From the Laboratory of Developmental Cell Biology, Van Andel Research Institute, Grand Rapids, Michigan 49503
Received for publication, November 4, 2002, and in revised form, December 13, 2002
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ABSTRACT |
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Anthrax lethal toxin produced by the bacterium
Bacillus anthracis is the major cause of death in
animals infected with anthrax. One component of this toxin, lethal
factor (LF), inactivates members of the mitogen-activated protein
kinase kinase or MEK family through proteolysis of their
NH2 termini. However, neither the substrate requirements
for LF cleavage nor the mechanism by which proteolysis inactivates MEK
have been demonstrated. By means of deletion mutant analysis and
site-directed mutagenesis, we have identified an LFIR (LF
interacting region) in the COOH-terminal kinase
domain of MEK1 adjacent to the proline-rich region, which is essential
for LF-mediated proteolysis of MEK. Point mutations in this region
block proteolysis but do not alter the kinase activity of MEK. Similar
mutations in MEK6 also prevent proteolysis, indicating that this region
is functionally conserved among MEKs. In addition,
NH2-terminal proteolysis of MEK1 by LF was found to reduce
not only the affinity of MEK1 for its substrate mitogen-activated
protein kinase but also its intrinsic kinase activity, indicating that
the NH2-terminal end of MEK is important not only for
substrate interaction but also for catalytic activity.
The lethal effects of Bacillus anthracis have been
attributed to an exotoxin, which it produces (1). This exotoxin is
composed of three proteins: protective antigen
(PA),1 edema factor, and
lethal factor (LF) (for recent reviews see Refs. 2 and 3). PA binds to
a cell surface receptor (4) and, upon proteolytic activation to a
63-kDa fragment, heptamerizes to form a membrane channel that mediates
the entry of three molecules of LF or edema factor into the cell
(5-7). Edema factor is an adenylate cyclase and, together with PA,
forms a toxin referred to as edema toxin (8). LF is a
Zn2+-metalloprotease, which together with PA forms a toxin
referred to as lethal toxin. Lethal toxin is the dominant virulence
factor produced by B. anthracis and is the major cause of
death in infected animals (9).
Although LF has been shown to cleave the NH2 termini of
select members of the mitogen-activated protein kinase kinase or MEK family (10-12), the substrate requirements that determine LF
specificity are unknown. Indirect evidence suggests that epitopes
distal to the cleavage site are required for LF-MEK interaction. Yeast
two-hybrid assays for binding partners of LF have isolated cDNA for
MEK2, which lacks the NH2-terminal cleavage site (13).
Moreover, although it has been demonstrated that LF-cleaved MEK1 as
well as recombinant MEK1, which lacks the seven
NH2-terminal residues that are removed by LF, has reduced
kinase activity (10), it is not clear how the absence of these residues
alters MEK activity. Therefore, to identify regions distal to the
cleavage site that are required for proteolysis, we have constructed a
series of internal and COOH-terminal deletion mutants of MEK1 and have
analyzed their cleavability by LF. The results reveal that a
functionally conserved COOH-terminal region located adjacent to a
proline-rich insert of MEK1 is essential for LF-mediated proteolysis of
MEKs. In addition, to determine the mechanism by which LF inactivates
MEKs, we have examined the kinase activity of MEK toward ERK at
rate-limiting or saturating concentrations. Our results show that
proteolysis by LF removes NH2-terminal epitopes of MEK that
are important not only for MEK-substrate association but also for
intrinsic kinase activity.
Materials--
Constitutively activated MEK (x7, Construction of Deletion and Point Mutants--
COOH-terminal
deletion mutants of MEK1 were made by digesting a plasmid encoding
His6-tagged human wild-type MEK1 (pMKK1) (14) with the
indicated restriction enzymes and ligating the resulting fragment into
the appropriate pRSET vector as follows:
Constructs containing MEK1 mutations in the
LF-interacting region (LFIR) were
generated by introducing the mutations into pMKK1 with the use of the
QuikChange site-directed mutagenesis kit. The primers used were
5'-CCAACAACTCAGCAATTGCCATGGG-3' for F310A,
5'-CCAACAAGTGAAAAATTGCCATGGG-3' for E311H,
5'-GGATTACATAGTCAACCACCCTCCTCC-3' for E319H,
5'-CCCATGGCAATTGCTCACTTGTTGG-3' for
Phe310-Glu311-Glu319 (FEE),
5'-ATTTTTGAGTTGGCGGATTACATAGTC-3' for L313A,
5'-TTGTTGGATTACGCAGTCAACGAGCCT-3' for I316A,
5'-TTGGATTACATAGCCAACGAGCCTCCT-3' for V317A,
5'-AACGAGCCTCCTGCAAAACTGCCCAGT-3' for P322A, and
5'-CCTCCTCCAAAAGCGCCCAGTGGAGTG-3' for L324A and their respective
complementary sequences. MEK6 was PCR-cloned from a plasmid containing
mouse MEK6 cDNA and ligated into a pRSET (NH2-terminal
His6-tagged) bacterial expression vector. Constructs containing MEK6 point mutations were generated by introducing the
mutations with the use of the QuikChange site-directed mutagenesis kit. The primers used were
5'-cgaaaccctggccttaaagacccaaaagaagcatttg-3'for I15D,
5'-CTCAAACAGGTGGCAGAGGAGCCATCGCCA-3' for V271A,
5'-GAGGAGCCATCGGCACAACTCCCAGCAGAC-3' for P276A, and
5'-CCATCGCCACAAGCCCCAGCAGACAAGTTC-3' for L278A and their
respective complementary sequences. ERK(CD) was made by site-directed
mutagenesis of the His6-tagged wild-type ERK2 expression
vector to introduce aspartate to asparagine substitutions at residues
321 and 324 with the primer
5'-ctggagcagtattataacccaagtaatgagcccattgctgaa-3'and its complementary
sequence. All of the sequences were confirmed by direct DNA sequencing.
Protein Expression and Purification--
Anthrax lethal factor
was produced in a non-toxigenic, sporulation-defective strain of
B. anthracis (BH445) as described elsewhere (15).
Recombinant MEK protein was expressed in Escherichia coli and purified by fast pressure liquid chromatography essentially as
described earlier (10). Wild-type ERK2 and ERK(CD) were expressed similarly with the exception that cultures were grown and induced overnight at 30 °C.
MEK Cleavage Analysis--
To measure MEK cleavage in a
cell-based assay, lysates of Xenopus laevis
oocytes were prepared as described previously (16). Recombinant MEK
proteins (0.5 µg) were added as indicated in the text to 50 µl of
oocyte lysate and diluted to a final volume of 0.5 ml with oocyte
extraction buffer (0.25 M sucrose, 0.1 M NaCl, 0.02 M Hepes (pH 7.5), 2.5 mM
MgCl2). MEK1(NT) antibody (5 µl) was added, and lysates
were incubated overnight on a rotator shaker at 4 °C. Immune
complexes were precipitated with protein A-agarose, washed, separated
by SDS-PAGE, and immunoblotted using polyclonal antibodies raised
against MEK1(NT).
Cleavage of MEK proteins in vitro was measured by adding 0.2 µg of MEK and 0.2 µg of LF to 2.5 µl of 4× assay buffer (20 mM NaCl, 20 mM EGTA, 320 mM
potassium phosphate buffer, pH 7.2), and distilled water was added to a
final volume of 10 µl. After incubation at 30 °C for 5-10 min,
proteins were separated by SDS-PAGE, blotted onto polyvinylidene
difluoride membrane, and probed with antibodies to the NH2
terminus of MEK1 (1:1000).
Kinase Assays--
To measure B-Raf phosphorylation of MEK
deletion mutants, 2 µl (0.4 units) of recombinant B-Raf, 0.2 µg of
MEK protein, 3 µl of AB (20 mM MOPS (pH 7.2), 25 mM 1. Identification of an LFIR--
To identify regions of MEK
that are distal to the NH2-terminal cleavage site and are
required for interaction with LF, we undertook an analysis of internal
and carboxyl-terminal deletion mutants of MEK1. Two internal and five
COOH-terminal deletion mutants (Fig.
1A) were generated and
analyzed each for their ability to be recognized and cleaved by LF. Of
the deletion mutants analyzed, only 2. The LFIR Is Functionally Conserved--
Because not only MEK1
but also MEKs 2-4 and MEK6 and MEK7 are cleaved by LF, we reasoned
that critical elements of the LFIR must be conserved among the MEKs.
Residues 292-306 are present at the COOH-terminal end of a
proline-rich insert, which is unique to MEKs 1 and 2, and thus are not
likely to play an important role in LF substrate recognition. However,
an analysis of a sequence alignment of MEKs in the region following the
proline-rich insert revealed the presence of conserved elements (Fig.
2A). We used site-directed
mutagenesis to determine the importance of these residues in LFIR
function. Neither the single mutations in Phe310,
Glu311, Glu319, and Leu313 nor a
triple mutation in FEE interfered with the ability of LF to cleave MEK1
(Fig. 2B). However, the substitution of alanine for
Ile316, Val317, Pro322, or
Leu324 inhibited the ability of LF to cleave MEK1,
indicating that these residues are critical for binding and/or cleavage
by LF. Moreover, the LFIR appears to be functionally conserved because
the introduction of similar mutations at Val271,
Pro276, or Leu278 in MEK6 also blocked
proteolysis by LF (Fig. 2C). These results indicate that a
conserved COOH-terminal region of MEKs is required for LF-mediated
proteolysis.
3. The LFIR Is Proximal to COOH-terminal Regulatory
Epitopes--
The COOH end of the proline-rich region of MEK1 is
necessary for the association of MEK with the MEK1 activator Raf
(17, 18), suggesting the possibility that the LFIR and Raf-binding elements in MEK1 may overlap. To further evaluate this possibility, we
assayed the ability of B-Raf to phosphorylate and activate 4. Mechanism of LF Inhibition of MEK Activity--
The preceding
observation raises the intriguing possibility that LF may inhibit the
activity of MEK by blocking its activation by Raf. However, LF(E687C),
a non-toxic inactive LF containing a single amino acid substitution in
the putative zinc binding site (20), was as able as wild-type LF to
inhibit B-Raf-mediated MEK phosphorylation (Fig. 3A).
Moreover, we have found that the ability of constitutively activated
MEK1 (
In the preceding experiments, phosphorylation was measured with MEK and
ERK present in approximately equimolar amounts. Thus, the extent of
phosphorylation reflected not only the affinity of MEK for ERK but also
the intrinsic kinase activity of MEK. However, the relative
contribution of each of these factors may be altered by varying the
ratio of substrate to kinase so that at relatively low concentrations
of ERK the rate of phosphorylation is limited by the affinity of MEK
for ERK, whereas at relatively high concentrations of ERK, the reaction
becomes saturated and the rate of phosphorylation largely reflects the
intrinsic kinase activity of MEK. Therefore, in the following
experiments, the level of MEK1 protein was kept constant at 1.5 µg
while the amount of substrate was varied from 0.25 to 12 µg. Rather
than conforming to classic Michaelis-Menten enzyme kinetics, a plot of
the extent of wild-type MEK1-mediated ERK phosphorylation
versus substrate concentration revealed a sigmoidal
activation of MEK, more closely resembling that of an allosteric enzyme
(Fig. 3D). Similar observations have been made in
Xenopus oocytes and were attributed to a combination of
protein synthesis-dependent positive feedback and the
intrinsic ultrasensitivity of the MEK-ERK kinase cascade (23, 24).
Because our results were obtained in vitro in the presence
of recombinant proteins, they indicate that ERK itself is capable of
positively regulating MEK activity. Consistent with the accepted view
that the NH2-terminal docking (D) domain of MEK1 binds
ERK2, treatment with LF lowered the affinity of MEK for ERK (Fig.
3C). However, LF treatment also decreased the extent of ERK
phosphorylation at saturating concentrations, indicating that the
NH2-terminal residues 1-7 of MEK1 play a role in
maintaining intrinsic kinase activity. To verify this result, we
repeated this assay, substituting ERK(CD) as the substrate (Fig.
3E). Under these conditions, LF did not alter the affinity
of MEK for its substrate but did decrease the extent of ERK(CD)
phosphorylation under saturating conditions. Thus, LF inhibits MEK
activity not only by lowering its affinity for ERK but also by
decreasing its intrinsic kinase activity.
To date, MEKs are the only identified physiological substrates of
LF. A comparison of the LF cleavage sites on MEKs1-4, MEK6, and MEK7
reveals elements of homology. In all cases, the cleavage site is
preceded by a series of basic residues and followed immediately by an
aliphatic residue. Also, with the exception of MEKs 3 and 4, the
cleavage site is preceded by one or more proline residues. Synthesizing
these results, the consensus site for LF cleavage fits the pattern
B(B/P)BP(X)2-3-Al,
where B represents a basic residue, P is proline,
X is the variable, and Al represents aliphatic residues.
This motif is similar to a described generic MAPK binding site or D
domain, consisting of a basic amino acid center, which is flanked by
hydrophobic residues on one or both sides (21). This raises the
possibility that LF may be a D domain protease, which targets both
activators and substrates of MAPKs. However, using in vitro
cleavage assays, we have been unable to detect LF-induced cleavage of
CL-100, c-Jun, or activating transcription factor-2, known
substrates of MAPKs that contain a D domain (data not shown). In
addition, yeast two-hybrid analyses for binding partners of LF have
isolated cDNA for MEK2, which lacks the NH2-terminal cleavage site (13). Thus, other regions of MEKs, in addition to the
NH2-terminal cleavage site, must be required for LF
substrate recognition.
Using mutational analysis, we have identified a functionally conserved
COOH-terminal region of MEKs that is essential for LF-mediated
proteolysis of MEK. The presence of a conserved region distal to the
cleavage site, which is necessary for binding and/or cleavage by LF,
may explain in part the failure to identify physiological LF substrates
other than MEKs. The fact that other MEK1-regulatory proteins, such as
B-Raf (17, 18), p21-activated kinase (25), and the scaffolding protein
MEK partner-1 (26, 27), also interact with MEK in this region
suggests that this region constitutes a key regulatory domain of MEK1
and perhaps other MEKs.
The presence of a shared regulatory domain may have functional
implications for LF toxicity because the presence of MEK-binding proteins may alter the access of LF to its substrates. Conversely, LF
might decrease MEK activity by competitively displacing positive regulators of MEKs. However, by itself, this mechanism seems
insufficient to explain how LF inactivates MEKs, because as noted in
the preceding section, LF can inhibit the activity of constitutively
activated MEK1. Because phosphorylation under these conditions is
dependent not only upon kinase activity but also substrate affinity, we reasoned that LF might inhibit MEK by either reducing its intrinsic kinase activity or decreasing its affinity for ERK. The latter seemed
more probable because MEK1 deletion mutants lacking the 32 NH2-terminal residues are deficient in their ability to
bind ERK (22, 28) and because mutations in the docking domain decrease the efficiency with which MEK1 activates ERK (21). However, we found
that as well as decreasing the affinity of MEK for its substrate, LF
also decreased the intrinsic kinase activity of MEK. The latter result
was not expected but is not without precedence. Based upon homology to
the A-helix of cAMP-dependent protein kinase and its
observation that NH2-terminal deletions and activation lip
substitutions synergize to activate MEK1, Ahn and colleagues (29) have
hypothesized that regions of the NH2 terminus form long
range interactions with the activation loop and that perturbation of
the structure within this region promotes conformational changes within
the activation loop that favor activation. In addition, the structural
analyses of the
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
n3/S222D)
was a kind gift of N. Ahn (University of Colorado, Boulder, CO).
Expression vectors encoding His6-tagged wild-type rat ERK2
as well as a chimeric protein consisting of the first two subdomains of
p38 MAPK fused to subdomains 2-10 of ERK2 (PIIE) were a kind gift from
M. Cobb (University of Texas Southwestern Medical Center, Dallas, TX). Mouse MEK6 cDNA was a kind gift from J. Han (Scripps Institute, La
Jolla, CA). CL100, c-Jun, activating factor-2, and B-Raf were obtained from Upstate Biotechnology (Lake Placid, NY). Polyclonal antibodies raised against the NH2 terminus of MEK1
(MEK1(NT)) were obtained from Upstate Biotechnology. Polyclonal
antibodies raised against the COOH terminus of MEK1 (C-18) as well as
the NH2-terminal His-tag (H-15) were obtained from Santa
Cruz Biotechnology (Santa Cruz, CA).
c1-(160-392)
EcoRI,
c2-(218-392) NheI/NcoI, and
c3-(229-392) NheI/AflIII. Additional
COOH-terminal deletion mutants of MEK1 were made by PCR
amplification of pMKK1 using PCR primers 5'-GGACAGCAAATGGGTCGGG-3' (corresponding to the multiple cloning site on pRSET) and
5'-CCGTATAAGCTTAGGGGCC-3' to introduce a novel HindIII site
at position 924. The resulting PCR product was digested with
BamHI and HindIII, gel-purified, and ligated into
BamHI/HindIII-digested pRSETA yielding a
construct encoding
c5-(292-392) as well as one encoding
c4-(261-392) because of a fortuitous error. Internal deletion
mutants of MEK1 were made by digesting pMKK1 with the indicated
restriction enzyme and re-ligating the gel-purified plasmid as follows:
i1-(20-318) HincI and
i2-(94-219) MscI.
To construct
i3-(292-318), we used the Stratagene QuikChange
site-directed Mutagenesis kit to introduce novel XhoI sites
into pMKK1 at position 910 using the primer
5'-GGCCAAGGACCCTCGAGAGGCCCCTTAGC-3' and its complementary sequence and
then at position 988 using the primer
5'-GTTGGATTACATAGTCCTCGAGCCTCCTC-3' and its complementary sequence. The resulting construct was digested with XhoI and
re-ligated following gel purification to generate
i3-(292-318). All
of the sequences were confirmed by direct DNA sequencing.
-glycerophosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM dithiothreitol),
and 3 µl of ATP mixture ([
-32P]ATP (10 mCi/ml, 3000 mCi/mmol, Amersham Biosciences) diluted 1:3 in 0.5 mM ATP,
75 mM MgCl2) were mixed with distilled water to
a final volume of 10 µl and incubated for 15 min at 30 °C. When
assaying the ability of B-raf to phosphorylate MEK1 in the absence or
presence of LF, B-raf was added last and the reaction was incubated for
5 min at 30 °C. Proteins were then separated by SDS-PAGE upon 10%
gels and processed for autoradiography. To measure MEK activity in the
presence of LF or inactive LF(E687C) (0.2 µg), samples were prepared
in a similar manner with the exception that ERK (wild type, PIIE,
or ERK(CD) was added. Proteins were then separated by SDS-PAGE on 14%
gels and processed for autoradiography.
RESULTS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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i2 was noticeably cleaved (Fig.
1, B-D, data not shown). This finding was surprising
considering that
i2 lacks residues 94-219, corresponding to the
amino-terminal kinase domain and suggests that the NH2
terminus may form a stable association with the COOH-terminal portion
of MEK1. Because neither
c5-(292-392) nor
i1-(20-318) was
cleaved by LF but
i2 was, we hypothesized that those regions absent
from
c5 and
i1 but present in
i2 must be necessary for binding
and/or cleavage. To test this hypothesis, we used site-directed
mutagenesis to introduce novel restriction sites in MEK1 to produce a
new deletion mutant,
i3, lacking residues 292-318. Analyses with
i3 showed that it was resistant to cleavage by LF (Fig.
1E). These results indicate that a region contained either
in whole or in part within residues 292-318 is necessary for binding
and/or cleavage by LF. We have called this region the
LF-interacting region or LFIR.
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Fig. 1.
Locating the LF-interacting region on
MEK1. A, a graphic representation of the MEK1 deletion
mutants (identified at the left of the boxed
regions) distinguishes those portions of wild type
(w.t.) MEK1 that has been deleted (in white) from
those that remain intact (in gray). The line at
the left of the boxed region indicates the
approximate site of cleavage, whereas the two open circles
above the boxed region are intended to represent activating
phosphorylation sites at residues 218 and 222. Because in
vitro cleavage assays might generate false-positive results
attributed to the presence of high concentrations of protease and
substrate and/or the presence of unstable translation products, we
elected instead to assay cleavage in a cell-like background. To do
this, we incubated recombinant MEK protein and LF in Xenopus
oocyte lysates and then immunoprecipitated with an antibody raised
against the NH2 terminus of MEK. This antibody does not
recognize MEK1 that has been cleaved by LF (10). The immunoprecipitates
were then analyzed by SDS-PAGE and immunoblotting with antibodies to
the NH2 terminus of MEK1 (MEK1-NT) (1:1000).
Selected Western blots of i1 (B),
i2 (C),
and
c5 (D) are shown. Lysates were mixed with nothing
(lane 1), active LF (lane 2), active LF (3) plus
a MEK deletion mutant (lane 3), inactive LF(E687C)
(lane 4), or inactive LF plus a MEK deletion mutant
(lane 5). E, His6-tagged wild-type
and
i3 MEK (0.2 µg) were incubated with LF (0.02 µg) in assay
buffer (4) at 30 °C for 5-10 min, and proteins were separated by
SDS-PAGE and immunoblotting as described above. Lanes 1 and
2, wild-type MEK in the absence or presence of LF,
respectively; lane 3 and 4,
i3 MEK in the
absence or presence of LF, respectively.
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Fig. 2.
Mutagenesis of the LF-binding region in
MEK1. A, conserved residues in the LFIR were identified
by multiple sequence alignment of MEKs 1-7 (19). Individual residues
in the LFIR of MEK1 (B) or MEK6 (C) were altered
by site-directed mutagenesis and assayed for cleavage by LF in
vitro. D, the ability of B-Raf (0.4 units) to
phosphorylate 0.2 µg of MEK proteins with mutations in the LFIR was
assessed using in vitro kinase assays.
i3 or
c5 MEK. Consistent with the hypothesis that LF interacts with MEK in
a region that contains a Raf-interacting site, in vitro
kinase assays demonstrated that B-Raf could phosphorylate neither
i3
nor
c5.2 Subsequent
analyses of the ability of B-Raf to phosphorylate MEK1 with point
mutations in the LFIR indicated that with the exception of
Phe310, alanine substitutions interfered with neither the
ability of B-Raf to phosphorylate (Fig. 2D) nor activate
MEK1.2 These results indicate that although the LFIR and
the B-Raf-interacting region of MEK1 are not identical, they are
adjacent or overlapping. If the LFIR and the B-Raf-interacting regions
of MEK1 do indeed overlap, LF should antagonize B-Raf-induced
activation of MEK. We tested this hypothesis by assaying the ability of
B-Raf to phosphorylate MEK1 in vitro in the presence of
increasing amounts of LF. The presence of an equimolar amount of LF but
not bovine serum albumin was sufficient to reduce the ability of B-Raf
to phosphorylate MEK1 by half (Fig.
3A). Collectively, these
results indicate that adjacent or overlapping epitopes of MEK1 are
required not only for cleavage by LF but also for B-Raf-mediated
phosphorylation.
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Fig. 3.
Inhibition of MEK1 by LF.
A, the ability of B-Raf (0.4 units) to phosphorylate
0.2 µg of His6-tagged wild-type MEK1 in the presence of
LF (0.04-4.0 µg, diagonal bars), inactive LF(E687C)
(0.04-4.0 µg, horizontal bars), or bovine serum albumin
(1 µg, hatched bar) was determined as described.
Phosphorylation was assessed by autoradiography and quantitated using a
Fujix BAS1000 (PhosphorImager) and MacBAS version 2.2 software. Results
are expressed relative myelin basic to control reactions in the
presence of B-Raf and MEK alone (open bar) as an average of
three experiments ± S.D. B, phosphorylation of ERK and
myelin basic protein in the presence of wild type (w.t.) or
constitutively active (x7) MEK1 and increasing amounts
(0.0-1.0 µg) of LF was measured in vitro. C,
the ability of wild-type MEK1 to phosphorylate ERK2, PIIE, and ERK(CD)
in the presence of LF (solid bars) or inactive LF(E687C)
(open bars) was assessed as described. Results are expressed
as an average of three experiments ± S.D. The saturation kinetics
of MEK1 activity in the presence of LF (open circles) or
inactive LF(E687C) (closed diamonds) were
determined by incubating 1.6 µg of MEK1 with increasing amounts
(0.25-12 µg) of ERK2 (D) or ERK(CD) (E). Each
graph shown is a representative sample of one of four
experiments.
n3/S222D) to phosphorylate and activate ERK2 is decreased in
the presence of LF (Fig. 3B). Because the kinase activity of
n3/S222D MEK1 is not dependent upon B-Raf-mediated activation, the
decrease in phosphorylation and activation of ERK associated with
exposure to LF must have resulted from a decrease in the intrinsic
kinase activity of MEK1 and/or a decrease in the ability of MEK1 to
interact with ERK. To test this hypothesis, we assayed the effects of
LF upon the ability of MEK1 to phosphorylate ERK2 protein containing
mutations at the common docking (CD) domain (D321N/D324N)
through which the NH2 terminus of MEK1 binds ERK2 (21). We
reasoned that if LF reduced the ability of MEK1 to interact with ERK
through its CD domain, LF should not inhibit MEK1-mediated
phosphorylation of ERK(CD). However, we found that although LF reduced
ERK2 phosphorylation by approximately two-thirds, it still reduced
ERK(CD) phosphorylation by half as much (Fig. 3C). Similar
results were obtained when we assayed phosphorylation of PIIE, a
chimeric protein consisting of the first two subdomains of p38 MAPK
fused to subdomains 2-10 of ERK2 and lacking a putative docking site
on ERK2 (Fig. 3C) (22). These results indicate that the loss
of MEK activity following proteolysis by LF cannot be attributed
entirely to a decreased ability to bind ERK.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit of the serine/threonine protein kinase
CKII, which is constitutively active, indicate that a cluster of basic
residues at its NH2 terminus stably associates with the
activation loop, keeping it in an open conformation (30, 31). By
analogy, we predict that the NH2 terminus of MEK1
associates with its activation loop to promote its activity.
NH2-terminal structure may also promote protein stability,
because we previously noted that the long term stability of MEKs is
decreased in cells treated with PA and LF (10).2 The
proximity of the D domain to the activation loop may coordinate MEK-ERK
interaction and facilitate ERK phosphorylation and activation.
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ACKNOWLEDGEMENTS |
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We thank D. Morrison for asking a good question to which we offer an answer in this paper. We also thank E. Xu, A. Alberts, G. Vande Woude, and T. Hunter for stimulating discussions as well as J. F. Bodart for comments on the paper.
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FOOTNOTES |
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* This work has been is supported in part by federal funds from the Department of Health and Human Services under contract number N01-C0-74101.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.
To whom correspondence should be addressed. Tel.: 616-234-5258;
Fax: 616-234-5259; E-mail: nick.duesbery@vai.org.
Published, JBC Papers in Press, January 9, 2003, DOI 10.1074/jbc.M211262200
2 N. S. Duesbery, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: PA, protective antigen; ERK, extracellular signal-regulated kinase; LF, lethal factor; LFIR, LF interacting region; MAPK, mitogen-activated protein kinase; MEK, MAPK/extracellular signal-regulated kinase kinase; NT, NH2 terminus; CD, common docking; MOPS, 4-morpholinepropanesulfonic acid; D, docking.
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