From the School of Biological Sciences, University of
Manchester, Manchester M13 9PT and the § School of
Biochemistry and Genetics, Medical School, University of Newcastle upon
Tyne, Newcastle upon Tyne NE2 4HH, United Kingdom
Received for publication, August 23, 2000, and in revised form, October 2, 2000
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ABSTRACT |
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MAPK pathways play important roles in
regulating the key cellular processes of proliferation,
differentiation, and apoptosis. There are multiple MAPK pathways, which
are subject to different regulatory cues. It is important that these
pathways maintain specificity in signaling to elicit the activation of
a specific program of gene expression. MAPK-docking domains in several
transcription factors have been shown to play important roles in
determining the specificity and efficiency of their phosphorylation by
MAPKs. Here we investigate the mechanisms by which MAPKs are targeted to the ETS domain transcription factor SAP-1. We demonstrate that SAP-1
contains two different domains that are required for its efficient
phosphorylation in vitro and activation in vivo
by ERK2 and a subset of p38 MAPKs. The D-domain is closely related to other MAPK-docking domains, but exhibits a novel specificity and serves
to promote selective targeting of ERK2, p38 Stringent controls are required to permit the transmission of
extracellular signals into a specific cellular response. Indeed, multiple mechanisms exist to ensure specificity in cellular signaling (reviewed in Ref. 1). The
MAPK1 pathways represent a
common route through which signals are transmitted into nuclear
responses. At least six parallel pathways exist in mammals (reviewed in
Ref. 2), the best studied pathways being the ERK, JNK, and p38
pathways. The p38 pathways can themselves be further subdivided into
different isoforms (p38 MAPK-docking sites were initially identified in c-Jun (16, 17) and
subsequently in a series of different transcription factors and
cytoplasmic substrates (reviewed in Refs. 14 and 15). The docking sites
found in transcription factors are typically <20 amino acids long and
show limited sequence similarity, but are characterized by a region
rich in basic amino acids, followed by either an LXL motif
and/or a triplet of hydrophobic amino acids. These docking domains
specify substrate phosphorylation by one (e.g. c-Jun-JNK,
MEF2A-p38 The ETS domain transcription factor SAP-1 belongs to the ternary
complex subfamily and is highly related to Elk-1 (reviewed in
Ref. 22). SAP-1 contains a domain that exhibits strong sequence similarity to the Elk-1 D-domain. However, it is not known whether this
domain functions in an analogous manner as a MAPK-docking domain. SAP-1
has been shown to be able to act as a target of the ERK, JNK, and p38
MAPK families (23-26). A direct comparison of Elk-1 and SAP-1
demonstrated that SAP-1 is preferentially phosphorylated by p38 More recently, a second type of MAPK-binding site was identified
conforming to the consensus sequence FXF, which plays an important role in ERK-mediated substrate phosphorylation (27, 28). This
motif is conserved between SAP-1 and Elk-1 and might also play a role
in determining the proficiency of SAP-1 as a MAPK substrate.
To further probe the mechanisms that establish specificity in MAPK
signaling, we have analyzed the specificity determinants in SAP-1 that
control its proficiency as a MAPK substrate. We demonstrate that the
D-domain of SAP-1 acts as a classical docking domain that recruits ERK2
and a subset of p38 MAPKs. This domain constitutes part of a MAPK
recognition module that also contains an FXF motif. This
second binding motif promotes SAP-1 phosphorylation by both the ERK and
p38 Plasmid Constructions--
The following plasmids were
constructed for expressing MBP and GST fusion proteins in
Escherichia coli. pAS777 (encoding MBP-SAP-1
The following plasmids were constructed for use in mammalian cell
transfections. pG5E1b-luc contains five GAL4 DNA-binding sites cloned
upstream of a minimal E1b promoter element and the firefly luciferase
gene (29). pSG424 (pAS243) encodes the GAL4 DNA-binding domain (30).
pAS1068 (pSG424-New) was derived from pSG424 with the insertion of a
new polylinker (oligonucleotides ADS673 and ADS674) to provide a
BamHI site whose reading frame is compatible with the MBP
system. The plasmids pCMV5 (pAS188), pCMV5-F-p38 Protein Expression and Purification--
The following procedure
was used to isolate MBP fusion proteins from 50-ml cultures.
Inoculation was carried out according to the manufacturer's
instructions (New England Biolabs Inc.). Cells were harvested, and the
pellet was resuspended in 4 ml of column buffer (10 mM
Tris-Cl, 200 mM NaCl, and 1 mM EDTA) including 200 µl of 100 mM phenylmethylsulfonyl fluoride (Sigma).
Then, the sample was sonicated (3 × 10-s bursts at 14 µ)
and spun down at 18,000 × g for 10 min at 4 °C. The
supernatant was transferred to a fresh tube containing 400 µl of 50%
reduced amylose resin beads (New England Biolabs Inc.). The sample was
gently mixed for 30 min at 4 °C and then pelleted briefly at
1000 × g. The pelleted beads were washed three times
with 10 ml of column buffer and resuspended in an equal amount of
column buffer (200 µl). The MBP fusion protein was eluted by adding
10 mM maltose (Sigma) to the purified protein, followed by
incubation for 30 min at 4 °C and removal of the beads by brief
centrifugation at 1000 × g. This process was repeated
to elute more protein, and the samples were pooled with 20% glycerol
and stored at Tissue Culture, Cell Transfection, and Reporter Gene
Assays--
COS-7 cells were maintained and transfection experiments
were carried out using Superfect reagent as described previously (21).
Active p38 MAPKs were isolated from COS-7 cells, cotransfected with
vectors encoding the constitutively active form of MKK6 (MKK6(E)) and
epitope-tagged p38 kinases as described previously (19). For reporter
gene assays, a luciferase reporter construct controlled by a
GAL4-driven promoter (pG5E1b-luc) was cotransfected with cytomegalovirus promoter-driven vectors encoding various GAL4 DNA-binding domain fusion proteins as described previously (21). The
activities of the GAL4 fusion proteins were measured in cotransfection assays in COS-7 cells using 1 µg of the reporter plasmid pG5E1b-luc and, where indicated, 250 ng of vectors encoding MAPK and
constitutively activated MAPK. Luciferase assays were carried using the
Tropix dual light system, and transfection efficiencies were monitored as described previously (19)
Protein Kinase Assays--
Recombinant active p38 Data Analysis and Presentation--
Figures were generated
electronically using Picture Publisher (Micrografix) or Adobe Photoshop
Version 5.5 and Powerpoint Version 7.0 (Microsoft) software. Data from
Western blots are computer-generated images (FluorS Max and Quantity
One, Bio-Rad). Phosphorimager data from kinase assays were quantified
using either Tina Version 2.08e software or Quantity One.
The SAP-1 D-domain Is Important for Phosphorylation by Specific p38
Isoforms--
SAP-1 is highly related to Elk-1 and exhibits a high
degree of sequence similarity within the conserved D-domain (see Fig. 4A). We therefore tested whether the D-domain of SAP-1 plays
a role in MAPK targeting as observed in Elk-1 and whether any
specificity in targeting occurs.
Initially, we constructed fusion proteins consisting of the MBP fused
to either the entire C terminus of SAP-1 (MBP-SAP-1) or the same region
lacking the D-domain (MBP-SAP-1 SAP-1 Is Selectively Activated by Specific p38 Isoforms in
Vivo--
One consequence of targeting MAPKs to substrates is thought
to be the promotion of their specificity of action in vivo.
To establish whether the selective targeting of p38 The SAP-1 D-domain Acts as a MAPK-docking Domain--
Peptide
competition assays were used to probe the potential roles of short
protein sequences as binding sites for protein kinases. The principle
behind these assays is that peptides that correspond to docking motifs
will competitively bind to the MAPKs and thereby block interactions
with docking sites on substrates and hence reduce the efficiency of
substrate phosphorylation by the kinase (e.g. Ref. 20). We
therefore analyzed the ability of a peptide corresponding to the SAP-1
D-domain (SAPD) (Fig. 3A) to
inhibit phosphorylation of SAP-1 by p38 MAPKs. In comparison, we
analyzed a peptide corresponding to a known p38-binding motif in MEF2A
and a mutant version of this peptide that can no longer inhibit
p38-mediated phosphorylation (MEFD and MEFmD, respectively) (Fig.
3A) (19).
Increasing concentrations of the SAPD peptide led to a
dose-dependent decrease in phosphorylation of SAP-1 by
p38
As SAP-1 can also be phosphorylated by ERK MAPK, we tested whether the
SAPD peptide acts as an inhibitor of ERK-mediated phosphorylation. The
SAPD peptide was compared with the ElkD peptide, which was derived from
the same region of Elk-1 and is a known ERK inhibitor (Fig.
4A) (20). Increasing
concentrations of the SAPD peptide resulted in a decrease in the
ability of ERK2 to phosphorylate SAP-1 (Fig. 4B, lanes
2 and 3). Similarly, the ElkD peptide, but not a mutant
version (ElkmD), led to reduced SAP-1 phosphorylation by ERK2 (Fig.
4B, lanes 4-7). These results suggest that the
SAP-1 D-domain acts as an ERK2-binding motif. To verify this
conclusion, we carried out kinase binding assays with either MBP-SAP-1
or MBP-Elk-1 fusion protein bound to agarose beads. Both MBP-SAP-1 and
MBP-Elk-1 were able to retain ERK2 on the beads (Fig. 4C, lanes 1 and 3). However, in contrast, ERK2
binding was significantly decreased upon deletion of the D-domain of
SAP-1 or Elk-1 (Fig. 4C, lanes 2 and
4). Collectively, these data demonstrate that, in addition
to its role in binding to p38 The "FXF Motif" Is Required for Efficient SAP-1 Phosphorylation
and Activation by ERK2 and Specific p38 MAPKs--
Recently, a novel
motif was identified that is required for efficient substrate
phosphorylation by MAPKs (27). This motif, which we have termed the
FXF motif, is also functionally conserved in Elk-1 and at
the sequence level (FQFP) in SAP-1. We therefore investigated whether
the FXF motif also plays a role in mediating MAPK
phosphorylation of SAP-1 in vitro and activation in
vivo in comparison with the same motif in Elk-1.
First, we created a series of GAL4 and MBP fusion proteins containing
either the intact C termini of Elk-1 and SAP-1 or mutant forms
either lacking the D-domain or containing point mutations in the
FXF motif (Fig.
5A). These proteins were then
tested for their proficiency as in vitro substrates for ERK2
(Fig. 5B) and for activation by the ERK pathway in
vivo (Fig. 5C).
In Elk-1, deletion of the D-domain or mutation of the FXF
motif led to a decrease in phosphorylation by ERK2 in vitro
(Fig. 5B, lanes 2 and 3). Furthermore,
the simultaneous disruption of both motifs led to a further drop in
ERK2-mediated Elk-1 phosphorylation (Fig. 5B,
lane 4). Similarly, individually deleting the
D-domain and FXF motif of SAP-1 led to decreases in in
vitro phosphorylation (Fig. 5B, lanes 6 and
7), and their simultaneous deletion led to a further drop in
phosphorylation by ERK2 (lane 8).
Transcriptional activation in vivo following activation of
the ERK pathway was monitored for each of the mutant proteins. In both
Elk-1 and SAP-1, deletion of the D-domain or FXF motif led
to a substantial drop in activation by the ERK pathway, and simultaneous disruption of these two motifs resulted in a further reduction (Fig. 5C, right panel, black
and white bars, respectively). These results essentially
mirror the in vitro situation (Fig. 5C, compare
with left panel) and demonstrate the importance of the
D-domain and FXF motif in ERK2 targeting to SAP-1 in
vivo.
Previously, the FXF motif was concluded to be specifically
involved in targeting ERK MAPKs to substrates (27). However, we tested
whether the FXF motif is also important in determining the
proficiency of SAP-1 as a p38 substrate in vitro and
in vivo. In vitro kinase assays demonstrated that, whereas
deletion of the D-domain led to a large decrease in SAP-1
phosphorylation by p38
We also analyzed the response of the mutant SAP-1 fusion protein to
activation by the p38 MAPK pathways in vivo (Fig.
6C). Deletion of the D-domain led to a large decrease in
p38
The D-domain of SAP-1 therefore plays a major role in promoting its
phosphorylation in vitro and activation in vivo
by p38 The FXF Motif Acts as a Binding Site for Subsets of MAPKs--
The
peptide competition assay was used to investigate whether the
FXF motif of SAP-1 acts as a MAPK-binding site. First, we tested the ability of a peptide encompassing the SAP-1 FXF
motif (SAPF) (Fig. 7A) to
inhibit its phosphorylation by ERK2 and p38 isoforms. The SAPF peptide
inhibited SAP-1 phosphorylation by both ERK2 and p38
We next compared the ability of peptides encompassing the
FXF motif and D-domain to inhibit substrate phosphorylation
by ERK2. Again, competition assays were used, but two different SAP-1
substrates were employed that lacked either the D-domain (SAP-1
Collectively, these results demonstrate that the D-domain and
FXF motif play important roles in mediating selective
substrate phosphorylation by MAPKs. However, peptide competition assays indicate that these motifs might function differently.
Peptides Containing the FXF Motif Act as Selective MAPK
Inhibitors--
The above results indicate that peptides containing
the FXF motif act as inhibitors of ERK2 and p38 The FXF Motif Promotes Kinase Binding and Substrate Phosphorylation
in Heterologous Contexts--
The D-domains of Elk-1 and MEF2A/C can
act in heterologous contexts to enhance substrate phosphorylation when
fused to different proteins (19, 20). To probe whether the
FXF motifs of Elk-1 and SAP-1 can function in an analogous
manner, we created a series of fusions with MEF2A (Fig.
9A) and tested them as
substrates for ERK2 and p38 isoforms.
MEF2A is a p38
As the Elk-1 D-domain can also target ERK2 to substrates (21, 27), we
also tested whether it can lead to enhanced MEF2A phosphorylation by
ERK2. However, in contrast to the effect of the FXF motif,
little enhancement of phosphorylation by ERK2 was observed upon fusion
of the Elk-1 D-domain to MEF2A (Fig. 9B, lane 6)
(21). Similarly, inclusion of the Elk-1 D-domain did not
strongly enhance MEF2A phosphorylation by p38
To assess whether the FXF motif is sufficient for ERK2
binding in these heterologous contexts, we carried out kinase binding assays. In these assays, MBP-SAP-1 was added as a substrate following the binding reaction with GST-MEF2 derivatives. MEF2A was unable to
bind to ERK2 (Fig. 9C, lane 1). However, the
introduction of an FXF motif promoted binding of ERK2,
whereas a mutant version of this motif was unable to impart this
activity (Fig. 9C, lanes 2 and 3).
Thus, the FXF motif is sufficient to promote ERK2 binding to
a heterologous substrate.
Collectively, these data show that the FXF motif can act in
an independent manner as a portable motif to impart differences in the
proficiency of heterologous proteins as ERK2 substrates. By contrast,
the Elk-1 D-domain is insufficient to promote MEF2A phosphorylation by
ERK2, although it can promote its phosphorylation by other MAPKs
(19).
The FXF Motif Acts in a Position-dependent
Manner--
The FXF motif in LIN-1, Elk-1, and SAP-1 is
located downstream from the key phosphoacceptor motifs, suggesting that
this spatial arrangement might be critical. We therefore introduced an
FXF motif into MEF2A upstream from the phosphoacceptor
motifs (F-MEF2A Specificity in cellular signaling is maintained by multiple
mechanisms that permit specific responses to be elicited in response to
activation of individual pathways (reviewed in Ref. 1). The MAPK
signaling pathways are subject to multiple levels of regulation. One
such regulatory event occurs in the nucleus, where the interactions of
the MAPKs with their substrates are regulated by specific docking sites
on transcription factors (reviewed in Refs. 14 and 15). These docking
motifs enhance the efficiency and efficacy of substrate phosphorylation
by MAPKs. Here we have identified a module, composed of two different
motifs, that determines the proficiency of SAP-1 as a substrate for
specific MAPK subtypes. One component of this module, the D-domain,
plays a key role in determining phosphorylation by ERK2, p38 MAPK-docking domains have been identified that specifically direct
substrate phosphorylation by one or more MAPKs. For example, the
The FXF motif is highly conserved between SAP-1 and Elk-1
(see Fig. 8A) and in the Caenorhabditis elegans
protein LIN-1 (27). Previously, it was thought that the FXF
motif acted specifically to promote substrate phosphorylation by ERK
MAPKs, hence the name DEF (docking site for
ERK, FXFP) for this motif (27).
However, here we demonstrate that the FXF motif also plays a
role in promoting substrate phosphorylation by p38 An important question is whether the FXF motif and D-domains
are functionally interchangeable. Although it is clear that both the
D-domains and FXF motif can bind to MAPKs, the results of peptide competition assays suggest that they may function in a different manner. Whereas the D-domain acts as a classical competitive inhibitor for binding to substrates containing a D-domain, peptides containing the FXF motif inhibit substrate phosphorylation,
irrespective of the presence of the FXF motif in the
substrate (Figs. 7 and 8) (27). This suggests that the two motifs bind
to different parts of the kinases. Indeed, due to the lack of sequence
similarity, it is unlikely that they bind to the same region of the
protein kinases. Thus, at least two regions exist on kinases for
substrate binding, one of which has recently been identified that
apparently binds to the basic regions of docking sites related to the
D-domains (34). The FXF motif might lie adjacent to this or,
alternatively, be completely separate. Interestingly, inhibitory
peptides bind to a hydrophobic groove in protein kinase A, which is
located adjacent to its catalytic site and permits insertion of a
extended region containing the phosphoacceptor motif into the active
site (35). It is tempting to speculate that the FXF motifs
might bind to the MAPKs in an analogous manner to substrate binding to
protein kinase A. As FXF motifs are hydrophobic in nature
and are located close to the phosphoacceptor motifs, this mode of action is a distinct possibility. Such a mode of action would explain
the inhibitory effects of the FXF motif, as binding of the
kinase by the peptide would likely obstruct access to the active site.
Furthermore, in support of this hypothesis, although the FXF
motif is sufficient to promote substrate phosphorylation, it does so
only in a position-dependent manner (Fig. 10), suggesting that the correct juxtapositioning of this motif relative to the phosphoacceptor sites is required. Future structural and mutagenic studies are required, however, to permit the identification of this
binding site on MAPKs.
Our results point to the existence of a complex MAPK recognition module
in SAP-1 (Fig. 11A) composed
of at least three determinants: a D-domain, a transcriptional
activation domain that contains the phosphoacceptor motifs, and an
FXF motif. A similar module has previously been proposed for
LIN-1 and Elk-1 (27). A comparison of Elk-1 and SAP-1 demonstrates how
the combination of domains can determine their proficiency as MAPK
substrates (Fig. 11B). For Elk-1, the D-domain alone
promotes phosphorylation by JNK, whereas ERK phosphorylation is
promoted by a combination of the D-domain and FXF motif.
Similarly, phosphorylation of SAP-1 by ERK is promoted by a combination
of the D-domain and FXF motif. However, whereas p38, and p38
2 to SAP-1. A second important region, the FXF motif, also
plays an important role in directing MAPKs to phosphorylate SAP-1. The FXF motif promotes targeting by ERK2 and, to a lesser
extent, p38
, but not p38
2. Our data therefore
demonstrate that a modular system of motifs is responsible for
directing specific MAPK subtypes to SAP-1, but also point to important
distinctions in the mechanism of action of the D-domain and
FXF motif.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, p38
2, p38
, and p38
)
that are subject to different regulation and that have different
substrate specificity (reviewed in Ref. 3; see Refs. 4-10). The MAPK
pathways are subject to multiple tiers of regulation (reviewed in Ref.
11), with cytoplasmic scaffolds representing one mechanism by which
particular cascades are assembled with the exclusion of components of
other related pathways (reviewed in Refs. 12 and 13). Within these
scaffolds, defined protein-protein interactions play key roles in
specifying the interaction of individual components. Similarly, in the
nucleus, the interactions of the kinases with their substrates are
regulated by specific docking sites on transcription factors (14, 15).
These docking motifs enhance the efficiency and efficacy of substrate
phosphorylation by MAPKs.
/p38
2) (16-19) or two (e.g.
Elk-1-ERK/JNK) (20) different classes of MAPKs. In the case of Elk-1,
for example, the D-domain specifies targeting by ERK and JNK MAPKs, but
does not appear important for p38 MAPKs (20, 21). Docking domains do,
however, exist in other proteins such as MEF2A and MEF2C, which specify
targeting by p38 MAPKs (19).
(25), indicating that specificity determinants exist. As the Elk-1
docking domain does not appear to target p38 MAPKs (20), this
difference in kinase selectivity might be determined by the SAP-1
D-domain.
MAPKs. Our data therefore demonstrate that a complex modular
system, consisting of the D-domain and FXF motif, directs
specific MAPK subtypes to SAP-1.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
D, SAP-1
amino acids 339-431), pAS785 (encoding MBP-SAP-1, SAP-1 amino acids
303-431), pAS1056 (encoding MBP-Elk-1, Elk-1 amino acids 310-428),
and pAS1057 (encoding MBP-Elk-1
D, Elk-1 amino acids 330-428) were
constructed by inserting a BamHI-HindIII-cleaved polymerase chain reaction-derived fragment into the same sites of
pMAL-C2 (New England Biolabs Inc.). pAS1080 (encoding MBP-SAP-1-mF) and
pAS1081 (encoding MBP-SAP-1
D-mF) were derived from pAS785 and
pAS777, respectively, with site-directed mutations Q399A/F400A. pAS1096
(encoding MBP-Elk-1-mF) and pAS1097 (encoding MBP-Elk-1
D-mF) were
derived from pAS1056 and pAS1057, respectively, with site-directed mutations Q396A/F397A. The plasmids pAS860 (encoding GST-MEF2A, MEF2A
amino acids 266-413), pAS861 (encoding GST-MEF2A
D, MEF2A amino
acids 283-413), pAS867 (encoding GST-MEF2C, MEF2C amino acids
249-378), and pAS873 (encoding GST-ElkD-MEF2A, Elk-1 amino acids
310-327 and MEF2A amino acids 283-413) were described previously (19). pAS1456 (encoding GST-ElkD-MEF2A-F), pAS1457
(GST-ElkD-MEF2A
D-mF), pAS1458 (encoding GST-MEF2A-F), and pAS1459
(encoding GST-MEF2A-mF) were generated by inserting a
BamHI-XbaI-cleaved polymerase chain reaction-derived fragment into the same sites of pAS867, whereas pAS1460 (encoding GST-MEF2A
D-F), pAS1492 (encoding GST-F-MEF2A
D), and pAS1461 (encoding GST-MEF2A
D-mF) were constructed by inserting a
BamHI-XbaI-cleaved polymerase chain
reaction-derived fragment into the same sites of pAS860.
(31),
pCDNA3-F-p38
2, pCDNA3-F-p38
(9),
pCDNA3-F-p38
(8), pCMV5-HA-ERK2 (32), pCDNA3-MKK6(E),
pCMV5-HA-MEK(E) (33), pAS900 (pCMV-GAL4-Elk-1), and pAS1351
(pCMV-GAL4-Elk-1
D) (19) have been described previously. pAS1069
(GAL4-SAP-1), pAS1070 (GAL4-SAP-1
D), pAS1092 (GAL4-SAP-1-mF),
pAS1093 (GAL4-SAP-1
D-mF), pAS1100 (GAL4-Elk-1-mF), and pAS1451
(GAL4-Elk-1
D-mF) were constructed by ligating the
BamHI-XbaI fragments from pAS785, pAS777,
pAS1080, pAS1081, pAS1096, and pAS1097, respectively, into the same
sites of pAS1068. pAS1071 (pCMV-GAL4-SAP-1), pAS1072
(pCMV-GAL4-SAP-1
D), pAS1094 (pCMV-GAL4-SAP-1-mF), pAS1095
(pCMV-GAL4-SAP-1
D-mF), pAS1452 (pCMV-GAL4-Elk-1-mF), and pAS1453
(pCMV-GAL4-Elk-1
D-mF) were constructed by ligating the
HindIII-XbaI fragments from pAS1069, pAS1070,
pAS1092, pAS1093, pAS1100, and pAS1451, respectively, into the same
sites of pCMV5. All plasmid constructs made by polymerase chain
reaction were verified by automated dideoxy sequencing.
80 °C. The concentrations of proteins were
determined after SDS-polyacrylamide gel electrophoresis and staining
with Coomassie Blue (Sigma) and by comparison with bovine serum albumin
as a standard. Purification of GST fusion proteins was performed as
described previously (21).
,
p38
2, p38
, and p38
were prepared from transfected
COS-7 cells as described above. Recombinant active ERK2 was obtained
from New England Biolabs Inc. Reactions were performed as described
previously (21). The phosphorylation of substrate proteins was examined
after SDS-polyacrylamide gel electrophoresis and quantified by
phosphorimaging (Fuji BAS1500 phosphorimager and Tina Version
2.08e software). Peptide competition assays were performed essentially
as described previously (21). However, no preincubation of the peptides
with the kinases was performed. Protein kinase assays and kinase
binding assays were carried out as described previously
(21).
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
D) (Fig.
1A). These fusion proteins
were subsequently tested as in vitro substrates for ERK and
p38 MAPKs (Fig. 1B), and the data were quantified (Fig.
1C). p38
could efficiently phosphorylate SAP-1 with
similar kinetics, irrespective of the presence of the D-domain. In
contrast, the efficiency of substrate phosphorylation by ERK2,
p38
2, and, to a lesser extent, p38
, was reduced upon
deletion of the D-domain. Phosphorylation of SAP-1 by p38
was
inefficient and occurred independently of the presence of the D-domain
(data not shown). These data therefore demonstrate that the SAP-1
D-domain plays an important role in determining its proficiency as an
ERK2, p38
, and p38
2 substrate, but that it does not
play a major role in determining its efficiency of phosphorylation by
p38
in vitro.
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Fig. 1.
Differential requirement of the D-domain for
SAP-1 phosphorylation by MAPKs. A, diagrammatic
representation of truncated SAP-1 and SAP-1 D proteins fused to MBP.
The locations of the D-domain (black boxes) and the
transcriptional activation domain (gray boxes) and the
numbers of the amino acids at the N and C termini of each construct are
indicated. B, protein kinase assays. Equimolar quantities (5 pmol) of SAP-1 (lanes 1-4) and SAP-1
D (lanes
5-8) were phosphorylated by ERK2, p38
, p38
2,
and p38
for the times indicated above each lane. C,
graphical representation of the results with data obtained from
B.
and
, MBP-SAP-1;
, MBP-SAP-1
D. Data are
presented relative to MBP-SAP-1 phosphorylation after 60 min taken as
100.
and
p38
2 isoforms to SAP-1 in vitro is reflected
by in vivo selectivity, we analyzed the activation of a
GAL4-SAP-1 fusion protein (Fig.
2A) by different p38 isoforms
in vivo. p38
2 (93-fold) and, to a lesser
extent, p38
(13-fold) efficiently activated SAP-1, whereas in
comparison, p38
(0.4-fold) and p38
(1.8-fold) were poor
activators (Fig. 2B). We also compared the activation of
SAP-1 by the p38
2 and ERK2 pathways (Fig.
2C). Similar levels of activation of SAP-1 were obtained,
although absolute comparisons are difficult, as differences in the
activities of the constitutively active upstream kinases cannot be
controlled. However, a good correlation exists between the importance
of the D-domain in in vitro phosphorylation (Fig. 1) of
SAP-1 and the degree of activation in vivo by ERK2 and
distinct p38 subfamily members.
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Fig. 2.
Differential activation of SAP-1 by p38 MAPKs
in vivo. A, a diagram is shown
illustrating the truncated SAP-1 protein fused to the GAL4 DNA-binding
domain. Annotations are as described in the legend to Fig.
1A. B and C, COS-7 cells were
cotransfected with expression vectors encoding GAL4-SAP-1, a
GAL4-driven luciferase reporter plasmid, a constitutively active form
of MKK6 (MKK6(E)) or MEK1 (MEK(DN)), and the indicated ERK and p38
MAPKs. Transfection efficiencies were monitored using the
-galactosidase expression vector pCH110. Values are
means ± S.E. from duplicate samples. The data in B are
representative of at least three independent experiments.
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Fig. 3.
The SAP-1 D-domain acts as a binding site for
p38 and
p38
2 MAPKs. A,
diagrammatic representation of the substrate (MBP-SAP-1) and sequence
comparison of the peptides used as competitors in the kinase assays.
The peptides SAPD and MEFD correspond to the D-domains of SAP-1 and
MEF2A, respectively (19). Identical or highly conserved amino acids are
shaded, and the changes in the mutant peptide (MEFmD) are
indicated in boldface. B, peptide competition
assays for phosphorylation of MBP-SAP-1 fusion protein by the indicated
MAPKs. The substrate (5 pmol) was phosphorylated by p38 MAPKs in the
absence (lane 1) or presence of competitor peptides (a
10-1000-fold excess over SAP-1 substrate) at 50 pmol (lanes
2, 5, and 8), 500 pmol (lanes 3,
6, and 9), and 5 nmol (lanes 4,
7, and 10). The reactions were performed for 30 min at 30 °C. Increases in the concentration of added peptides are
indicated schematically above each set of lanes. ND, not
done.
and p38
2, but had no effect on p38
(Fig.
3B, lanes 2-4). Similarly, the MEFD peptide
selectively inhibited SAP-1 phosphorylation by p38
and
p38
2 (Fig. 3B, lanes 5-7),
whereas a mutant form of this peptide (MEFmD) did not inhibit
phosphorylation by these kinases (lanes 8-10). Thus, like
the docking site in MEF2, the SAP-1 D-domain acts specifically to bind
to p38
and p38
2 MAPK isoforms.
and p38
2 MAPKs, the SAP-1 D-domain also recruits ERK MAPK.
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Fig. 4.
The SAP-1 D-domain acts as a binding site for
ERK2. A, diagrammatic representation of the substrates
and alignment of the sequences of the peptides used as competitors in
the kinase assays. The SAPD peptide corresponds to the D-domain of
SAP-1, whereas the ElkD peptide encompasses the Elk-1 D-domain (20).
Annotations to the peptides are as described in the legend to Fig.
3A. B, peptide competition assays for
phosphorylation of MBP-SAP-1 and MBP-Elk-1 fusion proteins by ERK2.
Phosphorylation of the proteins by ERK2 was performed in the absence
(lane 1) or presence of competitor peptides (a
500-1000-fold excess over substrate) at 2.5 nmol (lanes 2,
4, and 6) and 5 nmol (lanes 3,
5, and 7). The reactions were performed as
described in the legend to Fig. 3. ND represents assays that
were not performed. C, kinase binding assays for ERK2 with
MBP-SAP-1 and MBP-Elk-1 fusion proteins. The proteins (50 pmol) were
incubated with ERK2 (25 units) in kinase binding buffer for 4 h at
4 °C. Following extensive washing, the remaining kinase-substrate
complexes were used in kinase assays that were performed at 30 °C
for 90 min.
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Fig. 5.
Differential requirement of the D-domain and
FXF motif for SAP-1 phosphorylation in
vitro and transcriptional activation in vivo
by ERK2. A, schematic illustration of truncated
SAP-1 proteins fused to either MBP or the GAL4 DNA-binding domain.
Domains in SAP-1 (see Fig. 1) and the alanine substitutions within the
FXF motif of the proteins SAP-1-mF and SAP-1 D-mF are
indicated. B, phosphorylation of the indicated MBP-Elk-1
(lanes 1-4) and MBP-SAP-1 (lanes 5-8) fusion
proteins in vitro by ERK2. Kinase assays were performed for
30 min at 30 °C with equimolar concentration of all proteins (5 pmol) as substrates. C, quantification of in
vitro phosphorylation data from B (left
panel) and in vivo reporter gene activity (right
panel). COS-7 cells were cotransfected with expression vectors
encoding various GAL4-SAP-1 or GAL4-Elk-1 derivatives, a GAL4-driven
luciferase reporter plasmid, and a constitutively active form of MEK
and ERK2. The data presented were calculated as described in the legend
to Fig. 2B. WT, wild type.
and p38
2, mutation of the
FXF motif alone had either a small or virtually no effect on
its phosphorylation in vitro by p38
and
p38
2, respectively (Fig.
6B). Simultaneous disruption of the D-domain and FXF motif led to a further
decrease in the proficiency of SAP-1 as a p38
substrate (Fig.
6B, lane 4). In contrast,
phosphorylation of SAP-1 by p38
was barely affected by disruption of
these two regions. These data therefore demonstrate that, in
vitro, the FXF motif of SAP-1 plays a selective role in
determining its phosphorylation by the p38
isoform, although the
D-domain appears to be more important.
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Fig. 6.
Differential requirement of the D-domain and
FXF motif for SAP-1 phosphorylation in
vitro and transcriptional activation in vivo
by p38 MAPKs. A, schematic illustration of
truncated SAP-1 proteins fused to either MBP or the GAL4-DNA binding
domain (see the legend to Fig. 5 for details). B,
phosphorylation of the indicated MBP-SAP-1 fusion proteins in
vitro by p38 , p38
2, and p38
MAPKs. Kinase
assays were performed as described in the legend to Fig. 5.
C, activation of mutant SAP-1 proteins by p38 MAPK cascades
in vivo. COS-7 cells were cotransfected with expression
vectors encoding various GAL4-SAP-1 derivatives, a GAL4-driven
luciferase reporter plasmid, and a constitutively active form of MKK6
(MKK6(E)) and the indicated p38 MAPK. The data presented were
calculated as described in the legend to Fig. 2B.
- and p38
2-mediated transactivation. This is
consistent with the decreases in in vitro phosphorylation
observed with this mutant protein (Figs. 1B and
6B). Mutation of the FXF motif also led to a
large decrease in p38
-mediated transactivation, but barely affected p38
2-mediated transactivation (Fig. 6C).
Simultaneous disruption of the D-domain and FXF motif in
GAL4-SAP-1
D-mF led to a further decrease in p38
-mediated
transactivation, but did not lead to any further effects on
p38
2-mediated transactivation compared with deletion of
the D-domain alone (Fig. 6C). Again, these in vivo effects are fully consistent with the in vitro
phosphorylation data (Fig. 6B). Finally, as a control, we
tested the response of the SAP-1 derivatives to activation by
p38
in vivo, as neither motif appears to be important for
targeting of this kinase in vitro (Fig. 6B). As
predicted from this in vitro data, neither deletion of the
D-domain nor mutation of the FXF motif, either individually
or in combination, led to a significant decrease in p38
-mediated
transactivation (Fig. 6C). In contrast, the activity of the
fusion proteins was actually increased in the absence of the D-domain
(see "Discussion").
and p38
2. However, the FXF motif
plays a lesser role, which is only apparent for p38
. Further
specificity is implied by the observation that p38
is not targeted
to SAP-1 by these motifs.
, but did not
affect phosphorylation by p38
2 and p38
(Fig.
7B, lanes 2 and 3). A mutant peptide
containing two changes in the conserved phenylalanine residues (Fig.
7A) was unable to inhibit the activity of any of the ERK or
p38 MAPKs (Fig. 7B, lanes 4 and 5).
Thus, the SAP-1 FXF motif acts as a selective binding site
for subsets of MAPKs.
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Fig. 7.
The FXF motif peptides
function as inhibitors of ERK2 and p38
activity. A, diagram showing truncated forms of
SAP-1 protein fused to MBP. Annotations are as described in the legend
to Fig. 1. The numbers of the altered amino acids within the
FXF motif of the protein SAP-1-mF are also indicated. The
sequences of the peptide competitors are presented. The SAPF and SAPD
peptides correspond to the FXF motif and the D-domain of
SAP-1, respectively. The amino acids altered in the mutant peptide
(SAPmF) are indicated in boldface. B, peptide
competition assays for phosphorylation of MBP-SAP-1 fusion protein by
the indicated MAPKs. The protein was phosphorylated by the indicated
MAPKs for 30 min at 30 °C in the absence (lane 1) or
presence of competitor peptides (a 500-1000-fold excess over
substrate) at 2.5 nmol (lanes 2 and 4) and 5 nmol
(lanes 3 and 5). C, competition assays
were performed as described for B using 5 pmol of SAP-1
D
and SAP-1
D-mF as substrates. The proteins were phosphorylated
by ERK2 in the absence (lanes 1, 4, 7,
and 10) or presence of the SAPD (upper
panel) or SAPF (lower panel) competitor peptide (a
500-1000-fold excess over substrate) at 2.5 nmol (lanes 2,
5, 8, and 11) and 5 nmol
(lanes 3, 6, 9, and 12).
Increases in the concentration of added peptides are indicated
schematically above each set of lanes.
D) or
the FXF motif (SAP-1-mF). The predicted outcome of these
competition assays is that the peptides should only compete with the
kinase for substrate binding when the substrate contains a related
binding motif. As predicted, the SAPD peptide competed for ERK2 binding and inhibited SAP-1 phosphorylation in the presence of the D-domain in
the substrate, but not in its absence (Fig. 7C, lanes
1-6). However, in contrast, the SAPF peptide inhibited
phosphorylation of SAP-1 irrespective of the presence of the
FXF motif (Fig. 7C, lanes 7-12).
, but do
so in a substrate-independent manner. One way in which they might do
this is by binding to the kinase and blocking its catalytic activity,
either by a steric or allosteric mechanism (see "Discussion"). This
hypothesis predicts that these peptides will inhibit phosphorylation of
other substrates that lack FXF motifs. Indeed, both the SAPF
and ElkF peptides inhibited phosphorylation of myelin basic protein by
ERK2 with a similar potency to their ability to inhibit Elk-1
phosphorylation (Fig. 8B).
Furthermore, as the ElkF peptide was unable to inhibit the activity of
either the JNK or p38
2 MAPKs (Fig. 8B), their inhibitory effect appears to be kinase-specific. Finally, the SAPF
peptide could inhibit phosphorylation of MEF2C by p38
, despite the
lack of an FXF motif in this substrate (Fig. 8B).
Thus, peptides containing FXF motifs show selectivity in
their inhibition of protein kinases (ERK2 and p38
), but act in a
substrate-independent manner.
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Fig. 8.
The FXF motif peptides
specifically inhibit subsets of MAPKs in a substrate-independent
manner. A, sequence comparison of the peptides ElkF and
SAPF used in peptide competition assays. The SAPmF peptide represents a
mutant form of the SAPF peptide. The residues that were substituted
with alanines are indicated. B, peptide competition assays
for myelin basic protein, GST-MEF2C, and MBP-Elk-1. The proteins (5 pmol) were phosphorylated by the indicated MAPKs in the absence
(lane 1) or presence of competitor peptides (a
500-1000-fold excess over substrate) at 2.5 nmol (lanes 2,
4, and 6) and 5 nmol (lanes 3,
5, and 7) for 30 min at 30 °C. Increases in
the concentration of added peptides are indicated schematically above
each set of lanes. ND represents assays that were not
done.
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Fig. 9.
The FXF motif directs ERK2
to phosphorylate heterologous substrates. A, a
schematic illustration is shown of wild-type and chimeric MEF2A protein
substrates fused to GST. The hatched boxes represent the
MEF2 transcriptional activation domain. The MEF2 D-domain is indicated
by white boxes labeled with D, and the Elk-1
D-domain is depicted by black boxes. The insertion of a
wild-type (FQFP) or mutant (AQAP) FXF motif is indicated.
Numbers below the sequences represent MEF2-derived
sequences, whereas numbers above were derived from Elk-1.
B, protein kinase assays were carried out for 30 min using 5 pmol of substrate with the indicated GST fusion protein substrates and
protein kinases. Each panel contains data from a single gel that was
split into lanes for clarity of presentation. C, shown are
the results from kinase binding assays using the indicated GST-MEF2A
fusion proteins (lanes 1-3) to bind to ERK2. Prior to
initiating kinase reactions, the additional substrate MBP-SAP-1 was
added to detect bound kinases. The identities of the bands representing
the original (white arrow) and added (black
arrow) substrates are indicated.
and p38
2 substrate (19), and its
D-domain was required for efficient phosphorylation (Fig.
9B, compare lanes 8 and 15 and
lanes 11 and 18). In contrast, MEF2A was a poor
ERK2 substrate (Fig. 9B, lane 1),
possibly reflecting the absence of an FXF motif. However,
upon fusion of an FXF motif to MEF2A, MEF2A became a good
ERK2 substrate. In contrast, fusion of a mutated FXF motif
did not promote MEF2A phosphorylation by ERK2 (Fig. 9B,
compare lanes 2 and 3). Phosphorylation by p38
and p38
2 was virtually unaffected by the presence of the
FXF motif (Fig. 9B, lanes 9,
10, 16, and 17), presumably reflecting the dominance of the MEF2A D-domain in promoting phosphorylation by
these kinases. The effect of adding the FXF motif to MEF2A in the absence of its own D-domain was therefore tested (Fig. 9B, lanes 5, 12, 19, and
26). The presence of the FXF motif strongly enhanced MEF2A phosphorylation by ERK2 and slightly enhanced
phosphorylation by p38
, but did not affect its phosphorylation by
p38
2.
and
p38
2. In combination with the FXF motif, no
further enhancement of MEF2A phosphorylation by ERK2 was observed (Fig.
9B, compare lanes 5 and 7), and MEF2A
remained a poor substrate for p38
and p38
2 when its
D-domain was replaced with that of Elk-1. Together, these results are
consistent with the observation that the Elk-1 D-domain does not
represent a p38-binding motif and therefore cannot functionally replace
the p38-binding motif in MEF2A. This is in contrast to the observation
that the SAP-1 D-domain can fulfill this function as a p38-binding
motif (19). Finally, to confirm the specificity of the effects we
observed, phosphorylation of each of the chimeric MEF2A proteins by
p38
was compared. All the chimeric proteins were phosphorylated to
similar extents by p38
(Fig. 9B, lanes 22-28), in keeping with the observation that the Elk-1 and MEF2A D-domains and the FXF motif do not represent p38
-binding sites.
D) (Fig.
10A) and compared its
proficiency as an ERK substrate with MEF2A
D-F, in which the
FXF motif is located downstream from the phosphoacceptor
motifs. Whereas MEF2A
D-F represented a good ERK2 substrate,
F-MEF2A
D was a poor substrate (Fig. 10B, lanes
2 and 3). Thus, although the FXF motif is
sufficient for recruiting ERK2 to substrates, this is not sufficient to
promote their phosphorylation, and the correct spatial arrangement with
the phosphoacceptor motifs is required.
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Fig. 10.
Position-dependent activity of
the FXF motif. A, a schematic
illustration is shown of wild-type and chimeric MEF2A protein
substrates fused to GST. The hatched boxes represent the
MEF2 transcriptional activation domain. The position of insertion of an
FXF motif (FQFP) is indicated. Phosphoacceptor motifs are
represented by P. Numbers below the schematics
represent MEF2-derived sequences. B, protein kinase
assays were carried out as described in the legend to Fig. 9 with the
indicated GST fusion protein substrates and protein kinases.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, and
p38
2. A second component, the FXF motif, also
plays a role in directing phosphorylation by ERK2 and, to a lesser
extent, p38
, although this motif appears to be unimportant for
p38
2. However, the relative contribution of each motif
to determining the specificity of SAP-1 as a MAPK substrate differs,
and their mechanisms of action also appear to differ.
-domain of c-Jun specifically binds to the JNK MAPKs (16, 17),
whereas the docking domain of Elk-1 binds both the JNK and ERK MAPKs
(20). Here we demonstrate that the D-domain of SAP-1 exhibits a novel
specificity for MAPKs and is important for phosphorylation by ERK and a
subset of p38 MAPKs. Interestingly, the SAP-1 and Elk-1 D-domains are
highly conserved (see Fig. 4A), which is consistent with
their ability to act as ERK-docking sites. However, differences in the
amino acid sequences of these domains must be responsible for
determining the ability of the SAP-1 D-domain, but not the Elk-1
D-domain, to act as a p38-binding site. Other MAPKs such as p38
are
not targeted by this motif. Indeed, deletion of the D-domain of SAP-1
leads to enhanced transactivation by p38
in vivo. This
might reflect that the D-domain is also the target of a negatively
acting factor in vivo, whose effect is relieved upon
deletion of this domain. One attractive candidate for such an
inhibitory protein would be a protein phosphatase.
MAPKs.
Importantly though, this motif does not appear to be able to function
on its own with respect to p38 MAPKs and needs the presence of an
additional docking domain to promote kinase binding and substrate
phosphorylation. With ERK, however, the FXF motif is
sufficient to promote substrate phosphorylation (Fig. 9). Indeed, these
studies with chimeric proteins demonstrate that, like the D-domain, the
FXF motif is portable and sufficient to promote
phosphorylation of heterologous substrates. It appears, however, that
the FXF motif exhibits quite stringent kinase selectivity,
as it is unable to promote substrate phosphorylation by JNK and other
p38 isoforms, and peptides containing FXF motifs inhibit
only a subset of MAPKs.
is
influenced by the FXF motif, the D-domain is sufficient to
permit phosphorylation by p38
2. Thus, by using a
combination of motifs and by altering the sequence of the D-domains, differences in the specificity of MAPK phosphorylation can be elicited.
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Fig. 11.
MAPK recognition modules and targeting of
MAPKs. A, the MAPK recognition module in SAP-1. This
module is composed of a D-domain, an FXF motif, and the
intervening phosphoacceptor motifs within the transcriptional
activation domain (TAD). B, targeting of MAPKs to
Elk-1 and SAP-1. The involvement of the D-domain and FXF
motif in directing substrate phosphorylation by individual kinases is
indicated by arrows.
It is clear that the D-domain and FXF motif can act in concert to promote substrate phosphorylation with high specificity. However, mechanistically, they may act differently. For example, in proteins that contain composite regulatory modules, the D-domains might initially recruit the kinases to the substrates, and the FXF motifs then act as a further filter to determine whether the kinase becomes locked onto the phosphoacceptor motifs or not. Other proteins that lack additional docking domains might require these to be provided in trans for the initial recruitment phase. Further work is required, however, to substantiate this model and to determine the precise functions of the D-domains and FXF motifs.
In summary, we have identified a complex MAPK recognition module in
SAP-1. This module exhibits several novel features in comparison with
known modules and demonstrates how individual components can act either
alone or in concert to determine specificity in substrate
phosphorylation by MAPKs.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Linda Shore and Margaret Bell for excellent technical assistance. We also thank Alan Whitmarsh for comments on the manuscript. We are grateful to Alan Whitmarsh and Roger Davis for reagents.
![]() |
FOOTNOTES |
---|
* This work was supported in part by grants from the Wellcome Trust.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Recipient of a Jeffcock Ph.D. studentship from the University of Newcastle upon Tyne.
Supported by a Lister Institute of Preventative Medicine
Research fellowship. To whom correspondence should be addressed: School
of Biological Sciences, University of Manchester, 2.205 Stopford Bldg.,
Oxford Rd., Manchester M13 9PT, UK. Tel.: 44-161-275-5979; Fax:
44-161-275-5082; E-mail: a.d.sharrocks@man.ac.uk.
Published, JBC Papers in Press, October 11, 2000, DOI 10.1074/jbc.M007697200
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ABBREVIATIONS |
---|
The abbreviations used are: MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MBP, maltose-binding protein; GST, glutathione S-transferase; MKK, MAPK/ERK kinase kinase; MEK, MAPK/ERK kinase.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Pawson, T.,
and Nash, P.
(2000)
Genes Dev.
14,
1027-1047 |
2. | Robinson, M. J., and Cobb, M. H. (1997) Curr. Opin. Cell Biol. 9, 180-186[CrossRef][Medline] [Order article via Infotrieve] |
3. | Cohen, P. (1997) Trends Cell Biol. 7, 353-361[CrossRef] |
4. |
Jiang, Y.,
Gram, H.,
Zhao, M.,
New, L.,
Gu, J.,
Feng, L.,
Di Padova, F.,
Ulevitch, R. J.,
and Han, J.
(1997)
J. Biol. Chem.
272,
30122-30128 |
5. |
Jiang, Y.,
Chen, C.,
Li, Z.,
Guo, W.,
Gegner, J. A.,
Lin, S.,
and Han, J.
(1996)
J. Biol. Chem.
271,
17920-17926 |
6. |
Cuenda, A.,
Cohen, P.,
Buee-Scherrer, V.,
and Goedert, M.
(1997)
EMBO J.
16,
295-305 |
7. |
Goedert, M.,
Cuenda, A.,
Craxton, M.,
Jakes, R.,
and Cohen, P.
(1997)
EMBO J.
16,
3563-3571 |
8. |
Wang, X. S.,
Diener, K.,
Manthey, C. L.,
Wang, S.,
Rosenzweig, B.,
Bray, J.,
Delaney, J.,
Cole, C. N.,
Chan-Hui, P. Y.,
Mantlo, N.,
Lichenstein, H. S.,
Zukowski, M.,
and Yao, Z.
(1997)
J. Biol. Chem.
272,
23668-23674 |
9. |
Enslen, H.,
Raingeaud, J.,
and Davis, R. J.
(1998)
J. Biol. Chem.
273,
1741-1748 |
10. |
Enslen, H.,
Brancho, D. M.,
and Davis, R. J.
(2000)
EMBO J.
19,
1301-1311 |
11. | Madhani, H. D., and Fink, G. R. (1998) Trends Genet. 14, 151-155[CrossRef][Medline] [Order article via Infotrieve] |
12. | Whitmarsh, A. J., and Davis, R. J. (1998) Trends Biochem. Sci. 23, 481-485[CrossRef][Medline] [Order article via Infotrieve] |
13. | Garrington, T. P., and Johnson, G. L. (1999) Curr. Opin. Cell Biol. 11, 211-218[CrossRef][Medline] [Order article via Infotrieve] |
14. | Holland, P. M., and Cooper, J. A. (1999) Curr. Biol. 9, R329-R331[CrossRef][Medline] [Order article via Infotrieve] |
15. | Sharrocks, A. D., Galanis, A., and Yang, S.-H. (2000) Trends Biochem. Sci. 25, 448-453[CrossRef][Medline] [Order article via Infotrieve] |
16. | Hibi, M., Lin, A., Smeal, T., Minden, A., and Karin, M. (1993) Genes Dev. 7, 2135-2148[Abstract] |
17. | Dai, T., Rubie, E., Franklin, C. C., Kraft, A., Gillespie, D. A., Avruch, J., Kyriakis, J. M., and Woodgett, J. R. (1995) Oncogene 10, 849-855[Medline] [Order article via Infotrieve] |
18. | Kallunki, T., Su, B., Tsigelny, I., Sluss, H. K., Derijard, B., Moore, G., Davis, R. J., and Karin, M. (1994) Genes Dev. 8, 2996-3007[Abstract] |
19. |
Yang, S.-H.,
Galanis, A.,
and Sharrocks, A. D.
(1999)
Mol. Cell. Biol.
19,
4028-4038 |
20. |
Yang, S.-H.,
Whitmarsh, A. J.,
Davis, R. J.,
and Sharrocks, A. D.
(1998)
EMBO J.
17,
1740-1749 |
21. |
Yang, S.-H.,
Yates, P. R.,
Whitmarsh, A. J.,
Davis, R. J.,
and Sharrocks, A. D.
(1998)
Mol. Cell. Biol.
18,
710-720 |
22. | Yang, S.-H., Yates, P. R., Mo, Y., and Sharrocks, A. D. (1999) Gene Ther. Mol. Biol. 3, 355-371 |
23. | Janknecht, R., Ernst, W. H., and Nordheim, A. (1995) Oncogene 10, 1209-1216[Medline] [Order article via Infotrieve] |
24. | Price, M. A., Rogers, A. E., and Treisman, R. (1995) EMBO J. 14, 2589-2601[Abstract] |
25. |
Janknecht, R.,
and Hunter, T.
(1997)
EMBO J.
16,
1620-1627 |
26. |
Janknecht, R.,
and Hunter, T.
(1997)
J. Biol. Chem.
272,
4219-4224 |
27. |
Jacobs, D.,
Glossip, D.,
Xing, H.,
Muslin, A. J.,
and Kornfeld, K.
(1999)
Genes Dev.
13,
163-175 |
28. |
MacKenzie, S. J.,
Baillie, G. S.,
McPhee, I.,
Bolger, G. B.,
and Houslay, M. D.
(2000)
J. Biol. Chem.
275,
16609-16617 |
29. |
Seth, A.,
Gonzalez, F. A.,
Gupta, S.,
Raden, D. L.,
and Davis, R. J.
(1992)
J. Biol. Chem.
267,
24796-24804 |
30. | Sadowski, I., and Ptashne, M. (1989) Nucleic Acids Res. 17, 7539[Medline] [Order article via Infotrieve] |
31. |
Raingeaud, J.,
Gupta, S.,
Rogers, J. S.,
Dickens, M.,
Han, J.,
Ulevitch, R. J.,
and Davis, R. J.
(1995)
J. Biol. Chem.
270,
7420-7426 |
32. | Mansour, S. J., Matten, W. T., Hermann, A. S., Candia, J. M., Rong, S., Fukasawa, K., Vande-Woude, G. F., and Ahn, N. G. (1994) Science 265, 966-970[Medline] [Order article via Infotrieve] |
33. | Whitmarsh, A. J., Cavanagh, J., Tournier, C., Yasuda, J., and Davis, R. J. (1998) Science 17, 2360-2371 |
34. | Tanoue, T., Adachi, M., Moriguchi, T., and Nishida, E. (2000) Nat. Cell Biol. 2, 110-116[CrossRef][Medline] [Order article via Infotrieve] |
35. | Knighton, D. R., Zheng, J. H., Ten Eyck, L. F., Xuong, N. H., Taylor, S. S., and Sowadski, J. M. (1991) Science 253, 414-420[Medline] [Order article via Infotrieve] |