Phosphorylation of MAP Kinases by MAP/ERK Involves Multiple
Regions of MAP Kinases*
Julie L.
Wilsbacher
§,
Elizabeth J.
Goldsmith¶, and
Melanie H.
Cobb
From the Departments of
Pharmacology and
¶ Biochemistry, The University of Texas Southwestern
Medical Center, Dallas, Texas 75235-9041
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ABSTRACT |
Mitogen-activated protein (MAP) kinases are
activated with great specificity by MAP/ERK kinases (MEKs). The basis
for the specific activation is not understood. In this study chimeras composed of two MAP kinases, extracellular signal-regulated protein kinase 2 and p38, were assayed in vitro for phosphorylation
and activation by different MEK isoforms to probe the requirements for
productive interaction of MAP kinases with MEKs. Experimental results
and modeling support the conclusion that the specificity of MEK/MAP
kinase phosphorylation results from multiple contacts, including
surfaces in both the N- and C-terminal domains.
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INTRODUCTION |
Mitogen-activated protein
(MAP)1 kinase or
extracellular signal-regulated protein kinase (ERK) cascades are
present in all eukaryotes and are utilized in almost all signal
transduction pathways originating from receptors at the cell surface
(1, 2). A plethora of different stimuli, including growth factors, cytokines, heat shock, and ultraviolet light, can initiate signaling through these cascades. Each cascade consists of a three kinase module:
a MAP kinase, a MAP kinase/ERK kinase (MEK) that activates the MAP
kinase, and a MEK kinase (MEKK) that activates the MEK (3). The MAP
kinase in each cascade preferentially phosphorylates substrates with a
serine or threonine followed by a proline. There are three mammalian
MAP kinase modules that have been extensively studied. These include
the ERK1/2 module, the c-Jun N-terminal protein kinase/stress-activated
protein kinase module, and the p38 module. ERK3, ERK4, and ERK5 and
other p38 isoforms have also been identified, but the cascades leading
to activation of these kinases are not well characterized (4-11).
Like other protein kinases, the MAP kinases are folded into two domains
(12). The smaller N-terminal domain is composed mostly of
strands,
whereas the C-terminal domain is made up of
helices. ATP binds
between the two domains, and protein substrate is believed to bind on
the surface of the C-terminal domain. Alignment of the amino acid
sequences of many protein kinases reveals a common core catalytic
domain of 250-300 residues encoding the two-domain structure (13).
Protein kinases possess 12 conserved stretches of amino acids within
their catalytic domains known as subdomains (13-15). These conserved
elements as well as unique structures contribute to catalysis by and
regulation of protein kinases. The functions of several of the
conserved and unique structural motifs are known. A glycine-rich loop
in the N-terminal domain, termed the phosphate anchor ribbon, has a
role in binding ATP. Also in the N-terminal domain, subdomain III
encodes the C helix, which contains an invariant Glu involved in
binding MgATP (16, 17). This helix is important for maintaining an open domain conformation in unphosphorylated ERK2 (18), aligning catalytic
residues in Src (19) and Cdk2 (20), and binding to the cyclin
regulatory subunit in Cdk2 (21). The activation loop, known as the
phosphorylation lip in MAP kinases, is a poorly conserved element in
the C-terminal domain and contains the two MAP kinase phosphorylation
site residues within a TXY sequence. Also in the C-terminal
domain is the MAP kinase insert. It is composed of 32 residues and is
only found in the MAP kinases and the Cdks. The MAP kinase insert and a
loop consisting of residues 199-205 of ERK2 interact with the
phosphorylation lip in the unphosphorylated form of ERK2. Finally, a
loop at the C terminus of the MAP kinases, L16 or the C-terminal tail,
wraps around the back of the structure and interacts with the
N-terminal domain (18).
The crystal structures of unphosphorylated and phosphorylated ERK2 and
unphosphorylated p38 have been solved (18, 22-24). Several important
differences exist between the structures of the unphosphorylated forms
of ERK2 and p38. There is a wider domain separation in p38 than in
ERK2. The phosphorylation lip in p38 is six residues shorter and has a
different conformation. In p38 the lip is folded up between the two
domains, and the C-terminal portion of the lip forms a turn of helix
that blocks the P+1 specificity pocket. This pocket directs the proline
specificity of MAP kinases. The axis of the C helix is rotated, and the
N terminus of this helix is shifted by 6 Å relative to the helix in
ERK2. The helix at the end of L16 is extended by 7 Å, and there is an
increase in the hydrophilicity of residues that form contacts between
L16 and the N-terminal domain. This results in a less intimate
interaction between L16 and the N-terminal domain in p38 (22, 24).
Within MAP kinase cascades, the MEKs are the most specific enzymes.
These dual specificity kinases activate their respective MAP kinase
substrates by phosphorylating the threonine and tyrosine of the
specific TXY sequence located in the phosphorylation lip. The only known substrates of MEK1 and MEK2 are ERK1 and ERK2 (25). Other MEKs also phosphorylate only a small subset of the MAP kinase family. For example, MEK3 and MEK6 will only phosphorylate p38 isoforms, whereas MEK5 will only phosphorylate ERK5 (26-28). In addition, the MEKs require native MAP kinases as substrates; they will
not phosphorylate denatured proteins or peptides derived from the
phosphorylation lip (25). Little is known about the structural basis of
the specificity of interactions between MEKs and MAP kinases despite
information from the crystal structures of ERK2 and p38.
Previous in vitro studies concluded that neither the
phosphorylation lip length nor the residue between the phosphorylation sites is a critical element for directing MEK specificity (29, 30).
Brunet and Pouysségur (31) measured the in vivo
activities of chimeras of p38 and ERK1 after treatment of transfected
cells with stimuli that activated either p38 or ERK1. Based on these findings they proposed that a 40-residue stretch of amino acids within
subdomains III and IV of p38 was important for directing the
specificity of activation. In addition, they suggested that the C helix
within subdomain III was the key element for recognition of MAP kinases
by MEKs.
To define structural elements of the MAP kinases that are required to
direct specific recognition by MEKs, chimeras of ERK2 and p38 were
tested for phosphorylation and activation by five different MEKs
in vitro. Compilation of the data reveals multiple spatially
segregated contacts in the MEK/MAP kinase interface.
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EXPERIMENTAL PROCEDURES |
Generation of Mutants and Purification of
Proteins--
Restriction sites were inserted at the ends of
subdomains II and IV and at residue 326 of ERK2 and residue 322 of p38
with the QuikChange kit (Stratagene, La Jolla, CA). ERK2II
CT
contains all the restriction sites as well as sites at the ends of
subdomains III and V, and p38II contains the site at subdomain II. The
resulting plasmids were used to create ERK2-p38 chimeras.
His6-tagged proteins were expressed and purified as
described previously (32) or by batch binding to
Ni2+-nitrilotriacetic acid-agarose (Qiagen). Protein
concentrations were determined with the BCA assay (Pierce) and by
comparison of Coomassie Blue-stained bands after SDS-polyacrylamide gel
electrophoresis. Phosphorylated His6-MEK1, GST-MEK2, and
His6-MEK3 were purified from a previously described
coexpression system (33), and GST-MEK4 and GST-MEK6 were purified and
activated by GST-MEKK-C (34) essentially as described (29).
Phosphorylation and Activity Assays--
Phosphorylation and
activity assays were performed essentially as described (29). Activity
of ERK2, p38, or chimeras was determined using myelin basic protein
(Sigma) as substrate. One-dimensional phosphoamino acid analysis was
performed as described (35).
Time Courses of Phosphorylation and Activity--
Assays were
performed as described (29), but reactions were stopped at the times
indicated in Figs. 5 and 6 for MEK2 and MEK6, respectively. For MEKs 1, 3, and 4, 50-µl aliquots were removed from one large reaction mix and
stopped by addition to 15 µl of 5× SDS sample buffer.
Western Blotting--
20 ng of ERK2 or each chimera from the
time course reactions with MEK2 and MEK6 were resolved by
SDS-polyacrylamide gel electrophoresis and transferred to
nitrocellulose. Blots were probed with an antibody specific for
phosphorylated ERK2, V803 (Promega), and an antibody that recognizes
phosphorylated and unphosphorylated ERK2, Y691.
Phosphatase Treatment--
ERK2 or chimeras were incubated with
an equimolar amount of PTP1 at 30 °C for 30 min. Sodium
orthovanadate was added to one set of reactions at time 0 and to an
identical set at 30 min. Kinase assays with MEK2 were then performed as
above, but they were stopped after 1 min.
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RESULTS |
MEKs recognize the native MAP kinase structure with considerable
selectivity. The primary sequence surrounding the TXY
phosphorylation sites of the ERKs is not sufficient to determine MEK
recognition. Neither peptides derived from the double phosphorylation
site nor denatured proteins are phosphorylated by MEKs (25). These findings support the conclusion that MEKs require specific
conformations or secondary determinants for productive interaction with
their MAP kinase substrates. We wished to identify the structural
features of MAP kinases that allow MEKs to distinguish among them as
substrates. To do this we created chimeras between ERK2 and p38 that
contained selected intact structural elements from each enzyme (Fig.
1A). The chimeras were
composed of ERK2 and p38 because a related set of chimeras had been
tested in transfected cells (31), and the structures of both of these
MAP kinases were available for analysis (18, 22, 24). The chimeras were
expressed and purified and then tested in vitro for
activation by MEK family members, MEKs 1, 2, 3, 4, and 6, which
phosphorylate ERK2 or p38 in vitro. Measurements included
rate and extent of phosphorylation, phosphoamino acids, recognition by
antibodies selective for phosphorylated ERK2, and activated activity
relative to the wild type proteins. ERK2 and p38 proteins containing
inserted restriction sites were also purified and tested as
controls.

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Fig. 1.
Schematic and structural representation of
ERK2, p38, and chimeras. A, conserved subdomains I-XI
including VIa and VIb are indicated by blocks in ERK2 and
p38. Odd numbers are shown. Subdomains from ERK2 are
white, and subdomains from p38 are gray. The
chimeras were named according to the subdomains that were exchanged.
B, structural representation of MAP kinase domains using
ERK2 as the model. The C-terminal domain is shown in blue.
The N-terminal domain includes residues in white (subdomains
I and II), red (subdomains III and IV, which include the C
helix and strands 4 and 5), and yellow (L16). Chimeras
swapped the white, red, and yellow
structures as indicated in A and the text.
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The MAP kinase catalytic core contains two domains. The N-terminal
domain is composed of N-terminal sequence (white and
red in Fig. 1B) and the C-terminal tail, L16
(yellow in Fig. 1B), and is primarily involved in
binding ATP. In the MAP kinases L16 lies on the surface of the
N-terminal domain. The C-terminal domain (blue in Fig.
1B) binds the protein substrate. To identify important structural features, we first created a chimera, PIVECTP, which contained one of its two major structural domains from each kinase (Fig. 1). This chimera contained the larger C-terminal structural domain from ERK2 and the smaller N-terminal domain from p38. The p38
elements making up the N-terminal domain are the N terminus through
subdomain IV and L16 (shown in white, red, and
yellow in Fig. 1B).
We examined phosphorylation of this chimera by MEKs, the amino acids
phosphorylated, and its activity after phosphorylation. This chimera
was phosphorylated by MEK1 and MEK2 and by MEK3, MEK4, and MEK6,
although the stoichiometry of phosphorylation by MEK3/4/6 was lower
(Fig. 2). The rates of phosphorylation of the chimera by MEK2 and MEK1 were similar to the rates of
phosphorylation of wild type ERK2 by these MEKs (see Figs.
5A and 7A). Likewise, the rates of
phosphorylation of this chimera by MEK6 and MEK3 were similar to their
rates of phosphorylation of p38 (see Figs. 6A and
7B). In each case, both tyrosine and threonine were
phosphorylated (see Fig. 5C and 6C). These
results indicated that interaction determinants were present in both
domains. All five MEKs activated PIVECTP, and in each case the activity
was proportional to the extent of phosphorylation, with MEK4 being the
least effective (Figs. 2 and 3). The
activity of this unphosphorylated chimera was similar to that of wild
type ERK2. Maximal activity was about 15% of the activity of wild type
ERK2 (Fig. 3). This may be due to imperfect folding of the chimera.

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Fig. 2.
Phosphorylation of chimeras by MEK
isoforms. Relative stoichiometry of phosphate incorporation into
ERK2, PIVECTP, PIVE, PIIE, EIIPIVE, EIIP, and p38 by the indicated
MEKs. Data for each MEK are expressed relative to stoichiometry of
phosphorylation of ERK2 by MEK1 and MEK2 and of p38 by MEK3, MEK4, and
MEK6. Data are the means ± S.E. of three to five independent
experiments.
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Fig. 3.
Kinase activity of chimeras after
phosphorylation by MEK isoforms. Relative myelin basic protein
kinase activity of ERK2, PIVECTP, PIVE, PIIE, EIIPIVE, and EIIP after
phosphorylation by the indicated MEKs. Data are expressed relative to
specific activity of ERK2 after phosphorylation by MEK2, set to 1. Data
are the means ± S.E. of three to five independent
experiments.
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Inclusion of residues from p38 in the N-terminal domain of the chimera
allowed phosphorylation by MEK3/6 in addition to MEK1/2. We next wanted
to determine which structural motifs within the N-terminal domain were
responsible for this expanded interaction. Therefore, three additional
chimeras containing less of the N-terminal domain of p38 were
constructed and analyzed as described next.
To determine the contribution of L16 (yellow in Fig.
1B) to phosphorylation by MEK, the PIVE chimera, which had
all of the N-terminal domain except L16 from p38, was generated. It
contained p38 residues from the N terminus through subdomain IV and
ERK2 residues from subdomain V through the C terminus (Fig.
1A). Its activity in the unphosphorylated state was similar
to wild type ERK2. This chimera was phosphorylated by MEK1, MEK2, and
MEK6 but not by MEK3 or MEK4 (Fig. 2). All three MEKs that
phosphorylated PIVE activated it equally (Fig. 3). It had stimulated
protein kinase activity about 20% of that of ERK2. The rates of
phosphorylation of PIVE by MEK1, MEK2, and MEK6 were similar to the
rates of phosphorylation of the wild type proteins (see Figs.
5A, 6A, and 7A). These results suggested that the C-terminal tail within the N-terminal domain contributed to activation by MEK3.
EIIPIVE had only p38 subdomains III and IV, which contained the C helix
and
strands 4 and 5 (red in Fig. 1B). Without
phosphorylation by MEK, this chimera had slightly higher activity than
wild type ERK2. EIIPIVE was phosphorylated and activated by MEK1 and
MEK2 (Figs. 2 and 3). When phosphorylated, this chimera demonstrated decreased activity relative to wild type ERK2 (Fig. 3). MEK3, MEK4, and
MEK6 did not phosphorylate it. Therefore, the C helix,
4, and
5
of MAP kinases were not sufficient for phosphorylation of a MAP kinase
by these MEKs. EIIPIVE also was phosphorylated by MEK1 and MEK2 at
rates similar to the rates of phosphorylation of ERK2 (see Figs.
5A and 7A).
To determine the role of the first two subdomains (white in
Fig. 1B) in phosphorylation by MEKs, PIIE was tested. This
chimera contained p38 residues in the N terminus through subdomain II and ERK2 residues in subdomain III through the C terminus (Fig. 1A). This chimera had activity comparable with that of
unphosphorylated ERK2. Once again, only MEK1 and MEK2 phosphorylated
and activated this chimera (Figs. 2 and 3). The rates of
phosphorylation of PIIE by MEK1 and MEK2 were similar to the rates of
phosphorylation of wild type ERK2 by MEK1 and MEK2 (see Fig.
5A and 7A). Results from these four chimeras
indicated that many p38 residues must be present in the N-terminal
domain of each chimera for phosphorylation by the p38-specific MEKs.
We wanted to determine whether any differences existed in the pattern
of phosphorylation and activation by MEKs if the other MAP kinase was
at the N terminus of the chimera. Thus, we expressed a reciprocal
chimera, EIIP. EIIP contained ERK2 residues from the N terminus through
subdomain II (white in Fig. 1B) and p38 residues
from subdomain III through the C terminus. This chimera was
phosphorylated by MEK4 and MEK6 but not by MEK1, MEK2, or MEK3 (Fig.
2). The time courses of phosphorylation were slightly more rapid (see
Fig. 7C). This chimera had very low basal activity, less
than 1% of wild type ERK2, suggesting that it was poorly folded. After
phosphorylation, this chimera was inactive (Fig. 3). Analysis of its
phosphoamino acids after phosphorylation by MEK4 revealed that it was
inactive in part because there was little or no threonine
phosphorylated (Fig. 4). Domains I and II
of ERK2 alter the position of the C helix in the chimera, which may
account for the inability of MEKs to phosphorylate threonine.

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Fig. 4.
Phosphoamino acid analysis. Phosphoamino
acid analysis of p38 and p38II after incubation with MEK3 and EIIP
after incubation with MEK4. The positions of the three phosphoamino
acid standards are indicated.
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The two control proteins were also tested in the in vitro
kinase assays to determine whether any residues changed by the
insertion of the restriction sites caused the changes in interaction
with MEKs. The ERK2 control, ERK2II
CT was phosphorylated and
activated only by MEK1 and MEK2 (data not shown). Therefore, residues
that were changed by the insertion of restriction sites did not affect the productive interaction of ERK2 chimeras with MEKs. The p38 control,
p38II, was phosphorylated by MEK3, MEK4, and MEK6 (data not shown).
p38II was activated normally by MEK4/6 but less well by MEK3 (data not
shown). The phosphoamino acid analysis (Fig. 4) indicates that p38II is
not fully activated by MEK3 because it is poorly phosphorylated on threonine.
The maximum stoichiometries of phosphorylation of the chimeras by MEKs
1-4 and 6 were evaluated (Table I) and
were consistent with the extents of activation of the chimeras. With
the exception of EIIP, the time courses of phosphorylation of the
chimeras by individual MEK family members were similar to their rates
with their normal substrates, ERK2 or p38 (Figs.
5-7).
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Table I
Maximum stoichiometry of phosphorylation by MEKs
Maximum stoichiometry of phosphorylation of ERK2, p38, and each chimera
is listed as mol PO4/mol MAP kinase. Reactions were performed
at 30 °C for 1 h. There was no significant increase in the
stoichiometry of phosphorylation at longer times. Data are
representative of four or five experiments for each MAP kinase.
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Fig. 5.
Time course of phosphorylation and activation
of chimeras by MEK2. A, phosphate incorporation into
ERK2 ( ), PIIE ( ), PIVE ( ), PIVECTP ( ), and EIIPIVE ( ) by
MEK2 at 30 s and 1, 3, 5, 10, 20, 30, 60, and 90 min.
B, specific myelin basic protein kinase activity of ERK2
( ), PIIE ( ), PIVE ( ), PIVECTP ( ), and EIIPIVE ( ) after
incubation with MEK2 for the times listed in A.
C, immunoblots of ERK2 and chimeras after incubation with
MEK2 for the times listed in A. Top, blots were
probed with an antibody that specifically recognizes ERK2 or chimeras
phosphorylated on both threonine and tyrosine. Bottom, blots
were stripped and reprobed with an ERK antibody, Y691, which recognizes
both unphosphorylated and phosphorylated ERK2 or chimeras. The exposure
time for the PIIE pTEpY blot is longer to allow the bands to be
seen.
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Fig. 6.
Time course of phosphorylation and activation
of chimeras by MEK6. A, phosphate incorporation into
p38 ( ), PIVE ( ), and PIVECTP ( ) by MEK6 at 5, 10, 20, 30, 60, 90, 120, 180, and 240 min. B, specific myelin basic protein
kinase activity of p38 ( ), PIVE ( ), and PIVECTP ( ) after
incubation with MEK6 for the times listed in A.
C, immunoblots of chimeras after incubation with MEK6 for
the times listed in A. Top, blots were probed
with an antibody that specifically recognizes chimeras phosphorylated
on both threonine and tyrosine. Bottom, blots were stripped
and reprobed with an ERK antibody, Y691, which recognizes both
unphosphorylated and phosphorylated chimeras.
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Fig. 7.
Time course of phosphorylation and activation
of chimeras by MEK1, MEK3, and MEK4. A, phosphate
incorporation into ERK2 ( ), PIIE ( ), PIVE ( ), PIVECTP ( ),
and EIIPIVE ( ) after incubation with MEK1 for 5, 10, 15, 20, 30, 60, 90, 120, 180, and 240 min. B, phosphate incorporation into
p38 ( ) and PIVECTP ( ) after incubation with MEK3 for the times
listed in A. C, phosphate incorporation into p38
( ) and EIIP ( ) after incubation with MEK4 for the times listed in
A.
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To determine whether phosphorylation occurred on the predicted residues
(Thr183 and Tyr185), chimeras phosphorylated by
MEKs for different times were immunoblotted with antibodies that
selectively recognize the doubly phosphorylated ERK2 epitope. These
antibodies do not detect unphosphorylated or singly phosphorylated ERK2
at equivalent protein concentrations. The immunoblots reveal an
excellent parallel between increasing phosphorylation and appearance of
the phospho epitope (Figs. 5 and 6). Phosphorylation of the chimeras
saturated at the same times as did blotting intensity. This is
consistent with the conclusion that phosphorylation occurred largely on
Thr183 and Tyr185 and not on previously
unidentified residues.
These immunoblotting findings agree with phosphoamino acid analysis,
which showed the majority of phosphate on tyrosine and threonine.
However, PIVECTP, for example, apparently contained less
phosphotyrosine in these analyses than did wild type ERK2. To determine
whether this was due to prior autophosphorylation of the chimera, we
compared the stoichiometry of phosphorylation of wild type ERK2 and
three of the chimeras before and after dephosphorylation with the
tyrosine phosphatase PTP1 (Fig.
8A). The stoichiometry of
phosphorylation of ERK2 was unchanged by pretreatment with phosphatase;
on the other hand, phosphorylation of two of the chimeras increased by
20-30%. The increase was primarily due to increased tyrosine
phosphorylation as was apparent from the increase in phosphotyrosine
recovered in the phosphoamino acid analysis (Fig. 8, B and
C). This is consistent with the idea that the chimeras have
enhanced abilities to autophosphorylate on Tyr185 prior to
isolation from bacteria. Because this tyrosine is already partly
phosphorylated when the proteins are purified, less phosphate can be
transferred to tyrosine by MEKs.

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Fig. 8.
Autophosphorylation accounts for decreased
tyrosine phosphorylation of PIVE, PIVECTP, and EIIPIVE by MEK2.
A, relative stoichiometry of phosphate incorporation by MEK2
into ERK2, PIVE, PIVECTP, and EIIPIVE without or with pretreatment with
PTP1. Data are expressed as the means ± S.E. for three
independent experiments. B, phosphoamino acid analysis of
ERK2, PIVE, PIVECTP, and EIIPIVE not dephosphorylated with PTP1.
C, phosphoamino acid analysis of ERK2, PIVE, PIVECTP, and
ERK2IIPIVE first dephosphorylated with PTP1. For B and
C, the positions of the three phosphoamino acid standards
are indicated.
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DISCUSSION |
MEK isoforms recognize members of the MAP kinase family with
selectivity. The residues or structural motifs that are required for
this selective recognition have not been defined. ERK1/2 and p38
isoforms share about 40-45% sequence identity (5, 36, 37), and the
structures of the unphosphorylated forms of ERK2 and p38 are similar
(18, 22). The goal of this project was to use chimeras containing
swapped structural elements of ERK2 and p38 to identify regions of the
MAP kinases that direct interactions with particular MEKs.
Our findings using ERK2/p38 chimeras indicate that no single MAP kinase
structural motif forms a sufficient interaction surface with MEK. The
N-terminal structural domain likely forms part of the interface.
However, even the entire domain is not sufficient to restrict
phosphorylation by MEKs, because the chimera containing the N-terminal
domain from p38 was phosphorylated by MEK1 and MEK2 in addition to
MEK3/4/6. This demonstrates that there are interaction determinants in
the C-terminal domain. In addition, a fairly large surface area within
the N-terminal domain must be present for the interaction of the
chimeras with MEK3/4/6. Swapping only subdomains I and II or subdomains
III and IV was not enough to allow MEK3/4/6 to phosphorylate the
chimeras. Based on these data, it appears that MEKs require multiple
interacting sites within both domains to specifically phosphorylate
different MAP kinases.
Our results with ERK2/p38 chimeras are in part consistent with work
from Brunet and Pouysségur (31), who expressed p38/ERK1 chimeras
in mammalian cells. A p38/ERK1 chimera equivalent to PIVE was activated
by both growth factors and cell stress, whereas a chimera equivalent to
PIIE was only activated by growth factors. We have shown with further
subdivision of the swapped regions that in contrast to the suggestion
from the p38/ERK1 studies, the C helix alone is not the key element
determining which MEKs will phosphorylate a particular MAP kinase
in vitro.
Additional findings suggest that the MAP kinase insert is required for
phosphorylation by MEKs. To address the function of this insert, it was
deleted from wild type ERK2.2
Examination of the crystal structure indicated that deletion of these
residues would have a minimal effect on the tertiary structure of ERK2.
In the unphosphorylated state, the resulting protein had protein kinase
activity equivalent to unphosphorylated wild type ERK2. However, the
protein lacking the MAP kinase insert was no longer phosphorylated by
MEK2 in vitro.2 It is possible that the insert
aids in the correct folding of the phosphorylation lip, for binding to
MEKs, or that it may be directly involved in interaction with MEKs.
We mapped the currently available data for MEK specificity determinants
onto the structure of unphosphorylated ERK2 (Fig. 9). The data reported here indicate that
the C helix,
strands 4 and 5, and L16 of MAP kinase are important,
because when these motifs are from ERK2, phosphorylation by MEK3/4/6 is
lost. The findings noted above suggest a role for the MAP kinase
insert. In addition, previous work showed that mutating tyrosine 185 in the phosphorylation lip or deleting six residues from this loop of ERK2
resulted in significantly decreased phosphorylation by MEK1 and MEK2
(29, 32). Taken together, the regions of a MAP kinase that appear to be
important for the MEK/MAP kinase interaction include the vicinity of
the C helix and L16 from the N-terminal domain and include the
phosphorylation lip region and the MAP kinase insert from the
C-terminal domain. These experimental findings are in good agreement
with a visually generated model of the ERK2/MEK1 interface. The model
was created by mapping MEK1 residues onto the coordinates of the
cAMP-dependent protein kinase (14) and manually docking
with the ERK2 coordinates. The ERK2 phosphorylation lip was placed on
MEK1 in the position of the bound peptide substrate (16). The sites of
contact in this hypothetical ERK2/MEK1 model include residues from each
of the structural elements implicated experimentally, namely, the tip
of the C helix, the loop region of L16, the phosphorylation lip, and
the tip of the MAP kinase insert.

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Fig. 9.
Structural representation of ERK2 motifs that
are important for the MEK/MAP kinase interaction. ERK2 residues
that appear to be required for the interaction with MEK are
yellow. Residues not implicated in MEK binding in the
N-terminal domain are in green, and residues not implicated
in MEK binding in the C-terminal domain are in blue. The
figure was generated in InsightII (Molecular Biosystems).
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The proposed MEK/MAP kinase interface is involved in other
protein-protein interactions. Residues in this putative interface overlap with the ERK2 dimerization interface (38). In particular, L16
and the phosphorylation lip are intimately involved in both. Furthermore, it is intriguing that the proposed MEK/MAP kinase interface is similar to the interface between Cdk2 and cyclin A (21).
Structural elements of Cdk2 that participate in cyclin A binding
include the PSTAIRE helix region, which encompasses the helix
equivalent to the C helix of MAP kinases and the
4 and
5 strands.
In the C-terminal domain, contacts with cyclin A are made by residues
in the region of the T loop, equivalent to the ERK2 phosphorylation
lip, and a C-terminal helix, helix 7. The only motif that participates
in the proposed MEK/ERK interface but not in the Cdk2/cyclin A
interface is the MAP kinase insert.
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ACKNOWLEDGEMENTS |
We thank Megan Robinson and Meg Phillips for
helpful suggestions and comments about the manuscript and Tina Arikan
for assistance in its preparation.
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FOOTNOTES |
*
This work was supported by Grant I1243 from the Welch
Foundation and Grant DK34128 from the National Institutes of Health.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.
§
Supported by a predoctoral fellowship from the Howard Hughes
Medical Institute. This work was completed in partial fulfillment of
the requirements for the Ph.D. degree.
To whom correspondence should be addressed: Dept. of
Pharmacology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9041. Tel.: 214-648-3627; Fax:
214-648-3811.
2
J. L. Wilsbacher and M. H. Cobb,
unpublished results.
 |
ABBREVIATIONS |
The abbreviations used are:
MAP, mitogen-activated protein;
ERK, extracellular signal-regulated protein
kinase;
MEK, MAP/ERK kinase;
MEKK, MEK kinase;
PTP1, protein-tyrosine
phosphatase, type 1.
 |
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