JXB Advance Access published online on November 13, 2007
Journal of Experimental Botany, doi:10.1093/jxb/erm243
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© 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.
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RESEARCH PAPER |
Phosphatidic acid binds to and inhibits the activity of Arabidopsis CTR1
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
* To whom correspondence should be addressed. E-mail: testerink{at}science.uva.nl
Received 3 August 2007; Revised 13 September 2007 Accepted 14 September 2007
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Abstract |
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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.
Key words: Constitutive triple response 1, ethylene, lipid signalling, phosphatidic acid, plant stress signalling, protein kinase
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Introduction |
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Phosphatidic acid (PA) has been shown to represent an important lipid second messenger in several eukaryotic systems, including plants. It is produced via the phospholipase 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 PLD
1 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, PLD
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. PLD
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).
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 reticulum (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 transduction 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 response 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 CTR1 participates.
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Materials and methods |
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Generation of recombinant glutathione S-transferase (GST) fusion proteins and 35S-labelled proteins
The GST–CTR11–821 and GST–CTR1551–821 fusion 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 CTR1739–821 proteins 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-GCTTATGATGTGGCT-3' and 5'-AGAAAGCTGGGTTTTACAAATCCGAGCGGTT-3' for CTR1654–821, 5'-AAAAAGCAGGC-TGTATGGCTTATGATGTGGCT-3' and 5'-AGAAAGCTGGGTCAAGCTACCACAAGATGAC-3' for CTR1654–742, and 5'-AAAAAGCAGGCTGGGTCATCTTGTGGGAGCTT-3' and 5'-AGAAAGCTGGGTTTTACAAATCCGAGCGGTT-3' for CTR1739–821. The constructs were transformed 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 antibody.
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 µg of myelin basic protein (MBP) (Sigma Chemical, St Louis, MO, USA) for 30 min at 22 °C in a total volume of 30 µl. Reaction conditions consisted of 50 mM TRIS (pH 7.5), 2 mM DTT, 10 mM MgCl2, 10 µM non-radioactive ATP, and 5 µCi 
-[32P]ATP in the absence or presence of synthetic lipids (Avanti Polar Lipids). Following incubation, reactions were terminated by addition of 6x 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 µg of each fusion protein was used in a binding assay with 25 µl of CTR1551–821-radiolabelled protein either in the presence or absence of 100 nmol of PC or PA in a total volume of 400 µl. Assays were performed as previously described (Clark et al., 1998), except that samples were incubated for 16 h at 4 °C.
PA-binding assays
PA bead assays were performed according to Testerink et al. (2004). For this assay, 300 ng of purified protein was added to 3 µl of beads (containing 2.6 µmol 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 µm 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 µl 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 µl 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 (Molecular Dynamics).
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Results |
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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 the GST–CTR11–821 and GST–CTR1551–821 proteins bound PA (Fig. 1), with GST–CTR1551–821 having 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).
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To determine whether CTR1 specifically bound PA, lipid-binding specificity of GST–CTR11–821 was 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–CTR11–821 specifically 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 containing 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.
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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–CTR11–821 was incubated with myelin basic protein (MBP), which served as the substrate for CTR1,
-[32P]ATP, and the appropriate buffer. Following incubation, the samples were separated electrophoretically using an SDS–PAGE system and visualized by autoradiography. GST–CTR11–821 in 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 phosphorylation and autophosphorylation for GST–CTR11–821 (Fig. 3A), suggesting that PA is a negative regulator of CTR1 activity.
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It was tested whether acyl chain length is a determinant 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 moderate 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 elements.
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 phosphorylation (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 immunoprecipitated 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 protein.
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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 binding, the double (CTR1K601A,R604A) and triple (CTR1K601A,R602A,R604A) mutations had no effect on the strongly inhibitory effect of PA (Fig. 4C). Surprisingly, the CTR1K601A,R604A and CTR1K601A,R602A,R604A mutant 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, 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 CTR1654–821 fragments (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.
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Based on the above results, three new constructs were designed (Fig. 5A). The CTR1654–821 fragment, and two smaller fragments, CTR1654–742 and CTR1739–821, were produced as GST fusion proteins in E. coli and subsequently tested for their ability to bind liposomes containing increasing concentrations of PA. The GST–CTR1654–821 fragment was found to have affinity for PA (Fig. 5A), confirming the binding data of the in vitro-translated fragments. When GST–CTR1654–821 was subdivided into smaller fragments, mainly GST–CTR1654–742 and, to a lesser extent, GST–CTR1739–821 were found to contribute to PA binding.
CTR1739–821 had some affinity for PS- (0% PA in Fig. 5C) or PA-containing liposomes, but showed no specificity for PA, since binding did not increase upon increasing the concentration of PA in the liposomes. On the other hand, GST–CTR1654–742 bound PA specifically, although with lower affinity than the GST–CTR1654–821 fragment (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 CTR1654–742 fragment, although residues in the CTR1739–821 fragment may serve to increase the affinity for PA. Examination of the CTR1654–742 region did not reveal any highly basic amino acid motifs that could be responsible for binding.
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). 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 CTR153–568 was 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 CTR1551–821 in 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 association (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).
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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, 35S-labelled CTR1551–821 was 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 CTR1551–821 with these MBP fusions was not indiscriminate, for it was not possible to show any binding of CTR1551–821 to MBP–EER3 or MBP–EER4 (MJ Christians, LM Robles, PB Larsen, unpublished data).
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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 response phenotype (Hua and Meyerowitz, 1998). Nonetheless, 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 concentrations 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 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, CTR1654–821 was 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 CTR1654–821 fragment, 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 PP1C (Jones and Hannun, 2002).
Possible role of PA in stress-induced ethylene responses
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 binding, 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 complicated mechanism of CTR1 regulation.
Interestingly, the biochemical evidence presented here predicts a positive effect of stress-induced PA accumulation 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 PLD1-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.
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Acknowledgements |
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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 acknowledges the support of NWO-CW (grants Veni 700.52.401 and Vidi 700.56.429).
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Footnotes |
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These authors contributed equally to this work. 
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Abbreviations |
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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.
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References |
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