A Novel Tobacco Mitogen-activated Protein (MAP) Kinase Kinase, NtMEK1, Activates the Cell Cycle-regulated p43Ntf6 MAP Kinase*

Ornella CalderiniDagger §, Nathalie Glab||, Catherine Bergounioux||, Erwin Heberle-BorsDagger **, and Cathal WilsonDagger

From the Dagger  Institute of Microbiology and Genetics, University of Vienna, Dr. Bohrgasse 9, A-1030 Vienna, Austria, the § Istituto di Ricerche sul Miglioramento Genetico Piante Foraggere, Consiglio Nazionale delle Ricerche, via Madonna Alta 130, I-06128 Perugia, Italy, and the || Institut de Biotechnologie des Plantes, CNRS ERS 569, Université de Paris-Sud, Bât. 630, Plateau du Moulon, F-91405 Orsay Cedex, France

Received for publication, November 26, 2000, and in revised form, March 2, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Two-hybrid screening of a tobacco BY-2 cell suspension cDNA library using the p43Ntf6 mitogen-activated protein (MAP) kinase as bait resulted in the isolation of a cDNA encoding a protein with features characteristic of a MAP kinase kinase (MEK), which has been called NtMEK1. Two-hybrid interaction analysis and pull-down experiments showed a physical interaction between NtMEK1 and the tobacco MAP kinases p43Ntf6 and p45Ntf4, but not p43Ntf3. In kinase assays NtMEK1 preferentially phosphorylated p43Ntf6. Functional studies in yeast showed that p43Ntf6 could complement the yeast MAP kinase mutant mpk1 when co-expressed with NtMEK1, and that this complementation depended on the kinase activity of p43Ntf6. Expression analysis showed that the NtMEK1 and ntf6 genes are co-expressed both in plant tissues and following the induction of cell division in leaf pieces. These data suggest that NtMEK1 is an MEK for the p43Ntf6 MAP kinase.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Eukaryotic cells respond to a variety of extracellular stimuli via activation of specific MAP1 kinase cascades. A protein kinase cascade provides integration of different inputs, allows subtle changes in a stimulus to be transduced as a sharp switch in output, and allows the signal itself to reach different cellular compartments depending on the cascade (1, 2). The typical events of a MAP kinase cascade initiate at the plasma membrane with binding of a ligand to a receptor, which in turn leads to the activation of a G protein. The GTP-bound form of the G protein activates a MAP kinase kinase kinase (MAPKKK), which in turn phosphorylates and activates a MAP kinase kinase (MEK). The terminal component of the kinase module, the MAP kinase, is phosphorylated by MEK and subsequently translocates to the nucleus where it phosphorylates transcription factors, or targets cytosolic proteins such as other kinases or cytoskeletal-associated proteins (3, 4). G proteins, MAPKKKs, MEKs, and MAP kinases have been cloned in plants by homology-based screening, and many of the stimuli leading to the activation of the MAP kinases have been identified (reviewed in Ref. 5). However, building up different pathways by the identification of the relevant binding partner(s) of the cascade and, above all, substrates for MAP kinases is still at a relatively early stage in plants. A possible MAP kinase three-component module composed of ATMEKK1 (a MAPKKK), ATMKK2/MEK1 (two MEKs), and the MAP kinase ATMPK4 has been reported (6). MEK1 was also reported to interact with CTR1, a MAPKKK similar to the Raf protein kinase family (7). Two-hybrid screening identified a MEK that interacted with the stress-activated SIPK MAP kinase from tobacco, although SIPK could not be phosphorylated by the MEK in in vitro kinase assays (8).

In synchronized tobacco cell cultures, the p43Ntf6 MAP kinase (9) is activated at a late stage in mitosis, around the anaphase/early telophase transition, and localizes in the middle of two microtubule arrays characteristic of the phragmoplast (10), a plant-specific structure involved in laying down the new cell wall. The timing of kinase activation and its intracellular localization suggest that p43Ntf6 plays a role in cell plate formation during cell division. With the aim of identifying possible partners for p43Ntf6, we undertook a two-hybrid screen that identified a protein kinase whose characteristics suggest it is a MEK for the p43Ntf6 protein.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In Vitro Mutagenesis-- Loss-of-function (LOF) mutant proteins were created by mutating a conserved lysine residue in kinase subdomain II that is involved in the transfer of phosphate to the protein substrate. The ntf3 LOF mutant protein p43Ntf3-K61R has been described previously (9). LOF mutant constructs of ntf4 and ntf6 were made using the Altered Sites in vitro mutagenesis kit from Promega to produce the LOF mutant proteins p43Ntf6-K67R and p45Ntf4-K89R. A gain-of-function (GOF) mutant protein of p43Ntf6 was made as for the LOFs except the mutated site was from a glutamate at position 328 to asparagine, corresponding to the Drosophila sevenmaker GOF mutant, D334N (11). The GOF mutant protein was called p43Ntf6-E328N.

Plasmid Construction-- For two-hybrid screening, the ntf6 WT and GOF cDNAs were cloned into the SmaI site of the binding domain vector pBD-GAL4 Cam (Stratagene) to give pBD-ntf6WT and pBD-ntf6GOF. The ntf4, ntf3, and ntf6 LOF cDNAs were cloned into the same site of pBD-GAL4 Cam for two-hybrid interaction analysis. MAP kinase proteins were produced as glutathione S-transferase (GST) fusions in pGEX-2T'6 as described (9). The GST-NtMEK1 construct was obtained by PCR cloning of the cDNA into the BamHI-SalI sites of the vector pGEX-4-T1 (Amersham Pharmacia Biotech). For complementation studies in yeast, the MAP kinase cDNAs for ntf6, ntf6-LOF, ntf6-GOF, and ntf4 were cloned by PCR into the yeast expression vector pVT103U (12), which contains the constitutive alcohol dehydrogenase promoter and a uracil-selectable marker. Oligonucleotides were designed to change the nucleotides before the ATG codon of each cDNA to AAAA, which is a favored context for translation in yeast (13).

For in vitro translation, the NtMEK1 cDNA was amplified by PCR and cloned in the SmaI site of the pBAT vector (14) to give pBAT-NtMEK1. All plasmids produced by PCR cloning were checked by sequencing.

Yeast Two-hybrid Screening and Analysis-- The pBD-ntf6WT and the pBD-ntf6GOF plasmids were used as bait to screen a tobacco BY-2 cell suspension library constructed in the CLONTECH activation domain vector pACT2. Library and bait were co-transformed in the yeast strain HF7c according to the CLONTECH Matchmaker two-hybrid system protocol and transformants were plated on medium lacking histidine, tryptophan, and leucine. Approximately 1 million clones were screened for either bait. As the p43Ntf6-GAL4 fusion allowed some growth on medium lacking histidine, the colonies obtained after transformation were streaked on the same medium containing 3-aminotriazole (3-AT). The surviving colonies were further tested for the expression of beta -galactosidase by filter lift assays (CLONTECH). Plasmids were rescued following growth of the transformants in liquid medium lacking tryptophan and leucine, plasmid DNA isolation, and transformation of Escherichia coli. The plasmids recovered from E. coli were sequenced according to standard procedures.

Two-hybrid interaction analysis between NtMEK1 and the MAP kinases p43Ntf3, p45Ntf4, and p43Ntf6 (and the LOF and GOF versions of p43Ntf6) was tested on medium lacking histidine and containing 3-AT, and beta -galactosidase activity was measured in liquid assays (CLONTECH).

Complementation Studies in Yeast-- pVTntf6WT, -LOF, -GOF, pVTntf4, and pACTNtMEK1 were transformed into the yeast strain DL456 (15), which has a disrupted MPK1 (slt2) gene, and plated on selective medium. The yeast MPK1 gene was expressed in DL456 from the high copy number plasmid pFL44 (15). The different plasmid combinations were as reported in the text. Transformants were streaked on YPD medium (2% glucose, 2% peptone, 1% yeast extract, 2% agar) and on YPD medium containing M sorbitol, and the plates were incubated at 28 °C or 37 °C for 2 days (16). The pACTNtMEK1 plasmid was lost from pVTntf6WT/pACTNtMEK1 double transformants by growth in liquid minimal medium (2% glucose, 0.66% yeast nitrogen base) containing leucine (the selectable marker of the pACT2 plasmid), plated on the same solid medium, and then replica plated to solid minimal medium lacking leucine. The colonies that did not grow after replica plating were selected from the master plate to test for pACTNtMEK1-dependent complementation of the mpk1 mutant.

Protein Purification, Kinase Assays, and Immunoblots-- Expression and purification of GST fusion proteins were as described previously (9). Kinase-substrate reactions were carried out in 10 µl of kinase buffer (20 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM dithiothreitol, 25 µM ATP, 2 µCi of [gamma -32P]ATP (3000 Ci/mmol), at 30 °C for 30 min. Myelin basic protein was used at a final concentration of 2 µg/reaction. Approximately 1 µg of each GST fusion protein was used in the reactions, unless otherwise stated. Protein extraction from plant tissues and immunoblot analysis were as described in Ref. 10.

GST Pull-down Experiments-- [35S]Methionine-labeled NtMEK1 protein was synthesized from the pBAT-NtMEK1 vector in vitro using a coupled in vitro transcription/translation reaction system (TNT Coupled Reticulocyte Lysate System, Promega) according to the manufacturer's protocol. Approximately 2 µg of each GST-MAP kinase fusion protein or GST alone was incubated with one fifth of the [35S]methionine-labeled translation product in 100 µl of Nonidet P-40 buffer (1% Nonidet P-40, 150 mM NaCl, 50 mM Tris-HCl, pH 8). Binding was performed at 4 °C overnight, and then 20 µl of glutathione-Sepharose beads were added and incubated for 2 h. Finally, the reactions were washed three times with Nonidet P-40 buffer, resuspended in electrophoresis sample buffer, and separated by SDS-PAGE on 12% gels. The dried gels were then exposed to Kodak X-Omat AR films.

Tissue Culture-- Leaf discs of tobacco SR1 plants grown in vitro were treated according to Ref. 17 with slight modifications. Briefly, they were incubated in Murashige and Skoog liquid medium or Murashige and Skoog medium under the following conditions: (i) no addition, (ii) 1 µM naphthalene acetic acid (NAA), (iii) 2.2 µM 2,4-dichlorophenoxyacetic acid (2,4-D), (iv) 0.5 µM N6-benzyladenine (BA), (v) both NAA and BA, and (vi) both 2,4-D and BA. Samples were collected every 24 h over a 1-week period and further processed for RNA and protein extraction.

Northern Analysis-- Poly(A)+ RNA was isolated from different tissues of SR1 tobacco plants and from leaf pieces treated with hormones. RNA extraction was performed with Dynabeads Oligo(dT)25 (Dynal GmbH) according to the manufacturer's instructions. Approximately 10 µg of each RNA per sample were fractionated on 1% agarose gels and transferred to Hybond-N+ membranes (Amersham Pharmacia Biotech). After UV cross-linking, membranes were hybridized with 32P-labeled probes at 65 °C overnight in Church buffer (18).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Identification of a Novel Tobacco MAPKK by Two-hybrid Screening-- A two-hybrid interaction screen was used to search for molecules that interact with the tobacco MAP kinase p43Ntf6. Both the wild-type p43Ntf6 protein and a mutant construct of p43Ntf6 called p43Ntf6-E328N (see "Experimental Procedures"), analogous to the Drosophila sevenmaker (D334N, Ref. 11) and mammalian ERK2 (D319N, Ref. 19) gain-of-function (GOF) mutants, were used as bait. Both p43Ntf6 constructs were cloned into the two-hybrid interaction binding domain vector pBD-GAL4 Cam and used to screen a cDNA library prepared from a BY-2 cell suspension culture constructed in the interacting domain plasmid pACT2. Co-transformants were selected on media lacking histidine. Due to the growth of a large number of colonies on this media, they were replated on media containing 3-AT, which provides a more stringent selection for histidine-expressing cells. Of the 1000 colonies originally selected on medium lacking histidine, 250 were scored as being able to grow in the presence of 3-AT. These colonies were tested for beta -galactosidase production using a filter lift assay. Three colonies that were positive using this test turned out to harbor the same plasmid after rescue and sequencing. The open reading frame carried by all of these plasmids codes for a protein with a predicted molecular mass of 39.7 kDa and contains all the signature motifs of a protein kinase (Fig. 1). Data base searches showed that this protein, which we have named NtMEK1, had the highest homology to MAP kinase kinases (MAPKK) from plants (Fig. 1). The highest homology was to an Arabidopsis sequence identified in the EMBL data base (here called AtMEKhom, 83%, accession no. BAB09875), then ZmMEK1 82.7% (20); LeMEK1 68.1% (21); SIPKK 65% (8); AtMAP2Kbeta 63.4% (22); AtMEK1 60.2% (23). The signature sequence (AIVT)(GA)(TC)XX(YF)M(SAG)PER(IL) diagnostic of all MAPKKs (22) is present in subdomain VIII. However, the plant MAPKK signature described as (SQY)(LIV)(ILAV)LE(YF)M(DN)(GKQR)GSL(AE)(DG)(IFAL)(LHIV)(KIV) in subdomain V (22) differs in NtMEK1 in the third last and last amino acid of this motif (VIR). The MAPKK phosphorylation site motif ((S/T)XXXXX(S/T)) characteristic of plant MAPKKs is present between subdomains VII and VIII (Fig. 1).


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Fig. 1.   Sequence comparison of NtMEK1 with other plant MAPKKs. SIPKK, Nicotiana tabacum; LeMEK1, Lycopersicon esculentum; AtMEK1, AtMAP2Kbeta , AtMEKhom, Arabidopsis thaliana; ZmMEK1, Zea mays. Amino acid residues conserved in all sequences are indicated with an asterisk. The phosphorylation site motif of plant MAP kinase kinases is shown with arrows. The conserved domains of protein kinases are labeled with Roman numerals. Gaps are indicated by dashes.

NtMEK1 Preferentially Phosphorylates the p43Ntf6 MAP Kinase-- The NtMEK1 cDNA was cloned into the pGEX-4-T1 expression vector and expressed in E. coli to produce a GST fusion protein. Purification of the fusion protein by affinity chromatography resulted in a protein with a molecular mass that approximated the mass predicted from a fusion of GST (27 kDa) with the NtMEK1 protein (39.7 kDa; Fig. 2A, top panel). This fusion protein showed autophosphorylation activity and was able to phosphorylate MBP, confirming the isolation of an active protein kinase (Fig. 2A, bottom panel).


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Fig. 2.   NtMEK1 preferentially phosphorylates p43Nft6 in vitro. A, the tobacco MAP kinases p43Ntf3 (Ntf3), p45Ntf4 (Ntf4), and p43Ntf6 (Ntf6), and their respective loss-of-function derivatives (Ntf3K61R, Ntf4K89R, Ntf6K67R) and NtMEK1 (MEK) were expressed as GST fusion proteins in bacteria. After purification on glutathione-Sepharose beads, the individual proteins were tested for kinase activity using MBP as a substrate. The upper panel shows Coomassie Brilliant Blue R-250 staining of the gel, whereas the lower panel shows the kinase assays. Auto, autophosphorylation. Note that only one fifth of the amount of p45Ntf4 was loaded as it shows strong kinase activity that would mask the activity of the other kinases (9). Molecular size standards are shown on the right in kDa. B, kinase assays showing the phosphorylation of the MAP kinase loss-of-function proteins Ntf3K61R, Ntf4K89R, and Ntf6K67R by NtMEK1 (MEK). The top panel shows Coomassie Brilliant Blue R-250 staining of the gel, and the lower panel shows the kinase assays.

We tested the ability of NtMEK1 to use three tobacco MAP kinases as substrates. GST fusions with the tobacco MAP kinases, p43Ntf3, p45Ntf4, and p43Ntf6 (9) and with loss-of-function versions of the three kinases (p43Ntf3-K61R, p45Ntf4-K89R, and p43Ntf6-K67R) were produced in E. coli and affinity-purified by binding to glutathione-Sepharose beads. No autophosphorylation or phosphorylation of MBP by the loss-of-function MAP kinases was observed (Fig. 2A, bottom panel). This enabled us to test the ability of NtMEK1 to use these three MAP kinases as substrates. The three MAP kinase loss-of-function proteins were incubated with the NtMEK1 protein in kinase assays. Phosphorylation of both p45Ntf4-K89R and p43Ntf3-K61R by NtMEK1 was weak, while p43Ntf6-K67R was strongly phosphorylated (Fig. 2B). This differential phosphorylation of the MAP kinases shows that p43Ntf6 is the preferred in vitro substrate for NtMEK1.

In Vitro Interactions between NtMEK1 and MAP Kinases-- The ability of NtMEK1 to interact with the different MAP kinases was tested using a GST pull-down experiment. The NtMEK1 protein was synthesized and isotopically labeled in vitro, and the resulting protein was incubated on its own, with the GST protein, or with each of the three GST-MAP kinase wild-type fusion proteins. The protein mixtures were then bound to glutathione-Sepharose beads, washed, subjected to SDS-PAGE, and the resulting gel was then exposed. No signal was observed when GST alone was bound to the Sepharose, or when the reaction contained only the NtMEK1 protein. Strong signals were observed after incubation of NtMEK1 with p43Ntf6 and p45Ntf4 (Fig. 3). NtMEK1 pulled down more p45Ntf4 than p43Ntf6. This result suggests that both p43Ntf6 and p45Ntf4 physically interact with NtMEK1. A very weak signal, of uncertain significance, was also observed for p43Ntf3.


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Fig. 3.   GST pull-down assays show an interaction of NtMEK1 with p43Nft6 and p45Nft4. NtMEK1 (MEK) was synthesized and labeled in vitro and then incubated on its own, with GST, or with each of the GST-MAP kinase fusion proteins (Ntf3, Ntf4, Ntf6). After binding to glutathione-Sepharose beads, washing, and SDS-PAGE, the dried gel was exposed to x-ray film. Molecular size standards are shown on the right in kDa.

Two-hybrid Interactions between NtMEK1 and Tobacco MAP Kinases-- Since a physical interaction between NtMEK1 and both p43Ntf6 and p45Ntf4 was indicated by the GST pull-down experiment, but NtMEK1 preferentially phosphorylated p43Ntf6, we extended this study using the two-hybrid system to investigate the interaction between NtMEK1 and the three tobacco MAP kinases. The ntf3, ntf4, and ntf6 cDNAs, as well as the GOF and LOF versions of ntf6, were cloned into the pBD-GAL4 Cam plasmid and co-transformed either with the empty pACT2 plasmid or with pACT-NtMEK1 into yeast cells. Co-transformants were tested for growth in the absence of histidine. No growth occurred in the transformants containing an empty pACT2 plasmid in combination with any MAP kinase, in cells containing pACT-NtMEK1 with the pBD-GAL4 Cam plasmid, or in the pBDntf3/pACT-NtMEK1 combination (Fig. 4A). All ntf6 versions (pBD-ntf6WT, -K67R, and -E328N) grew in the absence of histidine when co-expressed with pACT-NtMEK1 (Fig. 4A). Interestingly, so did the combination of pACT-NtMEK1 and pBDntf4. In filter assays, only those combinations that could grow on media without histidine produced beta -galactosidase (data not shown). We measured the beta -galactosidase activity from the transformants to estimate the strength of the interaction between these proteins. The pBDntf4/pACT-NtMEK1 combination showed the strongest interaction of all (Fig. 4B). More curious was the observation that the loss-of-function version of p43Ntf6 interacted more strongly with NtMEK1 than either the wild-type or gain-of-function version of the protein, both of which resulted in roughly equivalent amounts of beta -galactosidase production (Fig. 4B).


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Fig. 4.   NtMEK1 interacts with p43Nft6 and p45Nft4 in a two-hybrid interaction assay. A, NtMEK1 (MEK) was expressed in yeast cells with the indicated MAP kinases or with the empty vector pBD-Gal4 Cam (pBDGal). Each MAP kinase was also expressed with the empty vector pACT2 (pACT) that was used to express NtMEK1. Co-transformants were plated on medium with histidine (-leu -trp) or without histidine but containing 3-AT (-leu -trp -his). B, beta -galactosidase activity (arbitrary units/mg of protein) was measured in extracts from the indicated co-transformants. Each measurement is the mean of three separate determinations.

Therefore, in vitro and in vivo interaction assays showed a strong physical interaction between NtMEK1 and both p43Ntf6 and p45Ntf4, even though p43Ntf6 was a preferred substrate for phosphorylation in vitro.

p43Ntf6 Complementation of the Yeast Mutant mpk1 Only Occurs in the Presence of NtMEK1-- A functional complementation assay was used to further investigate the interaction between NtMEK1 and p43Ntf6 and p45Ntf4. Disruption of the gene for MPK1, coding for a MAP kinase involved in cell wall biosynthesis, in yeast cells (here described as the mpk1 strain), results in a temperature-sensitive phenotype that can be rescued by growing cells in the presence of sorbitol as an osmotic stabilizer (15, 16). The NtMEK1, ntf6WT, ntf6K67R, ntf6E328N, and ntf4WT cDNAs were cloned into yeast expression vectors (see "Experimental Procedures") and used to transform the mpk1 strain. None of the single transformants could rescue the defect of mpk1 cells when grown at 37 °C in the absence of sorbitol (Fig. 5). However, co-transformation of mpk1 cells with NtMEK1 together with plasmids expressing either p43Ntf6-WT or p43Ntf6-E328N could complement the growth defect at 37 °C (Fig. 5). The growth of these double transformants was somewhat weaker than the growth of cells in which the wild-type MPK1 gene was expressed from a high copy number plasmid. By contrast, the cells expressing p45Ntf4 could not complement the mpk1 growth defect when co-expressed with NtMEK1. The complementation of mpk1 by p43Ntf6 depended on the kinase activity of the protein since co-expression of p43Ntf6-K67R with NtMEK1 did not result in growth at 37 °C (Fig. 5). No growth of the mpk1 transformants was observed after plasmid-losing experiments in which the pACT-NtMEK1 construct was eliminated from the pBD-ntf6WT/pACT-NtMEK1 co-transformant (see "Experimental Procedures"), confirming that NtMEK1 was necessary for the complementation (data not shown). Together, these data indicate that NtMEK1 can activate p43Ntf6.


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Fig. 5.   Complementation of the yeast mpk1 mutant by co-expressing NtMEK1 with p43Nft6 or p43Nft6E328N. Mutant yeast cells (mpk1) were transformed singly or doubly with the constructs shown. mpk1, untransformed cells; MPK1, the wild-type yeast gene MPK1 on a multicopy plasmid; vector, the empty cloning vector pVT103U. Other constructs are described under "Experimental Procedures." The yeast transformants were plated on medium with (+) or without (-) sorbitol and incubated at 37 °C.

Co-expression of ntf6 and NtMEK1 in Plant Tissues and after Induction of Cell Division-- Northern analysis of RNA isolated from different plant tissues was performed using ntf6 and NtMEK1 probes. They are expressed at a high level in cell suspension cultures. Expression of both ntf6 and NtMEK1 occurs in cotyledons and floral tissues, but not in roots or leaves (Fig. 6). Although NtMEK1 showed a similar expression profile to ntf6, no NtMEK1 signal was observed in young stems where weak ntf6 expression was observed.


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Fig. 6.   Northern analysis of NtMEK1 expression. RNA was extracted from the indicated tissues and used in Northern analysis using probes from NtMEK1, ntf6, or the constitutively expressed gene pCNT6 (39) as a loading control. Cell susp., cell suspension; buds-repro, flower buds from which anthers and ovaries have been removed.

The p43Ntf6 MAP kinase has been implicated in cell division processes in plant cells, showing activation during anaphase/telophase (10). To investigate whether NtMEK1 might be involved in this process, we induced cell division in leaf pieces by incubating them in media containing the plant hormones NAA and BA. The combination of these two plant hormones is absolutely required to induce cells to re-enter into the cell cycle (24). Samples were taken over a period of days, RNA extracted, and used for Northern analysis. A B-type cyclin gene (cycB1) was used as a probe to follow changes in the cell cycle in the leaf pieces over the time course of the experiment. The cycB gene is expressed during the M phase of the cell cycle (25). Two peaks of cycB transcript accumulation were observed 4 and 7 days after the induction of cell division in leaf pieces (Fig. 7), suggesting that a degree of cell cycle synchrony occurs after this inductive process. Both ntf6 and NtMEK1 showed a similar pattern of transcript accumulation, indicating that they are also being induced in a cell cycle-regulated manner (Fig. 7). To further corroborate the cell cycle-regulated expression of p43Ntf6, Western and kinase assays were performed using an anti-p43Ntf6 antibody with protein extracts from the leaf pieces induced to divide in a manner similar to that described above. In this instance we used a combination of NAA and BA or a combination of the synthetic auxin 2,4-D and BA. The p43Ntf6 protein kinase activity (Fig. 8A) and p43Ntf6 protein levels (Fig. 8B) followed a similar pattern to that observed with the ntf6, NtMEK1, and cycB transcript accumulation. This was observed in both hormone combinations (NAA with BA and 2,4-D with BA). No NtMEK1 or ntf6 transcripts (data not shown), nor protein or kinase activity for p43Ntf6 (Fig. 8, A and B), were observed without hormone addition or when only one of the hormones was added. These data support a role for cell cycle-regulated expression and activation of p43Ntf6, and the co-expression of NtMEK1.


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Fig. 7.   Cell cycle induction of NtMEK1 transcription. Cell division was induced in leaf pieces by the addition of plant hormones as indicated in the text and RNA was extracted on the days shown. After blotting the RNA to Hybond-N membranes, Northern analysis was performed with a NtMEK1, ntf6, cycB (an M phase-expressed cyclin gene), or pCNT6 (a constitutively expressed gene) probe.


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Fig. 8.   p43Nft6 expression and activity after inducing cell division in leaf pieces. Cell division was induced in leaf pieces by the addition of plant hormones as indicated (see also text). No hormone addition was used as a control. Protein extracts were prepared from samples taken over a period of days and immunoprecipitated with the anti-p43Ntf6 antibody and used in kinase assays with MBP as substrate (A) or used for immunoblot analysis with the anti-p43Ntf6 antibody (B).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The p43Ntf6 MAP kinase from tobacco appears to play a role in dividing cells (10). It is activated in mitosis and localizes to the phragmoplast, a plant-specific structure involved in laying down the new cell wall. It was therefore of interest to search for molecules that interact with p43Ntf6. We undertook a two-hybrid screen using p43Ntf6 as bait and isolated a cDNA that could encode a novel plant protein kinase that has been called NtMEK1 and presents all the characteristics of a MAP kinase kinase. A tobacco MAPKK was isolated previously by two-hybrid screening using the MAP kinase SIPK as a bait (8). These two proteins co-immunoprecipitated, but no phosphorylation of SIPK by SIPKK could be demonstrated. NtMEK1 and SIPKK are only 65% identical at the amino acid level.

In vitro and in vivo binding assays showed that NtMEK1 could bind two out of three tobacco MAP kinases, p43Ntf6 and p45Ntf4, but not p43Ntf3. Despite the preferential phosphorylation of p43Ntf6 over p45Ntf4 by NtMEK1, the latter protein showed apparently stronger binding to NtMEK1 as measured by the production of beta -galactosidase activity in a two-hybrid interaction assay. The observed weak phosphorylation of p45Ntf4 could be due to the slow release of phosphorylated p45Ntf4 from NtMEK1 resulting from this strong binding. MAPKKs may bind tightly to MAP kinases in their inactive form. The dissociation of the MAPKK and MAP kinase appears to be important for MAP kinase activation and subcellular localization. It has been suggested that the dissociation of MAPKK from a MAP kinase may involve the feedback phosphorylation of MAPKK by a MAP kinase (26, 27), and such phosphorylation of a MAPKK by a MAP kinase has been demonstrated (28). NtMEK1 might not be a substrate for p45Ntf4, thus preventing the feedback phosphorylation release mechanism that dissociates p45Ntf4 and NtMEK1 and thus the interaction of active p45Ntf4 with its substrates. In support of this was the observation that the p43Ntf6 LOF version also interacted more strongly with NtMEK1 than the wild-type or gain-of-function protein when measured as beta -galactosidase activity in a two-hybrid interaction assay. It has been shown previously that a MAPKK could bind to a MAP kinase but not activate it (29), and indeed could inhibit its activation (27).

A number of different docking domains between MAP kinases and MEKs have been described. One such domain located in the amino terminus of the protein is composed of a basic region followed by LXL (30). The amino terminus of NtMEK1 contains a sequence, also present in most of the MEKs shown in Fig. 1, which shows similarities to this pattern. Amino-terminal portions of MEKs have been shown to be sufficient to bind MAP kinases (31). The amino terminus before subdomain I of NtMEK1 and the remaining catalytic part of the protein were expressed separately from the pACT2 vector in combination with all of the MAP kinases (plus the p43Ntf6 LOF and GOF versions) expressed in pBD-Gal4 Cam in a two-hybrid interaction assay. No interactions were found for any of these combinations, except for the carboxy catalytic domain of NtMEK1 with p45Ntf4, and this interaction was much weaker than with the wild-type protein (data not shown). Therefore, the binding determinants of NtMEK1 are still unclear, and probably require multiple interacting sites to specifically interact with and phosphorylate different MAP kinases (32).

Significantly, p43Ntf6 was able to complement the mpk1 mutation of Saccharomyces cerevisiae while p45Ntf4 could not. This complementation depended on the presence of NtMEK1, and on a functional p43Ntf6 MAP kinase. Together, these data show that NtMEK1 can activate the p43Ntf6 MAP kinase. The data presented here are not sufficient to predict whether the complementation of mpk1 by p43Ntf6 is a reflection of functional conservation between the yeast and plant MAP kinases. An Arabidopsis MAP kinase kinase (MEK1) was shown to interact with and activate the MAP kinase ATMPK4 (33, 34). Similarly to the situation with p43Ntf6 and NtMEK1, ATMPK4 could complement mpk1 only when co-expressed with MEK1 (33). The alfalfa MMK2 MAP kinase, however, could complement mpk1 when expressed on its own (35). Therefore, although it is a useful tool for examining interaction and activation of different members of the MAP kinase cascade, it is not clear whether complementation of the yeast mutant reflects functional conservation. Nevertheless, it may be significant that all plant MAP kinases that have complemented the mpk1 mutation to date belong to subgroup 2 of the plant MAP kinases (36).

The MAP kinase MMK3 from alfalfa is closely related to p43Ntf6 and shows similar activation kinetics and localization and is expressed in actively dividing tissues (37). The p43Ntf6 MAP kinase has been shown to be activated in late anaphase/telophase in synchronized cell suspension cultures (10). In accordance with this, we have shown here that the induction of ntf6 gene transcription occurs in a manner that follows the expression of a cell cycle-regulated gene, cycB, in leaves induced to re-enter the cell cycle. Both p43Ntf6 protein and activity measurements showed similar induction kinetics. The induction of NtMEK1 gene transcription is similar to ntf6 and cycB induction, providing support for a role for NtMEK1 in activating p43Ntf6 in a cell cycle-regulated manner.

In summary, a number of lines of evidence suggest that NtMEK1 is a MAPKK for p43Ntf6 and not p45Ntf4. (i) In a two-hybrid screen, three positive clones were isolated, all of them containing the NtMEK1 cDNA. (ii) p43Ntf6 was a much better in vitro substrate for NtMEK1. NtMEK1 only weakly phosphorylated the p45Ntf4 protein. (iii) NtMEK1 could activate p43Ntf6 in vivo, as shown by the complementation of a mpk1 mutant. This complementation depended on the presence of NtMEK1 and an active p43Ntf6 kinase. p45Ntf4 could not complement mpk1. (iv) Both the NtMEK1 and ntf6 genes show similar expression patterns in plant tissues and after the induction of cell division. The ntf4 gene is expressed only in pollen and seeds (38). Future work on the activation and localization of NtMEK1 should provide further confirmation of the specificity of NtMEK1 for p43Ntf6.

    ACKNOWLEDGEMENTS

We thank Andrew J. Waskiewicz and Jonathan A. Cooper for helpful comments during the realization of the work.

    FOOTNOTES

* This work was supported by European Union Training and Mobility of Researchers Program RYPLOS Project FMRX-CT96-0007.The costs of publication of this article were defrayed in part by the payment of page charges. The 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 this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ302651 (NtMEK1).

Supported by EMBO Short Term Fellowship ASTF 9444.

** To whom correspondence should be addressed. Tel.: 43-1-4277-54603; Fax: 43-1-4277-9546; E-mail: erwin@gem.univie.ac.at.

Published, JBC Papers in Press, March 5, 2001, DOI 10.1074/jbc.M010621200

    ABBREVIATIONS

The abbreviations used are: MAP, mitogen-activated protein; WT, wild-type; GOF, gain-of-function; LOF, loss-of-function; MAPKK (MEK), mitogen-activated protein kinase kinase; MAPKKK, mitogen-activated protein kinase kinase kinase; GST, glutathione S-transferase; MBP, myelin basic protein; NAA, naphthalene acetic acid; BA, N6-benzyladenine; 2, 4-D, 2,4-dichlorophenoxyacetic acid; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; 3-AT, 3-aminotriazole; SIPK, salicylic acid-induced protein kinase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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