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First published online November 23, 2005; 10.1104/pp.105.066795 Plant Physiology 139:1914-1926 (2005) © 2005 American Society of Plant Biologists Involvement of PPS3 Phosphorylated by Elicitor-Responsive Mitogen-Activated Protein Kinases in the Regulation of Plant Cell Death1Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464–8601, Japan (S.K., H.Y., K.K., N.D.); Sainsbury Laboratory, John Innes Centre, Norwich NR4 7UH, United Kingdom (O.R., J.D.G.J.); and Developmental Regulation Laboratory, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464–8601, Japan (H.M.)
Mitogen-activated protein kinase (MAPK) cascades play pivotal roles in plant innate immunity. Overexpression of StMEK1DD, a constitutively active MAPK kinase that activates salicylic acid-induced protein kinase (SIPK) and wound-induced protein kinase (WIPK), provokes hypersensitive response-like cell death in Nicotiana benthamiana. Here we purified a 51-kD MAPK, which was activated in potato (Solanum tuberosum) tubers treated with hyphal wall elicitor of a plant pathogen, and isolated the cDNA designated StMPK1. The deduced amino acid sequence of the StMPK1 showed strong similarity to stress-responsive MAPKs, such as tobacco (Nicotiana tabacum) SIPK and Arabidopsis (Arabidopsis thaliana) AtMPK6. To investigate the downstream signaling of StMPK1, we identified several proteins phosphorylated by StMPK1 (PPSs) using an in vitro expression cloning method. To dissect the biological function of PPSs in the plant defense, we employed virus-induced gene silencing (VIGS) in N. benthamiana. VIGS of NbPPS3 significantly delayed cell death induced by the transient expression of StMEK1DD and treatment with hyphal wall elicitor. Furthermore, the mobility shift of NbPPS3 on SDS-polyacrylamide gel was induced by transient expression of StMEK1DD. The mobility shift of NbPPS3 induced by StMEK1DD was not compromised by VIGS of WIPK or SIPK alone, but drastically reduced by the silencing of both WIPK and SIPK. This work strongly supports the idea that PPS3 is a physiological substrate of StMPK1 and is involved in cell death activated by a MAPK cascade.
The mitogen-activated protein kinase (MAPK) cascade is one of the major and evolutionally conserved signaling pathways utilized to transduce extracellular stimuli into intracellular responses (MAPK Group, 2002  ). In the MAPK signal transduction cascade, a MAPK kinase (MAPKK) is activated by an upstream MAPKK kinase and in turn activates a MAPK. There are many reports showing that MAPKs are activated by pathogen stimuli in a wide range of plant species, either positively or negatively (Ligterink et al., 1997; Lebrun-Garcia et al., 1998; Zhang et al., 1998; Suzuki et al., 1999; Cardinale et al., 2000; Petersen et al., 2000; Asai et al., 2002; Ekengren et al., 2003; del Pozo et al., 2004; Lee et al., 2004; Nakagami et al., 2004). Treatment of tobacco (Nicotiana tabacum) plants expressing the Cf-9 resistance gene with a fungal Avr factor Avr9 from Cladosporium fulvum leads to the activation of salicylic acid-induced protein kinase (SIPK) and wound-induced protein kinase (WIPK; Romeis et al., 1999; Rivas et al., 2004). In addition, both MAPKs are activated by plant species-specific elicitors such as elicitins (Zhang et al., 2000) or tobacco mosaic virus infection (Zhang and Klessig, 1998), suggesting that SIPK and WIPK are convergent points in the signaling pathway of pathogens and pathogen-derived elicitors. In fact, the loss of function of either SIPK (or its Arabidopsis [Arabidopsis thaliana] ortholog AtMPK6) or WIPK compromised N gene-mediated gene-for-gene resistance to tobacco mosaic virus infection (Jin et al., 2003) and resistance to bacterial pathogens (Sharma et al., 2003; Menke et al., 2004).
Recently, it was reported that NtMEK2, a tobacco MAPKK, is upstream of both SIPK and WIPK (Yang et al., 2001
In mammals, MAPKs phosphorylate a wide range of proteins, including transcription factors, protein kinases, and phospholipase A2 (Davis, 1993
In a previous study, we reported that a 51-kD protein kinase (p51-PK) with the characteristics common to MAPKs was activated in potato tubers treated with hyphal wall components (HWC) elicitor from the late blight pathogen Phytophthora infestans (Katou et al., 1999
Purification of p51-PK
We reported that p51-PK activity peaked 3 h after treatment of potato tubers with hyphal wall elicitor and seemed to be inactivated by protein phosphatase(s) (Katou et al., 1999
Amino Acid Sequence Analysis of p51-PK and p46-PK Figure 1B shows the SDS-PAGE analysis followed by Coomassie Blue staining of purification steps. The pooled fraction from the PY20-Sepharose column contained three major bands (p47-1, p47-2, and p43). The p47-1 and p47-2 likely correspond to p51-PK, whereas p43 likely corresponds to p46-PK. Peptide mapping analyses of p47-1 and p47-2 were almost identical (data not shown), suggesting that they represent the same protein modified differently. N-terminal amino acid sequence of several internal tryptic peptides of p47-1 and p43 were identical (Fig. 2). These results suggest that p46-PK is a partial degradative product of p51-PK. However, we cannot rule out the possibility that they are distinct but highly related protein kinases.
cDNA Cloning of p51-PK Gene
Primers were designed from sequences of internal tryptic peptides 1 and 4, and a fragment of p51-PK cDNA was directly amplified from a potato cDNA library. A full-length cDNA clone, which contains all of the peptide sequences obtained by microsequencing of the purified protein, was obtained by screening of a cDNA library (Fig. 2). Analysis of the deduced protein sequence of the p51-PK cDNA indicates that it belongs to the MAPK family, so it was designated StMPK1. The calculated molecular mass and pI of StMPK1 are 45.6 kD and 5.42, respectively. The deduced amino acid sequence of StMPK1 shows strong similarity with other plant MAPKs, such as tobacco SIPK, alfalfa SIMK, and Arabidopsis AtMPK6, which are known to being activated in response to various stresses (Zhang et al., 1998
In addition to posttranslational activation of MAPKs in yeast, plants, and mammals, several plant MAPKs have been reported to be induced at the transcriptional level (Seo et al., 1995
In Vitro Activation of StMPK1 by StMEK1DD
A specific MAPKK, which phosphorylates and activates StMPK1, is likely required for activation of StMPK1. StMPK1 shares 94% amino acid sequences with tobacco SIPK (Fig. 2). The enzymatic activation profile of StMPK1 is also similar to that of SIPK, which is also not associated with an increase in transcript and protein upon elicitation (Zhang et al., 1998
Recombinant StMPK1 and StMEK1DD proteins were expressed in Escherichia coli and purified by nickel-affinity column chromatography. SDS-PAGE analysis followed by Coomassie staining of purified fractions showed that they were homogeneous (Fig. 4A). Phosphorylation of StMPK1 by StMEK1DD enhanced the activity of StMPK1 toward MBP about 50- to 100-fold (Fig. 4B). The specific activity of activated StMPK1 toward MBP was 400 to 600 nmol ATP min–1 mg–1. This value is comparable with that of SIPK purified from salicylic acid-treated tobacco suspension cultured cells (Zhang and Klessig, 1997
Identification of Proteins Phosphorylated by StMPK1 in Vitro
To identify the possible substrates of StMPK1, we screened proteins phosphorylated by StMPK1 in vitro using IVEC. The cDNA library, constructed from poly(A)+ RNAs of potato tubers treated with hyphal wall elicitor for 4 h (Yoshioka et al., 2001
Incubation of 35S-labeled protein transcribed and translated from PPS3 cDNA with the active StMPK1 resulted in the decreased mobility of protein band (Fig. 5A). Decreased mobility of the PPS3 is slight, suggesting that only few sites are phosphorylated by StMPK1. The Ser or Thr followed by Pro (S/TP) is a minimal consensus motif for MAPK phosphorylation (Sharrocks et al., 2000 ). Deduced amino acid sequence analysis of the PPS3 cDNA revealed 13 potential MAPK phosphorylation sites (Fig. 5C). To verify that PPS3 is indeed phosphorylated by StMPK1 in vitro, recombinant PPS3, expressed in E. coli, was incubated with active StMPK1 in the presence of [ -32P]ATP. As shown in Figure 5B, the thioredoxin (Trx)-fused PPS3 was highly phosphorylated by StMPK1 compared with Trx only. PPS3 was predicted to encode a 390-amino acid protein with a calculated molecular mass of 41.1 kD (Fig. 5C). A BLASTP search revealed that PPS3 contains a putative ZIM motif, which is found in a variety of plant transcription factors that include TIFF/YxG sequences (Fig. 5C), and may be involved in DNA binding (Nishii et al., 2000). An Arabidopsis hypothetical protein At3g17860 with 31% identity and 41% similarity to PPS3 also has TIFF/YxG sequences (Fig. 5C). RNA gel-blot analysis showed that the mRNA levels of PPS3 remained constant after elicitation by the hyphal wall elicitor (Fig. 5D). Genomic Southern-blot analysis indicated that PPS3 is single copy in the potato genome (data not shown).
VIGS of NbPPS3 Compromised StMEK1DD-Induced Cell Death
Recently, we reported that the expression of StMEK1DD induced HR-like cell death in N. benthamiana (Katou et al., 2003
The reduction of NbPPS3 mRNA was confirmed by RNA gel-blot analysis in triplicate samples (Fig. 6B). The other seven PPSs were also silenced by VIGS, but HR-like cell death caused by StMEK1DD was not compromised (data not shown).
A retardation of mobility on SDS-PAGE gel was observed when the PPS3 was phosphorylated by StMPK1 in vitro (Fig. 5A). To investigate the phosphorylation of PPS3 by StMPK1 in planta, we examined whether transient expression of StMEK1DD induced a mobility shift of NbPPS3 in N. benthamiana. We cloned a cDNA fragment of a N. benthamiana homolog of PPS3 (NbPPS3), of which the deduced amino acid sequence shows 78% identity and 83% similarity to PPS3 (Fig. 5C). The calculated molecular mass and pI of NbPPS3 are 40.3 kD and 9.28, respectively. Immunoblot analysis using anti-NbPPS3 antibody, which was raised against peptide corresponding from 111th to 129th amino acids of NbPPS3 (Fig. 5C), revealed that the mobility of NbPPS3 protein on a SDS-polyacrylamide gel was retarded by the transient expression of StMEK1DD (Fig. 7A). The mobility shift of NbPPS3 was accompanied by the activation of 48- and 44-kD kinases (Fig. 7B), which have been confirmed to be SIPK and WIPK, respectively, by immunocomplex kinase assays (Katou et al., 2003
Previous studies indicated that agroinfiltration alone scarcely induced WIPK and SIPK activities (Katou et al., 2003 ), suggesting that the mobility shift was caused by StMEK1DD activity. To confirm that the mobility shift of NbPPS3 resulted from phosphorylation by WIPK or SIPK, we investigated the effect of VIGS of WIPK, SIPK, and NbMEK2 on the StMEK1DD-induced mobility shift of NbPPS3. Complementary DNA fragments corresponding to WIPK, SIPK, and NbMEK2 were isolated from N. benthamiana and cloned into PVX binary vector pGR106 (PVX:WIPK, PVX:SIPK, PVX:WIPK/SIPK, or PVX:NbMEK2). The mobility shift of NbPPS3 in PVX control-inoculated leaves was induced by expression of StMEK1DD (Fig. 7A). VIGS of WIPK or SIPK specifically reduced the corresponding activity of these MAPK activities (Fig. 7B, top section), however, did not compromise the mobility shift of NbPPS3 (Fig. 7A). On the other hand, double silencing of WIPK/SIPK reduced both MAPK activities and compromised the mobility shift of NbPPS3, indicating that NbPPS3 may be phosphorylated by both WIPK and SIPK in vivo. However, the faint band was observed in the lane of double silencing (Fig. 7A) probably because of incomplete silencing of both kinases (Fig. 7B). To further confirm the possibility, StMEK1DD itself in addition to NbMEK2 were silenced by infection with PVX:NbMEK2. The NbMEK2 cDNA fragment used for VIGS showed 90% nucleotide identity to the corresponding region of StMEK1DD. The reduction of mRNA for StMEK1DD and NbMEK2 was confirmed by reverse transcription-PCR (data not shown). VIGS of NbMEK2 drastically reduced the mobility shift of NbPPS3 as well as WIPK and SIPK activities (Fig. 7, A and B). This result supports the idea that both SIPK and WIPK phosphorylate NbPPS3, and rules out the possible side effects of agroinfiltration and PVX infection on mobility shift of NbPPS3. Moreover, we conducted an in-gel kinase assay using recombinant NbPPS3 protein as a substrate (Fig. 7B, bottom section). The phosphorylation profile of the in-gel kinase assay is almost identical to the pattern of kinase activities obtained by using MBP as a substrate (Fig. 7B, top section), indicating that WIPK and SIPK also phosphorylate NbPPS3. In-gel kinase assay detected only weak NbWIPK activity. A likely explanation for this is that basal levels of SIPK are much higher than those of WIPK (Zhang and Klessig, 1998). Alternatively, WIPK proteins may be unstable under denaturing conditions in SDS-polyacrylamide gel.
To examine the effect of VIGS of NbPPS3 on HWC-induced cell death, N. benthamiana seedlings were infected with PVX or PVX:NbPPS3 by agroinfiltration in the same way as in Figure 6. The area of leaf infiltrated with HWC elicitor was collapsed within 3 d in the PVX-infected plants, whereas no cell death was observed in the plants infected with PVX:NbPPS3 (Fig. 8). This result indicates that NbPPS3 is involved in HWC-induced HR cell death.
IVEC Is Useful Tool to Identify the Substrate of Protein Kinase
MAPKs play pivotal roles in induced defense responses of plants. However, the regulatory mechanisms by which MAPKs induce defense responses are unclear. To obtain candidate substrates of StMPK1, we employed IVEC, which has been successfully used for identification of substrate proteins in Xenopus and human (Cryns et al., 1997
Among eight candidates identified by IVEC, we focused on PPS3 because its silencing in N. benthamiana significantly delayed StMEK1DD-induced HR-like cell death (Fig. 6). The S/TP is minimal consensus motif for MAPK phosphorylation. PPS3 and its N. benthamiana homolog NbPPS3 have more than 10 potential MAPK phosphorylation sequences (Fig. 5C), and recombinant protein of PPS3 is phosphorylated by StMPK1 in vitro (Fig. 5B). The mobility shift of NbPPS3 was also induced by transient expression of StMEK1DD in vivo (Fig. 7A). Interestingly, the mobility shift of NbPPS3 was not compromised by VIGS of WIPK or SIPK alone, but drastically reduced by the silencing of WIPK/SIPK or NbMEK2 (Fig. 7). SIPK and WIPK share same upstream MAPKK NtMEK2 (Yang et al., 2001
Several docking domains including
We assessed the function of PPS genes by employing VIGS in N. benthamiana and found that VIGS of NbPPS3 significantly delayed HWC-mediated HR cell death as well as StMEK1DD-induced HR-like cell death (Figs. 6 and 8). It was reported that SIPK is activated in tobacco leaves by HWC elicitor (Zhang and Klessig, 1998
Plant Materials, Elicitors, and Treatment Protocol
Tubers of the potato cultivar Irish cobbler (Solanum tuberosum) were harvested at Nagoya University farm and stored at 4°C in the dark until use. Nicotiana benthamiana were grown in environmentally controlled growth cabinets under a 16-h photoperiod and an 8-h dark period at 25°C. HWC elicitor was isolated from cultured mycelia of Phytophthora infestans according to a method previously described (Doke and Tomiyama, 1980
In-gel kinase assays were performed as described previously (Katou et al., 1999
Purification of p51-PK was performed by monitoring its activity with an in-gel kinase assay using MBP as substrate. Twelve hundred grams of potato tuber slices (2 mm thick) treated with HWC elicitor for 3 h were ground for 2 min with a mixer in 1.2 L of homogenization buffer (50 mM MOPS-KOH, pH 7.6, 0.5 M sorbitol, 5 mM EDTA, 5 mM EGTA, 0.1 M NaF, 100 µM Na3VO4, 10 µM [p-amidinophenyl] methanesulfonyl fluoride, 50 mM 2-mercaptoethanol, and 5% [w/v] Polyclar VT). The homogenate was filtered through two layers of nylon gauze, and Dowex 1X-2 (100–200 mesh, Cl– form, Dow Chemical) was added to a final volume of 7% (w/v). The homogenate was stirred for 15 min and filtered to remove Dowex. The homogenate was centrifuged at 14,000g for 30 min, and the resulting supernatant fraction was centrifuged at 275,000g for 30 min. The soluble supernatant fraction was applied to a Q-Sepharose high-performance column (25 mL, Pharmacia) equilibrated with buffer A (20 mM MOPS-KOH, pH 7.6, 50 mM NaF, 100 µM Na3VO4, and 7 mM 2-mercaptoethanol). Kinase activities were then eluted with 0.25 M NaCl in buffer A. Active fractions were pooled and (NH4)2SO4 was added to a concentration of 0.8 M, and loaded on a Resource phenyl column (6 mL, Pharmacia) equilibrated with 0.8 M (NH4)2SO4 in buffer A. Kinase activities were eluted with a 30-mL linear gradient of 0.8 to 0 M (NH4)2SO4 in buffer A. Active fractions were pooled, concentrated using concentrator (Centriplus YM10, Millipore), and applied to a Superdex 75 prep grade column (120 mL, Pharmacia) equilibrated with buffer A containing 0.2 M NaCl. Kinase activities were eluted with the same buffer. Active fractions were concentrated as above, and the concentrate was diluted 10-fold to reduce salt concentration. They were then applied to a Mono Q column (1 mL, Pharmacia), and kinase activities were eluted with a 30-mL linear gradient of 0.1 to 0.3 M NaCl in buffer A. Active fractions were concentrated and desalted using concentrator (Centriplus YM10, Millipore), and Triton X-100 was added to a final concentration of 0.5%. The pooled fractions were then applied to a PY20-Sepharose column (1 mL; prepared by coupling PY20 anti-phosphotyrosine monoclonal antibody to N-hydroxysuccimide-activated Sepharose, Pharmacia) equilibrated with 0.5% Triton X-100 in buffer B (buffer A without 2-mercaptoethanol). The column was washed with 5 mL of 0.5% Triton X-100 in buffer B and 2 mL of buffer B, respectively, and the kinase activities were eluted with 10 mM phosphotyrosine in buffer B.
Active fractions from the Mono Q column were concentrated using a concentrator (Centriplus YM10, Millipore), mixed with molecular mass standards (Pharmacia), and then applied to Superdex 75 prep grade column equilibrated with buffer A containing 0.2 M NaCl. The column was calibrated with the following markers: Blue Dextran (2,000 kD), bovine serum albumin (67 kD), ovalbumin (43 kD), chymotrypsinogen A (25 kD), and ribonuclease (13.7 kD).
Protein sequencing was performed as described previously (Hellman et al., 1995
A cDNA library constructed from poly(A)+ RNAs of potato tubers treated with HWC elicitor for 4 h (Yoshioka et al., 2001
Total RNA from potato tubers and N. benthamiana leaves was prepared as described previously (Kingston, 1987
The peptides for StMPK1-N (MDASAPQMDTMMPDVAAPAV) corresponding to the amino terminus of StMPK1 and NbPPS3-N (HYDAHAMHPSHGVRVLPVS) corresponding to amino acids 111 to 129 of the predicted NbPPS3 protein were synthesized and conjugated to keyhole limpet hemacyanin carrier. Polyclonal antiserum was raised in rabbits (Sawady) and purified by antigen-affinity chromatography. For immunoblot analyses, 10 ng each of recombinant StMPK1 and StMPK2 and 20 µg each of soluble protein from potato tubers and N. benthamiana leaves were separated on a 10% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane (Schleicher & Schuell). After blocking overnight in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl and 0.05% Tween 20 (TBS-T) containing 5% nonfat dry milk at 4°C, the membranes were incubated with anti-StMPK1-N or anti-NbPPS3-N antibody diluted with TBS-T (0.05 µg mL–1) at room temperature for 1 h. After washing with TBS-T, the membrane was incubated with horseradish peroxidase-conjugated anti-rabbit IgG antibody (Amersham) diluted with TBS-T (2,000-fold dilution). Antibody-antigen complex was detected using an enhanced chemiluminescence kit (Amersham).
Protein extract (400 µg) was incubated with anti-StMPK1-N antibody (2 µg) in immunoprecipitation buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 50 mM NaF, 10 mM
An MscI site for StMPK1 and StMPK2, a NcoI site for StMEK1DD, a BamHI site for PPS3, and an EcoRI site for NbPPS3 were introduced in front of the ATG start codons by PCR. Each of the amplified products except for NbPPS3 were ligated in frame into pET-32a(+) vector (Novagen); NbPPS3 was ligated in frame into pET-30a(+) vector (Novagen). The plasmids were transformed into E. coli strain BL21 (DE3) pLysS. Induction of fusion proteins with 1 mM isopropyl-
In vitro activation of StMPK1 by StMEK1DD was performed in buffer D (20 mM HEPES-KOH, pH 7.6, 10 mM MgCl2, 1 mM dithiothreitol, 100 µM Na3VO4, 5 mM NaF, 5 mM
Purified Trx-fused PPS3 or Trx only were incubated with 10 µg mL–1 StMPK1 in buffer C containing 1 mM EGTA, 50 µM ATP, and 50 µCi mL–1 [
IVEC was performed according to Gao et al. (2000)
To obtain the full-length cDNA of ortholog of PPS3 from N. benthamiana, NbPPS3 was amplified by PCR using phage DNA prepared from an oligo(dT)-primed Uni-ZAP XR library (Stratagene), which was constructed from poly(A)+ RNAs of N. benthamiana leaves treated for 6 h with HWC elicitor of P. infestans. Primers were designed from sequences of NbPPS3 cDNA fragment used for VIGS and the phage vector. The 3'-flanking regions of NbPPS3 were amplified using NbPPS3 (5'-CTCACGGAGTCAGAGTGCTCCC-3') and a vector primer T7 (5'-GTAATACGACTCACTATAGGGC-3'). The 5' terminus of NbPPS3 was amplified using a vector primer T3 (5'-AATTAACCCTCACTAAAGGG-3') and 5'-UTR primer (5'-TCTTGATCTTGTCTACATTATTTTGCCACC-3'). All PCR reactions were performed using a high fidelity taq polymerase (KOD+; Toyobo). Blunt-end PCR products were cloned into pCR-Blunt II-TOPO (Invitrogen). The overlapping portions of the sequences of these PCR products were identical.
VIGS was performed according to Ratcliff et al. (2001) Accession numbers for the StMPK1, StMPK2, PPS3, and NbPPS3 are AB062138, AB062139, AB111942, and AB111943, respectively. The accession numbers for the other sequences mentioned in this article are as follows: Arabidopsis (Arabidopsis thaliana) PPS3 homolog, AY085082; SIPK, AAB58396; Ntf4, Q40532; MMK1, Q07176; AtMPK6, AAB64027; and WIPK, BAB79636.
The authors deeply thank Phil Mullineaux and Roger Hellens of John Innes Centre for providing pGreen binary vector; David C. Baulcombe of Sainsbury Laboratory for pGR106; and Leaf Tobacco Research Center, Japan Tobacco for N. benthamiana seeds. The authors also thank the members of the Radioisotope Research Center (Nagoya University), and Naoki Ikeda and Miki Yoshioka of Nagoya University for technical assistance, and Scott, C. Peck of John Innes Centre for critical reading of this manuscript. Received June 6, 2005; returned for revision September 11, 2005; accepted October 10, 2005.
1 This work was supported by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists; by the Ministry of Education, Science and Culture of Japan (Grant-in-Aid for Scientific Research [S], grant no. 14104004); and by the Research for the Future Program of the Japan Society for the Promotion of Science (Grant-in-Aid for Scientific Research on Priority Area [A]). 
2 Present address: Department of Biology, Carleton University, Ottawa, Ontario, Canada K1S 5B6. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Hirofumi Yoshioka (hyoshiok{at}agr.nagoya-u.ac.jp). Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.066795. * Corresponding author; e-mail hyoshiok{at}agr.nagoya-u.ac.jp; fax: 81–52–789–5525.
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ABSTRACT
RESULTS
domain in c-Jun and LxL motif neighbored with basic and hydrophobic regions in many transcription factors have been identified in mammalian MAPK substrates (Sharrocks et al., 2000
-32P]dCTP and used to screen a potato cDNA library under high stringency. From 8 x 105 plaque-forming units, we isolated 13 positive clones. Positive phage plaques were excised directly in vivo into pBluescript SK(–) phagemid according to the manufacturer's instructions (Stratagene). Nucleotide sequences were determined for both strands with ABI Prism BigDye Terminator cycle sequencing kits and a DNA sequencing system (model 3100; Applied Biosystems). The nucleotide and the deduced amino acid sequence were analyzed with DNA analytical software (DNASIS; Hitachi Software). The alignment of amino acid sequences was made using ClustalW multiple sequence alignment program (version 1.7, June 1997; Thompson et al., 1994
-glycerophosphate, 1 mM Na3VO4, and 1% Triton X-100) at 4°C for 2 h with rotary shaking. Forty microliters of 50% protein A-Sepharose was added, and incubation was continued for another 2 h. Immunocomplex was collected by brief centrifugation and washed three times with immunoprecipitation buffer and then three times with buffer C (20 mM HEPES-KOH, pH 7.6, 10 mM MgCl2, and 1 mM dithiothreitol). Kinase activity was assayed at 25°C for 20 min in a volume of 25 µL buffer C containing 0.25 mg mL–1 MBP, 100 µM ATP, and 8 µCi mL–1 [