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(Received for publication, November 6, 1995; and in revised form, December 5, 1995) From the
Mitogen-activated protein (MAP) kinases require dual
phosphorylation on threonine and tyrosine residues in order to gain
enzymatic activity. This activation is carried out by a family of
enzymes known as MAP kinase kinases (MKKs or MEKs). It appears that
there are at least four subgroups in this family; MEK1/MEK2 subgroup
that activates ERK1/ERK2, MEK5 that activates ERK5/BMK1, MKK3 that
activates p38, and MKK4 that activates p38 and Jun kinase. Here we
describe the characteristics of a new MKK termed MKK6. The clones we
isolated encode two splice isoforms of human MKK6 comprised of 278 and
334 amino acids, respectively, and one murine MKK6 with 237 amino
acids. Sequence information derived from cDNA cloning indicated that
MKK6 is most closely related to MKK3. The functional data revealed from
co-transfection assays suggests that MKK6, like MKK3, selectively
phosphorylates p38. Unlike the previously described MKKs (or MEKs),
MKK6 exists in a variety of alternatively spliced isoforms with
distinct patterns of tissue expression. This suggests novel mechanisms
regulating activation and/or function of various forms of MKK6.
The signal transduction pathways that utilize mitogen-activated
protein (MAP) ( In mammalian cells p38, the Hog1p homologue is
activated by multiple stimuli acting through different receptors. For
example Lee et al.(20) showed that p38 is involved in
bacterial endotoxin (lipopolysaccharide)-induced cytokine production
through the use of pharmacologic inhibitors that are specific for
p38(20) . p38 is also activated by other bacterial components,
proinflammatory cytokines, and physical-chemical changes in the
extracellular milleau(21) . There have been several distinct
MKKs/MEKs identified in mammalian cells; one type, termed MEK1/MEK2,
does not phosphorylate or activate p38 or JNK while in contrast is a
strong activator of ERK1/ERK2(7, 9, 22) . In
contrast, two other MKKs known as MKK3 and MKK4 activate p38 but not
ERK(6, 7) ; MKK4 (also known as SEK1/JNKK1) also
activates JNK/SAPK(6, 7, 8) . MKK3 and MKK4
are most closely related to PBS2p, the upstream activator of
Hog1p(6, 7) . p38 and JNK are often activated in
parallel (21) but independent activation of p38 also has been
observed(23) . Simultaneous activation of ERK and p38 also
occurs when cells are exposed to a stimulus such as lipopolysaccharide
or increased extracellular osmolarity(14, 24) . Unlike
yeast MAP kinase pathways which appear to operate independently,
cross-talk seems to occur between the mammalian MAP kinase
pathways(6, 7, 25, 26) . Emerging
data suggest that the MAPK signal transduction pathways in mammalian
cells are much more complex than homologous systems in yeast. One level
of such complexity might exist at the level of the MAPK kinases. Indeed
multiple activators of p38 or JNK can be detected by fractionation of
the fibroblasts activated by hyperosmolar media(27) . Thus
there is a need to identify all of the p38 upstream activators in order
to fully understand the regulation of p38. To isolate additional MKKs
which function as activators of p38, we have employed a polymerase
chain reaction (PCR)-based strategy with degenerate oligonucleotides
based on conserved kinase domains present in the known members of the
MKK family. This approach resulted in the isolation of two human cDNAs
and one murine cDNA encoding closely related proteins of the MKK
family. These clones are the different splice forms of one gene which
we have designated MKK6, which is selectively expressed in different
tissues. Amino acid sequence comparison revealed that the proteins
encoded by these cDNAs are most closely related to MKK3, and
co-transfection studies show that MKK6, like MKK3, activates p38 but
fails to activate either ERK or JNK. In this article we also present
data showing that unlike MKK4, MKK3 and MKK6 are not regulated by Rac
or Cdc42.
Figure 1:
Primary structure of human MKK6, MKK6b,
and MuMKK6c obtained from cDNA cloning. A, the cDNA sequence
and deduced amino acid sequence of MKK6 and MKK6b. The protein sequence
are presented in single letter code. The first upstream in-frame
stop-codon is underlined. B, sequence comparison of
human MKK6 and MKK6b with the human MKKs. The identical and conserved
amino acid sequences are boxed. The PILE-UP program (Wisconsin
Genetics Computer Group) was used for the alignment; gaps were
introduced into sequences to optimize alignments. C, the
relation between members of the human MKK family is presented as a
dendrogram created by the unweighted pair group method with the use of
arithmetic averages (PILE-UP program). D, the cDNA sequence
and deduced amino acid sequence of murine MKK6c. The nucleotides are
numbered based on the alignment with human
MKK6/6b.
We believe that the cDNAs for MKK6 and MKK6b contain
full-coding regions because both clones contain in-frame stop codons 5`
upstream of the first ATG sequence. The first in-frame ATG of MKK6 has
been designated as position 1. Since it is the first ATG codon and the
typical Kozak sequence, it should be the authentic start site for MKK6.
The first ATG found in MKK6b is at position -168. Although not a
typical Kozak sequence, translation of MKK6b mRNA most likely begins at
this position. The murine clone (GenBank U93066, Fig. 1D) which is closely related to MKK6b has the
latter ATG sequence but not an equivalent ATG at position 1. Thus we
predict that MKK6 contains 278 amino acids while MKK6b contains 334
amino acid residues. Of interest are differences between MKK6 and MKK6b
in DNA sequence before position -154; this may be due to
differential splicing. A dendrogram created by progressive pairwise
alignment comparison of the MKK family is shown in Fig. 1C. When we compared the sequence of the murine
cDNA clone we isolated with the sequence of human MKK6 or MKK6b we
noted that the murine clone has a 62-base pair deletion covering the
region from residues 16 to 77. Due to this deletion, the ATG at
position 122 is most likely to be the starting codon. We predict that
this murine clone contains 237 amino acids. Although at the DNA level,
this murine clone is most closely related to human MKK6b, it has a
completely different 5` sequence beginning with position -219 and
a unique 3` sequence starting from position 1056 when compared with
MKK6b. This may result from differential splicing, although additional
studies are required to firmly establish this. On the basis of these
differences we term this murine cDNA, murine MKK6c. The sequence of
murine MKK6c is shown in Fig. 1D. Further investigation
is required to determine whether a comparable form exists in human.
Amino acid sequence comparison reveals that the murine MKK6c is 97.6%
identical to human MKK6/6b in the overlapping region. Conservation of
the amino acid sequence between species has been found in other MKKs
and may indicate the importance of this family
proteins(9, 22) .
Figure 2:
Tissue distribution of MKK6 mRNA detected
with different probes. A blot containing poly(A)
Figure 3:
MKK6/6b are p38 activators. COS-7 cells
were transfected with epitope-tagged p38, Erk1, or JNK1 together with
an expression vector encoding MKK3, MKK4, MKK6, MKK6b, or control DNA
represented by empty vector; or transfected with a signal expression
plasmid of MKK6 or MKK6 mutated at ATP binding site (M mutants),
phosphorylation sites (A mutant and E mutant), or empty vector. Some of
the cultured cells transfected with control empty plasmid were exposed
to UV radiation (50 J/m
Sequence
comparison of MKK6 with other MKKs suggested that Ser
Figure 4:
Regulation of the activation of MKK3,
MKK4, and MKK6. COS-7 cells were transfected with epitope-tagged MKK6,
MKK4, or MKK3 together with empty expression vector or an expression
vector encoding active-form MEKK1, Rac1, Cdc42, Ras, RhoA, and Raf.
Some of the cell cultures were exposed to UV radiation (50
J/m
Herein we describe the properties of a newly identified
member of a family of enzymes known as MAP kinase kinases (MKK).
Nucleotide sequence data indicates that the human cDNA clones we
isolated encode two proteins generated from one gene; the distinct mRNA
species are produced through alternative splicing from a single gene
encoding a MAP kinase kinase, here termed MKK6. There is
tissue-specific expression of the isoforms of MKK6. Functional studies
indicate that MKK6 can activate p38 but not ERK or JNK. In contrast
to the previously described members of this family, which are expressed
in almost all tissues with a single transcript at the mRNA
level(7, 8, 9, 22) , numerous splice
variants of this gene exist. One splice form, MKK6, appears restricted
to expression in skeletal muscle. In contrast, MKK6b is present in
various forms in multiple tissues. Thus this represents the first
report suggesting there are tissue-specific isoforms of the MKK family.
Unique functions for tissue-specific splice forms of MKK6 is not
provided by our work. However, demonstration of the presence of such
forms suggests the possibility of regulation that is tissue and/or cell
specific. Here we also determined that Ser Insofar as the MAP kinase
family is concerned it was not surprising that MKK6 and MKK3 are quite
similar in substrate specificity. MKK6 and MKK3 can be classified into
a subgroup distinct from others members of the MKK family. This is
justified based on sequence homologies and ability to activate p38
without activating ERK or JNK. Moreover here we showed that regulators
of MKK4, namely Rac1 and Cdc42, apparently do not control activation of
MKK6. Thus there may be two distinct pathways leading to p38
activation; one via MKK6/3 and another involving MKK4. Interestingly
the studies of Saito and colleagues (18, 19) with S. cerevisiae revealed two distinct pathways leading
to Hog1p activation. One involves a two-component histidine kinase
system leading to activation of the MKK encoded by the PBS2 gene (18) . In contrast, an alternative pathway leading to PBS2p
activation was discovered involving a membrane protein termed Sho1 that
directly activates PBS2 through SH3 interactions(19) . Given
the distinct expression patterns of MKK6, it is likely that this MKK
acts as a regulator of p38 activation depending on expression in a
given tissue and/or cell type. A future challenge will be to define the
specific function of individual p38 activators and how these proteins
interact with other components of the signal machinery to transduce
extracellular information into cellular responses. Gene targeting of
individual MKKs in cultured cells and mice should shed light on this
issue. We suggest that multiple signaling pathways can control
activation of p38. A model which takes into account the data presented
here as well as the findings of others (7, 8, 26, 34, 35) is
provided in Fig. 5. Investigations that may lead to the
elucidation of alternative pathways for p38 activation via MKK3 and/or
MKK6 are currently underway.
Figure 5:
Proposed signaling pathways for activation
of p38.
Volume 271,
Number 6,
Issue of February 9, 1996 pp. 2886-2891
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)kinases have an important role in a variety
of cellular responses including growth factor-induced proliferation,
gene expression, and compensation for alterations in the extracellular
milleau induced by heat shock, UV light, increased extracellular
osmolarity etc.(1, 2, 3, 4) . Four
MAP kinase pathways have been defined in yeast(5) ; these
pathways are functionally independent and are regulated by distinct
protein kinase cascades. Each pathway results in activation of a
separate MAPK by a unique MAPK kinase (MKK or MEK). It is now clear
that higher eukaryotes also have distinct MAP kinase pathways; such
pathways are comprised of kinase cascades leading to the activation of
discrete MKKs and
MAPKs(5, 6, 7, 8, 9) .
Specifically four subgroups of MAPKs have been identified(5) :
the ERKs (extracellular signal-regulated
kinase)(10, 11) , Jun kinases (c-Jun amino-terminal
kinases (JNK) or stress-activated protein kinase
(SAPK))(12, 13) , p38 MAP kinase(14) , and
BMK1/Erk5(15, 16) . The signal transduction pathway
leading to p38 activation is related, in part, to a pathway in yeast
leading to activation of a MAP kinase known as Hog1p. To date the
activation of this yeast pathway has been shown to occur principally in
response to increased extracellular osmolarity(17) . Recently
Saito and colleagues (18, 19) have defined two
distinct pathways leading to Hog1p activation in Saccharomyces
cerevisiae.
cDNA Cloning
Degenerate oligonucleotides
(AARYTNTGYGAYTTYGGNGT and ATNCKYTCNGGNGCCATRTANGG) were designed using
published information from the conserved kinase subdomain of known MKKs (7, 8, 22) ; these oligonucleotides were
employed as PCR primers to isolate fragments of MKK-related cDNAs from
a human placenta cDNA library. Comparison of the sequence of 48 clones
with the GenBank data base (Blast Fileserver, National Center of
Biotechnology Information) allowed identification of one clone that is
novel and exhibits a high level of homology with the MKK group. The PCR
fragment was labeled with P by random priming and used as
a probe to screen a
ZAPII human placenta library (Stratagene, La
Jolla, CA) and a
ZAPII murine jejunum library (provided by Dr. Z.
Chen, The Scripps Research Institute, La Jolla, CA). Two positive
clones were obtained from the human library after screening 2
10
phage. DNA sequencing of both strands of each clone was
performed by the Microchemistry Core Facility at The Scripps Research
Institute using a model 373A automated sequencer (Applied Biosystems,
Foster City, CA). The sequence data indicated these two clones are
identical and both contain a complete open reading frame. One positive
clone was isolated from the murine library after screening 5
10
phage. The sequencing of this clone was done as noted
above. The murine clone shows strong homology to the human clones
except at the 5` end. Nucleotide sequence comparison of the 5` end
sequence of the murine clone with the GenBank Data base reveals four
sets of nucleotide sequences (GenBank access number H08016, H06765,
T66783, and R15387) that have nearly identical sequence. These clones
are human fetal brain cDNAs that were sequenced by the Washington
University-Merck EST project. The clone R15387 was requested and
completely sequenced as noted above.Northern Blot Analysis
An RNA tissue blot was
purchased from ClonTech (San Francisco, CA). The blot contains 2 µg
of poly(A) RNA isolated from different human tissues,
fractionated by denaturing agarose gel and transferred onto a nylon
membrane. The blot was hybridized to a probe that was prepared by
labeling the 5` end fragment (-404 to -199 bp) of MKK6 with
[
-
P]dATP by random priming. The blot was
stripped by incubation for 2 min at 100 °C in water and reprobed
with a probe corresponding to the 5` end sequence (-453 to
-159 bp) of MKK6b/6c. The blot was stripped again and reprobed
with a probe containing the coding region and 3`-untranslated region
(-87 to 1560) of MKK6. Hybridization was performed overnight at
50 °C using 50% formamide, 5
SSPE (1 SSPE, 150
mM NaCl, 15 mM sodium citrate, pH 7.2), 5
Denhardt's, 1% SDS, and 200 µg/ml single-stranded fish sperm.
The blot was washed two times with 1 SSC (1 SSC, 150
mM NaCl, 10 mM NaH
PO
, 1
mM EDTA, pH 7.2), 0.1% SDS, and 1 mM EDTA and one
time with 0.1 SSC, 0.1% SDS, 1 mM EDTA at 65 °C
prior to autoradiography.cDNA Constructs
The A, E, or M mutants of MKK6
were created by the substitution of Ser and Thr
with Ala or Glu and Lys
with Met using a PCR-based
procedure as described(28) . The MKK6 cDNA and the mutants were
cloned into the expression vector pCDNA3 (Invitrogen, San Diego, CA)
with the HA epitope tag added so that the protein contained an HA-tag
in the amino-terminal region. The cloning sites used for these
constructs are HindIII and KpnI. The HA-tag sequence
is YPYDVPDYAGYPYDVPDYAGSYPYDVPDYAAA. The tag was added by a PCR
recombination as described(28) . The sequence of all constructs
were confirmed by DNA sequencing in the The Scripps Research Institute
Microchemistry Core Laboratory. p38 or JNK1 cDNA containing a
FLAG-epitope tag were prepared using a pCDNA3 vector (29) . The
constructs of Rac1(Q61L), Cdc42(Q61L), RhoA(Q63L), Ras(Q61L),
Raf
(22W), and Myc-tagged Erk1 have been
described(29) . The truncated form of MEKK1, here termed
MEKK1
, is in pCMV5(25) . The HA-tagged MKK3,
MKK4(SEK1/JNKK1) were prepared as described(6) . The expression
plasmid of GST-ATF2 was made by removing the 3` end part of the cDNA
with XbaI. The cDNA was then blunted and religated into
pGEX-2T vector (Pharmacia, Uppsala, Sweden). The recombinant GST-ATF2
protein contains 1-109 amino-terminal amino acids of ATF2.Proteins
GST-ATF2 was purified by affinity
chromatography on GSH-agarose (Sigma) as described(30) .
His-tagged p38 was purified by the nickel-chelate column as
described(31) . Myelin basic protein (MBP) was from Sigma.Transient Expression of Various cDNAs
COS-7 cells
were maintained in Dulbecco's modified Eagle's medium
supplemented with 5% fetal bovine serum. Cells on 35-mm plates were
transiently transfected with 1 µg (total) of plasmids DNA using
lipofectamine reagent (Life Technologies, Inc., Grand Island, NY)
according to the manufacturer's recommendations. After 48 h, the
cells treated with or without UV radiation as described(13) .
The transfection efficiency was evaluated by co-transfection with
plasmid pCMV
(ClonTech). The cell lysates were normalized
according to -galactosidase activity prior to immunoprecipitation.
-Galactosidase activity was measured as described(32) .Immunoprecipitation and Western Blot Analysis
The
cells were suspended in lysis buffer (25 mM HEPES (pH 7.5), 1%
Triton X-100, 130 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 0.1 mM Na
VO, and
1 mM phenylmethylsulfonyl fluoride) for 20 min with shaking at
4 °C and the lysate cleared by centrifugation at 130,000 g for 10 min. Monoclonal antibodies against the flag-epitope,
M2(IBI-Kodak), the HA-epitope, 12CA5 (kindly provided by Dr.Ian Wilson,
TSRI), or the c-Myc epitope, 9E10 (Santa Cruz Biotechnology, Santa
Cruz, CA), were prebound to protein G- or A-Sepharose beads,
respectively. 20 µl of 50% suspension of beads were added to 300
µl of cell lysates and gently shaken for 3-10 h at 4 °C.
The beads were washed six times with 1 ml of washing buffer (25 mM HEPES (pH 7.5), 50 mM NaCl, 0.1 mM EDTA, 0.05%
Triton X-100, 0.1 mM NaVO, 1 mM phenylmethylsulfonyl fluoride). The immunoprecipitates were used
in the kinase assay within 2 days. The immunoprecipitates were always
analyzed by Western blotting to ensure the precipitates contained the
targeted proteins(29) . The monoclonal antibodies M2, 12CA5, or
9E10 were used to detect epitope-tagged proteins.Protein Kinase Assays
Immuno-complex kinase assays
using anti-flag antibody M2, anti-HA antibody 12CA5, or anti-Myc
antibody 9E10 immunoprecipitates were performed at 37 °C for 20 min
using 2-5 µg of substrate, 20 µM ATP, and 10
µCi of [-
P]ATP in 20 µl of kinase
buffer (25 mM HEPES (pH 7.6), 20 mM MgCl
,
20 mM -glycerophosphate, 20 mMp-nitrophenyl phosphate, 0.1 mM NaVO, and 2 mM dithiothreitol).
The reactions were terminated with Laemmli sample buffer and the
products were resolved by 12% SDS-PAGE. The phosphorylated proteins
were visualized by autoradiography.
Isolation and Characterization of New MKK
cDNAs
We designed a set of degenerate oligonucleotides utilizing
known sequence information from the conserved kinase domains of
previously described MKKs(7, 8, 22) . These
oligonucleotides were used as PCR primers to isolate fragments of MKK
related cDNA from human placenta cDNA; 48 clones were sequenced. When
the sequences were analyzed using the GenBank data base one clone was
identified with a high degree of homology to other known MKKs but
nonetheless encoding what appeared to be a protein not previously
described. The PCR-derived fragment was used to screen a human placenta
or a murine jejunum library. Screening of the human library yielded 2
positive clones after screening 2 10 phage.
Sequencing revealed that the two clones were identical and contained a
complete open reading frame. Moreover it was ascertained that the
sequence of these clones was distinct from all other known MKKs. We
therefore termed the protein encoded by the cDNA as MKK6 (GenBank
U39064). The nucleotide and deduced protein sequence of MKK6 is shown
in Fig. 1A. A screen of a murine cDNA library for
MKK6-related clones resulted in identification of a single clone with a
high degree of sequence homology to human MKK6 except in the 5`-region.
When the 5`-end sequence of this murine clone was used to search
GenBank we found several clones with nearly identical sequences
deposited by the Washington University-Merck EST project. We obtained
one of these clones from the EST project, clone R15387. Sequencing
revealed a cDNA encoding a protein with some regions of complete
identity with MKK6 and other regions, primarily in the amino-terminal
region with marked differences compared to MKK6. Thus we believe this
form to be a splice variant of MKK6 and have termed it MKK6b (Fig. 1A). The protein sequence alignments (Fig. 1B) for MKK6, MKK6b, and other MKKs reveal
similarities and differences in the members of the family of enzymes.
In the overlapping regions MKK6 is about 80% identical to MKK3 and 40%
to MKK4.
Tissue Distribution of MKK6
Because of the
observed differences in nucleotide sequences in the 5`-region and
3`-region of the MKK6 cDNA clones we wondered if there is
tissue-specific expression of alternatively spliced forms. To do this
we made specific probes based upon unique sequences in the 5`-region of
MKK6 and MKK6b/6c and probed blots containing poly(A) RNA (Fig. 2). This experiment revealed a high level of
expression of MKK6 mRNA with size of 1.7 kilobase pairs in skeletal
muscle (Fig. 2A). The probe encompassing the specific
5`-end region of MKK6b detected multiple mRNA bands with the size of
1.8, 2.4, 4.5, and 11 kilobase pairs. The 11-kilobase pair band only
was noted in skeletal muscle and pancreas. We suspect that the multiple
bands represent additional differential splicing forms of MKK6. The
MKK6 mRNAs containing the sequence we used in this probe are enriched
in heart, skeletal muscle, pancreas, and liver and detectable in brain,
placenta, and lung (Fig. 2B). These data suggest that
tissue-specific splicing occurs and may play a role in the control of
MKK6 expression. Northern blot analysis of tissues for expression of
MKK6s with a probe encompassing the coding region which is most similar
in all MKK6 clones is shown in Fig. 2C. No additional
bands were noted with this probe; transcripts of MKK6 gene are most
abundant in the skeletal muscle.
RNA
isolated from various human tissues was hybridized with a probe
specific for the 5`-end of MKK6 (A), or the 5`-end of MKK6b/6c (B), or the region of the cDNA of all the splice forms of MKK
that displayed the highest degree of homology (C).
Enzymatic Activity of MAPK Kinases
We next
investigated one aspect of the substrate specificity of MKK6 by asking
if members of the MAP kinase family were activated by this enzyme. To
do this we coexpressed MKK6 or MKK6b in COS-7 cells by transient
transfection together with epitope-tagged forms of p38, JNK1, or ERK1.
At the same time we compared the activities of MKK3 and MKK4 with MKK6.
When specificities toward ERK1 were examined we used MBP as a substrate
for ERK1 or p38 (Fig. 3A). In contrast, ATF2 was used
as a substrate for JNK1 and p38 in a parallel set of studies (Fig. 3B). In this latter set of studies cotransfection
with MKK6b was not performed (Fig. 3B). The enzyme
activity of p38, JNK1, or ERK1 were determined by immunokinase assay
after immunoprecipitation with the anti-epitope antibody. The
overexpression of MKK6/6b in COS-7 cells caused increased activity of
p38 (Fig. 3, A and B). In contrast, the
co-expression of MKK6 does not enhance JNK1 (Fig. 3B)
or ERK1 activity (Fig. 3A). The activation of p38 by
MKK6 was similar to that of MKK3. The similar substrate specificity of
MKK3 and MKK6 is consistent with the high homology between these two
enzymes. In these studies we noticed that the migration of
JNK1-phosphorylated GST-ATF2 is slightly slower than p38-phosphorylated
GST-ATF2. This suggests differences in ATF2 phosphorylation by JNK1 and
p38. Further studies are underway to verify this.
); p38, Erk1, or JNK1 were isolated
by immunoprecipitation with anti-epitope antibody. The protein kinase
activity was measured in the immunocomplex with
[-
P]ATP and MBP (A) or GST-ATF2 (B) as substrate. The coupled kinase assay was used to test
the kinase activity of MKK6 mutants by including recombinant p38 and
MBP in the kinase reaction (C). The product of the
phosphorylation reactions was visualized after SDS-PAGE by
autoradiography.
and
Thr
may be phosphorylation sites required for enzymatic
activity. To investigate this we modified the MKK6 cDNA by replacing
Ser
and Thr
with Ala (A mutant) or Glu (E
mutant). An additional mutant was made by changing Lys
to
Met to delete the ATP binding site (M mutant). Epitope-tagged versions
of these mutants were made by adding an HA-tag to the amino-terminal of
MKK6; the proteins encoded by these cDNAs were expressed in COS-7
cells. HA-tagged proteins were immunoprecipitated using anti-HA
monoclonal antibody 12CA5. The immunoprecipitates were used in a
coupled MKK assay to determine the activity of MKK6 or its mutants by
including recombinant His-p38 and MBP in the kinase reaction mixture (Fig. 3C). All three mutants fail to activate p38;
previous studies with MEK where A and M mutants were created also
resulted in a loss of activity(33) . Here the E mutant also
failed to activate p38 while in contrast, the analogous structural
change in MEK produced an active enzyme(33) .
Regulation of MAPK Kinase Activation
We and others
recently reported that the low molecular weight GTP-binding
proteins(29, 34) Rac1 and Cdc42, possibly via
activation of p21-activated kinase (PAK) (29, 35) ,
regulate the activation of p38 and JNK. Other data suggest that a MKK
kinase, MEKK1, may lie downstream of these regulators and thus function
as an activator of the MKKs(34) . To examine whether MKK6 is
regulated in a similar manner we co-expressed dominant active forms of
Rac1, Cdc42, or MEKK1 with HA-tagged MKK6, MKK4, or MKK3 in COS-7
cells. The activity of MKKs were determined by immunokinase assay.
Overexpression of the active-form Rac1, Cdc42, or MEKK1 led to
activation of MKK4 only; we failed to observe activation of MKK6 or
MKK3 as detected by our assay system (Fig. 4). This observation
confirms the previous report that MKK4(JNKK1) is downstream of Rac1,
Cdc42, and MEKK1 (34) and further suggests that other
regulatory steps lead to activation MKK6 or MKK3.
). The MKK6, MKK4, or MKK3 was isolated by
immunoprecipitation with the use of anti-epitope antibody. The kinase
activity was measured in the immunocomplex with
[-
P]ATP and recombinant p38 as substrate.
The product of the phosphorylation reactions was visualized after
SDS-PAGE by autoradiography.
and
Thr
are likely to be important phosphorylation sites
since mutation of these residues to alanine prevented activation of
MKK6. This region is analogous to one established to be important by
others for MEK(33) . We also attempted to replace Ser
and Thr
with Glu to simulate the negative charge
resulting from phosphorylation. This approach did result in a
gain-of-function mutation for MEK(33) . Here we failed to
observe a similar effect. This may be due to the fact that the
phosphorylation sites of MEK are both Ser while in MKK6 the site is
determined by a Ser and Thr. The Glu may not mimic phosphothreonine in
the same way that it does phosphoserine.
)-terminal kinase or stress-activated protein kinase; Erk,
extracellular-regulated protein kinase; HA, hemagglutinin; PAGE,
polyacrylamide gel electrophoresis; PCR, polymerase chain reaction.
We thank Dr. R. J. Davis for the MKK3 and MKK4 cDNA,
Dr. M. Karin for the JNKK1(MKK4) construct, Dr. G. L. Johnson for the
MEKK1 construct, and Dr. G. M. Bokoch for many essential reagents. We
also thank Dr. J. C. Mathison for helpful discussion and Betty Chastain
for excellent secretarial assistance.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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 M. Shim and T. E. Eling Vitamin E succinate induces NAG-1 expression in a p38 kinase-dependent mechanism Mol. Cancer Ther., April 1, 2008; 7(4): 961 - 971. [Abstract] [Full Text] [PDF]
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X. Qi, N. M. Pohl, M. Loesch, S. Hou, R. Li, J.-Z. Qin, A. Cuenda, and G. Chen p38{alpha} Antagonizes p38{gamma} Activity through c-Jun-dependent Ubiquitin-proteasome Pathways in Regulating Ras Transformation and Stress Response J. Biol. Chem., October 26, 2007; 282(43): 31398 - 31408. [Abstract] [Full Text] [PDF]
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 J.F. Schindler, J.B. Monahan, and W.G. Smith p38 Pathway Kinases as Anti-inflammatory Drug Targets J. Dent. Res., September 1, 2007; 86(9): 800 - 811. [Abstract] [Full Text] [PDF]
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 J.F. Schindler, J.B. Monahan, and W.G. Smith p38 Pathway Kinases as Anti-inflammatory Drug Targets Journal of Dental Research, September 1, 2007; 86(9): 800 - 811. [Abstract] [Full Text] [PDF]
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 K. Jin, S. Park, D. Z. Ewton, and E. Friedman The Survival Kinase Mirk/Dyrk1B Is a Downstream Effector of Oncogenic K-ras in Pancreatic Cancer Cancer Res., August 1, 2007; 67(15): 7247 - 7255. [Abstract] [Full Text] [PDF]
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 E. V. Wattenberg Palytoxin: exploiting a novel skin tumor promoter to explore signal transduction and carcinogenesis Am J Physiol Cell Physiol, January 1, 2007; 292(1): C24 - C32. [Abstract] [Full Text] [PDF]
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 A. Jorgl, B. Platzer, S. Taschner, L. X. Heinz, B. Hocher, P. M. Reisner, F. Gobel, and H. Strobl Human Langerhans-cell activation triggered in vitro by conditionally expressed MKK6 is counterregulated by the downstream effector RelB Blood, January 1, 2007; 109(1): 185 - 193. [Abstract] [Full Text] [PDF]
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Y. J. Kang, A. Seit-Nebi, R. J. Davis, and J. Han Multiple Activation Mechanisms of p38{alpha} Mitogen-activated Protein Kinase J. Biol. Chem., September 8, 2006; 281(36): 26225 - 26234. [Abstract] [Full Text] [PDF]
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 S. Konig, A. Beguet, C. R. Bader, and L. Bernheim The calcineurin pathway links hyperpolarization (Kir2.1)-induced Ca2+ signals to human myoblast differentiation and fusion Development, August 15, 2006; 133(16): 3107 - 3114. [Abstract] [Full Text] [PDF]
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 S. Henness, E. van Thoor, Q. Ge, C. L. Armour, J. M. Hughes, and A. J. Ammit IL-17A acts via p38 MAPK to increase stability of TNF-{alpha}-induced IL-8 mRNA in human ASM Am J Physiol Lung Cell Mol Physiol, June 1, 2006; 290(6): L1283 - L1290. [Abstract] [Full Text] [PDF]
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 T. Inoue, D. L. Boyle, M. Corr, D. Hammaker, R. J. Davis, R. A. Flavell, and G. S. Firestein Mitogen-activated protein kinase kinase 3 is a pivotal pathway regulating p38 activation in inflammatory arthritis PNAS, April 4, 2006; 103(14): 5484 - 5489. [Abstract] [Full Text] [PDF]
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 J. Zhang, T. N. Bui, J. Xiang, and A. Lin Cyclic AMP Inhibits p38 Activation via CREB-Induced Dynein Light Chain Mol. Cell. Biol., February 15, 2006; 26(4): 1223 - 1234. [Abstract] [Full Text] [PDF]
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 J. Fu, Z. Yang, J. Wei, J. Han, and J. Gu Nuclear protein NP60 regulates p38 MAPK activity J. Cell Sci., January 1, 2006; 119(1): 115 - 123. [Abstract] [Full Text] [PDF]
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D. J. Vander Griend, M. Kocherginsky, J. A. Hickson, W. M. Stadler, A. Lin, and C. W. Rinker-Schaeffer Suppression of Metastatic Colonization by the Context-Dependent Activation of the c-Jun NH2-Terminal Kinase Kinases JNKK1/MKK4 and MKK7 Cancer Res., December 1, 2005; 65(23): 10984 - 10991. [Abstract] [Full Text] [PDF]
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 H. Wang, D. Gupta, X. Li, and R. Dziarski Peptidoglycan Recognition Protein 2 (N-Acetylmuramoyl-L-Ala Amidase) Is Induced in Keratinocytes by Bacteria through the p38 Kinase Pathway Infect. Immun., November 1, 2005; 73(11): 7216 - 7225. [Abstract] [Full Text] [PDF]
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K. Kim, J. H. Kim, J. Lee, H.-M. Jin, S.-H. Lee, D. E. Fisher, H. Kook, K. K. Kim, Y. Choi, and N. Kim Nuclear Factor of Activated T Cells c1 Induces Osteoclast-associated Receptor Gene Expression during Tumor Necrosis Factor-related Activation-induced Cytokine-mediated Osteoclastogenesis J. Biol. Chem., October 21, 2005; 280(42): 35209 - 35216. [Abstract] [Full Text] [PDF]
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F. Meng, Y. Yamagiwa, S. Taffetani, J. Han, and T. Patel IL-6 activates serum and glucocorticoid kinase via p38{alpha} mitogen-activated protein kinase pathway Am J Physiol Cell Physiol, October 1, 2005; 289(4): C971 - C981. [Abstract] [Full Text] [PDF]
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R. A. Deaton, C. Su, T. G. Valencia, and S. R. Grant Transforming Growth Factor-{beta}1-induced Expression of Smooth Muscle Marker Genes Involves Activation of PKN and p38 MAPK J. Biol. Chem., September 2, 2005; 280(35): 31172 - 31181. [Abstract] [Full Text] [PDF]
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J. Robidoux, W. Cao, H. Quan, K. W. Daniel, F. Moukdar, X. Bai, L. M. Floering, and S. Collins Selective Activation of Mitogen-Activated Protein (MAP) Kinase Kinase 3 and p38{alpha} MAP Kinase Is Essential for Cyclic AMP-Dependent UCP1 Expression in Adipocytes Mol. Cell. Biol., July 1, 2005; 25(13): 5466 - 5479. |