(Received for publication, June 26, 1995; and in revised form, September 18, 1995)
From the
The prototype mitogen-activated protein (MAP) kinase module is a
three-kinase cascade consisting of the MAP kinase, extracellular
signal-regulated protein kinase (ERK) 1 or ERK2, the MAP/ERK kinase
(MEK) MEK1 or MEK2, and the MEK kinase, Raf-1 or B-Raf. This and other
MAP kinase modules are thought to be critical signal transducers in
major cellular events including proliferation, differentiation, and
stress responses. To identify novel mammalian MAP kinase modules,
polymerase chain reaction was used to isolate a new MEK family member,
MEK5, from the rat. MEK5 is more closely related to MEK1 and MEK2 than
to the other known mammalian MEKs, MKK3 and MKK4. MEK5 is thought to
lie in an uncharacterized MAP kinase pathway, because MEK5 does not
phosphorylate the ERK/MAP kinase family members ERK1, ERK2, ERK3,
JNK/SAPK, or p38/HOG1, nor will Raf-1, c-Mos, or MEKK1 highly
phosphorylate it. Alternative splicing results in a 50-kDa and a
40-kDa
isoform of MEK5. MEK5
is ubiquitously distributed and
primarily cytosolic. MEK5
is expressed most highly in liver and
brain and is particulate. The 23 amino acids encoded by the 5` exon in
the larger
isoform are similar to a sequence found in certain
proteins believed to associate with the actin cytoskeleton; this
alternatively spliced modular domain may lead to the differential
subcellular localization of MEK5
.
A common element in many eukaryotic regulatory pathways is a
three-kinase cascade, known as a MAP kinase module. A
module consists of three protein kinases that act sequentially within a
pathway: a MAP kinase kinase kinase or MEKK (a MEK activator), a MAP
kinase/ERK kinase or MEK (a MAP kinase activator), and a MAP kinase or
ERK (extracellular signal-regulated protein kinase) homolog. First
recognized in yeast(1) , several MAP kinase modules have now
been identified in mammalian systems(2, 3) . This kind
of three-kinase regulatory cascade conveys information to target
effectors, coordinates incoming information from parallel signaling
pathways, confers a vast potential for amplification and specificity,
and incorporates multiple inactivation mechanisms. The first and best
studied is the MAP kinase pathway, made up of Raf-1 or B-Raf, MEK1 or
MEK2, and ERK1 or ERK2 (4) .
The closely related MAP kinases, ERK1 and ERK2(5, 6, 7, 8) , are ubiquitous signal transducers. They are activated by diverse extracellular stimuli and by proto-oncogene products that induce proliferation or enhance differentiation(7) . In addition to ERK1 and ERK2, other related mammalian enzymes have been detected including: several ERK3 isoforms (7, 9, 10) , ERK4(11) , Jun N-terminal kinases/stress-activated protein kinases (JNK/SAPKs)(12, 13) , p38/HOG1(14, 15, 16) , and p57 MAP kinases (17) . The presence of at least six MAP kinases in yeast (18) coupled with Southern analysis of rat DNA (7) suggest that there are numerous MAP kinase relatives and a corresponding number of MAPK kinase modules in mammals.
The two dual specificity MAP kinase kinases, MEK1 and MEK2(19, 20, 21, 22, 23, 24) , are the only known enzymes capable of phosphorylating and activating ERK1 and ERK2. Several laboratories have recently uncovered additional MEKs, for which some substrates have been defined. A mammalian homolog of a MEK first identified in Xenopus(25) is called MAP kinase kinase 4 (MKK4)(26) , SAPK/ERK kinase (SEK) (27) or JNK kinase (JNKK) (28) and activates JNK/SAPK and p38/HOG1, but not ERK1 or ERK2. Yet another newly cloned MEK, MKK3, selectively activates p38/HOG1 in transfected cells(26) . Thus, the second mammalian MAP kinase module to be defined apparently consists of MEKK1 (the MEKK)(29, 30, 31) , MKK4 (the MEK), and SAPK/JNK (the ERK), and a third module contains MKK3 and p38/HOG1. It is likely that the MEK in each module will confer much specificity in signaling, as evidence to date suggests that the MEKK and ERK components are more promiscuous enzymes(3) .
MAP kinase modules are thought to be critical participants in major
cellular events including proliferation, differentiation, and stress
responses(4, 32) . Thus, an important goal is to
identify and characterize other parallel cascades. Because of their
enzymatic specificity, we chose to search for additional members of the
MEK family. A PCR-based approach yielded a new MEK family member, MEK5,
which by sequence is slightly more related to MEK1, MEK2, and the
fission yeast MEK homolog Byr1 than to MKK3 or MKK4. None of the known
ERK/MAP kinase family members are substrates for MEK5 nor will Raf-1,
c-Mos, or MEKK1 highly phosphorylate it, suggesting that MEK5 is part
of a novel MAP kinase module. Consistent with this biochemical data,
MEK5 failed to complement deletions of MEKs in four yeast MAP kinase
pathways. MEK5 exists in at least two spliced forms; the smaller
isoform is ubiquitously distributed and is primarily cytosolic, while
the larger
isoform is expressed most highly in liver and brain
and is primarily particulate. The alternatively spliced exon in
MEK5
contains a sequence conserved in certain proteins thought to
interact with the actin cytoskeleton and is likely to account for its
differential localization. This is the first report of differential
intracellular targeting of proteins in MAP kinase modules directed by
alternative splicing.
One 150-bp PCR product was used to screen an oligo dT-primed
rat brain cDNA library (Stratagene). Two hybridizing clones were
isolated, neither of which contained a full-length cDNA. To obtain a
full-length clone, one of the partial clones was used to screen a
random-primed rat forebrain cDNA library (kindly provided by J.
Boulter, Salk Institute) with P-labeled, randomly primed
probes. Sequencing of six isolates was performed using the
dideoxynucleotide chain termination method (36) and an Applied
Biosystems automated sequencer.
To confirm the 3` end of the clone,
3` nested RACE was performed using oligo(dT)-primed first strand cDNA
from rat brain and testes as template. MEK5 5` primers were
CAGGATTTCGATTTGTACAG, GTGAAGCCTTCCAACATGC, and GAGGTTCTCGGAGCCGTTTG; 3`
primers were GACTCGAGTCGACATCGAT and GACTCGAGTCGACATCGAT.
Figure 1:
Sequence of MEK5. A,
full-length nucleotide and deduced amino acid sequence of rat MEK5.
Sequence corresponding to peptides used to make antisera N797
(N-terminal peptide) and L610 (C-terminal peptide) are underlined. Stop codons are denoted by asterisks. The
two alternatively spliced exons are boxed. The polyadenylation
signal is indicated by the dotted line. B, schematic
representations of MEK5 and MEK5
. Top schematic, the
inclusion of the 68-bp exon in the 5` untranslated sequence of
MEK5
results in an upstream change in frame usage. This adds 89
amino acids to the predicted sequence of MEK5
, yielding a protein
of 448 amino acids. Bottom schematic, in MEK5
, without
the 68-bp exon, the first methionine (doubly underlined in A) downstream of the exon splice site is used for inititation
yielding a protein of 359 amino acids. C, sequence alignment
of MEK catalytic domains. Rat MEK5 is aligned with human
MEK1(23) , human MKK4(26) , human MKK3(26) , S. cerevisiae MEKs, PBS2(48) , MKK1(47) , and
STE7(46) , and S. pombe MEKs, Wis1 (65) and
Byr1(50) . The sequence alignment was generated using the
PILEUP program (version 8.0; Wisconsin Genetics Computer Group).
Protein kinase subdomains (66) are indicated above the
sequence. The conserved, putative activating phosphorylation sites are
indicated by asterisks. D, comparison of MEK family
members. The dendrogram was produced using the PILEUP program. E, Northern analysis of MEK5. An autoradiogram of a blot,
containing 15 µg of poly(A
)-enriched RNA isolated
from the adult rat tissue indicated above each lane, probed with a PstI fragment of MEK5 is shown. The size markers are denatured
P-labeled HindIII fragments of bacteriophage
DNA. (A-) is 15 µg of
poly(A
)
RNA.
A second alternative splicing event was discovered when two clones
were isolated from the random-primed library that contained a second
alternatively spliced exon of 68 bp near the 5` end of MEK5. The
inclusion of this alternatively spliced 5` exon generates an initiation
site 5` to the spliced exon and to the initiation site in MEK5.
Initiation at the more 5` site produces a predicted protein, MEK5
(Fig. 1A), that is 89 amino acids longer at the N
terminus than MEK5
(Fig. 1B). This 68-bp exon (Fig. 1A) causes an upstream start codon to be in frame
with the kinase domain and contains no intervening in-frame stop
codons. Thus, the inclusion of an alternatively spliced 5` exon
generates a distinct initiation site in MEK5
. Alternative splicing
of the 5` and 3` exons may generate four forms of MEK5. We have
isolated MEK5
clones both with and without the subdomain IX-X
exon. It is not clear whether MEK5
exists in both forms.
Northern analysis suggests that MEK5 is expressed in all the tissues examined and that alternatively spliced variants exist. A major band of 2.5 kb was detected in brain and testes, and a slightly larger band of 2.8 kb was noted in liver and spleen (Fig. 1E). In testes an additional weakly hybridizing band of 3.8 kb was detected. Immunoblotting analysis indicates tissue selective expression of MEK5 isoforms (see below).
Figure 2:
Protein kinase activity of MEK5 and
mutants. Aspartic acid was introduced in place of the putative
phosphorylation sites, Ser-222 and Thr-226, in MEK5
by PCR with
Vent polymerase and oligomers that spanned these amino acids. The 3`
primer corresponding to the C terminus encoded a stop codon as well as
a HindIII site. Each resulting PCR product was subcloned into
the PvuII and HindIII sites of the MEK5 pGEX-KG
construct lacking the subdomain IX-X exon, and then a StuI-BamHI fragment from this form was subcloned into
the construct containing the IX-X exon. 30 µg/ml wild type
GST-MEK5
(with and without the subdomain IX-X exon) and the
indicated mutants of it were incubated either with or without 10
µg/ml MBP under phosphorylating conditions. Recombinant wild type
His
-MEK1 was incubated with and without MBP as a control.
Autoradiogram is shown.
Because of the low
activity of MEK5 and lack of information about its upstream activators,
efforts were made to activate MEK5 by changing its putative
phosphorylation sites to acidic residues. Comparable mutations resulted
in biochemical and functional activation of MEK1(41) . Thus,
aspartic acid was introduced into each of the phosphorylation sites
singly and together (double mutants) in MEK5 (Fig. 2). The
activity of these mutant proteins assayed with MBP was no greater than
that of the wild type proteins. A similar strategy also worked poorly
for MKK4.
Only a single MAP kinase module has been well characterized in
the fission yeast, Schistosaccharomyces pombe(18) .
This module, composed of the protein kinases Byr2 (MEKK), Byr1 (MEK),
and Spk1 (ERK) is required for mating pheromone responses(1) .
MEK5 was expressed from the strong S. pombe adh1 promoter in
mutants carrying null mutations of the byr1 gene(50) .
MEK5 failed to suppress the mating defect of a byr1S. pombe haploid strain and
the sporulation defect of a byr1
/byr1
diploid
mutant.
Figure 3:
Immunoblots of proteins from rat tissues
with anti-MEK5 antisera. A, specificity of MEK5 antibodies.
Duplicate blots of recombinant GST-MEK5 and GST-MEK5
were
probed with MEK5 antiserum N797 (left) and antiserum L610 (right). B, detection of endogenous MEK5
and
in rat tissues with anti-MEK5 antiserum L610. Left panels show clarified whole cell lysates (buffer A, as described under
``Materials and Methods''). Right panels show soluble
extracts (buffer B described under ``Materials and
Methods''). Upper panels, probed with a 1:500 dilution of
L610 antiserum; lower panels, probed with a 1:500 dilution of
L610 antiserum preincubated with 50 µg/ml of its antigenic peptide.
Immunoreactive bands specifically blocked by preincubation with the
peptide are indicated by the arrows and correspond to
MEK5
and
. Molecular size standards are indicated between the
blots. E. fat, epididymal fat; CHO, Chinese hamster
ovary cells. C, MEK5
is not cytosolic. Blots of whole
cell lysates (A) and supernatants (B) from rat liver
were probed with preimmune (left) or immune (right) N797 antiserum.
We have cloned multiple forms of a novel MEK family member,
MEK5. The smaller isoform is a ubiquitous cytosolic protein,
while the larger
isoform is particulate and has a more limited
expression in tissues and cells. The MEK5 catalytic domain has features
unique to the MEK protein kinase subfamily. The putative
phosphorylation sites (SIAKT) are in conserved positions
in the phosphorylation lip between subdomains VII and VIII and the lip
is of the same length as most other members of the family. MEK5 also
displays the MEK consensus sequence, CXXK, near the C terminus
of the catalytic domain, not present in other protein kinases.
A canonical MAP kinase module consists of three protein kinases that act sequentially within one pathway: a MEKK, a MEK, and an ERK/MAP kinase. The exquisite specificity of MEKs leads to the selective activation of a defined group of ERK-related enzymes. Pathway specificity is achieved in part by pairing of ERK-related enzymes with their MEK-like activators. MEK5 apparently lies in an, as yet, undescribed MAP kinase module, as it neither phosphorylates ERK1, ERK2, ERK3, JNK/SAPK, or p38/HOG1 nor is phosphorylated by known MEKKs, Raf-1, c-Mos, or MEKK1.
Data base searches (NCBI BLAST network services) using the
N-terminal sequence unique to MEK5 revealed a region of sequence
identity to the yeast putative GTP/GDP exchange factors scd1 (51) and CDC24(52) , the human p40
protein(53) , and the transmembrane tyrosine kinase,
Ros(54) . This region of identity exactly overlaps the
MEK5
alternative exon, implying that this sequence encodes a
modular domain utilized by a number of proteins to specify subcellular
localization. Of proteins with sequence related to the exon, Scd1 and
CDC24 are believed to encode guanine nucleotide exchange factors for
the yeast Rac/Rho family member Cdc42 (55, 56) in S. pombe and S. cerevisiae, respectively. The Rac/Rho
family of small G proteins has been linked to control of the actin
cytoskeleton in mammals(57, 58) . In yeast these
exchange factors appear to mediate certain Ras-regulated functions,
particularly cytoskeleton-dependent control of cell morphology.
p40
is an essential component of the leukocyte NADPH
oxidase system, which is directly regulated by Rac(53) . The
distinct localization of MEK5
that results from expression of the
longer N terminus is consistent with a role for sequence encoded by
this exon in cellular localization and subsequent signaling
capabilities.
Subcellular localization is recognized as an important, but poorly understood, contributor to signaling and enzymatic specificity(59, 60) . Most frequently, targeting of signaling molecules has been accomplished by either regulatory or dedicated targeting subunits. Well studied examples include the G subunit of phosphoprotein phosphatase I (61) and the AKAPs (A kinase anchor proteins), which serve to target cAMP-dependent protein kinase(62) . Recently, distinct isoforms of phosphoprotein phosphatases, including the tyrosine phosphatase PTP1(60, 63) , expressed as a consequence of alternative splicing, have been found localized to different subcellular compartments, including the cytoskeleton and the nucleus. Their concentration at these specific sites is a significant determinant of their substrate availability. A number of protein kinases are thought to exist in multiple alternatively spliced forms. However, MEK5 provides the first reported instance of differential intracellular targeting of a kinase in any MAP kinase module as a result of alternative splicing. Within MAP kinase modules, the current dogma is that the MAP kinase itself translocates from cytoplasm to nucleus under the control of the activating signal by an unknown mechanism. The MEK component, on the other hand, has been assigned a static role in the cytoplasm. Perhaps, in addition to its already highly selective enzymatic role, a MEK acts to direct and tether its respective MAP kinase module to various sites of action within the cytoplasm. In the case of MEK5 subcellular targeting also occurs in a tissue-specific manner. Targeting of the MEK might restrict substrates available to its otherwise promiscuous downstream ERK. Thus, alternative splicing may provide another mechanism for targeting signaling molecules to generate specificity in protein kinase pathways.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U37462[GenBank], U37463[GenBank], and U37464[GenBank].
Addendum-After
this work was completed, Zhou et al.(64) published
the sequence of the human MEK5 isoform. They also provided
additional evidence that MEK5 lies in a distinct MAP kinase module by
their cloning of a novel ERK family member (ERK5) in a two-hybrid
screen with MEK5(64) . Data concerning phosphorylation of any
substrates, including ERK5, by MEK5 were not reported as they also
could not measure MEK5 activity toward potential physiological
substrates.