From the 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
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ABSTRACT |
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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.
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.
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
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 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 1 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
[ 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).
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 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).
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.
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
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.
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.
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.
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 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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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.
-galactosidase activity was measured in liquid
assays (CLONTECH).
-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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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); AtMAP2K
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).
View larger version (81K):
[in a new window]
Fig. 1.
Sequence comparison of NtMEK1 with
other plant MAPKKs. SIPKK, Nicotiana
tabacum; LeMEK1, Lycopersicon esculentum;
AtMEK1, AtMAP2K , 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.
View larger version (36K):
[in a new window]
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.
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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.
-galactosidase (data not shown).
We measured the
-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
-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,
-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.
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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.
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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.
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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
-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
-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).
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ACKNOWLEDGEMENTS |
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We thank Andrew J. Waskiewicz and Jonathan A. Cooper for helpful comments during the realization of the work.
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FOOTNOTES |
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* 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
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ABBREVIATIONS |
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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.
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