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(Received for publication, January 25, 1996; and in revised form, March 4, 1996) From the
A novel protein kinase activity present in nuclear and cytosolic
extracts has been identified and partially purified as a consequence of
its tight binding to and phosphorylation of the extracellular
signal-regulated protein kinase (ERK) 3. This novel protein kinase is
inactivated by treatment with phosphoprotein phosphatase 2A. The ERK3
protein kinase was immunologically distinct from mitogen-activated
protein (MAP) kinase/ERK kinases (MEK) 1 and 2 which phosphorylate the
ERK3-related MAP kinases ERK1 and ERK2. This ERK3 kinase phosphorylated
a single site on ERK3, Ser
The ERK/MAP ( ERK1 and ERK2
are phosphorylated on two sites separated by a single residue in the
phosphorylation lip at the mouth of their active sites (10, 11) . Phosphorylation of both Tyr Much less is known about the protein
kinase ERK3. It was cloned in the same cDNA library screen as ERK1 and
ERK2 (16) and has greater sequence identity to ERK1 and ERK2
than do the JNK/SAPKs or p38 MAP kinase. However, three important
features distinguish ERK3 from the other family members. First, it
lacks the tyrosine phosphorylation site that is absolutely conserved
among those other related kinases. Second, ERK3 is a constitutively
nuclear protein kinase(17) . Third, it apparently has a very
restricted substrate specificity, because it does not phosphorylate any
of the known MAP kinase substrates. As no ERK3 substrates are known,
its regulation has been difficult to define. To understand more
about the regulation of ERK3, we have examined the phosphorylation of
ERK3 by MEK family members, and find that ERK3 is a poor substrate for
MEK1, MEK2, MKK4, and MEK5(18) . We have identified a novel
protein kinase activity in nuclear and cytosolic extracts that binds
very tightly to the catalytic domain of ERK3 and phosphorylates it
selectively. This ERK3 kinase phosphorylates a single site on ERK3,
Ser
Figure 1:
Chromatographic analysis of activities
that phosphorylate ERK3. A, rabbit muscle extracts were
fractionated on Q Sepharose and assayed for ERK2 (
Figure 2:
The ERK3 kinase binds to the catalytic
domain of ERK3. A, fractions of the rabbit muscle ERK3 kinase
partially purified on Q Sepharose and S Sepharose were mixed with GST,
GST-ERK3, GST-ERK3
Because it was difficult to elute the ERK3 kinase from the GST-ERK3
on glutathione-agarose beads, we ascertained if the activity that was
bound to GST-ERK3 on beads would phosphorylate exogenously added ERK3.
As shown in Fig. 2B, the ERK3 kinase bound to
GST-ERK3
Figure 3:
The ERK3 kinase activity is present in
both cytosolic and nuclear fractions of PC12 and 293 cells. The ERK3
kinase activity from nuclear (N) or cytosolic (S)
fractions was adsorbed to GST-ERK3
Figure 4:
Treatment of the ERK3 kinase with PP2A
decreases its protein kinase activity. The ERK3 kinase bound to
GST-ERK3
Figure 5:
The
ERK3 kinase phosphorylates Ser
Figure 6:
Tryptic phosphopeptide mapping of
phosphorylated ERK3. Autoradiograms of tryptic phosphopeptide maps of A, ERK3 phosphorylated by the ERK3 kinase; B,
ERK3
Figure 7:
Specificity of the ERK3 kinase. A, the ERK3 kinase bound to GST-ERK3
A concept that has developed from studies in yeast and
mammalian cells is that of the MAP kinase
module(1, 9, 28, 29) . A MAP kinase
module is a three-kinase cascade including a MAP kinase or ERK, a MEK,
and an activator of MEK, MEK kinase or MEKK. Thus far, studies indicate
that the MEK component has the greatest substrate specificity of
enzymes in the cascade(1, 14, 18) . The known
MEK family members selectively activate their designated MAP kinase
family members, by phosphorylating a threonine and a tyrosine that are
arranged with a single intervening residue. The three-dimensional
structure of the MAP kinase ERK2 contains the two-domain organization
characteristeric of the protein
kinases(11, 30, 31, 32) . The active
site is formed at the interface of these two domains. The two
regulatory phosphorylation sites in ERK2, Tyr A protein kinase that phosphorylates ERK3 and may serve
as an activator or MEK for ERK3 has been partially purified and is
characterized by its ability to bind to the catalytic domain of ERK3.
The ERK3 kinase is found in both the cytoplasm and nucleus of several
cell types, unlike MEK1 and MEK2 which are reported to be exclusively
in the cytoplasm (34) . Like known MEKs, this ERK3 kinase is
inactivated by dephosphorylation and is highly specific as demonstrated
by its inability to phosphorylate ERK1, ERK2, or ERK2 mutants that more
closely resemble ERK3 in the phosphorylation lip. Unlike known MEKs,
the ERK3 kinase will not phosphorylate tyrosine when tyrosine is
introduced into the appropriate position of the phosphorylation lip of
ERK3. Importantly, this ERK3 kinase phosphorylates Ser
Figure 8:
Potential mechanisms of ERK3 regulation.
ERK3 is a constitutively nuclear protein kinase. The protein kinase
activity of ERK3 may be regulated by the ERK3 kinase, which may respond
to extracellular or cytoplasmic cues and shuttle into the nucleus to
bind to and phosphorylate ERK3.
Volume 271,
Number 20,
Issue of May 17, 1996 pp. 12057-12062
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, comparable to
Thr
, one of the two activating phosphorylation sites of
ERK2. To test the specificity of the ERK3 kinase, mutants of ERK3 and
ERK2 were made in which the phosphorylated residues were exchanged. The
double mutant S189T,G191Y ERK3, in which the phosphorylated residues
from ERK2 replaced the comparable residues in ERK3, was phosphorylated
by the ERK3 kinase but only on threonine. The ERK3 kinase did not
phosphorylate ERK2 or ERK2 mutants. These findings indicate that
although the ERK3 kinase is highly specific for ERK3, it does not
recognize tyrosine, a feature that distinguishes it from MEKs that
phosphorylate other ERK/MAP kinase family members.
)kinase pathway is stimulated by
numerous hormones and growth factors and its activation is associated
with increased proliferative and differentiated functions of
cells(1, 2, 3, 4, 5) . The
importance of intracellular processes thought to be regulated by the
MAP kinases has focused attention on understanding the control of this
pathway. The MAP kinase kinases, also known as MAP/ERK kinases or MEK1
and MEK2, originally discovered by Ahn and Krebs, are dual-specificity
protein kinases known to activate the MAP kinases ERK1 and ERK2 in a
highly selective manner(6, 7, 8) . The MAP
kinases, on the other hand, are pleiotropic, phosphorylating many
substrates throughout the cell (reviewed in (3) ). Kinase
cascades containing a MEK and an ERK/MAP kinase are present in multiple
pathways in yeast and have been reiterated in mammalian
cells(1, 9) . Although mechanisms regulating the
similar, but parallel mammalian pathways are less well characterized,
the activation of a multipotential ERK/MAP kinase by a highly specific
MEK is the common feature of all the related cascades.
and Thr on ERK2 and comparable residues on ERK1,
catalyzed by the dual specificity protein kinases, MEK1 and MEK2, is
required for high
activity(10, 12, 13, 14, 15) .
Because of their exquisite specificity, MEK1 and MEK2 are not able to
phosphorylate other MAP kinase-related enzymes such as Jun-N-terminal
kinase/stress-activated protein kinase (JNK/SAPK) or p38 MAP kinase,
even though the phosphorylation sites are in comparable positions in
the sequence(1) ., which is comparable to Thr, one of the
activating phosphorylation sites of ERK2.
Cell Culture
PC12 and 293 cells were maintained
in Dulbecco's modified Eagle's medium supplemented with 10%
fetal bovine serum and 2 mM glutamine. Prior to mitogen
stimulation, PC12 and 293 cells were maintained in Dulbecco's
modified Eagle's medium without serum for 4 h and 18-20 h,
respectively, then treated with nerve growth factor (NGF, 100
nM, 15 min), epidermal growth factor (100 ng/ml, 5 min), or
phorbol ester (100 nM, 20 min, Sigma) as indicated.Bacterial Expression of ERK3, ERK2, and ERK
Mutants
Histidine-tagged and glutathione S-transferase
(GST) fusion proteins of ERK3, ERK3, without the C-terminal domain
(ERK3
Ct), and the following ERK3 mutants, S189A ERK3, S189E ERK3,
D171A ERK3, and S189T,G191Y ERK3, were expressed and purified as
described elsewhere(15, 17) . Histidine-tagged ERK2
and K52R ERK2 were expressed as described previously(15) .
T183S ERK2 and the double mutant T183S,Y185G ERK2 were constructed
using methods described earlier(15) . The mutant Y185G ERK2 was
made using the Chameleon double-stranded DNA mutagenesis kit
(Stratagene, La Jolla, CA).Subcellular Fractionation
Extracts from rabbit
muscle and from NGF-stimulated PC12 cells were prepared according to
the method of Seger et al.(14) for analysis of ERK1
and ERK2 phosphorylating activities. PC12 cells and 293 cells were
fractionated into cytosolic and nuclear fractions as described by
Dignam et al.(19) with modifications described
previously(17) . ERK3 was highly enriched in the nuclear
fraction.Purification of the Kinase Activity That Phosphorylates
ERK3
Extracts were fractionated by chromatography on Q
Sepharose, S Sepharose, Mono Q, or Mono S. Protein kinase activities in
fractions eluted with a 0-0.4 M NaCl gradient in MEK
purification buffer (14) were assayed with 10 µg/ml ERK2 or
ERK3 as substrate as described below. For affinity chromatography,
GST-ERK3 or GST-ERK3Ct bound to glutathione-agarose beads was
incubated with either crude cell extracts or fractions of the ERK3
kinase, partially purified from rabbit muscle or NGF-treated PC12
cells, for 2 h at 4 °C with shaking. The glutathione-agarose beads
were washed 5 
with 1 ml of 1 M NaCl, 20 mM HEPES, pH 7.5, 0.05% Triton X-100, 1 mM EDTA and once
with 1 ml of 50 mM HEPES pH 8.0. The ability of bound kinase
to phosphorylate ERK3 was measured by in vitro phosphorylation
assays (see below).Protein Kinase Assays
In vitro protein
kinase assays were performed in 30 mM HEPES, pH 8.0, 50
µM ATP ([
-
P]ATP to achieve
5-15 cpm/fmol), 10 mM MgCl
, 1 mM benzamidine, 1 mM dithiothreitol, 10 µg/ml ERK3,
ERK2, or mutants, and the ERK3 kinase at 30 °C for 30-60 min.
Reactions were stopped with 5 electrophoresis sample buffer and
analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) and
autoradiography(20) . MEK1 and MEK2 immunoprecipitated from
NGF-stimulated PC12 cells were assayed as described
elsewhere(21) .Treatment of the ERK3 Kinase with Phosphoprotein
Phosphatase 2A (PP2A)
The ERK3 kinase adsorbed to
GST-ERK3Ct on glutathione agarose beads was washed once with 1 ml
of phosphatase assay buffer (50 mM HEPES, pH 7.5, 0.5% bovine
serum albumin, 1 mM dithiothreitol). Aliquots of beads were
then mixed with 2.5 or 5 µg/ml of PP2A in phosphatase assay buffer
either without or with okadaic acid (5 µM, Moana
Bioproducts) at room temperature with shaking for 45 min. The reactions
were stopped with 10 mM sodium phosphate and 5 µM okadaic acid and the beads were washed twice with 1 ml of 50
mM HEPES, pH 8.0. The ERK3 kinase activity on the beads was
measured as described above.Other Methods and Reagents
Antibodies to ERK3 and
isoform selective antibodies recognizing MEK1 and MEK2 were reported
previously (17, 21) . Phosphopeptide mapping and
phosphoamino acid analysis were described before(22) . The
catalytic subunit of PP2A was purified from bovine heart(23) .
Protein kinase C was partially purified from rat brain as described
previously (24) which yields a mixture of isoforms. Recombinant
protein kinase C
purified from Sf9 cells infected with the
recombinant baculovirus was kindly provided by B. Singer (University of
Texas Southwestern).
Chromatographic Analysis of Activities That
Phosphorylate ERK3
Rabbit muscle extracts, a source of activated
MEK1 and MEK2 (25) , were fractionated on Q Sepharose and the
resulting fractions were assayed for their ability to phosphorylate
ERK3 and ERK2 (Fig. 1A). The predominant peak of kinase
activity phosphorylating ERK3 was found in fractions 42-52, and
lacked activity toward ERK2. An activity with similar specificity and
chromatographic behavior was also detected in Mono Q fractions
12-17 from extracts of NGF-stimulated PC12 cells (Fig. 1B). Q Sepharose fractions 47-53 containing
ERK3 phosphorylating activity from rabbit muscle extracts, referred to
hereafter as the ERK3 kinase, were pooled and the kinase was further
purified on Mono S (Fig. 1C). A single peak of activity
was detected. Q Sepharose fractions 20-42, which contained the
major peak of activity phosphorylating ERK2 from rabbit muscle
extracts, also displayed a small amount of activity toward ERK3. Other
studies indicated that MEK1 and MEK2 were both contained in the major
peak of activity phosphorylating ERK2 (26) . ()However, these enzymes contributed little of the ERK3
phosphorylating activity because MEK1 and MEK2 immunoprecipitated from
extracts of NGF-stimulated PC12 cells phosphorylated ERK2 but not ERK3
(data not shown). The activities in Q Sepharose fractions 32-40
from rabbit muscle extracts that phosphorylated ERK3 were not further
characterized, but may have been due to protein kinase C, because
protein kinase C eluted in this region of the gradient and was found to
phosphorylate ERK3 in vitro (data not shown).
) and ERK3
(
) phosphorylating activities as described under ``Material
and Methods.'' B, extracts of NGF-stimulated PC12 cells
were fractionated on Mono Q and assayed as in A. C, fractions
47-53 containing the ERK3 kinase activity from the Q Sepharose
profile in A were pooled and fractionated on Mono S, and the
resulting fractions were assayed as in A.
The ERK3 Kinase Binds Tightly to the ERK3 Catalytic
Domain
The ERK3 kinase could be distinguished from other protein
kinases based on its tight binding to the catalytic domain of ERK3 (Fig. 2A). GST-ERK3 or GST-ERK3Ct bound to
glutathione-agarose beads was incubated with the ERK3 kinase activity
from rabbit muscle that had first been partially purified on Q
Sepharose and S Sepharose. The bound protein kinase activity was
measured by its ability to phosphorylate GST-ERK3 or GST-ERK3Ct on
the beads (Fig. 2A). The ERK3 kinase activity was not
eluted with concentrations of NaCl up to 1 M, with 1% Triton
X-100 or with 1 M MgCl. The ERK3 kinase did not
bind to GST-ERK2. The tight association of the ERK3 kinase with ERK3
has a parallel in the binding of JNK/SAPK to c-Jun. The binding
requires only the kinase domain of ERK3 as deleting the C-terminal
domain did not eliminate binding to the ERK3 kinase. Because protein
kinase C was able to phosphorylate ERK3 in vitro, we tested
its capacity to bind to GST-ERK3. Neither rat brain protein kinase C
nor recombinant protein kinase C bound to GST-ERK3 (data not
shown), indicating that protein kinase C is not the ERK3 kinase.
Ct, and GST-D171A ERK3Ct as indicated above
the lanes. The bound material was collected on glutathione-agarose
beads and phosphorylation was measured. Phosphorylated GST-ERK3,
GST-ERK3Ct, and GST-D171A ERK3]Ct were resolved by
SDS-PAGE and an autoradiogram is shown. B. The ERK3 kinase bound to
GST-ERK3Ct on glutathione-agarose beads phosphorylated added
His
-ERK3Ct and His-D171A ERK3Ct as indicated
above the lanes. Phosphorylated GST-ERK3Ct,
His-ERK3Ct and mutants were resolved by SDS-PAGE and
an autoradiogram is shown. The molecular mass standards and the
mobilities of GST- and His-ERK3 are indicated. C.
Phosphoamino acid analysis of phosphorylated GST-ERK3, GST-ERK3Ct
and GST-D171A ERK3Ct. The positions of phosphoamino acid standards
are indicated.
Ct on beads phosphorylated not only bound GST-ERK3Ct
but also added His-ERK3Ct, which is different in size
from GST-ERK3Ct. It seemed unlikely that the ERK3 kinase was ERK3
itself because ERK3 autophosphorylation is intramolecular not
intermolecular (17) . However, it was possible that the protein
bound to ERK3 was not an ERK3 kinase but an activator that accelerated
ERK3 autophosphorylation. To demonstrate that the ERK3 kinase was not
ERK3 or an activator of ERK3 autophosphorylation, a catalytically
defective mutant, D171A ERK3, that neither autophosphorylates nor is
phosphorylated by wild type ERK3 in vitro(17) was
tested as a substrate for the ERK3 kinase. The ERK3 kinase bound
tightly to GST-D171A ERK3Ct and it phosphorylated GST-D171A
ERK3Ct or added His-D171A ERK3Ct as well as the
wild type protein (Fig. 2, A and B),
indicating that the protein bound to GST-ERK3 is an ERK3 protein
kinase. Phosphoamino acid analysis showed that the ERK3 kinase
phosphorylated ERK3 on serine (Fig. 2C).Subcellular Localization of the Kinase That
Phosphorylates ERK3
Because ERK3 is primarily in the
nucleus(17) , the subcellular distribution of the ERK3 kinase
was examined. Cytosolic and nuclear extracts from multiple cell types
were tested for the ERK3 kinase activity by binding to GST-ERK3Ct
on beads and assay of the bound material by phosphorylation of
GST-ERK3Ct. Activity that bound tightly to ERK3Ct and
phosphorylated it was found in both cytosolic and nuclear extracts of
PC12 and 293 cells (Fig. 3), and in extracts of other cell lines
such as NIH3T3, Cos, and Jurkat T cells (data not shown). The
distribution of the activity between cytosolic and nuclear fractions
was not changed by extracellular stimuli including epidermal growth
factor, NGF, or phorbol ester. These agents activate ERK1 and ERK2 and
cause their translocation to the nucleus (27) , but may not be
physiological regulators of ERK3.
Ct on glutathione-agarose beads
as described under ``Material and Methods.'' The mobilities
of GST-ERK3Ct and standard markers resolved as in Fig. 2are indicated.
Regulation of the ERK3 Kinase
To test the
possibility that the ERK3 kinase, like MEK1 and MEK2, is regulated by
phosphorylation, its activity was measured before and after treatment
with PP2A. The initial rate of ERK3 phosphorylation by the ERK3 kinase
was reduced 85-90% by a 45-min treatment with PP2A (Fig. 4). Preincubation of PP2A with okadaic acid blocked the
inactivation of the ERK3 kinase, demonstrating that loss of activity is
due to dephosphorylation of the ERK3 kinase preparation.
Ct on glutathione-agarose beads was untreated(-) or
treated with PP2A (+, 2.5 µg/ml; ++, 5 µg/ml),
or with PP2A plus 5 µM okadaic acid (OA), and its
ERK3 phosphorylating activity was then measured. Top,
GST-ERK3Ct was resolved by SDS-PAGE and an autoradiogram is shown.
The position of GST-ERK3Ct is indicated. Bottom, bar
graph quantitating the rate of phosphorylation of GST-ERK3Ct by
the bound ERK3 kinase before and after treatment with
PP2A.
Sites Phosphorylated on ERK3 by the ERK3 Kinase
We
determined previously that ERK3 autophosphorylated in vitro and was phosphorylated in intact cells on
Ser(17) , the residue comparable to
Thr, one of the two activating phosphorylation sites in
ERK2 (Fig. 5A). The stoichiometry of phosphorylation of
ERK3 by the ERK3 kinase was 0.7 mol phosphate/mol ERK3, consistent with
a single site of phosphorylation. In comparison, incorporation due to
autophosphorylation was never greater than 0.04 mol of phosphate/mol of
ERK3 even after overnight incubation. To determine if Ser was the site phosphorylated by the ERK3 kinase, this residue was
mutated to alanine (S189A ERK3) or glutamic acid (S189E ERK3) (Fig. 5A). The ERK3 kinase bound to GST-S189A ERK3 and
GST-S189E ERK3 on beads as determined by its ability to phosphorylate
added His-ERK3Ct (data not shown). However, it no
longer phosphorylated GST-S189A ERK3 or GST-S189E ERK3 to which it was
bound (Fig. 5, B and C). The ERK3 kinase also
did not phosphorylate added His-S189A or S189E ERK3Ct
(data not shown). These data support the conclusion that Ser is the site phosphorylated by the ERK3 kinase. To confirm that
the ERK3 kinase phosphorylated the same site on ERK3 that was
phosphorylated in intact cells, tryptic phosphopeptide maps of ERK3 and
ERK3Ct phosphorylated by the ERK3 kinase were compared to a map of
ERK3 phosphorylated in intact cells. Each map revealed a major
phosphopeptide (Fig. 6A-C) that migrated as a
single spot if tryptic phosphopeptides from ERK3 phosphorylated in
vitro were mixed with those from ERK3 phosphorylated in intact
cells (Fig. 6D). This major phosphopeptide was absent
from S189A ERK3 phosphorylated by the ERK3 kinase (Fig. 6E). The incorporation into S189A ERK3 was about
1-2% of that incorporated into wild type ERK3. The addition of
tryptic phosphopeptides from phosphorylated wild type ERK3 to those
from phosphorylated S189A ERK3 restored the major phosphopeptide (Fig. 6F). These data indicate that Ser of ERK3, the site phosphorylated in intact cells, is the major
site phosphorylated by the ERK3 kinase.
of ERK3. A,
comparison of the phosphorylation lips of ERK2 and ERK3. The ERK2 and
ERK3 mutants are indicated above and below the lip sequences. The sites
phosphorylated to activate ERK2 are marked with asterisks. Identical
residues between ERK2 and ERK3 are indicated with vertical bars. B, GST-ERK3Ct, GST-S189A ERK3Ct, and GST-S189E
ERK3Ct bound to glutathione-agarose beads were incubated with Mono
S fractions containing the ERK3 kinase activity from rabbit muscle.
After the beads were washed as described under ``Material and
Methods,'' the bound ERK3 kinase activity was measured by its
ability to phosphorylate GST-ERK3Ct, and mutants are as described
in Fig. 2. Top, an autoradiogram showing P
incorporation into GST-ERK3Ct and mutants. Bottom,
Coomassie Blue stain of GST-ERK3Ct and mutants. C,
phosphoamino acid analysis of phosphorylated GST-ERK3Ct and
GST-S189A ERK3Ct. Spots close to the origin were partially
hydrolyzed phosphorylated products. The positions of the phosphoamino
acid standards are indicated.
Ct phosphorylated by the ERK3 kinase; C, ERK3
phosphorylated in intact cells; D, mixture of ERK3
phosphorylated by the ERK3 kinase and in intact cells; E,
S189A ERK3 phosphorylated by the ERK3 kinase; F, mixture of
ERK3 and S189A ERK3 phosphorylated by the ERK3 kinase. Equal counts/min
were loaded onto each plate for mapping.
Specificity of the ERK3 Kinase
The specificity of
the ERK3 kinase was characterized. For these experiments, the ERK3
kinase was partially purified from rabbit muscle and then affinity
purified on GST-ERK3Ct bound to glutathione-agarose beads. Similar
results were obtained with the ERK3 kinase prior to binding to
GST-ERK3Ct on beads. His-ERK3Ct was
phosphorylated by the ERK3 kinase but less efficiently than bound
GST-ERK3Ct (Fig. 7A). In ERK2, Thr and Tyr are the activating phosphorylation sites.
These residues were interchanged with Ser and
Gly
, the comparable residues in ERK3. The double mutant
His-S189T, G191Y ERK3Ct was phosphorylated by the
ERK3 kinase primarily on threonine and to a lesser extent on serine but
not on tyrosine (Fig. 7, A and B). Thus,
unlike MEK1 and MEK2, the ERK3 kinase did not phosphorylate tyrosine in
the phosphorylation lip at a position equivalent to Tyr of ERK2. Further, the ERK3 kinase did not phosphorylate
His
-K52R ERK2 or the ERK3-like mutants T183S ERK2, Y185G
ERK2, and T183S,Y185G ERK2 (Fig. 7A).
Ct on
glutathione-agarose beads phosphorylated both GST-ERK3Ct and added
His-ERK3Ct or His-S189T, G191Y
ERK3Ct, but not added His-ERK2 mutants (all at 30
µg/ml). An autoradiogram is shown. The mobilities of
GST-ERK3Ct, His-ERK3Ct and His-ERK2
are indicated. Added His-ERK2 mutants T183S ERK2, Y185G
ERK2, and S183T,Y185G ERK2 displayed autophosphorylation rates higher
than ERK3 and different from each other. K52R ERK2 lacked the ability
to autophosphorylate. B, phosphoamino acid analysis of
phosphorylated S189T,G191Y ERK3Ct. Spots near the origin were
partially hydrolyzed phosphorylated products. The phosphoamino acid
standards are indicated.
and
Thr, are in a surface loop known as the phosphorylation
lip, that lies at the mouth of the active site. The phosphorylation lip
is an important but highly variable regulatory element of the protein
kinase family. Structural and biochemical studies indicate that
mutation of Tyr in ERK2 changes the conformation of the
phosphorylation lip and dramatically decreases ERK2
activity(33) ; thus Tyr is essential for correct
folding of this lip in both low and high activity forms. In ERK2,
Tyr faces the active site and can be partially
autophosphorylated. Unlike any other ERK/MAP kinase homologs, ERK3
lacks this tyrosine residue, in spite of the significant similarities
of the ERK3 phosphorylation lip in sequence and length to the
phosphorylation lip of ERK2. A single residue, Ser comparable to Thr of ERK2, is phosphorylated on
ERK3 in intact cells(17) . The essential nature of Tyr in ERK2 indicates that major differences in folding of the lip
may occur in ERK2 and ERK3. Replacement of Gly of ERK3
with tyrosine changes the autophosphorylated residue from serine to
tyrosine(17) . This suggests that tyrosine may also face the
active site in this ERK3 mutant. This mutant is a poor MEK substrate,
however, suggesting that portions of the protein that lie outside of
the phosphorylation lip are important determinants of MEK-ERK
recognition. of
ERK3, the site phosphorylated in intact cells. Thus, the ERK3 kinase
identified here may be the upstream regulator of ERK3. From a primarily
cytosolic location when inactive, ERK1 and ERK2 are translocated in
part to the nucleus upon activation, while the activating MEKs are
believed to remain cytosolic(27, 34) . In contrast,
ERK3 is found primarily in the nucleus and the ERK3 kinase is present
in both cytosolic and nuclear extracts. This suggests a regulatory
mechanism in which the ERK3 kinase may receive signals from membrane
bound or cytoplasmic cues and shuttle into the nucleus to phosphorylate
ERK3 (Fig. 8).
)Ct, without C-terminal
domain.)
We thank David Robbins (University of California, San
Francisco) for his input during the early stages of this work, Clark
Garcia and Peiqun Wu for preparation of some of the bacterial proteins
and cell extracts, Alphonsus Dang for MEK1 and MEK2
immunoprecipitation, and Jo Hicks for preparation of the manuscript.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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J. M. English, G. Pearson, R. Baer, and M. H. Cobb Identification of Substrates and Regulators of the Mitogen-activated Protein Kinase ERK5 Using Chimeric Protein Kinases J. Biol. Chem., February 13, 1998; 273(7): 3854 - 3860. [Abstract] [Full Text] [PDF]
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MeganJ. Robinson, M. Cheng, A. Khokhlatchev, D. Ebert, N. Ahn, K.-L. Guan, B. Stein, E. Goldsmith, and MelanieH. Cobb Contributions of the Mitogen-activated Protein (MAP) Kinase Backbone and Phosphorylation Loop to MEK Specificity J. Biol. Chem., November 22, 1996; 271(47): 29734 - 29739. [Abstract] [Full Text] [PDF]
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J. Zimmermann, N. Lamerant, R. Grossenbacher, and P. Furst Proteasome- and p38-dependent Regulation of ERK3 Expression J. Biol. Chem., March 30, 2001; 276(14): 10759 - 10766. [Abstract] [Full Text] [PDF]
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