From the Department of Vertebrate Genomics, Max Planck Institute for Molecular Genetics (MPI-MG), Ihnestr. 73, 14195 Berlin, Germany, ¶ Department of Stress and Developmental Biology, Leibniz Institute of Plant Biochemistry, Weinberg 3, 06120 Halle, Germany, || RZPD German Resource Centre for Genome Research GmbH, Heubnerweg 6, 14059 Berlin, Germany, gabi.rzpd.de/, and ** Business Unit Bioscience, Plant Research International, P O. Box 16, 6700 AA Wageningen, The Netherlands
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ABSTRACT |
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In our group, we have developed the first plant (20, 21) and bacterial microarrays (22) as well as a nonredundant human protein microarray (23) for analytical and functional studies. From this technical background, we aimed, in this study, to develop a protein microarray-based method for high throughput identification of potential protein kinase substrates and to apply this method to reveal novel substrates of Arabidopsis MAPKs.1
MAPKs are the terminal components of the "three-kinase" modules of MAPK cascades. These kinases are activated by phosphorylation: a MAPK kinase kinase (MAPKKK), which is a serine/threonine protein kinase, phosphorylates the subsequent dual specific MAPK kinase (MAPKK), which in turn activates the MAPK by phosphorylation of a threonine as well as tyrosine residue in the "activation loop" (24). Both residues are separated by one amino acid in this loop (Thr-X-Tyr) (25). Downstream of activated MAPKs, which are described as serine/threonine kinases, phosphorylation events occur and may influence the regulation of genes (26). However, the phosphorylation substrates of the activated MAPKs are, especially in plants, widely unexplored.
MAPK cascades are universal and highly conserved signal transduction modules in eucaryotes, including yeasts, animals, and plants, and mediate the intracellular transmission and amplification of extracellular stimuli, resulting in the induction of appropriate biochemical and physiological cellular responses (24, 27, 28). Activation of MAPK cascades is an important mechanism for stress adaptation by control of gene expression. In plants, MAPK signaling has been implicated in abiotic as well as biotic stress situations and is associated with various physiological, developmental, and hormonal responses (29, 30). In certain plant species, this activation is an important component in host and non-host resistance against several pathogens and exhibits strong similarity to the innate immune protection systems of mammals and Drosophila (24, 31). Roles for MAPK activation in triggering "early" defense gene expression have been demonstrated in Arabidopsis (32), parsley (33), and tobacco (25) in response to pathogens or pathogen-derived elicitors. In Arabidopsis, a complete signaling cascade following perception of a bacterial flagellin has been elucidated recently (32). Downstream of the flagellin receptor, a leucine-rich repeat (LRR) receptor kinase, the cascade consists of MEKK1 (MAPKKK), MKK4/MKK5 (MAPKKs), and MPK3/6 (MAPKs). Signaling via this cascade results in the up-regulation of WRKY22/WRKY29 transcription factor gene expression. Despite this effort, nothing is known about the phosphorylation events and their influence on gene expression downstream of activated MAPKs. Therefore, in this study we addressed the challenging task of identifying the protein substrates of two activated Arabidopsis MAPKs (MPK3 and MPK6) using a novel protein microarray technique as a powerful high throughput platform.
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EXPERIMENTAL PROCEDURES |
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The SuperScriptTM plasmid system for cDNA synthesis and cloning (Invitrogen) was used for cDNA library construction following the manufacturers protocol. The cDNA fragments were directionally ligated into the SalI/NotI cloning sites of the vector pQE30NASTattB (GenBankTM accession number AY386205) and transformed into competent Escherichia coli SCS1/pSE11 cells (35). Transformants were picked and kept in 384-well microtiter plates.
Selection and Rearray of Putative Expression Clones
To select putative expression clones, protein filters were generated from the arrayed cDNA library and screened with an anti-RGS-His6 antibody (Qiagen, Hilden, Germany) as described in detail previously (20, 35).
Sequence Analysis and Selection of Uniclones
cDNAs were sequenced from the 5'-end by AGOWA GmbH (Berlin, Germany) using the pQE65 vector primer. Raw sequence trace files were passed through the PHRED base calling program (36, 37), and sequence bases with confidence values <15 were masked. Cloning vector sequence removal was performed using pregap4 (Staden Package software, version 2001 (38)). Sequences with a minimum of one of the following characteristics were eliminated: (a) shorter than 100 nucleotides after vector and quality clipping and (b) sequences highly similar to E coli DNA (e-value > e20).
The remaining sequences were (i) analyzed by BLASTX 2.2.6 (39) with the "MIPS Arabidopsis Protein Database" as a reference (Munich Information Center for Protein Sequences, Munich, Germany, mips.gsf.de/), (ii) translated to the corresponding peptide sequences in the first forward frame using Emboss Transeq (40), and (iii) clustered using d2_cluster with default parameters (41) to screen for redundancy of the clone set. Using d2_cluster software sequences with a minimal overlap of 100 bp and at least 90% sequence identity are placed in the same cluster. Before clustering, the sequences were screened for interspersed repeats and low complexity DNA sequences using CrossMatch and RepBase database for repetitive Arabidopsis DNA sequence elements (42). The cDNA sequences were clustered together with the complete annotated Arabidopsis gene sequences lacking intron sequences and 5'- and 3'-untranslated sequences.
The Emboss Transeq output was screened for clones having a stop codon within the first 70 triplet codes, and the putative reading frames and the existence of 5'-UTRs were calculated from the BLASTX reports using scripts written in Perl (www.perl.com/). Clones identified as singletons by cluster analysis and at least one representative for each cluster were accepted for the uniclone set. For most of the clusters one representative each was selected. Selection criteria were: (i) full-length clone if available, (ii) smallest 5'-UTRs, and (iii) highest similarity to the corresponding gene. More than one sequence per cluster was selected for clusters that show a discrepancy between cluster and BLAST analysis, i.e. sequences composing one cluster with Arabidopsis Genome Initiative (AGI) gene codes, but BLAST found the highest similarity to an Arabidopsis gene not in the same cluster.
Recombinational Cloning in 96-well Format
cDNA inserts were transferred from GATEWAYTM entry vectors into E coli destination vector pQE30NASTDV by LR reaction (attL x attR recombination reaction in the GATEWAY cloning technology; for further details about this reaction please see the Invitrogen website). pQE-30NAST-DV is a derivative of pQE-30NAST (GenBankTM accession number AY386205 (pQE-30NAST-attB)) that has been modified to a GATEWAY destination vector by Invitrogen. LR reactions were carried out in 96-well format according to the manufacturers instructions with the exception that total volume and enzyme concentration were reduced by half. Transformation of E coli SCS1/pSE111 cells with 1 µl LR reaction mixture was performed by heat shock in 96-well format using 30 µl of cell suspension/well. Two clones per transformation were screened with colony PCR using vector primers. Recombinant clones were grown in 96-well microtiter plates. After adding glycerol to an end concentration of 20% (v/v), clones were stored at 80 °C.
Protein Expression and Purification in 96-well Format
Proteins were expressed in 1-ml cultures and purified (via metal chelate affinity chromatography) after lysis in denaturing lysis buffer (100 mM NaH2PO4, 10 mM Tris-HCl, 6 M guanidine hydrochloride, pH 8) as described previously (20). Purified proteins were analyzed visually for purity using a 15% polyacrylamide gel. Protein concentrations were determined by Bradford assay (43).
Purification and Activation of MPK3 and MPK6
The ORFs of MPK3 (At3g45640) and MPK6 (At2g43790) were amplified from first strand cDNA with modified gene-specific primers and cloned into pGEX4T-1 as BamHI/XhoI or BamHI/NotI fragments, respectively. Recombinant proteins were expressed in E coli cells and purified by glutathione-agarose chromatography according to the instructions of the supplier (Sigma-Aldrich Chemie). The purified proteins were dialyzed against two changes of water (4 °C, overnight). Myelin basic protein (MBP; Sigma-Aldrich Chemie) was used as an artificial substrate to evaluate the activity of the recombinant proteins. The inclusion of 1 mM MnCl2 in addition to 5 mM MgCl2 in microarray-based kinase assays (see reaction details below) was found to autoactivate the recombinant MPK3 to activity levels comparable to those extracted from elicited plant material.
Generation of Protein Microarrays
The purified His-tagged proteins were positioned on FASTTM slides (Schleicher & Schuell; Batch Numbers AOBZ001, AOBZ033, AOBZ035, and AOBZ645) alongside with positive and negative controls in an 11 x 11 spotting pattern in two identical fields at a relative humidity of 75% and at 20 °C. The protein-containing plates were cooled to 8 °C during arraying. The proteins were printed with an in-house modified and extended Genetix QArray microarrayer (Genetix LTD, New Milton, UK) equipped with an optimized 4 x 4 print head of Genetix stainless steel solid pins (X2777, tip diameter of 150 µm). Pins were inked in 20 µl of protein solution for 50 ms and stamped at one position once for 100 ms using a soft touch and a spotting distance of 1 mm. After every tenth transfer and before addressing a new inking position the solid pins were washed twice for 1 s with deionized water, washed once for 3 s with 80% technical ethanol (20% bidistilled water), and dried for 3 s with oil-free, pressured air supplied at 1 bar. A mixture of MBP (end concentration of 2,000 ng/µl; Sigma-Aldrich Chemie) and mouse anti-RGS-His6 antibody (end dilution of 1:10; Qiagen) in deionized water were chosen as positive controls for the phosphorylation experiments as well as for the detection of the proteins on the array surface. These positive controls were arranged as guide dots six times in each block of the array in an asymmetric fashion (schematic representation of the guide dots is given in Fig. 1) to assure correct orientation of each image for analysis and to justify the grids used for quantification. All purified proteins were spotted once in each of the identical fields in total twice on each array. In addition one spot position in each block was not addressed. Further controls (deionized water, PBS, 20 pmol/µl BSA, mouse anti-RGS-His6 antibody (Qiagen) diluted 1:10 in PBS, and rabbit anti-mouse IgG3-Cy3 (Dianova, Hamburg, Germany) diluted 1:25 in PBS) were spotted three times per field (six times on each array). After completion of the spotting run, a Genepix array list file, describing the positioning of all samples on the microarray, was generated.
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Microarray-based Kinase Assay
Protein microarrays, which were spotted the previous day, were washed in TBST for 1 h at room temperature with vigorous shaking to remove urea from the microarrays. Microarrays were blocked for 1 h at room temperature with 2% (w/v) BSA, TBST. All kinase incubations were then performed in the presence of [-33P]ATP (25 µCi/ml; Amersham Biosciences). Protein kinase A (PKA) reactions were carried out with 12.5 µg/ml PKA from mouse (New England Biolabs GmbH, Schwalbach, Germany) in PKA buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2) for 20 min. In the case of MAPKs the microarrays were incubated with 100 ng/µl MPK3 or 200 ng/µl MPK6, respectively, in MAPK buffer (25 mM Tris-HCl, pH 7.5, 1 mM EGTA, 1 mM DTT, 5 mM MgCl2, 1 mM MnCl2, 20 µM ATP) for 30 min. After incubation the microarrays were washed as follows: twice for 15 min in 2x PBST (PBS, 0.1% (v/v) Tween 20), twice for 15 min in 1x PBST, once for 30 min in 1x PBST, once for 30 min in 0.5x PBST, once for 30 min in 0.1x PBST, and once for 30 min in 0.1x PBS.
Microarrays were dried and transferred into an x-ray cassette (Hypercassette, Amersham Biosciences). The enzymatic phosphorylation of the immobilized proteins was detected on the dried and Saran-covered microarrays with BAS-SR0813 imaging screens (Fujifilm). After exposition (648 h, depending on the specific activity of the kinase under investigation) the screens were read out with an FLA-8,000 microarray scanner (Fujifilm) at 635 nm and a spatial resolution of 10 µm.
Evaluation of Radioactive Signals and Selection of Potential Targets
The resulting images of the screens were opened in Aida Array Metrix version 3.45 (Raytest, Straubenhardt, Germany; www.raytest.de). Subsequently the Genepix array list file, describing the positions of the samples on the array, was imported. The illustrated grid of the protein samples on the array was arranged on the images according to the positions of guide dots. The spotting pattern and the relative arrangement of identical fields were entered according to the users manual of the software package. These settings were saved as a template and used for analyzing all images. Subsequently the automatic grid-positioning feature was used, and the intensity of all spots was determined using a spot diameter of 320 µm. The average background of block spot was determined with the "mode of nonspot" evaluation mode. According to the manual the spot diameter, which is not considered for blockwise background correction, was enlarged by 4 µm. Subsequently the duplicate correlation of all spots was determined (Fig. 3). To avoid any artifacts resulting from the precipitation of labeled kinases, only spots deviating less than 25% from the average intensity of both spots (Fig. 3, region inside both green outer lines) were considered in the subsequent analysis. Signals (spots) deviating more than 25% from the average signal intensity were excluded from the subsequent analysis by marking them in the Aida Array Metrix software package. The reproducibility of the quantification of different microarrays was verified in Aida Array Compare version 3.53. Spots showing a deviation of the background corrected signals assigned to the same protein of more than a factor of 2 between two arrays were not included in the target identification. Finally only those spots exceeding the average signal intensity of the background by at least 10 times the deviation of the background signals were considered to identify potential targets of the kinases.
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Testing of Protein Solubility in 96-well Format
Protein expression and purification (via metal chelate affinity chromatography) were performed manually as described by Bussow et al. (44) with the exception that 50 mM Tris, pH 8.0 was substituted by 50 mM Hepes, pH 8.0 for cell lysis under native conditions. Aliquots of the following samples were collected for SDS-PAGE: (i) lysates, (ii) soluble cellular proteins (supernatant after centrifugation of the lysates), and (iii) purified protein (eluates after protein purification).
Kinase Assay in Solution Using Refolded Proteins
Purification of the potential targets for verification was performed as described above (see "Protein Expression and Purification in 96-well Format") except for a scale-up of the bacteria culture volume to 5 ml and of Ni-NTA-agarose (Qiagen) to 100 µl. After the last wash step of denaturing purification, the pH of the solution was adjusted by adding 33 µl of 1 M Tris (pH 7.5) to the 100 µl of remaining wash buffer. Consecutive dilutions by adding 130 µl, 260 µl, and 1 ml of refolding buffer (10 mM Tris, pH 7.5, containing 1 mM PMSF) were performed within a period of 30 min. The urea concentration was thus reduced to 0.5 M in the final step. After centrifugation, the buffer was removed, 1 ml of refolding buffer was added, and the samples were left on ice for another 2 h. 10 µl of the refolded proteins still attached to the Ni-NTA beads were then used for in-solution kinase assays as described previously (33).
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RESULTS |
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Sequence Analysis of the Library and Generation of a Uniclone Set
To create a uniclone set from this sublibrary, all of the 4,999 clones were sequenced from the 5'-end. Sequence data were passed through several analysis steps after vector clipping. 4,398 sequences remained after elimination of low quality sequences and sequences similar to E coli. All sequence data can be accessed via GenBankTM by accession numbers CK117511 to CK122014.
BLASTX analysis of the sequences revealed that 61.8% of the clone inserts are in the frame of the His tag, which is comparable to the rearrayed hEx1 library (55%) (23). Furthermore it was calculated that 39.4% of the clones from the ATM1 library contain the full-length coding sequence. The d2_cluster analysis computed 2,029 different sequence clusters of which 32% are singletons, whereas 68% of the sequences are members of 637 different clusters. 85% of these clusters had a size of less than six sequences. All sequence reads were translated to amino acid sequences in the frame of the His tag, and 1,442 clones were excluded from the uniclone set because a stop codon was detected within the first 70 triplet codes of the insert DNA. This ensured that only such clones were used that express proteins with a size of at least 8 kDa (23). 318 sequences were eliminated because their insert was calculated to be in the wrong frame by BLASTX analysis, and another 21 sequences were sorted out due to a lack of similarity to any Arabidopsis gene.
Finally 2,615 sequences passed all the preceding steps. 987 sequences computed to be singletons were picked out directly for the uniclone set. From the remaining 1,628 sequences belonging to 404 different clusters, 511 additional sequences were selected (one sequence from 384 clusters, and more than one from 20 clusters). These selections resulted in a uniclone set of altogether 1,498 clones. Sequence analysis results for the whole sequence dataset are provided in Supplemental Table 1 or in Supplemental Table 2 for the uniclone set.
Extension of the Uniclone Set
The uniclone set of 1,498 clones was extended by 192 full-length cDNA clones. 96 of them were described previously (21). The other 96 clones are transcription factor expression clones, which were generated from GATEWAY entry clones by LR reaction in this study. These 192 full-length clones together with the 1,498 uniclones compose the extended uniclone set of 1,690 clones.
Phosphorylation Studies with MPK3, MPK6, and PKA Using Arabidopsis Protein Microarrays
All 1,690 clones of the extended uniclone set were expressed in parallel in 96-well format. Proteins were purified by nickel chelate affinity chromatography under denaturing conditions. To control the quality of the purification 96 randomly chosen proteins were separated using 15% SDS-PAGE followed by CBB staining. 83% of the proteins were detected with a size range of 1450 kDa (data not shown).
To generate Arabidopsis protein microarrays, the 1,690 purified proteins of the extended uniclone set and controls were arrayed on FAST slides. The proteins were spotted once in each of the two identically fields. Each field consists of 16 blocks, and the proteins were arranged in an 11 x 11 spotting pattern in each block (see Fig. 1). The microarrays were screened with an anti-RGS-His6 antibody (Fig. 2, a and b) to detect recombinant proteins. Furthermore the microarrays were used for the identification of phosphorylation substrates of PKA (Fig. 2c), MPK3 (Fig. 2d), or MPK6 (Fig. 2e), each in the presence of radioactive ATP. Mouse PKA was included as an example of a kinase from a different family to validate the specificity of our method. In addition to other controls, we used a mixture of MBP and anti-RGS-His6 antibody as positive control and guide dot for both immunoscreening and phosphorylation studies. This control was spotted six times in each block in an asymmetric fashion as detailed in Fig. 1. This arrangement facilitates the grid adjustment for subsequent quantification. One position of every block was not addressed (Fig. 1).
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To select potential substrates of Arabidopsis MPK3 and MPK6 based on a quantitative system, we performed two independent experiments for both kinases including two microarrays each. The recombinant MPK6 was comparatively less active, probably as a result of poor folding in the E coli expression system, and hence was used at a higher concentration for the screen (see "Experimental Procedures"). Signal intensities were determined for every microarray with a high field-to-field correlation as demonstrated for one MPK3 microarray in Fig. 3. Only proteins that were identified as targets in both experiments according to our quantitative threshold-based criteria (see "Experimental Procedures") were defined as potential targets, resulting in a list of 48 or 39 for MPK3 (Table I) or MPK6 (Table II), respectively. 26 of them are common for both kinases as indicated with grey background in the tables. For comparison only, 35 substrates for mouse PKA have been identified in one experiment with two microarrays, and only a very low number of these targets overlap with the identified MAPK targets (three for MPK3, and four for MPK6) (data not shown).
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DISCUSSION |
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The general feasibility of protein microarrays to detect protein phosphorylation by kinases has been demonstrated previously using different surfaces for protein immobilization: BSA-N-hydroxysuccinimide monolayers to which substrates were covalently attached (17), protein microarrays bearing polydimethylsiloxane-coated microwells (19), or FAST slides, which are covered with a nitrocellulose-derived polymer for non-covalent protein attachment (20). However, in these previous studies, substrate identification was carried out only in a low/medium throughput manner with regard to the number of proteins analyzed for phosphorylation on one microarray (17, 19, 20) and for most instances without quantification of the microarray results (17, 20). Therefore, in this study we aimed to develop a high throughput method. Furthermore the method should allow the identification of substrates based on quantitative criteria. The combination of an 11 x 11 spotting pattern with a microarray design using duplicates of every protein on one array (Fig. 1) allows us to immobilize theoretically 1,936 different samples in two identical fields. With this we have developed a high density protein microarray-based kinase assay. Compared with other currently available microarray methods (17, 19, 20) our assay provides the highest number of proteins that may be analyzed for phosphorylation in parallel. The efficiency of protein expression and purification methods as well as of the technology used for protein transfer to the microarray surface has been demonstrated by identifying nearly all of the recombinant proteins after screening the microarrays with an anti-RGS-His6 antibody (Fig. 2a).
To detect the phosphorylation of immobilized proteins radioactively labeled ATP has been used. Initial studies were performed in which fluorescent dyes were applied for the identification of phosphorylated amino acids (46). Nevertheless the radioactive-based detection is still the most sensitive and robust detection method. Using phosphorimaging we were able to yield radioactive signals in the 11 x 11 pattern with sufficient resolution and a high field-to-field reproducibility (Fig. 3).
To manage substrate identification with a high significance, we performed two independent phosphorylation experiments for every kinase with two microarrays each. Only proteins that were detected in both experiments, taking into account the threshold-based quantitative criteria, were defined as potential targets thus reducing the number of false-positive results. We identified 48 potential substrates for MPK3 (Table I) and 39 for MPK6 (Table II). As expected, a large number of them (26 substrates) are common for both kinases. The lower number of MPK6 compared with MPK3 substrates probably reflects a lower representation of the MPK6 substrates in our set or a lower specific activity of the recombinant MPK6 used in the screen. To improve the significance of the method it would be recommendable for future studies to normalize the radioactive signals with respect to the concentrations of the different transferred proteins.
We demonstrated the suitability of our quantitative criteria for substrate selection exemplarily for one kinase (MPK3) by verification of nearly all MPK3 substrates using two different in vitro methods. 92% of the targets were confirmed with on-blot phosphorylation (data not shown), and 88% of the substrate candidates were verified by independent in vitro phosphorylation of refolded proteins in solution (Fig. 4). The failure to confirm some of the potential substrates may be due to low amounts of purified proteins or because of the loss of a potential linear phosphorylation site by refolding of the protein in the assay in solution.
The high specificity of the method has been proven comparing the phosphorylation pattern of kinases from the same as well as from different families (Fig. 2). As expected, kinases from different families yield clearly different patterns, resulting in a low number of common substrates that we identified for these kinases (e.g. three common substrates for MPK3 and PKA). The relatively high number of mouse PKA substrates identified within our set of Arabidopsis proteins (35 substrates, data not shown) may be due to the fact that several plant protein kinases belong to the same group as PKA, the AGC group (named after PKA, PKG, and PKC; see www.nih.go.jp/mirror/Kinases/pkr/pk_catalytic/pk_hanks_seq_align_long.html), and are sharing specific sequence motifs such as the FXXF hydrophobic motif in the C terminus (47).
The method described here is a valuable supplementation in the spectrum of existing in vitro methods for substrate identification such as solid-phase phosphorylation screening of phage cDNA expression libraries described by Fukunaga and Hunter (48) or the method developed by Shokat and colleagues (4951). The latter method uses kinases with modified ATP binding pockets that are accepting unnatural ATP analogues to display direct substrates of modified kinases in crude mixtures or cell lysates. In contrast to this method, no modification of the kinases is needed for our microarray-based screening. Compared with the screening of phage expression libraries, our method needs considerably smaller volumes of active kinase (200 µl for one incubation are sufficient using our method; this is 1,000 times less than for the screening of phage expression libraries (52)). The screening of proteins obtained from characterized arrayed clones in our study allows direct identification of substrate candidates because a positive result is directly linked to the sequence of the respective expression clone. Further verification experiments may be performed with proteins expressed from the same clone used for the screening experiments.
In general, our assay represents a rapid in vitro screening for potential substrates of protein kinases. We may obtain some false-positives because we use denatured proteins as targets. In the respective native-folded protein potential phosphorylation sites are possibly not accessible for the kinase. However, the confirmation experiment with the refolded proteins points to a low number of false-positives due to this reason. Furthermore it is possible that the substrate and the kinase can interact but will never associate in vivo, e.g. because they are localized in different cellular compartments. For that reason, potential substrates identified with our method have to be verified in subsequent in vivo experiments; e.g. protein-protein interaction studies in planta by innovative microspectroscopic approaches such as fluorescence resonance energy transfer and fluorescence lifetime imaging microscopy (53). To gain insights into the sites of phosphorylation, antibodies against phosphorylated protein epitopes may be used for detection (54) on protein microarrays. Furthermore peptide arrays (54, 55) or MS-based methods (56, 57) may be applied in this respect.
An excellent demonstration for the suitability of our method to find substrates with biological relevance is the identification of 1-aminocyclopropane-1-carboxylic acid synthase-6 (ACS-6) as an MPK6 substrate. ACS, the rate-limiting enzyme of ethylene biosynthesis, has been described very recently as the first plant MAPK substrate in vivo (58). Liu et al. (58) found that selected isoforms of ACS including ACS-6, which we identified in our study (Table II, number 16), are substrates of MPK6. Phosphorylation of ACS led to the accumulation of the protein and to ethylene production (58). Furthermore it has been speculated that plant stress-responsive MAPKs may phosphorylate transcription factors or transcription factor-regulating proteins (24, 32) similar to their mammalian orthologs (28). Several reports support this assumption, such as the increased nuclear localization of MAPKs in parsley cells after MAPK activation (45) or the up-regulation of WRKY22/WRKY29 transcription factor expression upon activation of the flagellin MAPK cascade (32). Asai et al. (32) suggested that not the WRKY transcription factors themselves but a specific WRKY inhibitor may be phosphorylated and inactivated by MAPKs. In agreement with these findings, we identified several transcription factors (Table I, numbers 3, 46, and 47; Table II, numbers 2, 22, 32, and 39) as well as a transcription regulator (Table II, number 14) as potential MAPK substrates in this study. Furthermore the phosphorylation of histones (Table I, numbers 6, 22, 28, 29, and 39; Table II, numbers 25 and 31) could be involved in regulating gene transcription as it has been shown that the tail domain of histones regulates chromatin structure and hence gene transcription (59).
Several substrates, which were identified in this study, or their mammalian homologues have been described previously to be involved in signal transduction (28) as indicated in bold in Tables I and II. The identified LRR family protein (Table I, number 13) may participate as a receptor in elicitor-induced MAPK cascades due to its LRR domain (32). Flowering locus T protein (Table I, number 27; Table II, number 23) is a putative membrane-associated protein with homology to human phosphatidylethanolamine-binding protein (60). This protein is identical to Raf kinase inhibitor protein that is involved in regulation of RAF/MEK/ERK signaling pathway (61).
The calmodulin-binding family protein (Table I, number 17) may be a component of the MAPK cascade because a calmodulin-binding protein has been described previously as a negative regulator for stress tolerance to sodium and osmotic stress (62). A further target that has been identified for both MPKs is a casein kinase (Table I, number 32; Table II, number 27). In HeLa cells, a direct interaction of p38 MAPK and casein kinase 2 was observed, and also a stress-induced activation of casein kinase 2 by this MAPK was shown (63). All these results support the assumption that regulation in response to MAPK signaling is very complex and not restricted to the transcriptional level (28).
In conclusion, we established a powerful generic test system for the in vitro identification of potential protein kinase substrates by high density protein arrays followed by independent verification with refolded proteins. The application of this test system for plant MAPKs resulted in a short list of candidates for further analysis. Follow-up experiments such as in vivo verification and the mapping of phosphorylation sites in substrates are essential to evaluate the physiological relevance of the targets in MAPK signaling.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Published, MCP Papers in Press, July 11, 2005, DOI 10.1074/mcp.M500007-MCP200
1 The abbreviations used are: MAPK, mitogen-activated protein kinase; ACS, 1-aminocyclopropane-1-carboxylic acid synthase; AGI, Arabidopsis Genome Initiative; ATM1, A. thaliana meristem 1; CBB, Coomassie Brilliant Blue; Cy3, indocarbocyanine; FAST, fluorescence array surface technology; LRR, leucine-rich repeat; LR reaction, recombination reaction in the GATEWAY cloning system (Invitrogen) to create expression clones, recombination of attL and attR sites; MAPKK, mitogen-activated protein kinase kinase; MAPKKK, mitogen-activated protein kinase kinase kinase; MBP, myelin basic protein; Ni-NTA, nickel nitriloacetic acid; PKA, protein kinase A; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; MKK, MAPK kinase; MEKK, MAPK/ERK kinase kinase.
* This work was funded by the Federal Ministry of Education and Research (Grants 0312274, 0312272, and 031U102D) and the Max Planck Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in the paper has been submitted to GenBankTM EBI Data Bank with accession number(s) CK117511 to CK122014.
S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material.
Present address: Centre for Human Proteomics, Royal College of Surgeons in Ireland, 121 St. Stephens Green, Dublin 2, Ireland.
Present address: Free University of Berlin, Königin-Luise-Str. 12-16, 14195 Berlin, Germany.
To whom correspondence should be addressed: Dept. Neuroproteomics, Max Delbrück Center for Molecular Medicine, Robert-Rössle-Str. 10, 13092 Berlin, Germany. Tel.: 49-30-9406-2636; Fax: 49-30-9406-2629; E-mail: b.kersten{at}mdc-berlin.de
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REFERENCES |
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