(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, 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.
The ERK/MAP ()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.
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 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) .
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, which is comparable to Thr
, one of the
activating phosphorylation sites of ERK2.
Figure 1:
Chromatographic analysis of activities
that phosphorylate ERK3. A, rabbit muscle extracts were
fractionated on Q Sepharose and assayed for ERK2 () 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.
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-ERK3Ct, and GST-D171A ERK3
Ct as indicated above
the lanes. The bound material was collected on glutathione-agarose
beads and phosphorylation was measured. Phosphorylated GST-ERK3,
GST-ERK3
Ct, and GST-D171A ERK3
]Ct were resolved by
SDS-PAGE and an autoradiogram is shown. B. The ERK3 kinase bound to
GST-ERK3
Ct on glutathione-agarose beads phosphorylated added
His
-ERK3
Ct and His-D171A ERK3
Ct as indicated
above the lanes. Phosphorylated GST-ERK3
Ct,
His
-ERK3
Ct 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-ERK3
Ct
and GST-D171A ERK3
Ct. The positions of phosphoamino acid standards
are indicated.
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-ERK3Ct on beads phosphorylated not only bound GST-ERK3
Ct
but also added His
-ERK3
Ct, which is different in size
from GST-ERK3
Ct. 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 ERK3
Ct and it phosphorylated GST-D171A
ERK3
Ct or added His
-D171A ERK3
Ct 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).
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-ERK3Ct on glutathione-agarose beads
as described under ``Material and Methods.'' The mobilities
of GST-ERK3
Ct and standard markers resolved as in Fig. 2are indicated.
Figure 4:
Treatment of the ERK3 kinase with PP2A
decreases its protein kinase activity. The ERK3 kinase bound to
GST-ERK3Ct 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-ERK3
Ct was resolved by SDS-PAGE and an autoradiogram is shown.
The position of GST-ERK3
Ct is indicated. Bottom, bar
graph quantitating the rate of phosphorylation of GST-ERK3
Ct by
the bound ERK3 kinase before and after treatment with
PP2A.
Figure 5:
The
ERK3 kinase phosphorylates Ser 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-ERK3
Ct, GST-S189A ERK3
Ct, and GST-S189E
ERK3
Ct 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-ERK3
Ct, and mutants are as described
in Fig. 2. Top, an autoradiogram showing
P
incorporation into GST-ERK3
Ct and mutants. Bottom,
Coomassie Blue stain of GST-ERK3
Ct and mutants. C,
phosphoamino acid analysis of phosphorylated GST-ERK3
Ct and
GST-S189A ERK3
Ct. Spots close to the origin were partially
hydrolyzed phosphorylated products. The positions of the phosphoamino
acid standards are indicated.
Figure 6:
Tryptic phosphopeptide mapping of
phosphorylated ERK3. Autoradiograms of tryptic phosphopeptide maps of A, ERK3 phosphorylated by the ERK3 kinase; B,
ERK3Ct 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.
Figure 7:
Specificity of the ERK3 kinase. A, the ERK3 kinase bound to GST-ERK3Ct on
glutathione-agarose beads phosphorylated both GST-ERK3
Ct and added
His
-ERK3
Ct or His
-S189T, G191Y
ERK3
Ct, but not added His
-ERK2 mutants (all at 30
µg/ml). An autoradiogram is shown. The mobilities of
GST-ERK3
Ct, His
-ERK3
Ct 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 ERK3
Ct. Spots near the origin were
partially hydrolyzed phosphorylated products. The phosphoamino acid
standards are indicated.
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 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.
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 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).
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.