From the
We have identified two components of a new protein kinase
signaling cascade, MAPK/ERK kinase 5 (MEK5) and extracellular
signal-regulated kinase 5 (ERK5). The MEK5 cDNA was isolated by
degenerate PCR and encodes a 444-amino acid protein, which has
approximately 40% identity to known MEKs. ERK5 was identified by a
specific interaction with the MEK5 mutants S311A/T315A and K195M in the
yeast two-hybrid system. The proteins were found to interact in an
in vitro binding assay as well. ERK5 did not interact with
MEK1 or MEK2. ERK5 is predicted to contain 815 amino acids and is
approximately twice the size of all known ERKs. The C terminus of ERK5
has sequences which suggest that it may be targeted to the
cytoskeleton. Sequences located in the N terminus of MEK5 may be
important in coupling GTPase signaling molecules to the MEK5 protein
kinase cascade. Both MEK5 and ERK5 are expressed in many adult tissue
and are abundant in heart and skeletal muscle. A recombinant GST-ERK5
kinase domain displays autophosphorylation on Ser/Thr and Tyr residues.
Some cell surface receptors respond to extracellular stimuli via
a sequential protein kinase cascade
(1, 2) . The kinase
signaling cascade involves multiple protein kinases, two of which are
extracellular signal-regulated kinase (ERK
We have
identified a new MEK, which we will refer to as MEK5. When MEK5 was
employed in the yeast two-hybrid system, we identified a new member of
the ERK family of protein kinases, which we have named ERK5. MEK5 and
ERK5 interacted specifically with one another and did not interact with
kinases in the MEK1/ERK1 signaling pathway, suggesting that the
MEK5/ERK5 protein cascade represents a novel signaling pathway in
higher eukaryotes. MEK5 contains unique amino acid sequences in its N
terminus, which suggest that this molecule may interact with GTPases
such as CDC42. In addition, the C terminus of ERK5 also contains
sequences which may target this kinase to the cytoskeletal elements
within the cell.
A human fetal brain cDNA library
was screened (Stratagene), using the cloned MEK5 PCR fragment labeled
with a multiprimer DNA labeling kit. Hybridization was carried out at
42 °C in 6
Two GST fusion proteins of ERK5 were created
for expression in E. coli, a full-length GST-ERK5 and a
truncated GST-ERK5 with the C-terminal 384 residues removed. This
truncated ERK5 corresponds to the kinase domain of the protein,
i.e. GST-ERK5(1-431). Both fusion proteins were purified
on glutathione-agarose affinity columns. The full-length GST-ERK5
protein had no detectable protein kinase activity (Fig. 5A and data not shown). The purified GST-ERK5(1-431) showed
basal autophosphorylation activity, as was evident from the radioactive
band of the predicted molecular weight following SDS-PAGE analysis
(Fig. 5A). Phosphoamino acid analysis of the radioactive
protein revealed phosphotyrosine, phosphothreonine, and phosphoserine
(Fig. 5B). These results suggest that the kinase domain
of the full-length ERK5 is rendered inactive by the presence of the
C-terminal 384 residues. Removal of the C-terminal domain results in
the ability of the functional ERK5 kinase domain to display its kinase
activity. The fact that ERK5, like ERK1, has a TEY motif and that
phospho-Ser/Thr and Tyr are observed in the analysis of labeled ERK5,
suggests that the catalytic properties of the two kinases may be
similar.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
We thank K.-L. Guan for plasmids encoding MEK1, MEK2
and ERK1. We also thank B. Yashar and R. Stone for critical reading of
the manuscript. We also acknowledge the help of J. Clemens, M. Wishart,
and C. F. Zheng with the yeast two-hybrid system.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
;
Refs. 3 and 4)
(
)
and MAPK/ERK kinase (MEK; Refs.
5 and 6).
(
)
Several ERK related proteins have
been identified in vertebrates including c-Jun N-terminal kinase (JNK;
Refs. 7 and 8)
(
)
and
p38
(9, 10, 11) . A variety of similar proteins
have also been identified in yeast
(12, 13, 14) .
All of the ERK kinases are activated by phosphorylation on Tyr and Thr
residues within a T
XY phosphorylation motif,
where ``X'' can be Glu, Pro, or Gly
(1). There are also a growing number of MEKs
(15, 16) .
In several cases, specific MEKs have been shown to phosphorylate
specific ERKs in a given pathway. MEK1 and MEK2 phosphorylate ERK1 and
ERK2 in a mitogenesis or differentiation response; MEK3 and p38 show
coordinate activation in the cytokine response, as do MEK4 (i.e. SEK1, the murine homolog of MEK4) and the JNKs in the stress
response. Sequential protein kinase cascades have been studied
extensively in yeast, where genetic tools have been invaluable in
assigning kinase cascades to specific signal transduction
pathways
(12, 13, 14) . In higher eukaryotic
systems, less is known about the specific stimuli responsible for
activation of individual signal transduction pathways. Although there
are clearly multiple genes encoding ERKs and MEKs, we have no good idea
about how many pathways there are in eukaryotic cells.
PCR and cDNA Screening
Two degenerate PCR
primers were synthesized based on the conserved amino acid sequences
present in MEKs from mammals and yeast
(6) . The sense primer was
5`-CTTGGATCCTA(C/T)ATA(C/T)GTNGGNTT(C/T)TA-3` and the antisense primer
was 5`-CTTGGATCC(G/T)TCNGGN(C/G)(A/T)CAT(A/G)TA-3`, which corresponds
to amino acid sequences YIVGFY and YMSPER, respectively. The underlined
BamHI sites were used for cloning the PCR product. Plasmid DNA
from a ZAP human fetal brain cDNA library was used as template.
The PCR was performed for 35 cycles at 94 °C for 1 min, 50 °C
for 2 min, and 72 °C for 2 min. The resultant 0.35-kb PCR products
were resolved by 3% Nusieve-agarose gel electrophoresis and subcloned
into a M13 vector and sequenced.
SSC (1
SSC is 0.15 M NaCl,
0.015 M sodium citrate, pH 7.0), 1
Denhardt's
solution (1
Denhardt's solution is 0.02% Ficoll 400,
0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone), 0.1 mg/ml
denatured salmon sperm DNA, 50% formamide. Both strands of the DNA
corresponding to the clone designated pBluescriptSK
MEK5 were sequenced.
The Yeast Two-hybrid Screen
The yeast strain Y190
(MATa, leu2-3, 112,
ura3-52 trp1-901 his3-200
ade2-101 gal4
gal 80
URA 3 GAL-lacZ LYS GAL-HIS3 cyh
) was a
generous gift of Stephen Elledge, Departments of Biochemistry and Human
and Molecular Genetics, Baylor College of Medicine. The NcoI
fragment encoding amino acid 12 through the stop codon of MEK5 were
subcloned into pAS1-CYH2 vector. Y190 was transformed with the
pAS1-CYH2-MEK5, pAS1-CYH2 S311A/T315A MEK5 mutant, or pAS1-CYH2 K195M
MEK5 mutant using the lithium acetate procedure
(17) . Plasmid
DNA from a HeLa cell Match Maker cDNA library was then transformed into
the yeast strain containing each ``bait'' plasmid.
Transformants were selected on SC yeast medium without Leu, Trp, His,
and with 35 mM 3-amino-1,2,4-triazole at 30 °C for
7-10 days
(17) . The transformants were lifted onto
nitrocellulose membrane and assayed for
-galactosidase
(17) . Plasmids were recovered from
-galactosidase positive clones and transformed into
Escherichia coli strain DH5
F` by electroporation. The
clone containing the longest ERK5 open reading frame was named A1.
In Vitro Binding Assay
A full-length cDNA encoding
the ERK5 protein sequence was constructed from two partial cDNAs into
pBluescriptSK (pBluescriptSK
ERK5).
This plasmid was used as template for in vitro transcription/translation employing T3 polymerase and a TNT kit
(Promega). The in vitro synthesized ERK5 was labeled with
[
S]Met, and incubated with
glutathione-agarose-immobilized GST, GST-MEK1, or GST-MEK5 at 4 °C
for 1-2 h in PBST (4 mM NaH
PO
,
16 mM Na
HPO
, 150 mM NaCl,
0.5% Triton X-100, pH 7.4), 0.05%
-mercaptoethanol, 10% glycerol,
0.2 mM phenymethylsulfonyl fluoride. Following SDS-PAGE, the
gel was treated with Amplify (Amersham Corp.) and autoradiographed.
Recombinant Proteins and Kinase Assays
GST-MEK5
was constructed by ligating a 2-kb NcoI fragment encoding
amino acid 12 through the stop codon of MEK5 into pGEX-KG
(18) .
GST-ERK5 was constructed by ligating the NcoI-BstEII
fragment of the cDNA clone 3-1 and the BstEII-XhoI
fragment of clone A1 (the XhoI site is in the vector) into
pGEX-KG digested with NcoI and SalI. GST-ERK5
(residues 1-431) was constructed by subcloning a 1.1-kb
NcoI fragment from pBluescriptSK ERK5
(encoding ERK5 amino acids 1-431) into pGEX-KG. GST-fusion
proteins were affinity-purified as described
(18) . The kinase
assays were performed at 30 °C for 10-40 min in a buffer
containing 18 mM Hepes (pH 7.5), 10 mM magnesium
acetate, 50 µM ATP, and 10 µCi of
[
-
P]ATP. Phosphoamino acid analysis was
carried out as described previously
(6) .
Isolation and Characterization of MEK5 cDNA
We
utilized PCR to identify additional proteins with sequence similarity
to MEK. Oligonucleotide primers for PCR amplification were based on the
conserved sequences from the yeast STE7 and byr1 genes and the partial amino acid sequence of the human
MEK1
(6) . A population of 0.35-kb fragments were produced by PCR
using human fetal brain cDNA as a template. The PCR products were
subcloned into an M13 vector. MEK1 cDNA was used subsequently as a
hybridization probe to eliminate MEK1 and MEK2 from the PCR clones. The
non-hybridizing clones were sequenced. Sequence analysis revealed
clones encoding MEK3 and MEK4
(16) as well as a novel MEK
homolog. We shall refer to the novel sequence as MEK5. The PCR fragment
of MEK5 was used to screen a human fetal brain cDNA library.
Twenty-four positive clones were analyzed by restriction mapping. The
longest insert (2.5 kb) was sequenced and yielded a predicted protein
sequence of 444 amino acids (Fig. 1). As shown in Fig. 1,
all 11 kinase subdomains are conserved for MEK5
(16) . The amino
acid sequence identity of MEK5 with known MEKs is approximately 40%.
The Raf-1 phosphorylation and activation motif of MEK1,
SXXXS
(19, 20) , was
also conserved as S
XXXT
in MEK5,
suggesting a similar regulatory mechanism. Like MEK3 and
MEK4
(15) , there was an amino acid sequence gap between domains
IX and X, resulting in deletion of the proline-rich region seen in MEK1
and MEK2. This region contains phosphorylation sites and negative
regulatory sequences
(5, 21) . The most divergent region
among the MEKs is upstream of kinase subdomain I. MEK5 is distinct from
other MEKs in that it contains a long N-terminal sequence. A data bank
search with the first 150 residues of MEK5 revealed sequence identity
with two proteins that have important roles in cell division, mating
and morphogenesis, namely the yeast cell division protein 24 (encoded
by Saccharomyces cerevisiaCDC24 gene
(22) and
the homologous protein encoded by the scd1 gene from
Schizosaccharomyces pombe(23) . The amino acid sequence
alignment of MEK5 with the encoded proteins of the scd1 gene
and CDC24 gene is shown in Fig. 2.
Figure 1:
Primary structure of MEK5. The primary
sequence of MEK5 was deduced from the sequence of cDNA clones isolated
from human fetal brain library. The PILEUP program (Wisconsin Genetics
Computation Group) was used to align the amino acid sequence of MEK5
with that of MEK1 and MEK2 (6), MKK3 and MKK4 (16), and SEK1 (15).
Romannumerals designate protein kinase domains; *,
conserved lysine in the ATP binding site and the activating
phosphorylation sites of MEK1; shading indicates identity of
all six sequences.
Figure 2:
Sequence alignment of MEK5 with the
scd1 gene and CDC24 gene encoded
proteins.
Genetic and
biochemical studies have demonstrated that the CDC24 encoded protein
has GDP release activity. When the CDC24 encoded protein binds to the
GTPase encoded by CDC42, it enhances the GTP/GDP exchange
(24) .
In a similar fashion, the scd1 encoded protein enhances the
nucleotide exchange of the corresponding S. pombe encoded
protein (cdc42sp). The binding domain of scd1 that recognizes cdc42sp
is located C-terminal to amino acid 671 of scd1 (23). It is this region
of scd1 that shows sequence identity to MEK5 as well as CDC24. Although
the function of this sequence is unknown, it is possible that the N
terminus of MEK5 interacts with the mammalian equivalent of CDC42. The
mammalian equivalent of yeast CDC42 is Rac (25). The amino acid
sequences in question may provide a mechanism for coupling GTPases
(i.e. CDC42 and Rac) to downstream protein kinase signaling
cascades.
A Partnership between MEK5 and ERK5
Mutations in
activating phosphorylation sites and the conserved lysine residue in
the ATP binding site of MEK1 result in dominant negative mutants that
interfere with the kinase function
(26, 27, 28) .
We anticipated that similar modifications in MEK5 might enhance its
interaction with upstream and/or downstream signaling molecules. To
enhance the enzyme-substrate interaction, mutations that led to the
dominant negative phenotype of MEK1 were made in MEK5, i.e. S311A/T315A and K195M
(20) . Using the yeast two-hybrid
system, three ``bait'' plasmids containing sequences encoding
wild-type MEK5, MEK5 S311A/T315A, or MEK5 K195M were used to screen a
HeLa cell library. From a pool of 4.2 10
transformants (2
10
from MEK5, 2
10
from S311A/T315A mutant, and 0.2
10
from K195M mutant), 6 clones were identified that encoded a novel
ERK, which we shall refer to as ERK5. Five clones in this pool were
obtained with the S311A/T315A mutant of MEK5 and one with the K195M
mutant. All clones contained the TEY activation motif. The product
encoded by the longest ERK5 clone, A1, interacted with MEK5 in the
yeast two-hybrid system, but not with MEK1 or MEK2 (Fig. 3,
A-C). In addition, MEK5 does not interact with p38 or
Raf A, B, or C in the yeast two-hybrid system. This result supports the
highly specific nature of the interactions between MEK5 and ERK5. It
should be noted that wild-type MEK5 also interacted with ERK5, although
we did not isolate any ERK5 clones from the initial yeast two-hybrid
screen. We anticipate that dominant negative MEKs may be useful in the
identification of additional upstream as well as downstream signaling
proteins in similar cascades.
Figure 3:
Specific interaction between MEK5 and
ERK5. The ERK5-containing plasmid A1 (ERK5 fusion with gal4 activation
domain) was cotransformed into yeast strain Y190 with vector pAS1-CYH2
or ``bait'' plasmids of MEK1, MEK2, MEK5, MEK5 S311A/T315A,
and MEK5 K195M. A, transformants were restreaked to SC medium
minus Leu, Trp, and His, plus 3-amino-1,2,4-triazole (35 mM)
to select for interaction. B, the transformants were also
restreaked to SC medium minus Leu and Trp and screened for
-galactosidase activity. C, diagram showing the
orientation of each transformant. D, in vitro translated ERK5 (1) was incubated with
glutathione-agarose-immobilized GST (2), GST-MEK1 (3), GST-MEK5 (4),
and GST-MEK5(K195M) (5) followed by washing and SDS-PAGE. After
treatment with Amplify, the SDS gel was autoradiographed with Kodak
x-ray film.
To obtain the full-length clone for
ERK5, the 2.5-kb insert of A1 was used to screen a human fetal brain
cDNA library. From 60 positive clones, two were used to complete
sequence analysis. The 2828-base pair sequence contained an open
reading frame of 2445 nucleotides encoding 815 amino acids
(Fig. 4). A number of residues important for the kinase activity
of ERKs are conserved, including the TEY activation motif
(1) .
ERK5 contains a 400-amino acid C-terminal domain, which houses two
proline-rich regions. The first proline-rich region, consisting of 32
amino acids, contains 16 prolines. The second proline-rich region (124
amino acids with 44 prolines) contained several small Pro-Ala repeats,
(PA), followed by Pro-Thr repeats, (PT)
, and
three repeats of PPGP. The (PA)
repeat is present
in myosin light chain kinase, and this sequence has been shown to
directly interact with actin, targeting the kinase to a specific
location in the cell
(29) . The unique ERK5 C-terminal sequence
may serve as a localization domain and/or a regulatory domain. It is
interesting to speculate that the (PA)
sequences
which target myosin light chain kinase to actin may also target ERK5 to
a similar location. Northern blot analysis of ERK5 mRNA (3.1 kb) and
MEK5 mRNA (2.7 kb) demonstrated they are expressed in many human
tissues and that the RNAs are most abundant in heart and skeletal
muscle (data not shown).
Figure 4:
The primary structure of ERK5.
A, the deduced amino acid sequence of ERK5 was
aligned with that of ERK1 and ERK2, JNK1 and human p38.*, lysine
residue in the conserved ATP binding site and important catalytic amino
acid residues; &cjs1219;, activating phosphorylation motif.
Proline-rich region 1 and proline-rich region 2 are
underlined.
To further document the specific
interaction between MEK5 and ERK5, in vitro binding assays
were conducted (Fig. 3D). In this experiment,
full-length ERK5 was synthesized in an in vitro transcription/translation system. As shown in
Fig. 3D, ERK5 binds to GST-MEK5 and the GST-MEK5 mutant
K195M, but not to GST alone or to GST-MEK1. Thus, MEK5 specifically
interacted with ERK5 in both our in vitro system and in the
yeast two-hybrid system.
Protein Kinase Activity of MEK5 and ERK5
GST
fusion protein containing full-length MEK5 could not be overexpressed
in E. coli. However, elimination of the N-terminal 11 residues
produced a MEK5 fusion protein which could be overexpressed and
affinity-purified. The purified protein showed no protein kinase
activity toward ERK1, JNK1, or GST-ERK5. However, a weak
autophosphorylation activity could be detected with the wild-type
enzyme but not with the MEK5 K195M mutant (data not shown). These
experiments suggested that additional modifications are required for
full kinase activity.
Figure 5:
Autophosphorylation of recombinant
GST-ERK5 (1-431). A, autophosphorylation of purified
full-length GST-ERK5 and GST-ERK5 (1-431). The samples were
resolved on a SDS-PAGE gel followed by autoradiography. The arrows indicate the position of the purified GST-ERK5 and GST-ERK5
(1-431). B, phosphoamino acid analysis of
autophosphorylated GST-ERK5 (1-431 shown in A. P-S,
phosphoserine; P-T, phosphothreonine; P-Y,
phosphotyrosine.
In summary, we have identified two novel kinases that
interact selectively with one another in what appears to represent a
new mammalian signal transduction pathway. Our results suggest that
there are no interactions between the proteins in the MEK1/ERK1 pathway
and the MEK5/ERK5 pathway, suggesting that the two signaling pathways
have either different or complementary functions. Our results also
suggest that the two different signaling pathways most likely do not
have overlapping functions in the cell. We are currently attempting to
identify both upstream activators of MEK5 as well as downstream
substrates of ERK5. The unusual length of the C-domain of ERK5 suggests
that the sequences located here may undergo additional
post-translational modifications as well as play roles in regulating or
targeting of ERK5.
/EMBL Data Bank with accession number(s) U25265 and
U25278.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.