(Received for publication, November 7, 1996, and in revised form, February 11, 1997)
From the Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9041
Mitogen-activated protein (MAP) kinase pathways
include a three-kinase cascade terminating in a MAP kinase family
member. The middle kinase in the cascade is a MAP/extracellular
signal-regulated kinase (ERK) kinase or MEK family member and is highly
specific for its MAP kinase target. The first kinase in the cascade, a MEK kinase (MEKK), is characterized by its ability to activate one or
more MEK family members. A two-plasmid bacterial expression system was
employed to express active forms of the following MEK and MAP kinase
family members: ERK1, ERK2, -SAPK, and p38 and their upstream
activators, MEK1, -2, -3, and -4. In each kinase module, the upstream
activator, a constitutively active mutant of MEK1 or MEKK1, was
expressed from a low copy plasmid, while one or two downstream effector
kinases were expressed from a high copy plasmid with different
antibiotic resistance genes and origins of replication. Consistent with
their high activity, ERK1 and ERK2 were doubly phosphorylated on Tyr
and Thr, were recognized by an antibody specific to the doubly
phosphorylated forms, and were inactivated by either phosphoprotein
phosphatase 2A or phosphotyrosine phosphatase type 1. Likewise,
activated p38 and
-stress-activated protein kinase could also be
inactivated by either phosphatase, and
-stress-activated protein
kinase was recognized by an antibody specific to the doubly
phosphorylated forms. These three purified, active MAP kinases have
specific activities in the range of 0.6-2.3 µmol/min/mg.
Coexpression of protein kinases with their substrates in bacteria is of
great value in the preparation of numerous phosphoproteins, heretofore
not possible in procaryotic expression systems.
Many extracellular stimuli transmit signals into eukaryotic cells in part through a conserved signal transduction mechanism, a mitogen-activated protein (MAP)1 kinase, or extracellular signal-regulated kinase (ERK) pathway. MAP kinase pathways are important pleiotropic signaling enzymes that regulate cytoplasmic enzymes, cytoskeletal processes, membrane proteins, gene activity, and other intracellular events. Although upstream regulatory mechanisms may vary, the overall organization of a MAP kinase pathway, which includes a three-kinase cascade, is conserved in evolution from yeast to metazoans; a MAP/ERK kinase kinase (MEKK) activates a MAP/ERK kinase (MEK), which activates an ERK or MAP kinase.
Currently, several different mammalian MAP kinase cascades have been defined, and three are relatively well characterized. One of them, the ERK module, is activated in response to stimuli that induce proliferation and differentiation. The c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38 MAP kinase modules are activated in response to environmental stresses such as osmotic shock or UV irradiation (reviewed in Refs. 1 and 2). Distinct MEK isoforms lie upstream in each cascade. MEK1 and -2 activate ERK1 and -2; MEK3 and -6 activate p38; MEK4 (also known as JNKK or SEK1) activates JNK/SAPK. MEKKs are a diverse group that include Raf isoforms, which are believed to act exclusively on MEK1 and MEK2, and MEKK1, which can activate MEK isoforms in all three modules.
One of the obstacles in studying the biochemical properties of individual MAP kinase modules and their constituents is the difficulty in obtaining sufficient quantities of pure active components. While most MAP kinases can be easily expressed in bacteria, they must be activated by corresponding MEKs, which must also be expressed and activated. Mutants with increased kinase activity have been described for some but not all MEK isoforms. Much less success has been achieved in activating MAP kinases, themselves, by mutagenesis (1, 3).
In this study, we coexpressed different components of the three best characterized MAP kinase modules in bacteria. We show that, in each case, if the upstream component is present in constitutively active form, coexpression results in the activation of the downstream component. Expression of a C-terminal, constitutively active fragment of MEKK1 allows us to activate MEK1, MEK2, MEK3, and MEK4 from all three modules and subsequently to activate the downstream MAP kinases. Coexpression of kinase and substrate provides a convenient method to generate highly purified milligram amounts of activated MAP kinases as well as other phosphoproteins for biochemical and structural studies.
cDNAs
encoding constitutively active MAP kinase cascade members were
subcloned under the control of an
isopropyl--D-thiogalactopyranoside-inducible Tac
promoter into the plasmid pBB131 (Fig. 1B). pBB131, a low copy plasmid with a p15A origin of replication and a kanamycin resistance marker (4), was kindly provided by Dr. J. Gordon (Washington
University, St. Louis, MO).
To subclone the constitutively active mutant of human MEK1 (kindly
provided by Dr. Natalie Ahn, University of Colorado at Boulder) into
pBB131, an NcoI restriction site was introduced at the start
ATG and an MluI site after the stop codon by polymerase chain reaction (PCR) with specific primers (5-GGGCCATGGCCAAGAAGAAGC-3
and 5
-CAAACGCGTCATGATCAACCACCGG-3
). The PCR product was digested with
MluI and partially with NcoI and ligated into
pBB131 digested with the same restriction enzymes. To subclone the
catalytic C-terminal fragment of rat MEKK1 (MEKK-C) (5) into pBB131, an
NcoI-EcoRI fragment of MEKK containing its
catalytic domain was ligated into pBB131 digested with the same
enzymes.
To express MEKs and ERKs in one plasmid, MEK1 was modified as follows.
Using PCR with the specific primers (MEK1,
5-CCCGCTAGCCATATGCCCAAGAAGAAGCCG-3
and
5
-CTCTTTGCATATGGGTACCTCCTTAGACGCCAGCAGCATGGGT-3
), an NdeI restriction site was introduced at the MEK starting ATG, and a ribosomal binding site was introduced after the MEK stop codon followed
by a second NdeI site. A similar approach was used for MEK2.
The resulting PCR products were inserted into pET His6ERK2, which contains rat ERK2 (6) at the NdeI site. pET
His6ERK2 is a derivative of pET-His6TEV (Life
Technologies, Inc.) from which the Tev protease cleavage site has been
deleted. The resulting plasmids direct expression of untagged MEK1 or
MEK2 and ERK2 with a His6 tag (Fig. 1B).
For the expression of human MEK4 (also known as JNKK or SEK1; Refs. 7
and 8) and rat -SAPK (9) from the same plasmid, a ribosomal binding
site and then an NcoI site were introduced immediately after
the
-SAPK stop codon using PCR with the T7 primer and a specific
primer, 5
-CCAGCCATGGTCTCCTTTCACAGACAAGTGCGCCATCTGCGAGGTTT-3
. The resulting PCR product was digested with NcoI and
ligated into NpT7-5 containing a full-length MEK4 cDNA (kindly
provided by M. Karin, University of California San Diego) digested with
the same enzyme. The resulting plasmid directed expression of untagged MEK4 and
-SAPK with a His6 tag (Fig. 1B).
For the expression of MEK4 and murine p38 from the same plasmid, MEK4
was excised from pGEX-KG with NcoI and HindIII
and cloned into pT7-5 (10). A ribosomal binding site and an
NcoI site were introduced after the p38 stop codon (11) by
PCR with the T7 primer and a specific primer,
5-CCAGCCATGGTCTCCTTTCAGGACTCCATTTCTTCTTGGTCAAGGGG-3
, using
pET14b-p38 as template. The PCR product was cleaved with NcoI and ligated into pT7-5 containing MEK4 that had been
digested with NcoI.
The resulting plasmids, one encoding kanamycin resistance with an
upstream constitutively active kinase and the other encoding ampicillin
resistance with downstream effector kinase(s), were transformed into
Escherichia coli strain BL21(DE3). Transformants were plated
on Luria broth plates containing 0.1 mg/ml of both carbenicillin and
kanamycin. From each plate, a minimum of five colonies were selected
for analysis. Expression was induced with 0.25 mM
isopropyl--D-thiogalactopyranoside (final concentration) for 7-12 h at 30 °C before harvesting. Cells were lysed by
sonication in 0.5 ml of buffer C (see below) and clarified by
sedimentation for 15 min at 15,000 × g at 4 °C.
Protein kinase activity in the lysates was assayed using the following
substrates: myelin basic protein for ERKs, GST-c-Jun
1-223 for
-SAPK, GST-ATF2
1-254 for p38 MAP kinase, K52R ERK2 for MEK1 and
MEK2, p38 for MEK3, and GST-SAPK
K55A for MEK4.
His6-tagged proteins were expressed and
purified essentially as described previously (5) with the following
modifications. Expression of recombinant protein was induced with 0.25 mM isopropyl--D-thiogalactopyranoside at
30 °C for 12-14 h. Cells were harvested and then either frozen in
liquid nitrogen and stored at
80 °C or lysed in buffer A (50 mM sodium phosphate, pH 8.0, 0.3 M NaCl, 0.5 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µM pepstatin, 1 mM benzamidine). The lysate
was clarified at 27,000 rpm for 30 min at 4 °C in a Ti35 rotor
(Beckman, Palo Alto, CA). The resulting supernatant was applied by
gravity flow to a 1-2-ml column of Ni2+-NTA resin (Qiagen
Inc., Chatsworth, CA) equilibrated in buffer A. The resin was washed
with 20 volumes of buffer B (20 mM Tris-HCl, pH 8.0, 0.3 M NaCl, 10 mM imidazole) and eluted with 0.25 M imidazole, pH 7.0, in buffer B. 1-ml fractions were
collected, and aliquots were analyzed by SDS-PAGE. Fractions containing
recombinant protein were pooled and dialyzed overnight against buffer C
(20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM dithiothreitol, 1 mM EGTA, 10 mM
benzamidine, 0.2 µM pepstatin, 0.5 mM
phenylmethylsulfonyl fluoride, and 20% glycerol). For fast protein
liquid chromatography purification, proteins were diluted 2-fold with
buffer C without glycerol, applied to a Mono Q HR5/5 column
equilibrated in the same buffer, and eluted with 500 mM
NaCl in buffer C. Fractions containing purified MAP kinases were
pooled, dialyzed against buffer C, frozen in liquid nitrogen, and
stored at
80 °C in small aliquots. GST-tagged proteins were
expressed as above and purified on reduced glutathione-Sepharose (Sigma) as described previously (12).
Protein concentration was
determined using the Bio-Rad dye reagent diluted according to the
manufacturer's recommendations with bovine serum albumin as a
standard. Protein kinases and substrates were incubated in 30 µl of
20 mM HEPES, pH 7.3, 10 mM MgCl2, 1 mM benzamidine, 1 mM dithiothreitol, and
100-200 µM ATP ([-32P]ATP to 10-30
cpm/fmol) for 15 or 30 min at 30 °C. Serial dilutions of active
enzymes were tested to guarantee that assays were in the linear range.
Reactions were stopped by either 1) the addition of 10 mg of bovine
serum albumin and 1 ml of 10% trichloroacetic acid followed by
collection of precipitates on glass filters or 2) the addition of 7.5 µl of 5 × Laemmli sample buffer followed by heating for 5 min
at 100 °C for analysis by SDS-PAGE.
Antisera raised against a C-terminal peptide of MEKK1, MEK1 (A2227), and MEK2 (A2228) were described previously (5). Antibodies to MEK3 were raised against a GST-MEK3 fusion protein (cDNA kindly provided by Dr. K. Guan, University of Michigan). Antibodies against MEK4 proteins were elicited with the synthetic peptide KRKALKLNFANPPVKSTART (residues 35-54 of MEK4), coupled to hemocyanin using methods described earlier (13). Antisera specific to phosphorylated forms of ERKs and JNK/SAPK were from Promega Corp.
Phosphatases and Phosphatase TreatmentThe catalytic subunit of protein phosphatase 2A (PP2A) was purified as described (14). A plasmid that encoded a truncated form of a protein-tyrosine phosphatase (PTP1) fused with GST was kindly provided by Dr. Guan. GST-PTP1 was expressed in E. coli, purified, and cleaved with thrombin, and PTP1 was separated from GST as described (15).
For inactivation with phosphatases, purified active ERK2 (1.7 µg/ml),
p38 (1.9 µg/ml), or -SAPK (1.65 µg/ml) was treated with 3 µg/ml PP2A in 20 mM Tris-HCl, pH 7.4, 2 mM
EGTA, 2 mM dithiothreitol, 2 mM
MgCl2, 0.5 mg/ml bovine serum albumin, 0.05% Brij-35, 5 µg/ml each of aprotinin and leupeptin, and 100 ng/ml
phenylmethylsulfonyl fluoride or with 7.4 µg/ml PTP1 in 50 mM Hepes, pH 7.5, and 0.1%
-mercaptoethanol for the
indicated times at 30 °C. For kinase assays, dephosphorylation
reactions were terminated by the addition of okadaic acid or vanadate
to final concentrations of 1 µM and 5 mM,
respectively. Alternatively, for immunoblotting (ECL kit, Amersham Life
Science, Inc.), reactions were terminated with electrophoresis sample
buffer.
It was shown previously that in eukaryotic cells ERK1 and ERK2 may be activated by both of the two closely related kinases, MEK1 and MEK2 (16, 17). This activation results in phosphorylation of two closely positioned residues between subdomains VII and VIII of ERKs, Thr202 and Tyr204 in ERK1 and Thr183 and Tyr185 in ERK2 (13, 18-20). MEKs also require two phosphorylations, both on serine or threonine, for high activity; thus, to activate ERKs efficiently, MEKs must be activated by phosphorylation by an upstream kinase (Refs. 18 and 19; Fig. 1A).
Constitutively active mutants of MEK1 and MEK2 have been described that
have several hundred-fold elevated activity toward ERKs (3). In the
absence of their own phosphorylation, these mutants can activate ERK1
and ERK2 in vitro or in intact cells (3, 21). We coexpressed
one of these highly activated MEK1 mutants, MEK1 N3 S218D S222D,
(referred to hereafter as MEK1-R4F) with wild-type ERK1 or ERK2 in
E. coli to see if we could produce activated ERKs. To
accomplish this, we initially employed the two-plasmid system used for
expression of N-myristoylated forms of G-protein
-subunits in E. coli (Ref. 4; Fig. 1B). Such coexpression of MEK1-R4F with either ERK1 or ERK2 results in their activation (data not shown).
We tested methods to increase the amount of active ERK produced by our
coexpression system. A single-plasmid system expressing both MEK1-R4F
and ERK2 from a single bicistronic mRNA under the control of the
bacteriophage T7 promoter yielded nearly complete conversion of ERK2 to
the reduced mobility, highly phosphorylated form. The proportion of
active ERK based on shifted mobility varied from 10% to close to
100%, depending on growth medium, conditions, and time of induction
(data not shown). The specific activity toward myelin basic protein of
ERK2 obtained from this single plasmid system and purified on
Ni2+-NTA-agarose and then on Mono Q varied from 1.6 to 2.3 µmol/min/mg protein in three ERK2 preparations. To demonstrate that
ERK2 expressed in this system is doubly phosphorylated on Tyr and Thr,
we treated the purified active kinase with protein phosphatases
selective for either the phosphotyrosine (PTP1) or the phosphothreonine (PP2A) residue. As shown in Fig. 2A,
treatment with either phosphatase, but not with phosphatases pretreated
with the corresponding inhibitors (vanadate and okadaic acid), results
in loss of ERK2 activity.
Characterization of Coexpressed and Phosphatase-treated ERK2 by a Specific Antiserum Directed against Phosphorylated ERKs
To characterize the coexpressed ERK proteins further, we used an antiserum raised against a doubly phosphorylated peptide from the ERK phosphorylation lip. The epitope was a 16-amino acid sequence encompassing phosphothreonine and phosphotyrosine residues that are required for ERK activation. This antibody does not recognize 10-20 ng of ERK2 expressed in the absence of an activating MEK (Fig. 2B). In contrast, if ERK2 has been coexpressed with MEK1-R4F, as little as 1-2 ng are recognized by this antibody (Fig. 2B). Treatment with PTP1 or PP2A greatly reduced ERK2 recognition in a time-dependent manner (Fig. 2C). Treatment with both phosphatases together nearly abolished recognition of 25 ng of ERK2 by the anti-active ERK antiserum (Fig. 2B). In some experiments, three bands corresponding to ERK2 with different electrophoretic mobilities could be resolved after phosphatase treatment: one corresponding to inactive, one to active, and the intermediate to inactive but singly phosphorylated forms. To generate the fastest migrating band, treatment with both phosphatases was required.
Coexpression of Constitutively Active MEKK-C with Wild-type MEK1 or MEK2 Results in MEK1 and MEK2 ActivationWe next employed the
coexpression system to activate the other components of the ERK module,
MEK1 and MEK2 (1). To achieve this, we used the C-terminal
constitutively active fragment of rat MEKK1, MEKK-C, shown previously
to activate MEKs in vitro and in transfected eukaryotic
cells (5, 22, 23). MEKK-C was subcloned into pBB131 and coexpressed
together with histidine-tagged wild-type human MEK1 and MEK2. As shown
in Fig. 3A, neither MEK1 nor MEK2 expressed
in the absence of an activator in bacteria phosphorylated ERK2 in
vitro. However, coexpression of MEK1 or MEK2 with MEKK-C greatly
increased the capacity of bacterial lysates containing them to
phosphorylate ERK2. Activated forms of MEK1 and MEK2 with reduced
electrophoretic mobility were detected on Western blots probed with
specific anti-MEK1 and anti-MEK2 antisera (Fig. 3A,
lower panel). Treatment with PP2A alone, but not with PP2A
pretreated with okadaic acid, resulted in the disappearance of the
upper band (Fig. 3A, lower panel).
The specific activities of coexpressed, histidine-tagged MEK1 and MEK2
after purification on Ni2+-NTA-agarose were in the range of
3-10 nmol/min/mg in three different MEK preparations (Fig.
4); however, MEKs purified by this single step are
relatively impure (20% or less, data not shown). Nevertheless, we were
able to achieve an average of 20-fold activation of MEK1 and 150-fold
activation of MEK2 using our coexpression system (Fig. 4).
Coexpression of Constitutively Active MEKK-C with Wild-type MEK3 and MEK4 Results in MEK3 and MEK4 Activation
MEK3 and MEK4 belong
to different MAP kinase modules (Fig. 1A) and are activated
by different stimuli (7, 8). Because MEKK1 can activate these other
modules after cotransfection into eukaryotic cells (5, 23, 24), we
wished to determine if MEK3 and MEK4 can be activated in E. coli by coexpression with MEKK-C. Histidine-tagged MEK3 and
GST-MEK4 were purified from bacterial cells expressing these kinases
together with MEKK-C and assayed for their ability to phosphorylate
SAPK and p38 MAP kinases. Coexpression of MEKK-C with MEK3 results
in the appearance of an activity capable of phosphorylating p38 MAP
kinase, a MEK3 substrate. Coexpression of MEK4 with MEKK-C results in
the appearance of an activity that phosphorylates both p38 MAP kinase
and SAPK
(Fig. 3B, upper panel). MEK3 and MEK4
expressed alone had little ability to phosphorylate p38 or SAPK
.
After purification of coexpressed MEK3 or MEK4 on Ni2+-NTA-agarose and glutathione-Sepharose, respectively, Western blotting with specific antibodies revealed forms with multiple electrophoretic mobilities (Fig. 3B, lower panel). Multiple forms, corresponding to low activity and activated MEK3, were also detected by Coomassie Blue staining of gels (data not shown). The expression of MEK3 and MEK4 with MEKK-C results in activation of approximately half of each kinase as deduced from their distribution between faster and slower migrating bands.
The specific activity of MEK3 after coexpression was lower than that of MEK4 using p38 as a substrate, despite the fact that in our protein preparations the low electrophoretic mobility, active form of MEK3 represents approximately 25% of the total protein, while active MEK4 is much less pure. We have observed equivalent differences in activity of MEK3 and MEK4 activated in vitro by a novel MEKK.2
Triple Coexpression of MEKK-C with MEKs and Downstream MAP Kinase Results in Activation of the MAP KinaseThe coexpression studies above suggested that each MAP kinase module component can be activated in bacteria by coexpression with a constitutively active upstream module component. Thus, we wished to determine if three components could be expressed simultaneously, leading to activation of the downstream enzymes. The wild-type forms of MAP kinases from three different modules were subcloned with an appropriate MEK on the same high copy plasmid in "head to tail" orientation under the control of the T7 promoter (Fig. 1B). As an activator for all three modules, we used MEKK-C expressed from the low-copy pBB131 plasmid, used previously for activation of MEK family members.
The expression of three kinases together resulted in activation of the
downstream kinase in the corresponding MAP kinase module, namely
-SAPK (Fig. 5A) and p38 (Fig.
5B). Expression of ERK2 with wild-type MEK1 or MEK2 and
MEKK-C also resulted in ERK2 activation (data not shown). The specific
activity of
-SAPK toward GST-c-Jun
1-223 from this coexpression
system and purified on Ni2+-NTA-agarose and then Mono Q
varied from 1 to 1.2 µmol/min/mg protein (Table I).
The activity of p38 with GST-ATF2
(1-254) as substrate varied from
0.6 to 0.9 µmol/min/mg protein. Expression of wild-type MEK1 or MEK2
with ERK2, MEK4 with
-SAPK, or MEK4 with p38 but without MEKK-C
resulted in little or no activation of downstream MAP kinases (data not
shown).
A, specific activity of -SAPK
expressed alone or coexpressed with MEK4 and MEKK-C, before and after
treatment with protein phosphatases. Purified
-SAPK (1.6 µg/ml)
expressed in E. coli alone or coexpressed with MEK4 and
MEKK-C was treated with the indicated protein phosphatases with or
without pretreatment with corresponding phosphatase inhibitors. 3 ng of SAPK were assayed with
GST-c-Jun
1-223 (40 µg/ml). Control experiments showed that both
protein phosphatases were completely inhibited and had no activity in
the GST-c-Jun phosphorylation assay (data not shown). A representative
experiment is shown. B, specific activity of p38 MAP kinase
expressed alone or coexpressed with MEK4 and MEKK-C after protein
phosphatase treatment. Purified p38 MAPK (1.9 µg/ml) expressed in
E. coli alone or coexpressed with MEK4 and MEKK-C was
treated with protein phosphatases as indicated. ~3 ng were assayed
with GST-ATF2
1-254 (0.18 mg/ml). A representative experiment of two
is shown. C, immunoreactivity of coexpressed
-SAPK with anti-phospho-SAPK antibody after protein phosphatase treatment. Purified
-SAPK (1.6 µg/ml) expressed in E. coli alone
or coexpressed with MEK4 and MEKK-C was treated with protein
phosphatases as indicated. An equivalent amount of
-SAPK (20 ng/lane) was probed with an antibody specific to phosphorylated SAPK
isoforms. Bottom, the same blot was stripped and reprobed
with anti-SAPK antibody O977. Shown here is a representative of two
experiments.
|
To demonstrate that -SAPK and p38 expressed in this system are
doubly phosphorylated on Tyr and Thr, we treated the purified active
kinases with PTP1 or PP2A. Treatment with either phosphatase results in
a time-dependent loss of
-SAPK (Fig. 5A) and
p38 activity (Fig. 5B) toward their substrates. Inactivation
was blocked by the phosphatase inhibitors (Fig. 5, A and
B).
To characterize the coexpressed -SAPK protein further, we used an
antiserum raised against a doubly phosphorylated peptide from the
JNK/SAPK phosphorylation lip sequence encompassing phosphothreonine and
phosphotyrosine residues that are required for their activation (7, 8).
This antibody did not recognize 20 ng of
-SAPK expressed in the
absence of activators (Fig. 5C, last lane). In contrast, if
-SAPK was coexpressed with MEK4 and MEKK-C, 7 ng of it
were recognized easily by this antibody (Fig. 5C,
lanes 1, 8, 16, and data not shown).
Treatment with PTP1 or PP2A greatly reduced
-SAPK recognition in a
time-dependent manner (Fig. 5C), although
treatment with PP2A did not eliminate
-SAPK immunoreactivity. Treatment with both phosphatases together completely abolished recognition of 20 ng of
-SAPK by the anti-active JNK/SAPK antiserum (Fig. 5C). Upon SDS-PAGE, the coexpressed kinase
preparations each contained major silver-stained bands that correspond
to their activated forms (not shown).
We describe the reconstitution of three different MAP kinase cascades in bacteria. The MEK and MAP kinase family members were activated by coexpression with MEKK-C or MEK1 R4F as the upstream activator. In both the two- and three-component systems, we observed a high degree of ERK2, JNK/SAPK, or p38 activation, concomitant with the conversion of the expressed kinases into the doubly phosphorylated forms. Thus, we demonstrate for the first time that bacteria can efficiently phosphorylate and activate expressed proteins if suitable modifying enzymes are present. This method has wide applicability in the production of significant quantities of selectively modified phosphoproteins.
One of the applications of the coexpression system described above is
the production of milligram amounts of active MAP kinases and MEKs for
biochemical and physical studies. As one example, we have recently
optimized this system for production of large amounts of phosphorylated
ERK2 that has led to the determination of its crystal structure. The
capacity to activate MAP kinase family members by expressing three
components of the kinase cascade simultaneously offers a significant
advance in efforts to obtain large quantities of downstream MAP kinases
even when a constitutively active MEK is not available. Using this
approach, we found that the specific activities of activated ERK2,
-SAPK, and p38 are similar, ~1-2 µmol/min/mg protein.
MEKs 1-4, which belong to the most extensively characterized MAP kinase modules, are activated by MEKK-C despite its low expression in E. coli. We were able to detect expressed MEKK-C after purification of GST-tagged or His6-tagged protein only on Western blots or by assay, not by protein staining. Although these findings suggest little selectivity of the catalytic domain of MEKK1 among MEK family members, we found that MEK5, a MEK from a different kinase module, cannot be activated by MEKK-C either in vitro or by coexpression in E. coli (12).3 Thus, MEKK1 has a restricted ability to phosphorylate MEK family members.
When we engineered bacteria to express three kinase cascade members simultaneously, we observed strong activation of the downstream enzyme in each MAP kinase module. This finding indicates that activation of downstream components of MAP kinase cascades does not require other components. Because MEKK1 phosphorylates several MEKs, the function of MEKK1 in a given context may not be due to its inherent enzymatic specificity. Perhaps components that exist only in mammalian cells, such as scaffolding proteins analogous to Ste5p found in the budding yeast S. cerevisiae, may confer specificity within a cellular context (25, 26). Specificity might also be achieved through other means of cascade compartmentalization. Indeed, we have found alternatively spliced forms of MEK5, which have different intracellular localizations that may lead to regulatory differences (12).
We thank Clark Garcia and Amanda Weitz for preparation of some of the bacterial proteins, Megan Robinson for critical reading of the text and instructions concerning preparation of figures, and Jo Hicks for preparation of the manuscript.