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(Received for publication, November 28, 1995; and in revised form, December 19, 1995) From the
To discern MEK1 and MEK2 specificity for their substrate,
extracellular signal-regulated kinase (ERK), site-directed mutagenesis
was performed on the amino acid residues flanking the regulatory
phosphorylation sites of ERK1. These ERK1 mutants were analyzed for the
ability to act as a substrate for MEK1 and MEK2. Based on both
phosphorylation and activation analyses, the mutants could be divided
into four classes: 1) dramatically decreased phosphorylation and
activation, 2) enhanced basal kinase activity, 3) preferentially
enhanced phosphorylation of tyrosine and decreased phosphorylation of
threonine, and 4) increased threonine phosphorylation with an increase
in activation. In general, the residues proximal to the regulatory
phosphorylation sites of ERK1 had greater influence on both
phosphorylation and activation. This is consistent with the highly
specific recognition of the ERK1 regulatory sites by MEK. Mutation of
Arg-208 or Thr-207 to an alanine residue significantly altered the
relative phosphorylation on Thr-202 and Tyr-204. The Arg-208 to alanine
mutant increased the phosphorylation of Tyr-204 approximately 4-fold
yet almost completely eliminated the phosphorylation on Thr-202. In
contrast, mutation of Gly-199 to alanine resulted in an increased
phosphorylation of Thr-202 relative to Tyr-204. This suggests that both
Gly-199 and Arg-208 play important roles in determining the relative
phosphorylation of Thr-202 and Tyr-204. Our results demonstrate that
residues in the phosphorylation lip of ERK play an important role in
the recognition and phosphorylation by MEK.
Mitogen-activated protein kinases (MAPKs) ( MAPKs are also
referred to as extracellular signal-regulated kinases (ERKs). The
upstream activator of ERK is a dual specific kinase called MAPK or ERK
kinase (MEK), which phosphorylates ERK2 on both threonine 183 and
tyrosine 185(15) . Mammalian isoforms of MEK, two of which
(MEK1 and MEK2) can phosphorylate and activate ERK, were identified by
molecular cloning
techniques(18, 19, 20, 21, 22, 23, 24) .
ERK is the only known substrate for MEK, with all other proteins tested
thus far being phosphorylated at least 1000-fold less efficiently by
MEK(25) . MEK is activated by a number of different
serine/threonine kinases including the proteins encoded by the
proto-oncogene c-raf(26, 27, 28, 29) . In fact, the
regulatory phosphorylation sites in MEK were mapped using
immunoprecipitated Raf from epidermal growth factor-stimulated Swiss
3T3 cells(30) . Activation was shown to occur through
phosphorylation of 2 serine residues at positions 218 and 222 of
MEK1(30) . Constitutively active MEK1 can be obtained by
mutation of the regulatory phosphorylation sites to glutamic
acid(31, 32) . Alternatively, if these sites are
mutated to alanine (S218A/S222A) the resulting MEK mutant is incapable
of being activated by Raf(30) . Phosphorylation and activation
of MEK occur in response to the same mitogenic stimuli leading to ERK
activation(2, 6, 33) , again providing
indirect evidence of MEK's substrate specificity for ERK. The
crystal structure of the unphosphorylated form of ERK2 reveals that
Thr-183 and Tyr-185 are contained within a loop structure(34) .
In this inactivated form, Tyr-185 is buried in a hydrophobic pocket and
Thr-183 is exposed to the surface of the molecule. Since the active
form of ERK2 is phosphorylated on both Tyr-185 and Thr-183, ERK2 must
undergo a conformational change in this loop structure upon association
with MEK. Although the exact mechanism of the MEK/ERK interaction is
unknown it is highly likely that some feature of the looped structure
is important for determining MEK specificity. Thus, it is quite
probable that the amino acid residues contained within this
phosphorylation lip contribute to both the conformational mobility of
ERK and the specificity of ERK recognition by MEK. In this study, we
have performed numerous site-directed mutations in both regions
flanking the regulatory phosphorylation sites of ERK1 to further define
MEK's specificity for its substrate, ERK. The ERK mutants were
tested for differences in phosphorylation and activation by GST-MEK1 or
GST-MEK2. Results in this paper indicate that four classes of mutants
have been obtained: the first exhibits dramatically decreased
phosphorylation and activation, the second shows enhanced basal kinase
activity of ERK, the third exhibits preferentially enhanced
phosphorylation of tyrosine and substantially decreased phosphorylation
of threonine, and the fourth revealed an increase in threonine
phosphorylation and an increase in activation. Results in this paper
further indicate that the specificity of MEK1 and MEK2 for their
substrate ERK1 is rendered, in part, by the amino acid residues
flanking the regulatory phosphorylation sites of ERK1. These flanking
residues not only influence the overall effectiveness of ERK1 as a
substrate of MEK1 and MEK2 but also affect the relative phosphorylation
levels of Thr-202 versus Tyr-204.
Purification of MEKs was performed as
stated above for the ERKs with the exception of the elimination of the
thrombin cleavage procedure. Instead, GST-MEKs were eluted from the
glutathione-agarose with 6 ml of Buffer C containing 5 mM glutathione. Samples were dialyzed with two exchanges of Buffer C.
GST-MEKs were concentrated by a Centricon 30 according to the
manufacturer's instructions. Purified proteins were frozen at
-80 °C in 10% glycerol, 1 mM phenylmethylsulfonyl
fluoride, and 5 mM EDTA (pH 8.0).
ERK is activated by a dual specific kinase (MEK) by
phosphorylation on specific threonine and tyrosine residues. The native
form of ERK is the only substrate identified thus far for
MEK(21) . This is based on the inability of MEK to
phosphorylate various proteins or peptides known to act as substrates
for either protein serine/threonine kinases or protein tyrosine
kinases. Furthermore, two-dimensional phosphopeptide mapping in
combination with mass spectrometry techniques has shown that, for ERK2,
only the regulatory residues Thr-183 and Tyr-185 are phosphorylated
upon activation by MEK (15) . The portion of the ERK2 sequence
containing the phosphorylation sites is conserved in ERK1 and thus the
same specificity is presumed to hold for residues Thr-202 and Tyr-204
of ERK1. The crystal structure of the unphosphorylated form of ERK2
reveals that Thr-183 and Tyr-185 are contained within a loop
structure(34) . The Tyr-185 is buried in a hydrophobic pocket,
and Thr-183 is exposed to the surface of the molecule. Since ERK2 is
more highly phosphorylated on Tyr-185 it has been suggested that the
protein must undergo both local and global conformational changes in
order for phosphorylation of this residue to occur upon association
with MEK(34) . Thus, it is conceivable that the amino acid
residues flanking the regulatory phosphorylation sites in both ERK1 and
ERK2 may contribute to interactions that are vital for their structural
mobility. Furthermore, it is plausible to speculate that these flanking
residues of the regulatory phosphorylation sites may contribute to the
specificity of ERK activation by MEK. We have undertaken mutation
assays of ERK1 to investigate this hypothesis.
Recombinant MEK proteins were
overexpressed as GST fusion proteins (GST-MEK1 or GST-MEK2) and
purified by glutathione-agarose affinity chromatography for use in ERK
activation studies. Since both MEKs have molecular weights similar to
that of ERK1, MEKs were not cleaved with thrombin but were purified as
GST fusion proteins. This aided in separation on SDS-PAGE, as GST-MEK1
or GST-MEK2 migrated at molecular masses of 69 and 70 kDa,
respectively(37) .
Figure 1:
Diagram of the mutations performed in
human ERK1. Site-directed mutagenesis was performed on the conserved
TEY sequence and both flanking sequences as illustrated.
Kinase-impaired mutants contain the conserved lysine (Lys-71)
substituted by an arginine (ERK1*). The phosphorylation target
threonine and tyrosine residues are denoted by asterisks.
ERK1* mutants were assayed for in vitro phosphorylation by GST-MEK1 or GST-MEK2. In vitro kinase assays were performed on catalytically active ERK1 mutants
to determine whether this activation by GST-MEK1 or GST-MEK2 correlated
with ERK1* phosphorylation. Mutants that revealed interesting
phosphorylation and activation results were further analyzed by
phosphoamino analysis.
Figure 2:
Phosphoamino acid analysis of ERK1*
mutants. Phosphorylated ERK1* mutants were resolved on SDS-PAGE and
transferred to an Immobilon-P membrane.
To determine whether the high substrate
specificity of MEK2 is influenced by residues flanking the
phosphorylation sites of ERK1, each amino acid in both flanking regions
was initially substituted with an alanine residue. In general, residues
in closer proximity to the phosphorylation sites have a more prominent
effect than distal residues (Fig. 3). The only exception to this
was mutant W209A, which had less than 30% phosphorylation. Mutants
L201A and V205A showed less than 50% phosphorylation; residues F200A,
E203Q, and Y204F had less than 25% phosphorylation. Interestingly,
mutation of residue Arg-208 to alanine dramatically increased the
phosphorylation of ERK1* by MEK2 approximately 4-fold. These results
indicated that residues flanking Thr-202 and Tyr-204 have a significant
influence on the ability of ERK1 to be phosphorylated by MEK2. However,
to determine the specific features of this flanking region important
for substrate recognition, further mutation analyses were performed on
selected residues.
Figure 3:
In vitro phosphorylation of ERK1*
mutants in the presence of GST-MEK2. Catalytically impaired ERK1*
mutants (0.3 µg) and GST-MEK2 (0.3 µg) in buffer containing 18
mM Hepes (pH 7.5), 20 µM ATP, and 10 mM magnesium acetate were incubated for 30 min at 30 °C in the
presence of [
Mutation of the glutamic acid at position 203 to
a glutamine (E203Q) was performed to determine what effect the negative
charge at this position has on the ability of MEK to phosphorylate
human ERK1. In addition, residue Glu-203 was mutated to an aspartic
acid (E203D) to determine the effects of side chain length. ERK1*E203Q
exhibited a 4-fold decrease in the level of phosphorylation, and even
the more conservative mutation, E203D, showed a similar 4-fold decrease
in phosphorylation. The dramatic effect of this relatively minor change
would seem to indicate a pivotal role for Glu-203 in ERK1
phosphorylation beyond simply providing a negatively charged center.
This central amino acid of the dual phosphorylation site motif is not
fully conserved among the MAPK family members. In the ERKs and yeast Saccharomyces cerevisiae proteins, encoded by the genes KSS1, FUS3, and MPK1, the motif is TEY (40, 41) . However, this site differs in the
stress-activated MAPKs, HOG1 and p38 (TGY) and JNK (TPY) (42, 43, 44) . With the
regulatory phosphorylation sites being invariant among the MAPK family
members, it is of interest that the residue in between these two sites
is not also highly conserved. It may be that this residue is at least
partly responsible for conferring specificity for the various MEK
isoforms. Due to the dramatic 4-fold decrease in the phosphorylation
level of ERK1*F200A it was decided to make an additional conservative
mutation of Phe-200 to a tyrosine in order to assess any role of the
aromatic side chain in this position. The ERK1*F200Y mutant also
exhibited a 3-fold decrease in the level of phosphorylation. The role
of Phe-200 is also unclear, but it may be involved in the
conformational integrity of ERK1 enabling Thr-202 and Tyr-204 to be
accessible for phosphorylation by MEK.
As expected, mutation of either
Thr-202 or Tyr-204 eliminated the activation of ERK1 by MEK2. The
phosphorylation level of T202S was nearly equal to that of ERK1*;
however, it was not an effective substrate for activation by MEK2.
These results were consistent with the 6-fold higher level of Tyr-204 versus Thr-202 phosphorylation in wild type ERK1. That is,
threonine phosphorylation represents only about 14% of the total ERK1
phosphorylation and will therefore have little effect on the overall
phosphorylation level of the protein. However, as phosphorylation of
both Thr-202 and Tyr-204 is necessary for activation, this lack of
threonine phosphorylation results in a marked decrease in activation
for the T202S mutant protein. The comparable substitutions of Ser-218
and Ser-222 of MEK1 by threonine did not eliminate phosphorylation of
MEK1 by Raf, indicating that MEK has a higher substrate specificity
than Raf(30) . In general, mutants revealed significantly
decreased activation by MEK2 in correlation with decreased
phosphorylation in vitro (Fig. 4). This attenuated
activation was also generally more severe than the phosphorylation
decrease. This may be explained by the fact that ERK must adopt an
altered conformation for MEK to bind, leading to subsequent
phosphorylation of both tyrosine and threonine. Thus, it may be
possible that even though phosphorylation was detected, indicating
interaction with MEK, the ERK1 mutants may not be able to adopt some
optimal conformation necessary for its full activity. However, some
notable exceptions were observed as described below.
Figure 4:
Activation of ERK1 mutants by GST-MEK2. To
activate ERK1, recombinant GST-MEK2 was incubated for 20 min at 30
°C in a 30-µl reaction volume containing 18 mM Hepes
(pH 7.5), 20 µM ATP, and 10 mM magnesium acetate.
To initiate the kinase reaction, myelin basic protein and
[
Figure 5:
Increased threonine phosphorylation in
ERK1*G199A and decreased threonine phosphorylation in ERK1*T207A and
ERK1*R208A mutants. Phosphorylated ERK1* mutants were resolved on
SDS-PAGE and transferred to an Immobilon-P membrane.
To determine whether
mutations at the phosphorylation sites and flanking residues of ERK1
have the same effect on both MEK1 and MEK2, ERK1 mutants were assayed
for phosphorylation and activation by MEK1 as described previously for
MEK2. The phosphorylation results obtained were similar to those for
MEK2 (Fig. 6); mutation of the flanking residues generally
decreased the phosphorylation of ERK1* by MEK1. These data suggest that
a common sequence motif in ERK is recognized by both MEK1 and MEK2;
however, noticeable differences were observed. For example, the
ERK1*R208A mutant showed an increase in phosphorylation by MEK2 of
4-fold as compared with 2-fold for MEK1.
Figure 6:
In vitro phosphorylation of ERK1*
mutants in the presence of GST-MEK1. Catalytically impaired ERK1*
mutants (0.3 µg) and GST-MEK1 (0.3 µg) in buffer containing 18
mM Hepes (pH 7.5), 20 µM ATP, and 10 mM magnesium acetate were incubated for 30 min at 30 °C in the
presence of [
Activation of ERK1 mutants
by MEK1 was also comparable with that by MEK2 (Fig. 7). Mutation
of flanking residues dramatically reduced the activation potential of
ERK1 by both MEK1 and MEK2. Among all the mutants, G199A was the only
one to exhibit less than a 70% decrease in activation by MEK1.
Figure 7:
Activation of ERK1 mutants by GST-MEK1. To
activate ERK1, recombinant GST-MEK1 was incubated for 20 min at 30
°C in a 30-µl reaction volume containing 18 mM Hepes
(pH 7.5), 20 µM ATP, and 10 mM magnesium acetate.
To initiate the kinase reaction myelin basic protein and
[
Activation of MAPKs is a common event in many signal transduction
pathways. At least six identified pathways in the budding yeast S.
cerevisiae utilize different MAPK family members for their signal
transduction(50) . These pathways have very unique functions,
which include the mating pheromone response, osmolarity regulation,
cell wall construction, pseudohyphal development and spore formation in
diploid strains, and invasiveness in haploid strains(50) . The
number of MAPKs and their upstream activators involved in such a
variety of vital functions of the organism raises important questions
on how the different isoforms of MAPK are regulated. The results of the
mutation experiments presented in this paper provide additional
information toward understanding the mechanisms of MAPK activation and
the selectivity of MEK. Furthermore, these data will provide valuable
insight toward the complete interpretation of the crystal structures of
the tyrosine-phosphorylated and dual phosphorylated MAPK in terms of
the mechanisms of MAPK dual phosphorylation. Understanding the
mechanisms of activation for the different isoforms of MAPK and the
specificity of the various activators will indeed lead to an improved
general understanding of the mechanisms of regulation involved in the
cell signaling pathways in mammalian systems.
Volume 271,
Number 8,
Issue of February 23, 1996 pp. 4230-4235
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)are
important components of various signaling pathways, phosphorylate
numerous substrates, and may be key components in linking growth factor
receptor activation to serine/threonine protein
phosphorylation(1, 2, 3) . MAPK activation
can be induced by numerous mitogenic stimuli including phorbol esters,
cytokines, T-cell antigens, and growth factors with tyrosine kinase
receptors such as insulin, epidermal growth factor, fibroblast growth
factor, and platelet-derived growth
factor(3, 4, 5, 6) . Stimulation of
MAPK in various non-lymphoid cell types leads to a multitude of
cellular responses including phosphorylation of microtubule-associated
proteins involved in microtubule rearrangement (7, 8) and phosphorylation resulting in subsequent
activation of transcription factors TCF and
STAT(9, 10, 11) . In lymphoid cells,
activation of MAPK results in stimulation of T-cells to produce
cytokines(12, 13, 14) . The involvement of
MAPK in such a variety of cells and cellular processes emphasizes a
potentially vital role of MAPK in mediating cellular signal
transduction. One of the most unique features of MAPK is that it must
be phosphorylated on both threonine and tyrosine to exhibit full
enzymatic activity(15) . Dephosphorylation of either
phosphothreonine or phosphotyrosine completely inactivates MAPK,
emphasizing the importance of both residues for
activation(16, 17, 18) .
Materials
Buffers
Buffer A consisted of 20 mM phosphate buffer (pH 7.3) containing 0.15 M NaCl, 1%
Triton X-100 (Sigma), 0.1% 2-mercaptoethanol, and 0.5 mM EDTA.
Buffer B consisted of 50 mM Tris (pH 8.0) containing 0.15 M NaCl, 2.5 mM CaCl
, and 0.1%
2-mercaptoethanol. Buffer C consisted of 25 mM HEPES (pH 8.0)
containing 0.5 mM EDTA and 0.1% 2-mercaptoethanol. Buffer D
consisted of 18 mM HEPES (pH 7.5) containing 10 mM magnesium acetate and 20 µM ATP. Buffer E consisted
of 5.9% (v/v) glacial acetic acid (pH 2.5), 0.8% (v/v) formic acid
(88%), 0.3% (v/v) pyridine, and 0.3 mM EDTA (35) .Methods
Bacterial Purification of GST-MEKs and
ERKs
Cells from a 1-liter culture were pelleted by
centrifugation at 3500 rpm for 20 min. The supernatant was discarded
and the pellets were resuspended in 20 ml of Buffer A. Cells were lysed
by two passes through a French press at 1200 p.s.i. The lysate was
centrifuged at 12,000 rpm for 10 min to pellet the cell debris. The
supernatant was then added to 3 ml of glutathione-agarose beads and
mixed gently on ice for 1 h. The samples were briefly centrifuged at
3000 rpm for 5 min, and the supernatant was discarded. The matrix was
transferred to a 10-ml polypropylene column and washed with 40 ml of
Buffer A. The matrix was then equilibrated with 10 ml of Buffer B.
GST-ERKs were cleaved using the thrombin cleavage method described by
Guan and Dixon(36) . Samples were concentrated by a Centricon
30 (Amicon, Inc.) according to the manufacturer's instructions.
Purified proteins were frozen at -80 °C in a final
concentration of 10% glycerol, 1 mM phenylmethylsulfonyl
fluoride, and 5 mM EDTA (pH 8.0). Proteins were separated on
10% SDS-PAGE and quantitated by a densitometric scanner using bovine
serum albumin as a standard.Site-directed Mutagenesis of ERK1
Human ERK1
plasmid was a generous gift from Dr. S. Pelech, University of British
Columbia. The entire 5`-coding region of human ERK1 to the BglII site was cloned in the pAlter-1 vector (Promega). The
mutagenesis was performed according to the manufacturer's
instructions using synthetic oligonucleotides that contain the mutated
bases. Mutations were confirmed by Sanger dideoxy DNA
sequencing(51) . The mutated human ERK1s were then subcloned
into pGEX-2T vector in frame with GST (36) .MEK Expression Vector
BamHI sites were
introduced into the 5`- and 3`-ends of the coding sequence of MEK by
polymerase chain reaction(24) . These polymerase chain reaction
products were then subcloned, in frame with GST, into the BamHI site of pGEX-KG(24) .In Vitro Phosphorylation Assays
Human ERK1 mutants
used in the phosphorylation assays contained the conserved lysine
(Lys-71) mutated to an arginine residue. Human ERK1 (0.3 µg)
mutants were incubated with 0.3 µg of either GST-MEK1 or GST-MEK2
(0.3 µg) in the presence of Buffer D and
[-
P]ATP (5000 cpm/pmol). Samples were
incubated at 30 °C for 30 min. Reactions were terminated by
addition of SDS sample buffer and separated by 10% SDS-PAGE. The gel
was then transferred to Immobilon-P and visualized by autoradiography.
Bands were quantitated by a Molecular Dynamics, Inc. PhosphorImager.
In Vitro Activation Assays
The activation of ERK1
was performed by incubation with recombinant GST-MEK for 20 min at 30
°C in a 30-µl reaction volume containing 18 mM HEPES
(pH 7.5), 20 µM ATP, and 10 mM magnesium acetate.
To initiate the kinase reaction, myelin basic protein (20 µg) and
[-
P]ATP (5000 cpm/pmol) were added in the
same reaction buffer and incubated for an additional 30 min at 30
°C. Half of the reaction volume (20 µl) was applied to P81
phosphocellulose filters. The filters were washed with 180 mM phosphoric acid (5 times) and rinsed with 95% ethanol. Filters
were quantitated by liquid scintillation counting.
Phosphoamino Acid Analysis
Phosphorylated ERK1
mutants were resolved on 10% SDS-PAGE and transferred to an Immobilon-P
membrane. P incorporated bands were excised and hydrolyzed
in 6 N HCl for 2 h at 110 °C. Samples were dried and
washed in distilled water (2
1 ml) and dissolved in 10 µl
of distilled water. Phosphoamino acid analysis was performed by
separation in Buffer E using one-dimensional electrophoresis on
cellulose plates (Eastman Kodak Co.) and visualized by autoradiography.
Amino acids were quantitated by a Molecular Dynamics, Inc.
PhosphorImager.
Expression and Purification of Recombinant Human ERK1
Mutants
Recombinant ERK1 proteins were overexpressed as GST
fusion proteins and purified by glutathione-agarose affinity
chromatography(36) . Purified ERK1 mutants were then cleaved
with thrombin as stated in the experimental procedures. Upon analysis
by SDS-PAGE, all 32 ERK1 mutants were approximately 85% pure and
migrated as a doublet at 44 kDa (data not shown). The amount of
purified protein obtained from a 1-liter culture was approximately
1-5 mg for all ERK1 mutants.Mutational Analysis of Human ERK1
In an effort to
discern the substrate specificity of MEK for ERK1, in vitro site-directed mutagenesis studies were performed on the conserved
``TEY'' sequence and both amino acid flanking regions of
ERK1, residues 197-209 (Fig. 1). Mutational analyses
initially concentrated on these regions surrounding the regulatory
phosphorylation sites of ERK1 due to their probable role in imparting
MEK substrate specificity (21, 37) and the inferred
ability of this part of the molecule to form a conformationally mobile
``looped structure''(34) . To investigate this
specificity, mutations were performed on both wild type kinase-active
ERK1 and the catalytically impaired enzyme (ERK1*) made by mutation of
the conserved lysine (Lys-71) to an arginine. Residue Lys-71 is located
in subdomain II and is known to be essential for kinase
activity(38, 39) .
Phosphorylation of ERK1* in the Presence of
GST-MEK2
Phosphorylation sites Thr-202 and Tyr-204 were
initially substituted with residues incapable of being phosphorylated.
As expected, in vitro phosphorylation by GST-MEK2 followed by
phosphoamino acid analysis of mutants ERK1*T202A (data not shown) and
ERK1*Y204F (Fig. 2) revealed that MEK2 only phosphorylated the
ERK1 mutants on tyrosine or threonine, respectively, results that are
consistent with previous observations(25) . This result
demonstrates that phosphorylation of Thr-202 and Tyr-204 can occur
independently. These same two residues were also mutated to serine
residues that could potentially be phosphorylated. However, serine
phosphorylation was not observed for either ERK1*T202S or ERK1*Y204S (Fig. 2). This inability to phosphorylate serine residues at the
regulatory sites further illustrates the extremely high specificity for
substrate recognition of MEK. These data demonstrate that in order for
phosphorylation by MEK to occur, residues 202 and 204 of ERK1 must be
Thr and Tyr, respectively.
P incorporated
bands were excised and hydrolyzed in 6 N HCl for 2 h at 110
°C. Phosphoamino acid analysis was performed by separation using
one-dimensional electrophoresis on cellulose plates (Kodak) and
visualized by autoradiography. Phosphothreonine, phosphotyrosine,
phosphoserine, origin, and unincorporated phosphate are indicated by pT, pY, pS, O, and Pi,
respectively. Lanes correspond to the following proteins: lane 1, ERK1*; lane 2, ERK1*Y204S; lane 3,
ERK1*T202S; and lane 4, ERK1*Y204F. The weak spot between
P
and phosphotyrosine in ERK1*T202S did not co-migrate with
the phosphoserine standard.
-
P]ATP. Samples were analyzed
by SDS-PAGE, transferred to Immobilon-P, and visualized by
autoradiography. Bands were quantitated by a Molecular Dynamics, Inc.
PhosphorImager. The percent of phosphorylation for ERK1*R208A was 406
± 131. Error bars denote standard deviations of two to
four experiments.
Activation Assays of Wild Type Kinase-active ERK1 Mutants
by MEK2
All but three mutants had basal kinase activities for
phosphorylation of myelin basic protein comparable with wild type
kinase-active ERK1 (data not shown). Mutants T198A and F200Y exhibited
increased basal kinase activities of 8- and 2-fold, respectively. Thus,
the activation of T198A mutant may be underestimated because of the
increase in basal kinase activity. This observation was of interest
since a constitutively active form of ERK has not been reported but
would be extremely useful to study the roles of the different ERK
isoforms in signal transduction. Unfortunately, neither of these
mutants had sufficiently high basal activity to be considered
constitutively active. In addition, a double mutation of T198A and
F200Y did not exhibit an increase in basal activity in vitro compared with either Thr-198 or Phe-200 mutation alone (data not
shown). However, it would be of interest to determine whether any of
the mutants that showed an increase in basal activity in vitro could function as a constitutively active mutant in the
transformation of cultured cells. An additional mutant, W209A, had no
detectable basal activity. Therefore, this residue appears to be
crucial to either the structural or mechanistic integrity of ERK1,
regardless of MEK activation.
-
P]ATP (5000 cpm/pmol) were added in the
same reaction buffer and incubated for an additional 30 min at 30
°C. Half of the reaction volume (20 µl) was applied to P81
phosphocellulose filters. The filters were washed with 180 mM phosphoric acid (5 times) and rinsed with 95% ethanol. Filters
were quantitated by liquid scintillation counting. Each sample was
performed in duplicate; error bars denote the standard
deviation of two to four experiments.
Mutation of Glycine 199 Enhances the Phosphorylation of
Threonine
One mutant, G199A, actually showed an increased
activation of nearly 2-fold. This is in contrast to the slight decrease
in phosphorylation observed for the corresponding ERK1*G199A mutant.
Phosphoamino acid analysis of ERK1*G199A revealed an increase in the
level of threonine phosphorylation relative to tyrosine (1:3 versus 1:6 in wild type, Fig. 5A). This may explain the
overall increase in activation since phosphorylation of both threonine
and tyrosine on a single molecule is necessary for full enzymatic
activity of ERK(21, 45, 46) . In wild type
ERK, the limiting factor for activation is threonine phosphorylation of
the same molecule containing a phosphorylated tyrosine residue.
Therefore, by increasing the level of threonine phosphorylation, a
larger number of molecules will be phosphorylated on both threonine and
tyrosine residues. That is, although the overall level of
phosphorylation decreased, the larger proportion of diphosphorylated
molecules results in an overall increase in ERK kinase activity.
P
incorporated bands were excised and hydrolyzed in 6 N HCl for
2 h at 110 °C. Phosphoamino acid analysis was performed by
separation using one-dimensional electrophoresis on cellulose plates
(Kodak) and visualized by autoradiography. Phosphothreonine,
phosphotyrosine, phosphoserine, origin and unincorporated phosphate are
indicated by pT, pY, pS, O, and Pi, respectively. Lanes correspond to the following
proteins: A) lane 1, ERK1*G199A, lane 2,
ERK1*; and B) lane 1, ERK1*, lane 2,
ERK1*T207A, and lane 3, ERK1*R208A. Similar results were
obtained in duplicate experiments.
Mutation of Threonine 207 or Arginine 208 Decreases the
Phosphorylation of Threonine 202 but Increases Phosphorylation on
Tyrosine 204
One of the most perplexing results was obtained
with the R208A mutant, which showed a 400% increase in the level of
phosphorylation, yet the kinase activation was almost completely
abolished ( Fig. 3and Fig. 4). Similar results were
observed with the T207A mutant, which had only a slight decrease in
total phosphorylation while less than 2% activation was observed.
Phosphoamino acid analysis of these mutants was performed to determine
the molecular basis for this lack of correlation between
phosphorylation and activation. Surprisingly, mutant R208A was
exclusively phosphorylated on tyrosine, while threonine phosphorylation
was not observed (Fig. 5B). Similarly, mutant T207A
retained only residual threonine phosphorylation, while the ratio of
phosphothreonine to phosphotyrosine was increased to 1:50 versus 1:6 for ERK1* (Fig. 5B). These observations
demonstrate that Thr-207 and Arg-208 play a critical role in the
recognition of Thr-202 of ERK1 by MEK. The drastic effects on
phosphorylation or basal kinase activity of distal residues Thr-207,
Arg-208, and Trp-209 suggest an important function for this region in
the mechanism of ERK activation.Effect of ERK1 Mutations on Phosphorylation and
Activation by MEK1
MEK1 and MEK2 are two closely related kinases
that have been identified as ERK
activators(21, 45, 46) . The functional
difference between MEK1 and MEK2 is unclear, although MEK2 shows higher
basal activity toward both ERK1 and ERK2(37) . These
differences were also detected in interactions with Raf. MEK1, but not
MEK2, associated with c-Raf(47) . Disruption of this MEK1-Raf
complex reduced activation of MEK1 by Raf family members in both in
vitro and in vivo studies(48) . A unique
proline-rich sequence has been identified and shown to be important for
this MEK1 and Raf interaction (48) . Differences in MEK
activation by A-Raf were also detected (49) .
Immunoprecipitated A-Raf preferentially activated MEK1 versus MEK2(49) . Thus, a significant component of specificity of
the ERK isoforms for various signaling pathways may be contributed by
differences in their upstream activators, MEKs.
-
P]ATP. Samples were analyzed
by SDS-PAGE, transferred to Immobilon-P, and visualized by
autoradiography. Bands were quantitated by a Molecular Dynamics, Inc.
PhosphorImager. The percent of phosphorylation for ERK1*R208A was 248
± 79. Error bars denote standard deviations of two to
eight experiments.
-
P]ATP (5000 cpm/pmol) were added in the
same reaction buffer and incubated for an additional 30 min at 30
°C. Half of the reaction volume (20 µl) was applied to P81
phosphocellulose filters. The filters were washed with 180 mM phosphoric acid (5 times) and rinsed with 95% ethanol. Filters
were quantitated by liquid scintillation counting. Each sample was
performed in duplicate; error bars denote the standard
deviation of two to three experiments.
)
We thank Dr. S. Pelech (University of British
Columbia) for the human ERK1 cDNA, Dr. C.-F. Zheng for the MEK
expression constructs, and Dr. M. Uhler for helpful suggestions.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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