(Received for publication, November 30, 1995; and in revised form, January 30, 1996)
From the
The ERK3 cDNA predicts a protein of 62,000 in size with a
C-terminal domain that extends 180 amino acids beyond the conserved
core of ERK family protein kinases. Immunoblotting with antibodies
raised to recombinant protein and to peptides from the catalytic core
and three regions of the C-terminal tail revealed that ERK3 is the
expected size and is ubiquitously expressed in a variety of cell lines
and tissues. ERK3, unlike the MAP kinases ERK1 and ERK2, is localized
in the nucleus in exponentially growing, quiescent, and growth
factor-stimulated cells. If the 180 amino acids at its C terminus are
deleted, the resulting ERK3 fragment of 45 kDa is still found primarily
in the nucleus, indicating that the C terminus is not required for its
localization. Recombinant ERK3 expressed in mammalian cells or in
bacteria is a protein kinase, as deduced from its capacity to
autophosphorylate. Mutation of a conserved residue (Asp)
expected to be involved in catalysis eliminated autophosphorylation.
Ser
of ERK3, which corresponds to Thr
, one
of the activating phosphorylation sites of ERK2, is autophosphorylated in vitro and phosphorylated in vivo. Despite marked
similarities to ERK1 and ERK2, ERK3 does not phosphorylate typical MAP
kinase substrates, indicating that it has distinct functions.
ERKs (extracellular signal-regulated protein kinases) ()are a subfamily of the protein kinases involved in
hormonal signal transduction. Two closely related members of this
subfamily, the mitogen-activated protein (MAP) kinases ERK1 and ERK2,
are regulated by growth factors and products of the protooncogenes Ras
and Raf (reviewed in (1, 2, 3) ). Blockade of
the MAP kinases using dominant interfering mutations inhibits growth
factor and oncogene-induced cell proliferation(4) . Other ERK
relatives include the c-Jun N-terminal protein kinase/stress-activated
protein kinases, which phosphorylate and activate
c-Jun(5, 6) , and
p38(7, 8, 9) , which is required for
lipopolysaccharide-induced translation of tumor necrosis
factor(7) . These enzymes have been suggested to play a role in
the response of cells to stress. ERK3, the third member of the ERK
family to be cloned(10) , is 50% identical to ERKs 1 and 2
within its catalytic domain. Its cDNA from rat predicts a protein of 62
kDa with a C-terminal domain that extends 180 amino acids beyond the
conserved ERK core. Given the important roles of other members of this
subfamily of protein kinases in cellular regulation, it is likely that
ERK3 will also have important, albeit unknown, functions.
A human ERK3-related enzyme was cloned (11) that is 72% identical to rat ERK3 in the kinase domain. The tail domains of ERK3 and the ERK3-related enzyme are less similar (only 28% identical) and are unrelated over the last 65 amino acids. More recently, Flier and colleagues have found the human ERK3 homolog. It is nearly identical to rat ERK3 throughout the catalytic core as well as the tail, and appears to be alternatively spliced, generating a form that contains an additional 178 residues at the C terminus(12) . Southern analysis suggests at least three ERK3-related genes, supporting the idea that there are multiple ERK3-like proteins(10) . One unusual feature of ERK3 and the related kinase is that they contain an arginine in place of the subdomain VIII glutamate that is highly conserved among the protein kinases(10, 11, 12, 13) . Analysis of crystal structures of several protein kinases including ERK2 suggests that this residue is involved not in catalysis but in stabilizing the structure of the C-terminal fold of the protein kinase(14, 15) . Members of the casein kinase I family also lack this glutamate(16) . The three-dimensional structure of a yeast casein kinase I indicates a different mechanism to stabilize the protein structure(17) , suggesting that this arginine in ERK3 is compatible with its protein kinase activity. To test this directly, we expressed and immunopurified ERK3 to measure its catalytic activity.
In addition, we wished to confirm the existence of ERK3 protein by examining its expression and distribution in cells and tissues. ERK3-specific antibodies demonstrated that ERK3 was widely distributed and, unlike ERK1 and ERK2, ERK3 was localized to the nucleus in the absence of serum factors.
Figure 1:
A, ERK3 structure and location of
peptide antigens. The region included in ERK3Ct is indicated by
the white bar. D175 and D176 were raised to an epitope absent
from the ERK3-related kinase. B, specificity of anti-ERK3
peptide antibodies D175 and D176. Cell extracts (50 µg) from 293
and PC12 cells were resolved in duplicate on 10% acrylamide gels and
transferred to nitrocellulose for immunoblotting. Anti-ERK3 peptide
antibody D175 (1:1000 dilution) and D176 (1:1000 dilution) were used
for immunoblotting. The molecular mass standards are
indicated.
To express ERK3 as a GST fusion protein, oligonucleotides
were used for PCR amplification of either ERK3Ct, or full-length
ERK3 and the PCR products were subcloned into EcoRI and SalI sites of pGEX-KG(20) . Expression was as
described except that cultures were grown in Terrific Broth (21) to an OD
of 0.5-0.6 and then induced
with 0.4 mM isopropyl-
-D-thiogalactoside for
12-14 h. Cells were washed once with 50 mM Tris, pH 8.0,
25% sucrose, and 10 mM EDTA and then lysed in 40 ml of 10
mM Tris, pH 7.4, 1 mM EDTA, 1 mM DTT, 0.15 M NaCl, 1% Triton X-100, 1 mM phenylmethylsulfonyl
fluoride (PMSF), 1 µg/ml leupeptin, 1 µg/ml pepstatin, 2
µg/ml aprotinin, and 3 mg/ml lysozyme. The lysates were sonicated
for 2 min to shear DNA and clarified by ultracentrifugation at 25,000
rpm for 30 min in a Beckman Ti45 rotor. The supernatants were mixed
with glutathione-agarose beads for 2 h, and the beads were washed four
times with 20 mM HEPES, pH 7.6, 0.1 M KCl, 20%
glycerol, 1 mM DTT, 1 mM EDTA, 1 mM PMSF.
GST-ERK3 and GST-ERK3
Ct were eluted with 5 mM reduced
glutathione (Sigma), dialyzed against ERK purification buffer, and
stored at -80 °C.
COS7 and Jurkat T cells were fractionated into
cytosolic and nuclear fractions as described(24, 25) .
Nuclear proteins were extracted with nuclear extraction buffer (0.42 M NaCl, 20 mM HEPES, pH 7.9, 1.5 mM MgCl, 0.2 mM EDTA, 25% glycerol, 0.5 mM DTT, 1 mMp-nitrophenyl phosphate, 1 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 2 µg/ml
aprotinin). Equal amounts of protein from each fraction were subjected
to sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE), followed by immunoblotting with anti-ERK3 antibodies.
293
cells were changed to serum-free Krebs-Ringer bicarbonate containing 2%
bovine serum albumin (26) for 1 h prior to addition of
[P]orthophosphate (1 mCi/ml) for 2 h. Cells were
washed twice with ice-cold phosphate-buffered saline, lysed in ERK
lysis buffer supplemented with 0.15 M NaCl, 1% Triton X-100,
0.5% deoxycholate, 3 mM MgCl
, 5 mM EGTA,
1 mM DTT, and 0.1% SDS for 30 min at 4 °C. After
centrifugation for 30 min in an Eppendorf microcentrifuge, the cell
lysates were incubated with 2 µl anti-HA monoclonal antibody 12CA5
(BabCo) and 30 µl of protein A-Sepharose 4B (Sigma) for 2 h at 4
°C. The immunoprecipitates were washed three times with 20 mM Tris
HCl, pH 7.5, 50 mM NaF, 1 mM sodium
vanadate, 1 mM EGTA, 0.15 M NaCl, 1% Triton X-100,
0.1% SDS; twice with 0.5 M LiCl, 0.1 M Tris
HCl,
pH 7.5; and once with 50 mM Tris
HCl, pH 7.6, 1 mM EGTA, 0.1 M NaCl. The immunoprecipitates were analyzed by
SDS-PAGE and autoradiography.
We examined the distribution and relative abundance of ERK3 in rat tissues using antisera D175 and A654, the latter an anti-ERK3 peptide antibody to subdomain XI. All tissues contained an immunoreactive band of 62 kDa, corresponding to ERK3 that were recognized by both antibodies (Fig. 2). A654 also revealed a significant band slightly smaller than ERK3 with a distinct tissue distribution, as well as minor bands of 43 and 41 kDa, corresponding to ERK1 and ERK2, consistent with the similarity of their sequences to ERK3 in subdomain XI. Both A654 and D175 also recognized proteins of 160 and 95 kDa in rat tissues, suggesting the existence of ERK3-related proteins in these tissues. The 95-kDa protein may be the alternatively spliced form of ERK3 reported by Zhu et al.(12) because it is found most highly in muscle.
Figure 2: Expression of ERK3 in rat tissues. Nuclear extracts from rat tissues (100 µg) were resolved on 10% acrylamide gels and transferred to nitrocellulose for immunoblotting. D175 (1:1000 dilution) and A654 (1:2000 dilution) were used for immunoblotting. The molecular mass standards are indicated.
Figure 3: Subcellular distribution of ERK1, ERK2, and ERK3 in Jurkat cells. Nuclear (N) and cytosolic (S) fractions were prepared from Jurkat cells untreated(-) or treated with phorbol 12-myristate 13-acetate (+), 10 ng/ml, 20 min. After electrophoresis, proteins were transferred to nitrocellulose and probed with anti-ERK1 peptide antibody Y691 (blots ERKs 1 and 2) and anti-ERK3 peptide antibody A654 (blots ERK3 and to a lesser extent ERK1 and ERK2).
The nuclear localization of ERK3 was confirmed by indirect immunofluorescence with the specific anti-ERK3 peptide antibody D175. ERK3 was found in the nuclei of log phase REF52 cells (Fig. 4, A and B). The punctate, nuclear staining was blocked by preabsorbing the antibody with antigenic peptide (Fig. 4, C and D) or recombinant GST-ERK3 (data not shown). Preimmune serum detected no signal in these cells (data not shown). Similar results were also obtained with several other cell lines, including COS, NIH3T3, Rat1, Jurkat, and P19 cells. In quiescent cells and cells stimulated to activate MAP kinase activity, ERK3 was also largely in the nucleus (Fig. 4, E and F). For comparison, the subcellular localization of ERK1 and ERK2 was examined with antibody Y691. In quiescent cells, ERK1 and ERK2 were predominantly in the cytoplasm (Fig. 4G); after 10 min of serum stimulation, they accumulated in and around the nucleus (Fig. 4H) (24) although they were also clearly visible in the cytoplasm.
Figure 4: Immunofluorescent localization of ERK3 in REF52 cells. A, cells stained with anti-ERK3 peptide antibody D175 (1:100 dilution); B, the same cells stained with 4,6 diamidino-2-phenylindole(27) . C, cells stained with D175 preincubated with 500 µM antigenic peptide; D, the same cells stained with 4,6 diamidino-2-phenylindole. E, ERK3 in REF52 cells serum-starved for 18 h. F, same as E, except starved cells were restimulated with 10% FBS for 10 min. G, ERK1 and ERK2 detected with antibody Y691 in REF52 cells serum-starved for 18 h. H, same as G, except starved cells were restimulated with 10% FBS for 10 min. Exposures for panels A and C were 30 s. Exposures for panels B, D, and E-H were 15 s.
The C-terminal tail of ERK3 has several standard nuclear
localization sequences that might account for its constitutive presence
in the nucleus. To determine if the C-terminal domain was responsible
for the nuclear localization of ERK3, both the full-length protein and
ERK3 lacking the C-terminal 180 amino acids, ERK3Ct, were
expressed in COS7 cells. The transfected cells were examined by
immunofluorescent staining with the anti-Myc peptide monoclonal
antibody 9E10 and immunoblotting with anti-ERK3 antibody A654 after
cell fractionation. Immunostaining showed that, like the endogenous
protein, Myc-ERK3 was found primarily in the nucleus of the transfected
cells (Fig. 5A); Myc-ERK3
Ct was also detected in
the nucleus as well as scattered in the cytoplasm (Fig. 5B). Immunoblotting of subcellular fractions also
showed that endogenous ERK3 and both transfected Myc-ERK3 and
Myc-ERK3
Ct were predominantly in the nuclear fractions (Fig. 5C).
Figure 5:
The
nuclear localization of ERK3 is independent of its C-terminal region.
Both pCMV5-Myc-ERK3 and pCMV5-Myc-ERK3Ct plasmids were transiently
transfected into COS7 cells. A, Myc-ERK3; B,
Myc-ERK3
Ct detected in transfected cells with anti-Myc monoclonal
antibody 9E10. Exposures for panels A and B were 15
s, the same as for panels E-H of Fig. 4. C, COS 7 cells transfected as indicated, fractionated into
nuclear (N) and cytosolic (S) fractions, and
immunoblotted with A654 (1:2000 dilution). Myc-ERK3 and Myc-ERK3
Ct
are indicated. The prominent band at 62 kDa in the nuclear fractions is
endogenous ERK3.
In the absence of
substrates, we tested the ability of ERK3 to autophosphorylate. Both
ERK3 and ERK3Ct underwent autophosphorylation (Fig. 6A), confirming that ERK3 is a protein kinase. A
conserved aspartate (Asp
) important for catalytic
activity (32) was mutated to alanine to create D171A ERK3. This
mutant did not autophosphorylate (Fig. 6D), ruling out
the possibility that an associated kinase phosphorylated ERK3.
Figure 6:
Autophosphorylation of ERK3. A,
autophosphorylation of GST-ERK3 and GST-ERK3Ct in vitro. B, phosphoamino acid analysis of autophosphorylated GST-ERK3
and GST-ERK3
Ct. The phosphoamino acid standards are indicated. C, comparison of the phosphorylation lips of ERK2 and ERK3;
ERK3 mutants are indicated. The phosphorylation sites of ERK2 are
indicated with asterisks. The identical residues between ERK2
and ERK3 are marked with vertical bars. D, top, autophosphorylation of histidine-tagged ERK3
Ct and
mutants in vitro; bottom, Coomassie Blue staining. E, phosphoamino acid analysis of autophosphorylated
ERK3
Ct and mutants. The phosphoamino acid standards are indicated.
In A, B, D, and E, autoradiograms
are shown.
Three
kinds of results suggest that ERK3 autophosphorylation was an
intramolecular reaction. First, ERK3 autophosphorylated in a
concentration-independent manner (data not shown). Second, ERK3 did not
phosphorylate D171A ERK3. In this experiment, GST-D171A ERK3 was mixed
with histidine-tagged wild type ERK3 and histidine-tagged D171A ERK3
was mixed with GST-ERK3. In neither case was autophosphorylation
detected (data not shown). Third, ERK3 and D171A ERK3 were not
phosphorylated by ERK3 immunoprecipitated from 293 cells with anti-ERK3
antibody D175. Phosphoamino acid analysis showed that both the ERK3 and
ERK3Ct autophosphorylate on serine (Fig. 6B) and
in both cases, the stoichiometry was less than 0.05 mol of
phosphate/mol of ERK3 even after 8 h.
Figure 7:
Peptide mapping of ERK3 and mutants
autophosphorylated. Figure shows autoradiograms of tryptic peptide map
of autophosphorylated GST-ERK3 (A), autophosphorylated
GST-ERK3Ct (B), autophosphorylated GST-S189A ERK3 (C), and mixture of autophosphorylated GST-ERK3 and GST-S189A
ERK3 (D).
Figure 8:
Phosphorylation of ERK3 in intact cells. A, pCEP4-HA and pCEP4-HA-ERK3 were transiently transfected
into 293 cells. HA-ERK3 was immunoprecipitated from P-labeled cell extracts with anti-HA peptide antibody
12CA5 and resolved on 10% acrylamide gels. An autoradiogram is shown. B, phosphoamino acid analysis of phosphorylated HA-ERK3. The
phosphoamino acid standards are indicated. C, pCEP4-HA,
pCEP4-HA-ERK3, and pCEP4-HA-S189A ERK3 were transiently transfected
into 293 cells. HA-ERK3 and the mutant were immunoprecipitated from
P-labeled cell extracts with 12CA5 and resolved on 10%
acrylamide gels. An autoradiogram is shown.
ERK3 is a 62-kDa protein, ubiquitously expressed in a variety
of rat tissues and mammalian cell lines. Two human ERK3 isoforms have
been cloned, one of which appears to be the homolog of rat
ERK3(11, 12) . The human homolog is alternatively
spliced resulting in ERK3 proteins of 62 and 97 kDa, the latter of
which has an additional 178 residues at its C terminus. In a variety of
cultured cell lines, the 62-kDa form was the only form detected by
immunoblotting with antibodies to different epitopes. Zhu et al.(12) also found only the 62-kDa form in cultured cells by
immunoblotting. However, in rat tissues two larger species, one of
97 kDa, were also found, indicating that multiple ERK3-related
proteins are expressed.
ERK3 is categorized as a member of the ERK subfamily based on characteristic features of its sequence. For example, subdomain V of ERK3 is 83% identical to ERK1 and subdomain IX is 72% identical. In contrast, subdomain V of cAMP-dependent protein kinase is only 17% identical to ERK1. Also striking is the similarity in lengths of inserts between conserved subdomains. On the other hand, ERK3 appears to lie on a distinct evolutionary branch from the rest of the ERK/MAP kinase family. Several properties of ERK3 distinguish it from all other known ERK homologs. ERK3 does not retain the activating tyrosine phosphorylation site, a hallmark of this family. It does not phosphorylate substrates recognized by other MAP kinases. ERK3 is constitutively nuclear; it is not translocated in response to a stimulus. However, it retains a much greater similarity to ERK1 and ERK2, particularly in the phosphorylation lip, than other known family members.
ERK3 does not phosphorylate MAP kinase substrates or
substrates of other MAP kinase homologs, including nuclear proteins
such as c-Jun. Songyang and Cantley examined the specificity of ERK3 by
screening peptide libraries as they have for other protein
kinases(35) . ERK3 phosphorylated peptides in the library,
further verifying that it has protein kinase activity. However, the
phosphopeptides represented too small a fraction to be purified,
preventing a determination of the phosphorylated sequences. ()These findings suggest that ERK3 has a very restricted
substrate specificity. Mutation of Tyr
of ERK2 to
glycine, as exists normally in that position of ERK3, greatly impairs
the ability of ERK2 to phosphorylate its substrates, suggesting that
the tyrosine residue may be a key factor in protein substrate
interactions in this kinase family. (
)
In contrast to our
results, the 97-kDa form of ERK3 isolated in immune complexes, was
reported to phosphorylate histone H1 and myelin basic
protein(12) . One explanation for this apparent discrepancy is
that the 97-kDa form of ERK3 may be a more active protein kinase. A
second possibility is that the ERK3 immunoprecipitates were
contaminated with another protein kinase. We have found at least one
protein kinase that binds ERK3 very tightly and phosphorylates not only
ERK3 but also myelin basic protein.
ERK3, unlike ERK1 and ERK2, is localized to the nucleus in the absence of serum factors. Similar results were obtained by immunofluorescence and by subcellular fractionation. The latter method allowed us to confirm that the protein detected was ERK3 based on its correct size. ERK3 appears to be tightly bound to nuclear sites, because almost all of it remained with nuclei during subcellular fractionation and required high salt or detergent for extraction. It appears that the C-terminal 180 residues stabilize but are not required for its nuclear localization. ERK3 lacking these C-terminal residues retains a primarily nuclear distribution, although some of the protein is found in the cytoplasm. This may be due to a reduction in its size, as the truncated protein is only 45 kDa and may diffuse more readily from the nucleus than the intact protein.
Peptide mapping indicated a single major phosphopeptide in
autophosphorylated ERK3. The presence of a comigrating phosphopeptide
in ERK3Ct indicates that this site is within the catalytic domain.
Ser
is the deduced phosphorylation site, based on the
loss of the phosphopeptide in an ERK3 mutant lacking this site.
Ser
lies in the phosphorylation lip between subdomains
VII and VIII and corresponds to the threonine regulatory
phosphorylation site in the MAP kinases. This suggests that ERK3 may be
regulated in a manner analogous to other MAP kinase family members.
ERK3 is poorly phosphorylated by MEK1 or MEK2, but is phosphorylated by
a partially purified activity present in both soluble and nuclear
extracts of cells.
ERK3 autophosphorylates on this site as
well but very weakly; thus, phosphorylation of this site in intact
cells is likely to be mediated by a regulatory enzyme such as the one
whose activity we have detected. Differences in both upstream
regulators and substrates suggest that ERK3 is not in the MAP kinase
cascade but in a distinct pathway. Because no ERK3 substrates have yet
been identified, it is not possible to test effects of Ser
phosphorylation on ERK3 activity. Thus, we have been unable to
ascertain if this phosphorylation regulates its activity. Current
efforts are directed toward the identification of ERK3 substrates that
will allow us to understand more about the function and regulation of
this unusual protein kinase.