ABSTRACT
Mitogen-activated protein kinase (MPK) cascades are essential signal transduction components that control a variety of cellular responses in all eukaryotes. MPKs convert extracellular stimuli into cellular responses by the phosphorylation of downstream substrates. Although MPK cascades are predicted to be very complex, only limited numbers of MPK substrates have been identified in plants. Here, we used the kinase client (KiC) assay to identify novel substrates of MPK3 and MPK6. Recombinant MPK3 or MPK6 were tested against a large synthetic peptide library representing in vivo phosphorylation sites, and phosphorylated peptides were identified by high-resolution tandem mass spectrometry. From this screen, we identified 23 and 21 putative client peptides of MPK3 and MPK6, respectively. To verify the phosphorylation of putative client peptides, we performed in vitro kinase assay with recombinant fusion proteins of isolated client peptides. We found that 13 and 9 recombinant proteins were phosphorylated by MPK3 and MPK6. Among them, 11 proteins were proven to be the novel substrates of two MPKs. This study suggests that the KiC assay is a useful method to identify new substrates of MPKs.
KEYWORDS: MPKs, Phosphorylation, Substrates, signaling
Introduction
Mitogen-activated protein kinase (MPK) cascades have been known to be highly conserved signal transduction modules in all eukaryotes including plants. MPK cascades are typically composed of three classes of protein kinases, MPK kinase kinase (MPKKK), MPK kinase (MPKK), and MPK. MPK cascades transduce extracellular signals to intracellular compartments to result in cellular response through the phosphorylation of downstream targets1–3. MPKs are activated by consecutive activation of MPKKKs and MPKKs in response to external signals. Activated MPKs directly phosphorylate the conserved motif of substrates, Ser/Thr residues followed by a Pro residue (S/T-P).4,5 Phosphorylation of substrates by MPKs is known to generate appropriate cellular responses by changing subcellular localization, stability, transcriptional activity, and interaction with other proteins.6,7
Among 20 MPKs in Arabidopsis, MPK3 and MPK6 have emerged as two key MPKs of primary interest because they are mainly involved in a diverse range of biotic and abiotic stresses.8–11 Moreover, Arabidopsis MPK3 and MPK6 have been shown to play important roles in plant developments, such as stomatal patterning and lateral root formation.12–14 In uncovering the intricate roles of MPKs in plants, the identification of new substrates of MPKs is important. To identify substrates of MPKs, several methods including protein microarray, affinity chromatography, solid-phase screening, and phosphoproteomic analysis have been attempted.15–20 However, it is believed that a large number of MPKs substrates have not been identified yet because the possible combinations of MPKKK-MPKK-MPK cascades are very complex, thereby the specific downstream substrates of possible combinations would be required to result in appropriate responses.
The kinase client (KiC) assay was developed as a new method for identifying the substrates of kinases through in vitro phosphorylation of synthetic peptide library coupled with tandem mass spectrometry.21 This method has potential application for high throughput characterization of kinases–substrates interaction. Successfully, 23 proteins were identified as putative substrates of 17 different protein kinases using the KiC assay.22 In addition, novel substrates of P2K1 (DORN1) and ILK1 were identified by the KiC assay.23,24 Eventually, the biological and biochemical functions of RBOHD and ILK5 isolated by the KiC assay were extensively investigated as new substrates of P2K1.24,25 In this study, we identified potential substrates of MPK3 and MPK6 by the KiC assay. Furthermore, these peptides were verified through in vitro kinase assay with recombinant proteins. As a result, we confirmed that a half of potential substrates were phosphorylated by MPKs. Finally, we identified 11 novel substrates of MPKs that would provide clues for understanding MPK-mediated signaling in Arabidopsis. Conclusively, this research suggests that the KiC assay is a useful tool for the identification of novel MPK substrates in plants.
Materials and methods
Construction of synthetic peptide library
Based on the results obtained from in vivo phosphoproteomic analysis in Arabidopsis thaliana, a library (PEPscreen, sigma, St. Louis, MO, USA) consisting of approximately 2,100 10 to 20-mer peptides was synthesized. Stock peptide solutions were prepared by dissolving the peptides in 80% (v/v) dimethylformamide in water to a final concentration of 8 mM. Samples from the stock solutions were then diluted into the KiC assay.21,26 These synthetic peptides were then used as the basis for an integrated experimental strategy for the identification of kinase-client proteins.
Expression of recombinant his-MPK3 and MPK6 in E. coli
Full-length MPK3 and MPK6 cDNA were amplified by PCR from a cDNA library of Arabidopsis seedlings using gene-specific primers (Table S1) and subcloned into pQE30 for the expression of 6×His-fused MPK3 and MPK6. The 6×His-tag fusion proteins were expressed in Escherichia coli (M15) and purified using Ni-NTA agarose beads (Qiagen, Hilden, Germany).
Phosphorylation of synthetic peptides by MPK3 and MPK6
The KiC assay was performed as previously described with slight modifications.26 In vitro kinase reaction of a mixture of synthetic peptides containing 2,100 different peptides was conducted using recombinant MPK3 or MPK6 at 37°C for 1 h with shaking. Kinase reaction was stopped by adding one volume of 1% formic acid/99% acetonitrile, evaporated to near dryness in a centrifugal evaporator, and then stored at −20°C until mass spectrometry.
Liquid chromatography and mass spectrometry
Freeze-dried peptides were dissolved by adding 40 µL of 0.1% formic acid. Samples were loaded into 96-well plates which were then placed onto a pre-chilled 10°C auto-sampler. 10 µL of each sample was analyzed using Finnigan Surveyor liquid chromatography (LC) system attached to either a stand-alone LTQ-XL or a LTQ Orbitrap XL ETD mass spectrometer (Thermo Fisher, San Jose, CA, USA). Peptides are separated on a C18 microcapillary column using a mobile phase.
Analysis of the synthetic peptides screen was performed using a stand-alone LTQ-XL. Analysis of the recombinant protein kinase client assay was performed using a LTQ Orbitrap XL ETD. The mass spectra were collected using nanospray ionization in the positive ion mode. Detailed mass spectrometry was performed as previously described.22 Data-dependent ions fragment with ETD has a mass exclusion width of 10 ppm. Decision tree settings were previously described.27 The reagent ion source settings, including temperature, emission current, energy level, and CI pressure were 160°C, 50 µA, −70 V, and 17.5 psi, respectively. The activation time was 100 ms and supplemental activation mode was enabled.
Bioinformatics analysis
The raw MS files were searched against a decoy database consisting of the random complement of the sequences comprising the peptide library, using SEQUEST algorithms (Proteome Discoverer 1.0, Thermo Fisher). Instrument and search parameter settings have been previously described.22,26 Identification data were evaluated using the XCorr function of SEQUEST, and phosphorylation-site localization was accomplished using phosphoRS (Proteome Discoverer, v. 1.0.3, Thermo Fisher). The XCorr values for each charge state were set to default and no decoy hits were allowed. Peptide mass deviation was 10 ppm and a setting of two PSMs/protein was used to further filter the data. For final validation, each spectrum was inspected manually and accepted only when the phospho-peptide has the highest pRS site probability, pRS score, XCorr value, and site-determining fragment ions allowed unambiguous localization of the phosphorylation site. Phospho-peptides with a pRS score ≥ 50 and/or a pRS site probability of ≥ 55% were accepted.
Expression of recombinant MPK3 and MPK6 substrate proteins in E. coli
Full-length cDNAs of substrate were amplified by PCR from a cDNA library of Arabidopsis seedlings using gene-specific primers (Table S1) and cloned into the pGEM-T Easy Vector (Promega, USA). Verified inserts by sequencing were excised with proper restriction enzymes and subcloned into pGEX 5X–1 for the expression of Glutathione S-transferase (GST)-fused proteins. GST-fusion proteins were expressed in E. coli BL21(DE3) and purified using glutathione Sepharose-4B beads (GE Healthcare, Piscataway, NJ, USA).
In vitro kinase assay
In vitro kinase assays were performed as previously described11 by incubating GST-fused substrate proteins (3 μg) and 6×His-MPK3 (2 μg) or 6×His-MPK6 (2 μg) in kinase buffer (25 mM Tris-HCl, pH 7.5, 1 mM DTT, 20 mM MgCl2, 2 mM MnCl2, and 50 μM [γ-32P] ATP) of 20 μL. GST (1 μg; negative control) and myelin basic protein, MBP (0.5 μg; positive control) were used as negative and positive controls, respectively. The reactions were initiated using 1 μCi [γ-32P] ATP, allowed to proceed at 30°C for 30 min, and stopped by the addition of 4×SDS-loading buffer. Phosphorylated substrates were visualized by autoradiography after electrophoresis on 12.5% polyacrylamide gels.
Results
Autophosphorylation site mapping of recombinant MPKs produced in E. coli
MPKs are known to be fully activated by the phosphorylation of Thr-X-Tyr (T-X-Y) motif in activation loop by upstream MPKKs.29 Since many studies showed that recombinant MPKs produced in E. coli had basal activity,9,11,15,30 in this study, we performed the KiC assay by using recombinant MPKs purified from E. coli without the activation by MPKKs. Expectedly, purified recombinant MPKs had basal kinase activities. These results indicate that recombinant MPKs produced in E. coli may have basal activities through autophosphorylation. However, the autophosphorylation sites involved in the basal activations of MPKs are poorly identified. Therefore, we analyzed the autophosphorylation site of recombinant MPK3 and MPK6 that were produced in E. coli in the absence or presence of ATP. Quantitative mass spectrometry revealed that autophosphorylation occurred on multiple residues of recombinant MPKs (Figure 1). Unexpectedly, T-X-Y motif in MPK3 (Thr196 and Tyr198) and MPK6 (Thr221 and Tyr223) were already autophosphorylated before the addition of ATP. Interestingly, the autophosphorylations of Thr196 and Tyr198 residues in MPK3 and Tyr223 residue in MPK6 significantly enhanced by the addition of ATP (Figure 1). Furthermore, Tyr,37 Thr58, Ser157, Tyr274, Ser291 and Ser406 residues of MPK3 also were autophosphorylated only in the presence of ATP (Figure 1). This result indicates that recombinant MPKs produced in E. coli have their basal activities through autophosphorylation.
Figure 1.
Autophosphorylation sites of recombinant MPK3 and MPK6.
Quantitative mass spectrometry for identified phosphorylated sites of recombinant (a) MPK3 and (b) MPK6. Spectral count data presented are means of three replicates. Dark and white bars represent the presence or absence of ATP, respectively.
Identification of potential substrates of MPK3 and MPK6 using the KiC assay
To identify novel putative substrates of MPK3 and MPK6, we performed the KiC assay according to the standard procedure.26 Purified recombinant MPK3 or MPK6 was incubated with a peptide library comprised of approximately 2,100 synthetic peptides (representing in vivo phosphorylation events) and phosphorylated peptides were analyzed by tandem mass spectrometry. As results, we identified 23 and 21 peptides as potential client peptides of MPK3 (Table 1) and MPK6 (Table 2), respectively. Among these client peptides, eight client peptides (AT1g07630, AT1g15400, AT1g53050, AT2g38280, AT3g04470, AT4g02300, AT4g12770, and AT4g42590) were isolated by both MPK3 and MPK6, whereas 15 and 13 client peptides were specifically isolated by either MPK3 and MPK6, respectively (Figure 2A).
Table 1.
List of identified client peptides by KiC assay using a synthetic peptide library with recombinant MPK3.
| NO. | Accession | Phospho-peptide | pRS scorea | pRS site probability (%)b |
|---|---|---|---|---|
| 1 | At1g01550 | S*MGASTLQATSPKKAAG | 143 | S(1): 100.0; S(5): 0.0 |
| 2 | At4g02300 | T*MIMFIGDGIGKTVIKAN | 135 | T(1): 100.0; T(13): 0.0 |
| 3 | At1g53050 | RQT*QPLTSRVVTLWY | 81 | T(3): 98.7; T(7): 1.1 |
| 4 | At1g15400 | TTGRVSPAVDPPSPRIS*S* | 80 | S(17): 93.0; S(18): 99.4 |
| 5 | At1g07630 | PIVLGSGPIERGFLS*GPIER | 66 | S(6): 0.0; S(15): 100.0 |
| 6 | At2g38280 | VRPIS*PKSPVASASAF | 63 | S(5): 97.1; S(8): 2.7 |
| 7 | At5g13240 | Y*LGKSSDTDSSSPVDLLLSR | 61 | Y(1): 94.8; S(5): 4.9 |
| 8 | At5g42590 | SVSKMNSY*VSGK | 59 | Y(8): 98.6; S(10): 0.7 |
| 9 | At3g04470 | AAEEEFS*TPPSSPVFHDAK | 50 | S(7): 88.4; T(8): 11.2 |
| 10 | At3g19330 | VASTSRNDASISSPTFNLS*R | 48 | T(15): 0.4; S(19): 95.3 |
| 11 | At3g05090 | KTVFQRGGS*FLAGNLS*F | 38 | S(9): 99.8; S(16): 100.0 |
| 12 | At4g30480 | IES*SES*EDEILIKNEPK | 32 | S(3): 79.4; S(6): 93.4 |
| 13 | At4g39680 | EAQITNSATPT*T*T*PRSTGL | 28 | T(11): 52.1; T(12): 63.5; T(13): 59.0 |
| 14 | At1g73980 | LS*LDDDTVSSPKEALSRASV | 25 | S(2): 87.4; T(7): 3.7 |
| 15 | At2g21150 | GSDEDDGENKSS*GT*GNLR | 23 | S(12): 90.3; T(14): 90.3 |
| 16 | At1g26150 | ESSSPRS*DS*ALLKT*QS*SA | 23 | S(7): 66.1; S(9): 66.1; T(14): 66.1 |
| 17 | At3g62700 | MAS*PITQRSIS*IES*PR | 22 | S(3): 91.0; S(11): 87.7; S(14): 87.7 |
| 18 | At3g01160 | SSHVESEEES*ESELKVASLD | 22 | S(2): 47.4; S(10): 78.1 |
| 19 | At4g12770 | VPSSGRASVNsPTAS*QMDEL | 20 | S(11): 65.0; S(15): 73.0 |
| 20 | At1g18670 | NAS*GNKQPLT*SRVVTLW | 16 | S(3): 99.3; T(10): 97.7 |
| 21 | At2g04235 | VFARRSPEGNTNS*EIEGS*L | 15 | S(13): 99.8; S(18): 98.8 |
| 22 | At4g18950 | VKKLDDEVLS* | 50 | S(10): 100.0 |
| 23 | At2g36570 | QFELDDLLKASAEMLGKGS* | 23 | S(11): 24.2; S(19): 75.8 |
*Amino acid residues that could potentially be phosphorylated by MPK3.
bpRS score is based on the cumulative binomial probability that the observed match is a random event. Potential phosphorylation sites were predicted by pRS score.
cpRS site probabilities are estimations of the probability (0 ~ 100%) for the respective site being truly phosphorylated.
Table 2.
List of identified client peptides by KiC assay using a synthetic peptide library with recombinant MPK6.
| NO. | Accession | Phospho-peptide | pRS scorea | pRS site probabilityb |
|---|---|---|---|---|
| 1 | At4g02300 | T*MIMFIGDGIGKTVIKAN | 126 | T(1): 100.0; T(13): 0.0 |
| 2 | At1g11360 | SPTVVTVQPSS*PRFPISTPT | 100 | S(11): 81.5; S(17): 6.0; |
| 3 | At1g53050 | RQT*QPLTSRVVTLWY | 82 | T(3): 99.8; T(7): 0.2 |
| 4 | At3g06480 | SRS*YSRSPSPVYE | 63 | S(1): 7.0; S(3): 85.2 |
| 5 | At2g38280 | VRPIS*PKSPVASASAF | 59 | S(5): 97.3; S(8): 2.7 |
| 6 | At1g15400 | TTGRVSPAVDPPSPRIS*S* | 56 | S(17): 93.4; S(18): 93.4 |
| 7 | At2g21300 | HSDDDLEEEMSPRHS*GDQSE | 50 | S(2): 0.5; S(11): 99.1 |
| 8 | At1g07630 | PIVLGS*GPIERGFLSGPIER | 50 | S(6): 92.7; S(15): 7.3 |
| 9 | At3g55270 | VHAFPLS*PTSLLRMY | 47 | S(7): 72.5; T(9): 12.5 |
| 10 | At3g04470 | AAEEEFS*TPPSSPVFHDAK | 41 | S(7): 90.3; T(8): 9.3 |
| 11 | At5g42590 | SVSKMNSY*VSGK | 37 | Y(8): 94.2; S(10): 2.8 |
| 12 | At2g16850 | AAIKALAS*FRSNPTN | 37 | S(8): 99.5; S(11): 0.5 |
| 13 | At1g11180 | GYGRTVDIPLDRPGS*GAQDL | 35 | T(5): 4.5; S(15): 91.1 |
| 14 | At3g46450 | SRNS*MMATVSSGKELLP | 31 | S(1): 5.7; S(4): 93.7 |
| 15 | At2g41830 | GLPRS*LSRTASVFSSSAALF | 30 | S(5): 70.4; S(7): 9.5 |
| 16 | At1g08420 | GALGGMVRQLS*IDQFENEGR | 30 | S(11): 100.0 |
| 17 | At1g09770 | ATRALLANYSQT*PRQGMT*P | 22 | T(12): 72.0; T(18): 72.0 |
| 18 | At2g27060 | SSTPSLPKIQNS*PDNPTS*R | 22 | S(12): 80.8; S(18): 71.3 |
| 19 | At4g12770 | VPSSGRASVNS*PTAS*QMDEL | 21 | S(11): 67.7; S(15): 73.7 |
| 20 | At1g63730 | TMLEDLPQSIRLWS*GLQV | 18 | S(9): 3.0; S(14): 95.4 |
| 21 | At5g20280 | LDVGQGLDDARS*S*PS*LLL | 15 | S(12): 100.0; S(13):100.0; S(15):100.0 |
*Amino acid residues that could potentially be phosphorylated by MPK6.
bpRS score is based on the cumulative binomial probability that the observed match is a random event. Potential phosphorylation sites were predicted by pRS score.
cpRS site probabilities are estimations of the probability (0 ~ 100%) for the respective site being truly phosphorylated.
Figure 2.
Venn diagram analysis of MPKs client peptides identified by the KiC assay.
(a) Venn diagram showed the overlap between the identified MPK3 and MPK6 client peptides. (b) Venn diagram showed the overlap between the identified MPK3 client peptides and previously published datasets. (c) Venn diagram showed the overlap between the identified MPK6 client peptides and previously published datasets. The numbers 325 and 2,209 represent the number of reported substrates of MPK3 or MPK6 by various methods, respectively (Figure S1)15–20. The overlap segments indicate the numbers of the same substrates.
Previously, 325 putative substrates of MPK3 and 2,209 putative substrates of MPK6 were identified through various methods such as protein microarray, primary sequence specificity, affinity chromatography and phosphoproteome analysis (Figure S1).15–20 To evaluate our results, we compared the isolated client peptides by the KiC assay with previously reported putative substrates of MPK3 and MPK6. As results, we found that 4 of 23 isolated client peptides (At1g15400, At2g38280, AT4g39680, and At4g12770) and 10 of 21 isolated client peptides (At1g08420, At1g09770, At1g11360, At1g15400, At2g38280, AT2g41830, At3g04470, At3g55270, At4g12770, and At5g20280) were previously reported as bona fide substrates of MPK3 and MPK6, respectively (Figure 2b, c).
Nonconserved motifs are phosphorylated by MPKs in the KiC assay
It is well reported that MPKs mostly phosphorylate their substrates within a simple conserved motif, Ser or Thr residues followed by Pro residue (S/T-P).4,5 In this study, we confidently identified phosphorylation sites of isolated client peptides by tandem mass spectrometry (Tables 1,2). Almost all mapped client peptides were phosphorylated on either Ser or Thr residues. However, the Tyr residues of some client peptides were surprisingly phosphorylated by MPK3 and MPK6 (Figure 3a). Meanwhile, four and eight isolated client peptides were phosphorylated on the conserved motif by MPK3 and MPK6, respectively. However, 19 and 13 isolated client peptides were unexpectedly phosphorylated within nonconserved motifs by MPK3 and MPK6, respectively (Figure 3b). These results suggest that MPKs can phosphorylate nonconserved Ser or Thr residues and even Tyr residues in in vitro reconstituted experiments.
Figure 3.
Analysis of phosphorylation sites on phosphorylated peptides.
(a) Compare phosphorylated peptide sequences. The MPK3 phosphorylates on Ser (70%), Thr (24%) and Tyr (6%) residues of peptides and MPK6 phosphorylates on Ser (82%), Thr (14%), and Tyr (4%) residues of peptides. Putative phosphorylation sites were aligned on position 0. (b) Number of client peptides phosphorylated by MPKs on conserved motif or nonconserved motifs. Among 23 client peptides, 19 were phosphorylated on nonconserved motifs, whereas 4 were phosphorylated on conserved motif by MPK3. Among 21 client peptides, 13 were phosphorylated on nonconserved motifs, whereas 8 were phosphorylated on conserved motif by MPK6.
Verification of the substrates using recombinant fusion protein
Because the client peptides in the KiC assay were 10-to 20-mer short synthetic peptides, it is required to verify whether the full-length fusion proteins of isolated client peptides can also be phosphorylated by MPKs. To examine the phosphorylation of full-length proteins by in vitro kinase assay, we expressed and purified GST-fused full-length proteins of isolated client peptides. In the case of AT3g62700, we constructed a partial cDNA clone encoding the partial fusion protein including the potential phosphorylation site because we failed to produce its full-length fusion protein. As results, we found that 13 of 23 fusion proteins were phosphorylated by MPK3 (Figure 4A). Similarly, 9 of 21 fusion proteins were phosphorylated by MPK6 (Figure 4B). Overall, these results demonstrate that approximately half of client peptides isolated by the KiC assay were verified as the potential substrates of MPKs in vitro. Interestingly, 4 of 13 isolated substrate proteins have already been reported as MPK3 substrates (Table 3) and 7 of 9 isolated substrate proteins were already reported as MPK6 substrates (Table 4). Among these substrates, three substrates (AT1g15400, AT2g38280, and AT4g12770) were phosphorylated by both MPK3 and MPK6, while five substrates (AT1g08420, AT1g11360, AT3g55270, AT4g39680, and AT5g20280) were specifically phosphorylated by either MPK3 or MPK6. We compared the phosphorylation sites identified by the KiC assay with those identified in the previous reports.15–20 The phosphorylation sites of four substrates were same with those previously reported, but the phosphorylation sites of four other substrates were different from those previously reported (Table S2). These results indicate that not only the same phosphorylation sites but also the new phosphorylation sites can be identified from the same substrates by the KiC assay. Conclusively, based on the KiC assay we could identify nine and two novel putative substrates of MPK3 and MPK6, respectively.
Figure 4.
Verification of putative substrates using in vitro kinase assay.
In vitro kinase performed with (a) 6XHis-MPK3 (2 µg) or (B) 6XHis-MPK6 (2 µg) and the recombinant substrate protein (3 µg) in the presence of [γ-32P] ATP. After SDS-PAGE, the proteins were visualized by Coomassie blue staining (Staining). The phosphorylated proteins were visualized by autoradiography (Autorad). The asterisks represent phosphorylated substrate proteins, and the arrow represents the position of substrate proteins in SDS-PAGE. The numbers represent the fusion protein of MPK3 or MPK6 client peptides (Tables 1 and 2). MBP and GST were used as positive and negative control, respectively.
Table 3.
List of phosphorylated recombinant substrates by MPK3 using in vitro kinase assay.
| NO. | Accession | Annotationa | Phosphorylationb | Subcellular localizationc | Reference |
|---|---|---|---|---|---|
| 1 | At1g01550 | BYPASS1 | ++ | Nucleus | |
| 2 | At4g02300 | PECTIN METHYLESTERASE 39 | - | Cell wall | |
| 3 | At1g53050 | Protein kinase superfamily protein | - | Nucleus | |
| 4 | At1g15400 | Unknown protein | ++ | Nucleus | Sörensson et al. 201217 |
| 5 | At1g07630 | POL-like5 | - | Nucleus | |
| 6 | At2g38280 | Embryonic factor1 | + | Nucleus | Hoehenwarter et al. 201318 |
| 7 | At5g13240 | Transcription regulator | - | Nucleus | |
| 8 | At5g42590 | CYP71A16 | - | Extracellular | |
| 9 | At3g04470 | Ankyrin repeat family protein | + | Plasma membrane | |
| 10 | At3g19330 | DUF677 | + | Nucleus | |
| 11 | At3g05090 | Lateral root stimulator | + | Nucleus | |
| 12 | At4g30480 | AtTPR1 | + | Cytosol | |
| 13 | At4g39680 | SAP domain-containing protein | ++ | Nucleus | Hoehenwarter et al. 201318 |
| 14 | At1g73980 | Phosphoribulokinase | ++ | Cytosol | |
| 15 | At2g21150 | XAP5 | - | Nucleus | |
| 16 | At1g26150 | AtPERK10 | - | Plasma membrane | |
| 17 | At3g62700 | ATP-binding cassette C14 | + | Plasma membrane | |
| 18 | At3g01160 | Unknown protein | - | Nucleus | |
| 19 | At4g12770 | Chaperon DnaJ-domain superfamily protein | + | Cytosol | Hoehenwarter et al. 201318 |
| 20 | At1g18670 | IBS1 | - | Nucleus | |
| 21 | At2g04235 | Unknown protein | + | Nucleus | |
| 22 | At4g18950 | Integrin-linked protein kinase family | + | Nucleus | |
| 23 | At2g36570 | PXC1 | - | Plasma membrane |
aProtein annotation from TAIR database.
bThe number of + indicates the intensity of phosphorylation by MPK3, and – indicates no phosphorylation by MPK3.
cProtein subcellular localization extracted from TAIR database.
Table 4.
List of phosphorylated recombinant substrates by MPK6 using in vitro kinase assay.
| NO. | Accession | Annotationa | Phosphorylationb | Subcellular localizationc | Reference |
|---|---|---|---|---|---|
| 1 | AT4g02300 | PECTIN METHYLESTERASE 39 | - | Cell wall | |
| 2 | AT1g11360 | Adenine nucleotide alpha hydrolases-like superfamily | ++ | Nucleus | Hoehenwarter et al. 201318; Sörensson et al. 201217; Wang et al. 202020 |
| 3 | AT1g53050 | Protein kinase superfamily protein | - | Nucleus | |
| 4 | AT3g06480 | DEAD box RNA helicase family protein | - | Nucleus | |
| 5 | AT2g38280 | Embryonic factor 1 | + | Nucleus | Hoehenwarter et al. 201318; Wang et al. 202020 |
| 6 | AT1g15400 | Unknown protein | + | Nucleus | Sörensson et al. 201217 |
| 7 | AT2g21300 | ATP binding microtubule motor family protein | + | Cytosol | |
| 8 | AT1g07630 | POL-LIKE 5 | - | Nucleus | |
| 9 | AT3g55270 | AtMKP1 | ++ | Nucleus | Hoehenwarter et al. 201318; Wang et al. 202020 |
| 10 | AT3g04470 | Ankyrin repeat family protein | - | Plasma membrane | Wang et al. 202020 |
| 11 | AT5g42590 | CYP71A16 | - | Extracellular | |
| 12 | AT2g16850 | PIP2;8 | ++ | Cytosol | |
| 13 | AT1g11180 | Secretory carrier membrane protein 2 | - | Plasma membrane | |
| 14 | AT3g46450 | SEC14 cytosolic factor family protein | - | Cytosol | |
| 15 | AT2g41830 | Uncharacterized protein | - | Plasma membrane | |
| 16 | AT1g08420 | BSL2/BRI1 suppressor 1 (BSU1)-like 2 | ++ | Nucleus | Wang et al. 202020 |
| 17 | AT1g09770 | AtCDC5 | - | Nucleus | Wang et al. 202020 |
| 18 | AT2g27060 | Leucine-rich repeat receptor kinase family protein | - | Plasma membrane | |
| 19 | AT4g12770 | Chaperone DnaJ-domain superfamily protein | + | Cytosol | Hoehenwarter et al. 201318 |
| 20 | AT1g63730 | Disease resistance protein (TIR-NBS-LRR class) family | - | Unknown | |
| 21 | AT5g20280 | Sucrose phosphate synthase 1F | + | Plasma membrane | Wang et al. 202020 |
aProtein annotation from TAIR database.
bThe number of + indicates the intensity of phosphorylation by MPK6, and – indicates no phosphorylation by MPK6.
cProtein subcellular localization extracted from TAIR database.
Discussion
Identification of new substrates of MPKs using the KiC assay
MPK cascades in plants are believed to be composed of a large number of downstream substrates because they play a numerous variety of roles in the cellular responses to various external stimuli.1–3 To understand the novel functions of MPK cascades in many signaling pathways, isolation of new substrates of MPKs is essential. In this study, we applied the KiC assay to identify novel substrates of MPKs. As a result, 23 and 21 client peptides were identified as putative substrates of MPK3 and MPK6, respectively (Tables 1, 2). Interestingly, eight of identified client peptides were overlapped (Figure 2A), indicating that the targets of MPK3 and MPK6 are redundant in some signaling pathways. 13 client peptides of MPK3 and 9 client peptides of MPK6 were verified as substrates of MPKs by in vitro kinase assay of their fusion proteins. In conclusion, we identified nine novel substrates of MPK3 and two novel substrates of MPK6 in this study (Figure 4). Using the same KiC assay, 23 putative substrates of P2K1 were identified and some of them were extensively studied.24 Conclusively, the new signaling pathway of plant immune response controlled by P2K1 was elucidated by the phosphorylation studies of those isolated substrates.24,25,32 Thus, we suggest that the KiC assay is a useful method to identify novel substrates of MPKs and the novel signaling pathway of MPKs through these substrates may be revealed by subsequent research.
Validation of the KiC assay using in vitro kinase assay
Generally, it is known that MPKs could phosphorylate the Ser or Thr residues on the S/T-P motif of their substrates.4,5 Interestingly, most isolated client peptides were phosphorylated on Ser or Thr residues, but two isolated client peptides (AT5g13240 and AT5g42590) were phosphorylated on Tyr residues by the KiC assay (Figure 3A). However, subsequent in vitro kinase assay of fusion proteins showed that these recombinant proteins were not phosphorylated by MPKs (Figure 4). These results indicate that the KiC assay results included nonspecific phosphorylation. Since we confirmed isolated client peptides by the KiC assay through in vitro kinase assay of their recombinant proteins (Figure 4), the novel substrates identified in this study would be bona fide substrates of MPKs.
We speculate that nonspecific phosphorylation in the KiC assay is possibly due to the excess amount of peptides and kinases employed. To improve the accuracy of the KiC assay, it is necessary to obtain results by comparing a series of different dilution reactions of peptides and kinases. Through this series of reactions can minimize nonspecific phosphorylation while maintaining efficient phosphorylation of substrates.
Further research of novel identified MPKs-substrate modules
In this study, we identified 11 novel substrates of MPKs by in vitro kinase assay (Figure 4, Table 3, 4). Among them, AT1g01550 and AT3g05090 have been suggested to be involved in plant growth by participating in auxin responses,28,33 and AT3g73980, AT4g12700, AT4g30480, and AT2g16850 have been reported to be involved in abiotic stress response.31,34–37 To identify the biological roles of their phosphorylation by MPKs, it is necessary to test whether the phenotypes of these knock-out mutants can be rescued by the expression of the non-phosphorylation form. In the case of unknown substrates (AT2g04235, AT2g21300, AT3g04470, AT3g19330, and AT4g18950), the biological function of them would be identified first, and then the biological function of their phosphorylation should be investigated. In this report, we mainly focused on the usefulness of the KiC assay for the identification of novel substrates of MPKs by the assay in vitro. Therefore, independent more detail functional and biochemical researches including in vivo phosphorylation of the identified substrates by MPKs should be performed.
Supplementary Material
Funding Statement
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education [2020R1A6A1A03044344, 2023R1A2C1005572 and RS-2023-00235511].
Disclosure statement
No potential conflict of interest was reported by the author(s).
Author contributions
S.B., N. A. and W.S.C. designed, planned, and organized the experiment. S.B., N.A., J.A., S.H.K. and Z.R. performed experiments and data analysis. S.B., N.A., J.C.H., J.J.T. and W.S.C. wrote the manuscript with feedback from all authors.
Supplementary material
Supplemental data for this article can be accessed online at https://doi.org/10.1080/15592324.2024.2326238
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