(Received for publication, December 19, 1995; and in revised form, February 26, 1996)
From the
Mitogen-activated protein kinases are members of a conserved cascade of kinases involved in many signal transduction pathways. They stimulate phosphorylation of transcription factors in response to extracellular signals such as growth factors, cytokines, ultraviolet light, and stress-inducing agents. A novel mitogen-activated protein kinase kinase, MEK6, was cloned and characterized. The complete MEK6 cDNA was isolated by polymerase chain reaction. It encodes a 334-amino acid protein with 82% identity to MKK3. MEK6 is highly expressed in skeletal muscle like many other members of this family, but in contrast to MKK3 its expression in leukocytes is very low. MEK6 is a member of the p38 kinase cascade and efficiently phosphorylates p38 but not c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) family members in direct kinase assays. Coupled kinase assays demonstrated that MEK6 induces phosphorylation of ATF2 by p38 but does not phosphorylate ATF2 directly. MEK6 is strongly activated by UV, anisomycin, and osmotic shock but not by phorbol esters, nerve growth factor, and epidermal growth factor. This separates MEK6 from the ERK subgroup of protein kinases. MEK6 is only a poor substrate for MEKK, a mitogen-activated protein kinase kinase kinase that efficiently phosphorylates the related family member JNKK.
Protein phosphorylation plays a major role in many signal
transduction pathways. Stress-activated or mitogen-activated protein
kinases (MAPKs) ()are members of a conserved cascade of
kinases that stimulate phosphorylation of transcription factors and
other targets in response to extracellular signals such as growth
factors, cytokines, ultraviolet light, and stress-inducing agents. In
higher eukaryotes the physiological role of MAPK signaling has been
correlated with cellular events such as proliferation, oncogenesis,
development and differentiation, and cell cycle. Several MAPK cascades
have been identified in yeast and vertebrates (for review see (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) ).
Each cascade consists of several modules. Most of the mammalian MAPK
modules have been identified by analogy with yeast protein kinase
cascades. We are following the nomenclature of Seger and Krebs (10) and name the individual levels of kinases regardless of
their pathway MAPK, MAPKK, and MAPKKK. The extracellular
signal-regulated kinase (ERK) subgroup of MAPKs has been examined in
detail(11, 12, 13, 14, 15, 16) .
The c-Jun N-terminal kinase (JNK) subgroup (also known as
stress-activated protein kinase, or SAPK) was first described as a
kinase cascade leading to the phosphorylation of
Jun(17, 18, 19) . More recently the p38 MAPK
pathway has been identified in analogy to the yeast HOG1
pathway(20, 21) .
We were interested in identifying novel members of the MAPKK family that use p38 as substrate. So far only two of these kinases have been identified in humans: MKK3 and JNKK (MKK4, SEK). JNKK is a substrate for MEKK1 (22, 23, 24) and phosphorylates and activates JNK1, JNK2, and p38 in vivo and in vitro(23, 25) . In contrast, MKK3 phosphorylates and activates exclusively p38(25) ; however, the upstream MAPKKK for MKK3 is unknown, since MEKK1 is not an activator of p38 in vivo(22) . In an attempt to find novel members of the p38 MAP kinase cascade we cloned and characterized a new human MAPKK, which we named MEK6. MEK6 differs from its closest homologue, MKK3, most significantly in the N-terminal and C-terminal amino acid regions. MEK6 efficiently phosphorylates p38 but not JNK or ERK. MEK6 is strongly activated by UV, anisomycin, and osmotic shock.
Human p38
(GenBank accession number U10871) was cloned by PCR
amplification of a Jurkat cDNA library with primers against the 5` end
(5`-CCAACCATGGCTCAGGAGAG-3`) and 3` end (5`-CGGTACCTTCAGGACTCCATCT-3`)
of the published human p38 sequence. Each strand of the PCR fragment
was sequenced several times with an ABI 373 automated sequencer.
Figure 1: Primary structure of MEK6. The primary amino acid sequence of MEK6 was deduced from the sequence of the cDNA clone isolated from a human MOLT-4 cDNA library. The MacVector program (Kodak-IBI) was used to align the amino acid sequence of MEK6 with that of MKK3(25) . Asterisks indicate the conserved lysine in the ATP binding site and the dual phosphorylation motif. The accession number for the MEK6 sequence is U49732.
The expression of human MKK3 and MEK6 was examined by Northern blot
analysis of RNA isolated from various human tissues. MKK3 is widely
expressed in many adult human tissues with highest levels in skeletal
muscle and leukocytes (Fig. 2A). In contrast, MEK6 is
predominantly expressed in skeletal muscle and at lower levels in heart
and pancreas (Fig. 2B). All tissue samples expressed
similar levels of -actin mRNA (data not shown).
Figure 2: Tissue distribution of MKK3 and MEK6. The expression of human MKK3 (A) and human MEK6 (B) mRNA in selected adult human tissues was investigated by Northern blot analysis. The position of RNA size markers in kb is shown on the left.
Figure 3:
Substrate specificity of MEK6. A,
recombinant, purified GST or GST-MEK6 was used in a kinase reaction
with 1 µg of purified recombinant MAPK substrate (GST, GST-JNK2,
GST-p38, His-ERK1(K52R)) as described under ``Experimental
Procedures.'' B, coupled kinase assay. Purified GST or
GST-MEK6 were incubated with purified GST-JNK2, GST-p38, or GST in the
presence of JNKK buffer and 100 µM ATP. The proteins were
isolated by binding to GSH-Sepharose and, after washing, incubated with
GST-c-Jun(1-79) or GST-ATF2 in the presence of JNK buffer and
[-
P]ATP. Reactions were separated by
SDS-polyacrylamide gel electrophoresis and visualized by
autoradiography. The position of protein molecular mass markers in kDa
is shown on the left.
Figure 4:
Time course of MEK6 induction. HeLa cells
were transiently transfected with epitope-tagged MEK6 and treated with
anisomycin (50 ng/ml) or UV (254 nm; 120 J/m) for the times
indicated. Cell lysates were prepared and used in an immune complex
kinase assay with GST-p38 substrate as described under
``Experimental Procedures.'' Reactions were separated by
SDS-polyacrylamide gel electrophoresis and quantitated with a
PhosphorImager and ImageQuant software. The relative level of MEK6
activity in untreated cells was arbitrarily assigned 1. The presence of
equal amounts of MEK6 in all kinase reactions was confirmed by Western
blot analysis of the cell lysates with an anti-HA antibody (data not
shown).
Figure 5:
UV
dose response of MEK6. HeLa cells were transiently transfected with
epitope-tagged MEK6 and activated for 45 min with 20-120
J/m UV (254 nm) as indicated. Cell lysates were used in an
immune complex kinase assay with GST-p38 substrate as described under
``Experimental Procedures.'' Reactions were separated by
SDS-polyacrylamide gel electrophoresis and visualized by
autoradiography. The positions of protein molecular weight markers in
kDa are shown on the left. MEK6 activity was quantitated with
a PhosphorImager and ImageQuant software and is shown in a bar
graph. The presence of equal amounts of MEK6 in all kinase
reactions was confirmed by Western blot analysis of the cell lysates
with an anti-HA antibody (data not shown).
To determine whether the increase
in p38 phosphorylation by activated MEK6 augments p38 kinase activity,
a coupled immune complex kinase assay was performed. Epitope-tagged
MEK6 was isolated from anisomycin-treated HeLa cells and subjected to
two subsequent kinase reactions using recombinant p38, ATF2, and GST
alone. In support of our in vitro results, anisomycin
treatment caused increased phosphorylation of ATF2 only when MEK6 and
p38 were present (Fig. 6, compare lanes 5 and 6 with lanes 7 and 8). Similar results have been
found with MEK6 activated by UV treatment of cells (data not shown). No
inducible phosphorylation of p38 or ATF2 was observed in HeLa cells
transfected with the empty expression vector SR3 (Fig. 6,
compare lanes 5 and 6 with lanes 13 and 14). This clearly indicates that the inducible phosphorylation
of ATF2 depends on a kinase cascade comprised of MEK6 and p38.
Interestingly, p38 also phosphorylated weakly a protein with a mobility
slightly faster than ATF2 (indicated by an asterisk in Fig. 6). This phosphorylation event was slightly augmented by
anisomycin in the presence of MEK6 (Fig. 6, compare lanes 3 and 4 with lanes 11 and 12).
Figure 6:
Coupled kinase assay in HeLa cells. HeLa
cells were transiently transfected with epitope-tagged MEK6 (lanes
1-8) or the empty expression vector SR3 (lanes 9 and 16) and treated for 45 min with anisomycin (An., 50 ng/ml) or left untreated (ctrl) as
indicated. Cell lysates were prepared and used in a coupled immune
complex kinase assay as described under ``Experimental
Procedures'' and the legend to Fig. 3. The positions of
protein molecular mass markers in kDa are shown on the left.
The positions of p38, ATF2, and an unknown protein (*) are indicated on
the right.
Figure 7:
Stimulators of MEK6 in vivo. HeLa (A) or COS cells (B) were transiently transfected
with epitope-tagged MEK6 (lanes 1-12) or the empty
expression vector SR3 (lanes 13-16) and treated for
45 min with interleukin-1
(IL-1
, 10 ng/ml; R & D
Systems), tumor necrosis factor-
(TNF-
, 10 ng/ml; R
& D Systems), EGF (50 ng/ml; Life Technologies, Inc.), NGF (50
ng/ml; Life Technologies, Inc.), phorbol 12-myristate 13-acetate (PMA, 50 ng/ml; Sigma), anisomycin (50 ng/ml; Sigma),
cycloheximide (CX, 50 ng/ml; Sigma), arsenite (Arsen., 200 µM; Sigma), NaCl (200 mM;
Sigma), or UV (254 nm; 120 J/m
) or cotransfected with 1000
ng CMV5-MEKK as indicated. Cell lysates were used in an immune complex
kinase assay with GST-p38 substrate as described under
``Experimental Procedures.'' The positions of protein
molecular mass markers in kDa are shown on the left. MEK6
activity was quantitated with a PhosphorImager and ImageQuant software.
The presence of equal amounts of MEK6 in all kinase reactions was
confirmed by Western blot analysis (data not
shown).
Figure 8: Effect of MEKK on MEK6 and JNKK activity. COS cells were transiently transfected with epitope-tagged MEK6 (lanes 1-7) or JNKK (lanes 8-12) and increasing amounts of CMV5-MEKK expression vector as indicated. The total amount of transfected DNA was kept constant by adding empty CMV expression vector. Cell lysates were used in an immune complex kinase assay with GST-p38 (lanes 1-7) or GST-JNK2 (lanes 8-12) substrate as described under ``Experimental Procedures.''
In this report, we describe the cloning and characterization
of a novel member of the MAPKK family of dual specificity protein
kinases, which we named MEK6. MEK6 was first identified in a BLAST
homology search of the EST subdivision of NCBI GenBank as
a 223-bp partial cDNA fragment. The full-length cDNA was obtained by
PCR amplification of Jurkat and MOLT-4 cDNA libraries with
gene-specific primers. We minimized the chance of introducing errors in
the amplified sequence by using a DNA polymerase mixture with
proofreading function. Further, we sequenced three independent clones
that were identical. The cDNA has a long open reading frame that is
preceded by about 250 bp of sequence with stop codons in all three
reading frames. The size determination of in vitro translated
protein indicates that translation starts at the identified start codon
of MEK6 cDNA. Further, Northern blot analysis revealed that the size of
MEK6 mRNA corresponds to the size of MEK6 cDNA. This suggests that we
cloned the full-length cDNA of MEK6. MEK6 has significant homology on
the amino acid level to MKK3 (82% amino acid identity) but is not
significantly related to MKK3 at the DNA level. This clearly
demonstrates that MEK6 is encoded by a novel gene. MEK6 differs from
MKK3 most significantly in the C terminus and N terminus, which has an
additional 18 amino acids. All relevant kinase subdomains, the ATP
acceptor site, and phosphorylation sites are conserved.
Many members
of the MAPK cascades are expressed at high levels in skeletal
muscle(18, 25, 33) , which prompted us to
compare the expression of MEK6 and its closest homologue MKK3 in 16
different adult human tissues. We observed very high expression of MEK6
mRNA in skeletal muscle, consistent with the high levels of other MAPK
family members, but surprising was the absence of MEK6 mRNA in spleen,
thymus, prostate, ovary, small intestine, colon, and leukocytes.
Analysis of the same blot showed that MKK3 is widely expressed, and all
16 tissues expressed equal amounts of -actin mRNA. We assume that
MEK6 is expressed in these tissues at levels that are below the
detection limit of Northern blot analysis. The fact that we isolated
MEK6 cDNA from human T cells implies that activated T cells can express
MEK6. Interestingly, some of the tissues expressed an MEK6-related mRNA
of about 4.2 kb. This band was not observed when we used a
MEK6-specific probe that was directed against the 3` end of MEK6 cDNA.
Similarities between MEK6 and MKK3 prompted us to investigate
whether MEK6 is able to utilize p38 as substrate. In vitro MEK6 efficiently phosphorylated p38 but not ERK and JNK, although
in parallel experiments the phosphorylation of JNK by JNKK was observed
(data not shown). This indicates that MEK6, like MKK3, has substrate
selectivity for the p38 subgroup of MAPK. Activation of p38 requires
phosphorylation of Thr and
Tyr
(20) . We subjected p38 that has been
phosphorylated by MEK6 to a second kinase reaction with ATF2 as
substrate. MEK6 induced phosphorylation of ATF2 by p38 but did not
directly phosphorylate ATF2. These experiments were carried out with
MEK6 prepared in bacteria as GST fusion protein and with epitope-tagged
MEK6 isolated from HeLa cells after stimulation with anisomycin or UV.
These results demonstrate that MEK6 specifically phosphorylates p38,
resulting in its activation.
In some of the coupled kinase assays we also observed extra bands migrating slower and faster than ATF2. Some of these bands represent degradation products of ATF2 and phosphorylated forms of p38 and MEK6, as indicated in the figures. Additionally, we observed weak phosphorylation of a protein with a migration slightly faster than ATF2. This protein was not observed in in vitro kinase assays and therefore is most likely a contamination of the immunoprecipitation.
MEK6 is strongly activated by stress-inducing and DNA-damaging agents, anisomycin, UV, and also osmotic shock. Phorbol esters, NGF, and EGF, strong stimulators of the ERK pathway in the same cell lines analyzed (11, 12, 14) , did not stimulate MEK6. Similarly, cycloheximide, a stimulator of p54 kinase (JNK2) (35) and of the ERK pathway(36) , did not significantly activate MEK6. Anisomycin and cycloheximide are known to be effective protein synthesis inhibitors, yet the described effects on the MAPK pathways occur at concentrations that only marginally affect protein synthesis. Interestingly, we noted in our in vivo kinase assays with lysates prepared from HeLa cells but not from COS cells two bands of variable intensity that were stimulated by NGF and EGF. These bands most likely represent contaminants in the immunoprecipitate that became phosphorylated by ERK family members.
A time course
experiment revealed that the induction of MEK6 by UV lagged behind the
anisomycin induction by about 10-15 min. Further, in contrast to
the transient activation by anisomycin, UV-induced MEK6 stayed active
for at least 120 min. The slight reduction of MEK6 activity at the
90-min time point was not observed in other experiments. This time
course experiment suggests that different pathways are used for
anisomycin versus UV stimulation of MEK6. The time course
correlates well with the activation of JNK1 by UV (18) .
Additionally we tested different doses of UV. Consistent with the
dose-response curves of JNK1 activation(18) , we observed an
increase in MEK6 stimulation with UV doses up to 120 J/m.
MAPKK are activated by phosphorylation through MAPKKK. MEKK(37) , the upstream activator for JNKK, activates the JNK pathway much more efficiently than it does the ERK pathway(23) . Overexpression of MEKK activates MEK1 and MEK2, but this does not cause activation of ERK2 (38) . p38 activity was only weakly potentiated by cotransfected MEKK vector (in the presence or absence of the JNKK vector) in either COS or HeLa cells(22) . In vitro, however, extracts from MEKK-transfected cells activated p38 as efficiently as they activated JNK1(22) . Therefore, at physiological concentrations JNKK seems to be the only substrate for MEKK.
Studies in yeast have shown the existence of a scaffolding protein (STE5) that independently associates with all members of the MAPKKK, MAPKK, and MAPK module(39, 40) . Although the precise function of STE5 is still open to speculation, researchers propose that mammalian cells also have a protein(s) that act(s) as scaffold for the MAP kinase module. Additionally, it is possible that this protein acts as a kinase itself and phosphorylates other MAP kinases. The existence of different MAP kinase modules held together by scaffolding protein(s) may contribute to signal and tissue specificity and may restrict the cross-talk between MAP kinase cascades as illustrated with the MEKK example.
This prompted us to study whether MEKK can activate MEK6 in COS cells. We performed a titration experiment cotransfecting increasing amounts of the expression vector for MEKK with a fixed amount of the expression vector for epitope-tagged MEK6 or JNKK, respectively. JNKK activity as assayed in vitro by phosphorylation of JNK2 was already strongly stimulated with 125 ng of MEKK, while similar levels of stimulation of MEK6 as assayed in vitro by phosphorylation of p38 needed 1000 ng of MEKK. This suggests that MEK6 is not a physiological substrate for MEKK, although we cannot exclude the possibility that in vivo scaffolding proteins are involved in bringing together a signaling complex containing MEKK, MEK6, and p38. A schematic presentation of the MAPK cascades based on our information and published information is presented in Fig. 9.
Figure 9: MAPK pathways. Structure of MAPK signal transduction pathways in mammals.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U49732[GenBank].