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Mitogen-activated protein kinase (MAP kinase, MAPK) cascades play pivotal roles in signal transduction of extracellular stimuli, such as environmental stresses and growth regulators, in various organisms. Arabidopsis thaliana MAP kinases constitute a gene family, but stimulatory signals for each MAP kinase have not been elucidated. Here we show that environmental stresses such as low temperature, low humidity, hyper-osmolarity, touch and wounding induce rapid and transient activation of the Arabidopsis MAP kinases ATMPK4 and ATMPK6. Activation of ATMPK4 and ATMPK6 was associated with tyrosine phosphorylation but not with the amounts of mRNA or protein. Kinetics during activation differ between these two MAP kinases. These results suggest that ATMPK4 and ATMPK6 are involved in distinct signal transduction pathways responding to these environmental stresses.
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The growth of land plants is greatly affected by a variety of environmental stresses, such as dehydration, low temperature, heat, mechanical perturbation (touch), wounding and pathogen infection. Environmental stresses induce various biochemical, physiological and molecular responses, including gene expression (for reviews, see Bowles, 1993; Braam et al., 1997; Ingram and Bartels, 1996; Lamb and Dixon, 1997; Shinozaki and Yamaguchi-Shinozaki, 1999; Yang et al., 1997). It is thought that these responses are promoted as a result of the perception of environmental stimuli and signal transduction in plant cells, but the mechanisms by which plants sense and transduce signals in response to environmental stresses are largely unknown.
MAPK homologues have been isolated from various plant species (Ligterink, 2000; Mizoguchi et al., 1997). Recently, it has been reported that specific MAPKs are activated in response to environmental stimuli and phytohormones. Biotic and abiotic stress-induced activation of tobacco MAPKs, salicylic-acid-induced protein kinase (SIPK) and wound-induced protein kinase (WIPK) has been thoroughly studied. SIPK was initially identified as a protein kinase induced by salicylic acid in tobacco cell suspensions (Zhang and Klessig, 1997). Extensive study of SIPK showed that various environmental stresses such as elicitors (Romeis et al., 1999; Zhang et al., 1998), wounding (Zhang and Klessig, 1998a), tobacco mosaic virus (TMV) infection (Zhang and Klessig, 1998b) and hyper-osmolarity (Hoyos and Zhang, 2000; Mikołajczyk et al., 2000) induced activation of SIPK. The activity of SIPK correlates with its tyrosyl phosphorylation, but not with transcriptional and translational levels. A putative activator of SIPK, SIPK kinase (SIPKK), has been identified as an SIPK-interacting protein by screening using a yeast two-hybrid system (Liu et al., 2000).
The WIPK gene was originally isolated as a wound-inducible gene by differential screening (Seo et al., 1995). Biochemical analyses using a specific antibody against WIPK showed an increase in WIPK activity by TMV infection (Zhang and Klessig, 1998b), elicitors (Romeis et al., 1999) and wounding (Seo et al., 1999). Transcript levels of WIPK are also elevated by stress treatments. In alfalfa, environmental stress activates two kinds of MAPK. One is a stress-activated MAPK (SAMK, also known as MMK4), related to WIPK, that responds in both activity and transcription to environmental stresses such as low temperature and drought (Jonak et al., 1996), mechanical perturbation (Bögre et al., 1996) and wounding (Bögre et al., 1997). The other is a salt-stress-inducible MAPK (SIMK, also known as MsERK1, MsK7 and MMK1). SIMK is structurally highly similar to tobacco SIPK and responds to hyper-osmotic stress (Munnik et al., 1999). SIMK is constitutively localized in the nucleus before and after activation. These results suggest that plant MAPKs are ubiquitous messengers of biotic and abiotic stress stimuli, but no MAPK cascade has had its constituents and function in plants clarified.
An Arabidopsis MAPK, ATMPK6, has a high structural similarity to tobacco SIPK and was recently found to be activated by elicitors (Nühse et al., 2000). Kovtun et al. (2000) also showed H2O2-induced activation of ATMPK3 and ATMPK6 through Arabidopsis NPK1-like protein kinases (ANPs (MAPKKKs); Nishihama et al., 1997) using a protoplast transient expression assay. However, whether ATMPK6 responds to other stimuli, as SIPK does, remains to be resolved. Moreover, no report has studied in detail the activation of multiple MAPKs by different environmental stresses.
In this study, we show that various abiotic stresses induce activation of at least three Arabidopsis MAPKs: ATMPK4, ATMPK6 and a 44 kDa MAPK. Activation of ATMPK4 and ATMPK6 did not correlated with their transcript or protein levels. The kinetics upon activation of the two MAPKs were not identical. The possible functions of these MAPKs in the stress response and their cascades are discussed.
Environmental stresses induce activation of Arabidopsis MAPKs
Initially we studied whether environmental stresses, such as low temperature, dehydration, touch and wounding, induce activation of MAPKs. Arabidopsis plants or detached leaves were subjected to environmental stress treatments, and protein extracts were prepared. Protein kinase activity was analysed by in-gel kinase assay using bovine myelin basic protein (MBP) as an artificial substrate. Low temperature, low humidity, touch and wounding induced activation of protein kinases of 47 and 44 kDa (Figure 1a). To examine whether these activated protein kinases are related to MAPKs, we immunoprecipitated them with E10 monoclonal antibody, which is specific to doubly phosphorylated TEY residues. In mammalian cells, the activation of MAPKs is correlated with their dual phosphorylation on threonine and tyrosine residues within the TxY motif (Ray and Sturgill, 1988). Plant MAPKs contain this conserved phosphorylation motif as TEY or TDY. The 49 and 44 kDa protein kinases were recovered in the immunoprecipitate (Figure 1b), but protein A–Sepharose alone failed to recover them (Figure 1c). These data provide evidence for the activation of MAPKs in response to environmental stresses.
Antibodies raised against ATMPK4 and ATMPK6 specifically recognize ATMPK4 and ATMPK6 proteins, respectively
We have already identified a possible Arabidopsis MAPK cascade, ATMEKK1 → MEK1/ATMKK2 → ATMPK4, by yeast two-hybrid analysis and complementation analysis of yeast mutants (Ichimura et al., 1998; Mizoguchi et al., 1998). According to our model, ATMPK4 can function downstream of ATMEKK1. Transcription of the ATMEKK1 gene was rapidly increased by low-temperature, high-salinity and mechanical stresses (Mizoguchi et al., 1996). This finding suggests that ATMEKK1 and its downstream factors function in a signal transduction pathway for environmental stresses in Arabidopsis. We were also interested in ATMPK6 because it is structurally most similar to tobacco SIPK, which is activated by various environmental stresses.
To analyse the activation of ATMPK4 and ATMPK6 under environmental stress conditions, we raised polyclonal antibodies against the C-terminal 16 amino acids of ATMPK4 and the N-terminal 25 amino acids of ATMPK6, named Ab4CT1 and Ab6NT1, respectively. The specificity of these antibodies was assessed by immunoblotting and immunoprecipitation analyses against various Arabidopsis MAPKs. Eight Arabidopsis MAPKs (ATMPK1–8; Mizoguchi et al., 1993; Mizoguchi et al., 1997) were expressed as a glutathione S-transferase (GST) fusion protein in Escherichia coli and were affinity-purified. The GST fusion proteins and protein extracts of Arabidopsis leaves were separated by SDS–PAGE (Figure 2a) and immunoblotted with Ab4CT1 or Ab6NT1. Ab4CT1 and Ab6NT1 specifically recognized the GST–ATMPK4 and GST–ATMPK6 proteins, respectively (Figure 2b,c). In leaf extracts, Ab4CT1 and Ab6NT1 reacted with a 43 kDa protein and a 47 kDa protein, respectively (Figure 2b,c). These molecular masses are consistent with the calculated masses deduced from their amino acid sequences. Ab4CT1 also bound a 50 kDa leaf protein in a cross-reaction with the large subunit of Rubisco. (Figure 2b). These results suggest that Ab4CT1 and Ab6NT1 specifically bind to ATMPK4 and ATMPK6, respectively, on immunoblotting.
We also verified the specificity of the antibodies upon immunoprecipitation. Each antibody was incubated with GST–ATMPK proteins 1–8 and immunoprecipitated. The sedimented immunocomplex was resolved by SDS–PAGE and blotted onto a membrane. GST fusion proteins were detected by immunoblotting with anti-GST antibody. Ab4CT1 and Ab6NT1 specifically immunoprecipitated GST–ATMPK4 and GST–ATMPK6, respectively (Figure 2d,e).
ATMPK4 and ATMPK6 are rapidly activated by low-temperature, dehydration, touch, wounding and hyper-osmotic stresses, but not by heat stress or ABA treatment
Using specific antibodies, we examined the kinase activity of ATMPK4 and ATMPK6 under stress conditions. Activities of ATMPK4 and ATMPK6 were analysed by in-gel kinase assay coupled with immunoprecipitation with Ab4CT1 and Ab6NT1. ATMPK4 was recovered as a 43 kDa protein kinase and was activated by low temperature, low humidity, touch and wounding (Figure 3a–d, middle panels). ATMPK6 was immunopurified as a 49 kDa protein kinase and was also activated by the same environmental stresses as ATMPK4 (Figure 3a–d, bottom panels). Low temperature induced activation of ATMPK4 and ATMPK6 within 2 min (Figure 3a). Maximum activity of ATMPK4 was reached at 60 min, whereas ATMPK6 was fully activated at 10 min. Activation of ATMPK4 lasted at least 120 min. Low humidity also induced activation of ATMPK4 and ATMPK6 within a few minutes, and their activities peaked within 30 min (Figure 3b). Touch and wounding rapidly activated ATMPK4 and ATMPK6, with maximum activity by around 5–10 min (Figure 3c,d).
We also examined the activation of ATMPK4 and ATMPK6 by heat treatment and abscisic acid (ABA). Arabidopsis plants were exposed to heat shock (37°C) or sprayed with 100 µm ABA, and then leaf protein extracts were prepared. ATMPK4 and ATMPK6 were immunoprecipitated, and their activities were examined by in-gel assay. No activation was observed after heat shock (Figure 3e,f, top panels). ABA spray slightly activated both ATMPK4 and ATMPK6 (Figure 3e,f, middle panels). However, spraying with 0.1% ethanol solution as a control also slightly activated them (Figure 3e,f, bottom panels). These results indicate that ATMPK4 and ATMPK6 were not activated by ABA, and their slight activation was due to the mechanical stimulus of spraying, because both ATMPK4 and ATMPK6 are activated in response to touch (Figure 3c).
To check the specificity of immunoprecipitation, we performed immunoprecipitation in the presence or absence of antigen peptides for Ab4CT1 and Ab6NT1 (Figure 3g). Pre-incubation with an excess amount of the peptides completely blocked immunoprecipitation of ATMPK4 and ATMPK6. This shows that the antibodies against ATMPK4 (Ab4CT1) and ATMPK6 (Ab6NT1) can specifically immunoprecipitate endogenous ATMPK4 and ATMPK6 proteins.
The activation pattern of ATMPK4 (Figure 3a–d, middle panels) was not identical to that of the 44 kDa MAPK detected in total protein extract (Figure 3a–d, top panels). This result suggests that ATMPK4 is different from the 44 kDa MAPK. ATMPK6 was immunopurified as a 49 kDa protein kinase (Figure 3a–d, bottom panels). In contrast with ATMPK4, the kinetics of ATMPK6 activation were identical to those of the 47 kDa MAPK in total leaf extract (Figure 3a–d, top panels), which suggests that ATMPK6 corresponds to the 47 kDa MAPK. To confirm whether ATMPK4 is not the 44 kDa MAPK and ATMPK6 is the 47 kDa MAPK, we performed immunodepletion of ATMPK4 and ATMPK6 from total Arabidopsis protein extract prepared 10 min after touch stimulation. In the extract immunodepleted of ATMPK4, the 44 kDa MAPK remains visible as a strong signal (Figure 3h, lane 2) whereas ATMPK4 is certainly immunoprecipitated (Figure 3h, lane 3) and the 43 kDa band of ATMPK4 is significantly reduced on immunoblotting (Figure 3h, lane 5). In the extract that has been immunodepleted of ATMPK6, the 47 kDa band is strongly reduced (Figure 3h, lane 7) compared with an untreated sample, while the 44 kDa band remains unchanged. These results indicate that ATMPK4 is different from the 44 kDa MAPK, and that ATMPK6 is the 47 kDa MAPK in the in-gel assay. The discrepancy in the mobility of ATMPK6 may be due to a large amount of the large subunit of Rubisco, with a similar molecular weight, in the total protein extract.
We further analysed whether osmotic stress activates ATMPK4 and ATMPK6 (Figure 4). High-salinity and hyper-osmotic stresses rapidly induced transient activation of ATMPK4 and ATMPK6 (Figure 4). To test whether activation of ATMPK4 and ATMPK6 depends on salt and sorbitol concentration, Arabidopsis leaves were treated with different concentrations of NaCl and sorbitol. ATMPK4 and ATMPK6 were activated in a dose-dependent manner, but their dose dependencies differed (Figure 4). These results show that ATMPK4 and ATMPK6 were activated by hyper-osmotic stress.
Expression of the ATMPK4 and ATMPK6 genes and their proteins does not correlate with activation of their protein kinase activities under stress conditions
To investigate whether stress-induced activation of ATMPK4 and ATMPK6 is regulated at transcriptional or post-translational levels, we examined the mRNA levels of ATMPK4 and ATMPK6 by Northern blot analysis. Transcripts of the two genes remained unchanged after low-temperature, low-humidity, touch and wounding treatments (Figure 5). In the same blots, expression of the marker genes rd29A (Yamaguchi-Shinozaki and Shinozaki, 1993), TCH3 (Braam and Davis, 1990) and Atvsp (Berger et al., 1995) was stimulated by cold and low humidity, by touch and by wounding stresses, respectively (Figure 5a–d). Little or no stress-induced gene expression of ATMPK4 and ATMPK6 was observed in any of the treatments.
To measure levels of the ATMPK4 and ATMPK6 proteins during activation, we carried out immunoblot analyses with the Ab4CT1 and Ab6NT1 antibodies. Aliquots of leaf protein extracts were subjected to SDS–PAGE and immunoblot analysis. Although activation of ATMPK4 and ATMPK6 was detected after exposure to various environmental stresses, the amounts of the proteins remained constant during the stress conditions (Figure 5e).
Phosphorylation of tyrosine residue correlates with the activation of ATMPK4 and ATMPK6
Levels of the ATMPK4 and ATMPK6 proteins and their transcripts were unchanged under stress conditions. This indicates that post-translational modification was responsible for activation of ATMPK4 and ATMPK6. Because yeast and mammalian MAPKs are activated by dual phosphorylation of the TxY motif between sub-domains VII and VIII of the catalytic domain, we tested whether tyrosine phosphorylation was associated with the activation of ATMPK4 and ATMPK6. For detection of tyrosine phosphorylation of ATMPK4, we used T87 cells instead of Arabidopsis plants, because we could not detect tyrosine phosphorylation of ATMPK4 in protein extracts prepared from Arabidopsis plants because of the low protein content (data not shown). Protein extracts from low-temperature-treated T87 Arabidopsis cells were immunoprecipitated with Ab4CT1, and those from low-temperature-treated Arabidopsis plants were immunoprecipitated with Ab6NT1. The sedimented immunocomplexes were then subjected to immunoblot analysis with an anti-phosphotyrosine monoclonal antibody, 4G10. The specificity of this immunoprecipitation was monitored by adding the competitor peptide. As anticipated, tyrosine phosphorylation was associated with the activation of ATMPK4 by low temperature (Figure 6a). Pre-incubation of Ab4CT1 with excess antigen peptide completely blocked both recovery of ATMPK4 (Figure 6a, upper panel) and detection of phosphotyrosine in the 43 kDa immunoprecipitate (Figure 6a, lower panel). Tyrosine phosphorylation of ATMPK6 was also correlated with its activity under low temperature (Figure 6b). Incubation of Ab6NT1 with excess antigen peptide abolished detection of both protein kinase activity and phosphotyrosine in the immunoprecipitate (Figure 6b). These results suggest that ATMPK4 and ATMPK6 are activated by phosphorylation on their tyrosine residues in the TEY motif.
In this study, we show that two Arabidopsis MAPKs, ATMPK4 and ATMPK6, are activated by treatment with environmental stresses, such as low temperature, low humidity, hyper-osmolarity, touch and wounding. ATMPK4 may be activated through its upstream factors, ATMEKK1, MEK1 and ATMKK2 (Figure 7), because ATMPK4 specifically interacts with them and can be activated by MEK1 in yeast and in vitro (Huang et al., 2000; Mizoguchi et al., 1998). These stresses also induced activation of 47 and 44 kDa MAPKs in total leaf extract. We have concluded that the 47 kDa MAPK is ATMPK6 because the activation profile of the 47 kDa MAPK was identical to that of ATMPK6 (Figure 3a–d), and Ab6NT1, a specific antibody against ATMPK6, specifically immunodepleted the 47 kDa MAPK in total leaf extract (Figure 3h). We also concluded that ATMPK6 and the 44 kDa MAPK may share the same MAPKK (Figure 7), because the activation patterns of ATMPK6 and the 44 kDa MAPK are identical to each other (Figure 3a–d). In contrast to ATMPK6, the time course of ATMPK4 activation was dissimilar to that of the 44 kDa MAPK (Figure 3a–d). In addition to this observation, we showed that activity of the 44 kDa MAPK is still detectable as a strong signal in the ATMPK4 immunodepleted protein extract (Figure 3h). This indicates that ATMPK4 is not the 44 kDa MAPK. These results provide evidence indicating that environmental stresses induce activation of at least three different MAPKs in Arabidopsis (Figure 7). Similarly, hydrogen peroxide induced activation of ATMPK3 and ATMPK6 (Kovtun et al., 2000). Simultaneous activation of two tobacco MAPKs, WIPK and SIPK, by the avirulence gene product Avr9 was also reported by Romeis et al. (1999). WIPK and SIPK have high sequence similarity with Arabidopsis ATMPK3 and ATMPK6, respectively. This raises the possibility that the 44 kDa MAPK may be ATMPK3, because ATMPK3 and ATMPK6 are specifically activated with ANP-dependent manner (Kovtun et al., 2000). We prepared anti-ATMPK3-specific antibody, and then studied whether ATMPK3 is activated by low temperature, low humidity, touch or wounding. However, no activation of ATMPK3 was detected (data not shown). Similar results have been reported by Nühse et al. (2000). This suggests that ATMPK3 is activated by a specific stimulus and does not respond to other stresses in spite of the responsiveness of its gene expression, and that a much higher affinity to anti-ATMPK3 antibody is required to detect ATMPK3 activity. Thus, it remains to be resolved whether the 44 kDa MAPK is ATMPK3.
The next question concerns the similarity or differences of the roles of ATMPK4 and ATMPK6 in environmental stress responses. We consider that the roles of ATMPK4 and ATMPK6 are not completely identical for four reasons.
1ATMPK4 and ATMPK6 are structurally dissimilar; they are classified into different subgroups. The structural difference may cause the different specificity of enzyme–substrate interactions.
2ATMPK4 specifically interacts with ATMEKK1, MEK1 and ATMKK2, but ATMPK6 does not (Ichimura et al., 1998).
3Co-expression of ATMPK4 and MEK1 in Saccharo myces cerevisiae suppressed caffeine- and temperature-sensitive mpk1 phenotypes, but co-expression of ATMPK6 and MEK1 did not (unpublished results).
4The kinetics upon activation of ATMPK4 and ATMPK6 are not identical; clear differences can be detected in the low-temperature stress response (Figure 3a).
The last question is how ubiquitously activated MAPKs generate specific and biologically appropriate cellular responses? There are two possibilities concerning the role of MAPK in response to different environmental stresses. First, the duration of the activation state of MAPK can play an important role in the generation of specific biological responses. This is especially apparent in mammalian PC12 cells, where ERK (extracellular signal-regulated kinase) activation can initiate two opposing biological programmes, cell growth and cell differentiation (Marshall, 1995). Sustained ERK activation leads to neurite outgrowth, whereas transient activation of ERKs induces cell growth. Thus, activation of the ERK cascade can lead to contrasting physiological responses in the same cellular context. This suggests that signal specificity is also determined by the duration of the activation state of MAPK other than the selective activation of a MAPK module. This may be the case with ATMPK4, because low-temperature stress treatment induces sustained activation of ATMPK4, whereas other stress treatments induce transient activation (Figure 3). A similar result was found for elicitor-induced MAPK activation in plants. In cultured tobacco cells, a cell-wall-derived elicitor induced transient activation of MAPK but not cell death, but proteinaceous elicitors such as elicitins and xylanase induced prolonged activation and cell death (Suzuki et al., 1999; Zhang et al., 1998). This suggests that the duration of the activated state of MAPK can determine stimulus-dependent responses.
Second, complex formation may determine signal specificity. In S. cerevisiae, complexes have been identified in certain MAPK components involved in a pheromone mating-response pathway and an osmoregulatory pathway (for review, Whitmarsh and Davis, 1998). The Ste5 protein serves as a scaffold in the pheromone mating-response pathway and binds components of the MAPK cascade, Ste11 (MAPKKK), Ste7 (MAPKK) and Fus3/Kss1 (MAPKs). In the osmoregulatory pathway, the Pbs2 protein kinase functions not only as a MAPKK but also as a scaffold protein. Pbs2 interacts with Ste11 (MAPKKK) and Hog1 (MAPK). Although Ste11 is involved in both pathways, there is no cross-talk between the pathways. Thus, complex formation increases the local concentration of the components involved in the specific cascade, and minimizes interference among signalling pathways (Yashar et al., 1995). Our previous results show that ATMPK4, ATMEKK1 and MEK1/ATMKK2 may form a complex and constitute a MAPK cascade in Arabidopsis (Ichimura et al., 1998; Mizoguchi et al., 1998). ATMPK4 and ATMPK6 may form different complexes and transduce different signals under different conditions.
To clarify the in vivo role of the plant MAPK cascade, analyses of gene knock-out mutants and transgenic plants that over-express or repress genes for factors in the MAPK cascade will help to elucidate the molecular mechanisms of the stress response and of adaptations to environmental changes in higher plants.
Arabidopsis thaliana (Columbia ecotype) was grown in soil at 22°C under continuous light. Arabidopsis T87 cultured cells were sub-cultured at 1-week intervals in JPL medium (Axelos et al., 1992).
Stresses and phytohormone treatments
Six- to eight-week-old Arabidopsis plants were allowed to rest for at least 2 h in the laboratory.
Low temperature and heat treatments. Plants were shifted to 4°C or 37°C, respectively.
Low humidity treatment. The top of a pot was wrapped with plastic film for 2 days, then the plant was exposed to low humidity by removing the film.
Touch treatment. The plant was gently moved back and forth for 15 sec by a gloved hand.
Wounding treatment. A rosette leaf was isolated and allowed to stand for 24 h in water. The detached leaf was mechanically wounded by nipping twice with forceps.
NaCl and sorbitol treatments. A rosette leaf was isolated and allowed to stand for 24 h in 10 ml water. Then an equal volume of NaCl or sorbitol solution was added.
ABA treatment. ABA treatment was performed by spraying 50 ml of 100 µm ABA solution on plants. As a control, 0.1% ethanol solution was also sprayed. At different times, two rosette leaves per plant were isolated and immediately frozen in liquid nitrogen, and then stored at −80°C.
Treatment of Arabidopsis T87 cells. For low-temperature treatment of Arabidopsis T87 cells, 7-day-cultured cells (325 ml) were transferred to a 1 litre beaker and shaken for at least 2 h. Beaker of cultured cells was incubated in ice water. At different times, 25 ml of cells were harvested by filtration. The cells were quickly frozen in liquid nitrogen and stored at −80°C.
Preparation of protein extracts
Frozen leaves and cultured cells were ground in liquid nitrogen, then thawed in extraction buffer (50 mm HEPES–KOH pH 7.5, 5 mm EDTA, 5 mm EGTA, 2 mm DTT, 25 mm NaF, 1 mm Na3VO4, 50 mmβ-glycerophosphate, 20% glycerol, 2 µg ml−1 leupeptin, 2 µg ml−1 pepstatin A, 2 mm PMSF). After centrifugation at 15 000 g for 30 min at 4°C, supernatants were transferred into clean tubes, frozen in liquid nitrogen, and stored at −80°C. The protein concentration was determined by a turbidimetric procedure (Vera, 1988).
Preparation of GST fusion proteins
Expression and affinity purification of glutathione S-transferase (GST) fusion proteins (ATMPK1–8, Mizoguchi et al., 1993; Mizoguchi et al., 1997) were performed as follows. E. coli JM109 cells transformed with the GST fusion constructs were grown at 37°C overnight and were sub-cultured until the OD600 reached 0.5. Expression of the GST–ATMPK fusion proteins was induced with 0.5 mm isopropyl-β-d-thiogalactopyranoside (IPTG). Cells were incubated at 16°C overnight, then harvested by centrifugation at 5000 g for 15 min at 4°C. The pellets were re-suspended in ice-cold 1 × PBS with 2 mm DTT, 2 µg ml−1 leupeptin, 2 µg ml−1 pepstatin A, 2 mm PMSF and 5 mg ml−1 lysozyme. The bacterial cells were disrupted by sonication. Triton X-100 was added to a final concentration of 1%. The samples were gently inverted for 30 min at 4°C, and cell debris was removed by centrifugation at 12 000 g for 10 min at 4°C. Beads of glutathione–Sepharose 4B (Pharmacia Biotech, Uppsala, Sweden) were added to the supernatant and incubated with gentle inverting for 30 min at 4°C. The Sepharose beads were washed three times with 1 × PBS. The GST–ATMPK fusion proteins were eluted from the Sepharose beads three times using 10 mm reduced glutathione in 50 mm Tris–HCl (pH 8.0). The eluted fractions were combined. Exchange of buffer with TTBS (20 mm Tris–HCl pH 7.5, 150 mm NaCl, 0.1% v/v Tween 20) and protein concentration were simultaneously performed on a Microcon-30 column (Millipore, Boston, MA, USA).
Antibody production and immunoblot analysis
ATMPK4- and ATMPK6-specific antibodies (Ab4CT1 and Ab6NT1) were produced against synthetic peptides corresponding to the C-terminus of ATMPK4 (ELIYRETVKFNPQDSV) and the N-terminus of ATMPK6 (MDGGSGQPAADTEMTEAPGGFPGGFPAAAPS), respectively. The synthesized peptides were conjugated with keyhole limpet hemocyanin carrier. Polyclonal antisera were raised in rabbits and purified by affinity chromatography (Sawady Technology Inc, Tokyo, Japan).
For immunoblot analysis, 20 ng of each GST fusion protein and 20 µg of Arabidopsis leaf total protein were separated on a 10% polyacrylamide gel, and transferred to PVDF membrane by semi-dry electroblotting. After blocking for 1 h in TTBS buffer containing 5% non-fat dried milk (Yukijirushi, Sapporo, Japan) at room temperature, the membrane was incubated in TTBS buffer with either Ab4CT1 (1/5000 dilution) or Ab6NT1 (1/1000) for 1 h at room temperature. After washing twice in TTBS buffer, the blots were incubated with a horseradish peroxidase-conjugated secondary antibody (Amersham, Buckinghamshire, UK), and the complexes were made visible by ECL (Enhanced Chemiluminescence) (Amersham) following the manufacturer's instructions.
Crude extracts of leaves and cell cultures were adjusted to a protein concentration of 1 mg ml−1 and gently agitated in IP buffer (50 mm HEPES–KOH pH 7.5, 5 mm EDTA, 5 mm EGTA, 2 mm DTT, 25 mm NaF, 1 mm Na3VO4, 50 mmβ-glycerophosphate, 20% glycerol, 150 mm NaCl, 1% Nonidet P-40) with 20 µl of a 50% suspension of protein A–Sepharose CL-4B (Pharmacia) for 1 h at 4°C. For immunoprecipitation of Ab4CT1 and Ab6NT1, 500 µg and 100 µg of crude extracts were used, respectively. The mixture was centrifuged at 12 000 g for 5 min at 4°C and the supernatant was transferred to a new tube containing 1/50 volume of antibody. The mixture was gently agitated at 4°C overnight. After adding 20 µl of the protein A beads, the mixture was agitated for 1 h at 4°C. The protein A beads were collected by centrifugation at 4000 g for 1 min, washed three times with 500 µl IP buffer, and then suspended in SDS sample buffer.
In-gel kinase assay
The in-gel kinase assay was performed as described previously (Romeis et al., 1999; Zhang and Klessig, 1997). In brief, 20 µg of protein per lane was subjected to electrophoresis on a 10% polyacrylamide gel that contained SDS and 0.25 mg of bovine brain myelin basic protein (MBP; Sigma-Aldrich, St Louis, MI, USA) per ml. After electrophoresis, the gel was washed three times with washing buffer (25 mm Tris–HCl pH 7.5, 0.5 mm DTT, 0.1 mm Na3VO4, 5 mm NaF, 0.5 mg ml−1 BSA, 0.1% Triton X-100) for 30 min at room temperature, followed by three washes with renaturation buffer (25 mm Tris–HCl pH 7.5, 1 mm DTT, 0.1 mm Na3VO4, 5 mm NaF) at 4°C overnight. The gel was washed with reaction buffer (25 mm Tris–HCl pH 7.5, 2 mm EGTA, 12 mm MgCl2, 1 mm DTT, 0.1 mm Na3VO4) for 30 min at room temperature and then incubated in 12.5 ml of reaction buffer with 250 nm ATP plus 1.85 MBq [γ-32P]ATP (3000 Ci mmol−1) for 90 min at room temperature. The gel was washed five times with washing solution (5% TCA, 1% pyrophosphoric acid) and once with 5% glycerol. The gel was dried on Whatman 3MM paper and exposed to RX-U film (Fuji Photo Film, Tokyo, Japan) or a BAS2500 imaging plate (Fuji Photo Film).
We thank Dr Takashi Hirayama for critical reading of the manuscript. This work was supported in part by the Special Coordination Fund of the Science and Technology Agency of Japan, by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan, and by a grant from the Program for Promotion of Basic Research Activities for Innovative Bioscience, all to K.S. It was also supported by a grant from the Biodesign Research Programs from RIKEN to K.S. and T.M. K.I., R.Y. and T.Y. were supported by postdoctoral fellowships from the Special Postdoctoral Researchers' Program of RIKEN.