11–821in
order to determine the effectiveness of short-chain (di-C8:0) compared with long-chain (C16:0, C18:1) PA for inhibition of CTR1 activity. (C) The
inhibitory effect of PA was also determined for GST–CTR1551–821, with the effectiveness of 1 nmol PC or PA (C16:0, C18:1) being tested, using the
in vitro kinase assay described. (D) As a control, GFP–SnRK2.10 in vitro kinase activity was measured in the absence or presence of PC, PS, or PA
(C16:0, C18:1) as described above for GST–CTR1. MBP phosphorylation is indicated.
Phosphatidic acid regulates CTR1 activity 3909
Consequently, it was determined whether PA affected this
intramolecular association. In order to test this, a fusion
protein of maltose binding protein (MBP) combined with
CTR153–568was produced using a bacterial expression
system. Subsequently, either MBP or MBP-CTR153–568
were incubated for 18 h at 4 ?C with [35S]methionine-
labelled CTR1551–821in the presence or absence of 1-
palmitoyl-2-oleoyl-PC or PA. Samples were washed to
remove unbound probe, separated by SDS–PAGE, and
visualized by autoradiography. As described earlier,
CTR1’s amino terminus clearly bound the radiolabelled
CTR1 kinase. Addition of PC had no effect on this, while
PA almost completely blocked the intramolecular associ-
ation (Fig. 6). This disruption is at least partly dependent
on chain length since short-chain PA (di-C8:0) had only
a limited effect on the CTR1 intramolecular association
(data not shown).
In addition to binding to its amino-terminal domain, it
has also been found that CTR1’s kinase associates with
the cytoplasmic portion of the ethylene receptor, ETR1.
Using the same in vitro binding assay,
CTR1551–821was incubated in the presence or absence of
100 nmol 1-palmitoyl-2-oleoyl-PC or PA with MBP or
MBP-ETR1293–729. Analysis revealed that, as with the
CTR1 intramolecular association, addition of PA, but not
PC, significantly reduced the association of CTR1551–821
with MBP-ETR1293–729. Although addition of 100 nmol
of PA slightly reduced binding to MBP alone, the
inhibition of CTR1 binding to the MBP fusions was far
more severe. It should also be noted that the association of
CTR1551–821with these MBP fusions was not indiscrimi-
nate, for it was not possible to show any binding of
CTR1551–821to MBP–EER3 or MBP–EER4 (MJ Christians,
LM Robles, PB Larsen, unpublished data).
Fig. 4. The conserved Raf-1 PA-binding motif in CTR1 is not required for PA-binding or PA-dependent inhibition of kinase activity. (A) Schematic
representation of Arabidopsis CTR1 and the mutations introduced. (B) Binding of GST–CTR1 and GST–CTR1K601A,R602A,R604Ato PA-containing
liposomes. Protein (1250 ng) was incubated with 640 nmol of liposomes. Bound proteins were separated by SDS–PAGE and stained using colloidal
Coomassie brilliant blue. 0% PA, PC/PS 80/20; 5% PA, PC/PS/PA 80/15/5; 20% PA, PC/PA 80/20. (C) The effects of the CTR1K601A,R604A
and CTR1K601A,R602A,R604Amutations on the inhibitory effect of PA on CTR1 kinase activity. Fifty nanograms of baculovirus generated
GST–CTR1K601A,R604Aand GST–CTR1K601A,R602A,R604Awere incubated with 5 lg of myelin basic protein (MBP) and 5 lCi c-[32P]ATP in the
presence or absence of 1 nmol PA. Samples were subsequently analysed by SDS–PAGE and visualized by autoradiography. As with wt GST–CTR1–FL, PA
dramatically inhibited the autophosphorylation and substrate phosphorylation of both GST–CTR1K601A,R604Aand GST–CTR1K601A,R602A,R604A. (D)
Mutations in the conserved Raf-1 PA binding site of CTR1 reduce its intrinsic kinase activity. Fifty nanograms of wt GST–CTR1, GST–CTR1K601A,R604A
and GST–CTR1K601A,R602A,R604Awere incubated with 5 lg of MBP and 5 lCi c-[32P]ATP for 30 min. Proteins were then analysed by SDS–PAGE and
visualized by autoradiography.
3910 Testerink et al.
Inhibition of CTR1 kinase activity by PA through a novel
PA-binding region in the kinase domain
Ethylene is a hormone that is essential for regulating plant
growth and development along with its responses to biotic
and abiotic stresses. Genetic analysis has identified several
components of the ethylene signal transduction cascade,
including a family of five ethylene receptors that all
operate via the Raf-1-like kinase CTR1, which functions
as a repressor of ethylene responses. Yet the biochemical
mechanisms fundamental to propagation of the signal
following ethylene binding remain elusive. In recent
years, regulation of CTR1 activity has received substantial
attention. The results suggest that CTR1 directly interacts
with the ethylene receptors at the ER and that this
interaction is required to maintain the repression of
ethylene responses in the absence of ethylene (Clark
et al., 1998; Gao et al., 2003; Huang et al., 2003). For
example, the progressive mutational loss of ethylene
receptors leads to a profound constitutive ethylene re-
sponse phenotype (Hua and Meyerowitz, 1998). Nonethe-
less, it is not known how the receptor/CTR1 interaction
regulates CTR1 activity.
In this report, it is shown that the lipid second messenger
PA can regulate CTR1 activity by binding to CTR1’s kinase
domain. CTR1 bound both to pure PA coupled to Sepharose
beads and to lipid bilayers supplemented with low concen-
trations of PA. The capacity for PA association is specific to
the kinase domain, since this domain alone also specifically
bound PA. While binding of the kinase domain was similar
to the full-length protein on PA liposomes, removal of
CTR1’s amino-terminal regulatory domain increased its
affinity for the PA beads. Thus, there might be a negative
effect of the N-terminus on PA-binding, depending on the
conditions used to test lipid binding. Binding appears to be
important for regulation of CTR1 activity since PA inhibited
CTR1 kinase activity and prevented the kinase domain from
interacting with the N-terminus. PA also inhibited the
activity of the kinase domain alone. This implies that it is
not the inhibition of the intramolecular interaction that
results in reduced kinase activity, but rather a direct effect of
PA on the kinase domain.
PA also reduced binding of CTR1’s kinase domain to the
ethylene receptor, ETR1, which is an association that has
Fig. 5. Identification of the PA-binding region in CTR1. (A) Schematic representation of Arabidopsis CTR1 and the deletion constructs used. The
position of the CTR1K601A,R602A,R604Amutation is indicated by ***. (B) Binding of in vitro-translated35S-labelled CTR1 fragments to liposomes
containing 5% PA (PC/PS/PA, 80/15/5). (C) Binding of GST–CTR1654–821, GST–CTR1654–742, or GST–CTR1739–821and GST alone to liposomes
containing increasing concentrations of PA. GST-tagged protein (1250 ng) was incubated with 640 nmol of liposomes. Bound proteins were
separated on SDS–PAGE and visualized by colloidal Coomassie brilliant blue staining and quantified. Lipid mixtures: 0% PA, PC/PS 80:20; 2% PA,
PC/PS/PA 80/18/2; 5% PA, PC/PS/PA 80/15/5; 20% PA, PC/PA 80/20.
Fig. 6. PA inhibits intra- and intermolecular interactions of the CTR1
kinase domain. Using an in vitro binding assay in which
radiolabelled CTR1551–821was generated by in vitro translation and
tested for an association with maltose binding protein fusion proteins
associated with amylose resin, it was previously found that CTR1’s
kinase has high affinity for the CTR1 amino-terminal domain. Using the
same in vitro binding assay, either with or without 100 nmol PC or PA,
25 ll of35S-radiolabelled CTR1551–821was incubated for 16 h at 4 ?C
with 5 lg of maltose binding protein (MBP), MBP–CTR153–568, or
MBP–ETR1293–729in 400 ll total volume, after which samples were
washed, separated by SDS–PAGE, and visualized by autoradiography.
Phosphatidic acid regulates CTR1 activity 3911
never been demonstrated before, although the conditions used
to determine this association had not been attempted in the
past (Clark et al., 1998; Larsen and Cancel, 2003).
Interestingly, the interaction of CTR1 with ETR1 is
predicted to activate CTR1, suggesting that PA may function
as a step in the inactivation of CTR1 in vivo. Therefore, PA
may be a negative regulator of CTR1 kinase activity, either
acting independently of ethylene to enhance its effects or as
part of a multi-step mechanism that is triggered by ethylene
binding and required for shutting off CTR1.
Raf-1’s PA-binding motif represents a cluster of basic
amino acids that coordinate binding of the negatively
charged phosphate group of PA (Ghosh et al., 2003).
A similar motif is present in CTR1’s kinase and so double
and triple mutants of CTR1 were generated to destroy this
site. By contrast to the Raf-1 paradigm, the mutations did
not affect either PA binding or PA-dependent inhibition of
kinase activity. On the other hand, they severely reduced
the intrinsic kinase activity of CTR1, indicating that these
amino acids make some critical yet unknown contribution
to the activation and/or maintenance of CTR1 activity.
The equivalent mutations in Raf-1 that prevent PA
binding have not been checked for their effect on kinase
activity (Ghosh et al., 2003).
Since the Raf-1 homologous site was not responsible for
PA-binding by CTR1, deletion fragments of CTR1 were
produced and tested for PA-binding to identify the
binding site. From this analysis, CTR1654–821was found
to be essential and sufficient for specific PA-binding. This
fragment indeed lacks the highly basic amino acid motif,
thus confirming that the Raf-1 homologous site is not
involved. Within the CTR1654–821fragment, CTR1654–742
conferred selective binding to PA, while CTR1739–821
showed non-selective binding to both PA and PS. The
Raf-1 protein was shown to contain a PS-binding site, but
this site is located in the N-terminus of the protein, a part
that has no sequence homology to CTR1 (Ghosh et al.,
1996). Currently, the CTR1 PA-binding region is being
analysed to identify potential PA-binding sites, and
a mutagenesis approach will be continued in order to
disrupt PA association.
CTR1 is the first protein kinase that has been found to
be inhibited by PA. Activation by PA has been reported
for protein kinase Ce, AtPDK1, and CDPK (Lopez-
Andreo et al., 2003; Anthony et al., 2004; Szczegielniak
et al., 2005), as well as for the protein tyrosine
phosphatase SHP-1 (Frank et al., 1999), while inhibition
has been found for the phosphatases ABI1 (Zhang et al.,
2004) and PP1Cc (Jones and Hannun, 2002).
Possible role of PA in stress-induced ethylene
The current model for ethylene signal transduction
predicts that, in the absence of ethylene, the ethylene
receptors at the ER activate CTR1’s kinase activity which
suppresses ethylene responses. Following ethylene bind-
ing, CTR1 would dissociate from the receptors and
become inactive, resulting in the induction of ethylene
responses (Guo and Ecker, 2004; Chen et al., 2005). PA
could play a role here, as it is present at low levels in the
ER at all times as an intermediate in lipid biosynthesis.
Thus, basal levels of PA might have a function in the
inhibition of CTR1 activity as part of the likely compli-
cated mechanism of CTR1 regulation.
Interestingly, the biochemical evidence presented here
predicts a positive effect of stress-induced PA accumula-
tion on ethylene responses, which would be consistent
with, and provide an explanation for, several observations
described in the literature. Various stress stimuli, such as
wounding, pathogen elicitors and osmotic stress, are
known to induce ethylene responses (Felix et al., 1991,
2000; Abeles et al., 1992; O’Donnell et al., 1996; Liu and
Zhang, 2004). It is unclear, however, how the ethylene
signalling pathway is initially activated in the absence of
ethylene. The same biotic and abiotic stresses have been
shown to trigger a very rapid PA response (Testerink and
Munnik, 2005). Based on the present data, which show
that PA inhibits CTR1 activity, we hypothesize that PA
formed in response to wounding, elicitors, or salt can
induce ethylene responses through membrane recruitment
and subsequent inhibition of CTR1’s kinase. Interestingly,
as PA directly affects CTR1 activity, the predicted
pathway could function in the early response to quickly
turn on the ethylene-signalling pathway before ethylene
itself is generated. Of course, the present model, based on
biochemical evidence, still needs to be tested in planta. In
support, analysis of a PLDa1-silenced line suggests a role
for PA in ethylene responses (Fan et al., 1997). Future
work should focus on elucidating the function of the
described PA association on CTR1 function and ethylene
responses, through identification of the PA binding site
and subsequent evaluation of the necessity of this site for
regulation of CTR1 activity and ethylene signalling.
We thank Chris Loewen for advice on the liposome assays and
Jesse D Cancel for technical assistance. We are grateful to Alan
Musgrave for critically reading the manuscript. TM’s laboratory is
financially supported by the Netherlands Organization for Scientific
Research (NWO; grants ALW 863.04.004 and Vidi 864.05.001),
the European Commission (HPRN-CT-2002–00251), and the Royal
Netherlands Academy of Arts and Sciences (KNAW). CT acknowl-
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