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J. Biol. Chem., Vol. 278, Issue 35, 32662-32672, August 29, 2003
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From the Department of Developmental and Cell Biology, University of California, Irvine, California 92697
Received for publication, April 22, 2003 , and in revised form, June 2, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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, and p38
to MKK4. Mutations that altered
conserved residues in this docking site reduced JNK/p38 binding. In addition,
a peptide version of this docking site, as well as a peptide version of the
JNK-binding site of the JIP-1 scaffold protein, inhibited both MKK4/JNK
binding and MKK4-mediated phosphorylation of JNK1. These same peptides also
inhibited JNK2-mediated phosphorylation of c-Jun and ATF2, suggesting that
transcription factors, MKK4, and the JIP scaffold compete for docking to JNK.
Finally, the selectivity of the MKK4, MEK1, and MEK2 D-sites for JNK
versus ERK was quantified. The MEK1 and MEK2 D-sites displayed a
strong selectivity for their cognate MAPK (ERK2) versus a non-cognate
MAPK (JNK). In contrast, the MKK4 D-site exhibited only limited selectivity
for JNK versus ERK. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Central to these cascades is a MAPK kinase (MKK) that phosphorylates and thereby activates the MAPK. Activated MAPKs phosphorylate multiple targets, including transcription factors, other kinases, and other enzymes. In mammals, there are three major MAPK pathways (the extracellular signal-regulated kinase (ERK)-1/2, c-Jun N-terminal kinase (JNK), and p38 modules) that each contain two to four different MAPKs regulated by two different MKKs (2). (Some MAPKs are also called ERKs, and some MKKs are also called MAPK/ERK kinases (MEKs).) In general, the MKKs in one pathway do not phosphorylate MAPKs in other pathways. How this selectivity is achieved is incompletely understood.
The JNK pathway is activated primarily in response to cytokines and environmental stress and plays an important role in the regulation of stress-induced apoptosis (reviewed in Refs. 3–5). Key targets phosphorylated by JNK include components of the AP-1 transcription factor such as c-Jun and ATF2. Current thinking suggests that the JNK cascade and AP-1 can promote proliferation or differentiation and survival or death depending on the cell type, the nature and duration of the stimulus, and the activity of other signaling pathways (6, 7). The JNK cascade has been implicated in the pathogenesis of many human diseases, including cancer (8), obesity and insulin resistance (9), muscular dystrophy (10), arthritis (11), aspects of heart disease (12), Parkinson's disease (13), and other neurological disorders characterized by abnormal cell death (14).
There are three different JNK proteins (JNK1, JNK2, and JNK3) encoded by
separate genes. Two protein kinases that activate JNK have been identified:
MKK4 (also known as JNKK1 and SEK1) and MKK7/JNKK2. Although both MKK4 and
MKK7 activate JNK, they are functionally distinct in several ways
(4). First, although both
kinases activate JNK by dual phosphorylation at Thr and Tyr, MKK4 prefers Tyr,
and MKK7 prefers Thr (15).
Second, MKK4 is activated primarily by environmental stress, and MKK7 by
cytokines. Third, MKK4 (but not MKK7) can also activate the p38 MAPK. Finally,
MKK4 (but not yet MKK7) is a candidate tumor/metastasis suppressor, as
inactivating mutations in the MKK4 gene (MAP2K4) have been found in
5% of a wide variety of tumor types
(16–18).
As in other MAPK pathways, the formation of stable complexes between MKKs
and MAPKs is thought to contribute to signal transmission and specificity in
the JNK pathway (19,
20). Full-length MKK4 has been
shown to form complexes with JNK1/SAPK
(20), JNK2/SAPK
(21), and JNK3/SAPK
(22,
23). Like other MKKs, MKK4
consists of a highly conserved catalytic domain and an N-terminal extension
that exhibits substantially less conservation (see
Fig. 1A). The
N-terminal extension has been found to mediate MAPK binding by many MKKs,
including yeast Ste7 (24) as
well as mammalian MEK1 (25),
MEK2 (26), MKK3/MKK6
(27), and MKK7
(28). In keeping with this
pattern, the N-terminal extension of MKK4 has been shown to be both necessary
and sufficient for JNK binding
(20,
23).
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The N termini of many MKKs contain a conserved MAPK-docking site (consensus sequence (K/R)2–3-X1–6-(L/I)-X-(L/I), herein referred to as the "D-site") that was first recognized based on sequence similarity to yeast Ste7 (26, 29). Indeed, the D-site in Ste7 (24, 26) and those in mammalian MEK1 (26, 30), MEK2 (26), and MKK3 and MKK6 (27) have been shown to mediate high affinity binding to their cognate MAPKs. Similar and related D-sites were also found in the transcription factors c-Jun (31) and Elk-1 (32) as well as in other transcription factors, phosphatases, scaffolds, other kinases, and other proteins (see Fig. 1B) (reviewed in Refs. 33 and 34). D-sites appear to be portable modular motifs that mediate the interaction of MAPKs with multiple binding partners, contributing to both signal transmission and specificity. Yet, the fact that distinct docking sites share a core consensus sequence suggests that there might be limits to the specificity intrinsic to docking interactions.
The D-sites in MEK1, MEK2, MKK3, and MKK6 have been shown to be substrates for cleavage by the lethal factor protease of Bacillus anthracis (anthrax) (35); furthermore, lethal factor cleavage has been shown to compromise the ability of some of these enzymes to activate their cognate MAPKs (36, 37). MKK4 is also cleaved by lethal factor at two sites in its N terminus (see Fig. 1A) (35), but whether one or both of these cleavage sites are also MAPK-docking sites has not heretofore been examined.
A putative D-site that fits the consensus sequence (K/R)2–3-X1–6-(L/I)-X-(L/I) has been identified in MKK4 (26), but has not previously been shown to be functional; this D-site corresponds to the first of the two lethal factor cleavage sites. Here, we show that this D-site is indeed functional, necessary, and sufficient for JNK binding; quantify its selectivity for JNK versus ERK2; and provide evidence that it competes with D-site-containing substrates for binding to JNK.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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1 (MAPK10; accession number
NM_ 002753), p38 (MAPK14; L35253
[GenBank]
), and p38
(MAPK11; accession number NM_002751
[GenBank]
).
Proteins and Antisera—Several purified or partially purified
proteins used in this study were purchased from Cell Signaling Technology, Upstate Biotechnology, Inc., Calbiochem, New England Biolabs Inc., and Santa
Cruz Biotechnology. These included fusions of glutathione
S-transferase (GST) to human c-Jun-(1–89), ATF2-(19–96),
and Elk-1-(307–428) (all from Cell Signaling Technology; the
GenBankTM/EBI accession numbers of the corresponding full-length human
proteins are NM_002228
[GenBank]
, NM_001880
[GenBank]
, and NM_005229
[GenBank]
, respectively); inactive
(K52R mutation) mouse ERK2 (Cell Signaling Technology; accession number
BAA01733
[GenBank]
); unactivated rat ERK2 (Calbiochem; accession number M64300
[GenBank]
);
unactivated human JNK11 (Upstate Biotechnology, Inc. or Santa Cruz
Biotechnology; accession number L26318
[GenBank]
), JNK11 (Calbiochem; accession
number U35004
[GenBank]
), and JNK22 (Upstate Biotechnology, Inc.; accession
number L31951
[GenBank]
); activated human JNK22 (Upstate Biotechnology, Inc.;
accession number L31951
[GenBank]
) and mouse ERK2 (New England Biolabs Inc.; accession
number BAA01733
[GenBank]
); and activated human MEK2 (Upstate Biotechnology, Inc.;
accession number A46723
[GenBank]
) and mouse MKK4-(35–397) (Upstate Biotechnology,
Inc.; accession number U18310
[GenBank]
). Anti-ERK2 and anti-JNK antisera was from Santa
Cruz Biotechnology.
Plasmids for in Vitro Transcription/Translation—To
construct pGEM-JNK3 (a gift from Douglas M. Molina of this laboratory), the
JNK31 coding region was amplified by high fidelity PCR using
Pfu DNA polymerase, primers LB119 and LB120 (see
Table I), and a cDNA clone
obtained from the IMAGE consortium
(38) as the template (IMAGE ID
632588, GenBankTM/EBI accession numbers AA191055
[GenBank]
and AA188639
[GenBank]
). The PCR
product was digested with BamHI and SalI and inserted into
the corresponding sites of pGEM4Z (Promega).
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The MKK4 constructs were generated using pcDNA3-FLAG-MKK4 (a gift from Roger Davis, University of Massachusetts Medical School) as the template for PCR and MKK4endRNew as the reverse primer. For pGEM-MKK4 (full-length), the forward primer used was MKK4ForBeg. For pGEM-MKK4-(37–399) and pGEM-MKK4-(47–399), MKK4_37–94F and MKK4_47endFNew were used as the forward primers, respectively. All three MKK4 PCR products were digested with BamHI and SalI before being inserted into the corresponding sites of pGEM4Z.
The p38 open reading frame was amplified by PCR using primers LB117
and LB118 and a cDNA clone obtained from Invitrogen (Genestorm clone ID
RG000027) as the template. The resulting PCR product was cut with
BamHI and XhoI and inserted into pGEM4Z that had been cut
with BamHI and SalI, yielding pGEM-p38. Plasmid
pGEM-p38 was constructed following a similar strategy, using primers
p38Up and p38Down, Invitrogen Genestorm clone ID RG002657 as the
template, and cutting with EcoRI and SalI.
Plasmids for the Production of GST Fusion Proteins—The vector used for generating the GST fusion proteins was pGEXLB (26), a derivative of pGEX-4T-1 (Amersham Biosciences). In pGEXLB, an encoded Pro residue is replaced with a Gly-Gly-Gly-Gly-Gly-Ser-Gly sequence to promote the independent functioning of the GST and fusion moieties.
To construct pGEXLB-JNK3 (a gift from Douglas M. Molina), the JNK3 coding region was excised from pGEM4Z-JNK3 by restriction digest with BamHI and SalI and inserted into the corresponding sites of pGEXLB. The MEK1-(1–60) coding sequence was amplified by PCR with primers SP6PL (26) and JB804 (see Table I) using pGEM-MEK1 (26) as the template; digested with BamHI and SalI; and inserted into the corresponding sites of pGEXLB, yielding pGEXLB-MEK1-(1–60). Plasmid pGEXLB-MEK2-(1–64), containing MEK2 residues 1–64, was generated by a similar procedure using primers SP6PL and JB805, pGEM-MEK2 (26) as the template, and cutting with EcoRI and SalI.
Plasmid pGEXLB-MKK4-(1–94) was generated by PCR amplification using
primers DH700 and DH701 and the template pSR3-JNKK1 (a gift from
Michael Karin, University of California at San Diego). The PCR product was
subsequently digested with BamHI and SalI and inserted into
the corresponding sites of pGEXLB. The pGEXLB-MKK4-(37–94) wild-type and
mutant constructs were generated using the pGEM-MKK4-(1–94) construct as
the template. The wild-type construct was generated using the forward primer
MKK4_37–94F, whereas the mutant construct was generated using the
forward primer MKK4_37–94FMut; in both cases, DH701 was the reverse
primer. Both MKK4 PCR products were digested with BamHI and
SalI before being inserted into the corresponding sites of
pGEM4Z.
To generate the MKK4 D-site mutant constructs, an adaptor oligonucleotide
approach was utilized. Site-directed mutagenesis using oligonucleotide
pGEXBssHIIDestroyFwd and its complement was carried out to destroy a
BssHII site at bases 4102–4107 within the -lactamase gene
of pGEXLB-MKK4-(1–94). A unique BssHII site was then introduced
by silent substitution at codons 57 and 58 of the MKK4-(1–94) open
reading frame; this was accomplished by site-directed mutagenesis using
oligonucleotide pGEXBssHIIFormingFwd and its complement. This new plasmid,
pGEX-2SDM-MKK4-(1–94), was then digested with BamHI and
BssHII to excise a 168-bp fragment encoding residues 1–58. The
excised fragment was replaced with annealed oligonucleotide pairs encoding
residues 37–58 and containing BamHI and BssHII sticky
ends. These oligonucleotide pairs contained the particular docking site
mutations. For example, the K40A mutant of the pGEXLB-MKK4-(37–94)
mutant construct was generated using oligonucleotides M4ForMut1 and M4RevMut1
(Table I). The other mutants
(see Fig. 5A) were
constructed by the same procedure, except that oligonucleotides encoding the
indicated substitutions were employed.
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Transcription and Translation in Vitro—Proteins labeled with [35S]methionine were produced by coupled transcription and translation reactions (SPT3, Novagen). Translation products were partially purified by ammonium sulfate precipitation and quantified as described previously (24, 26).
Binding Assays—GST fusion proteins were expressed in
bacteria, purified by affinity chromatography using glutathione-Sepharose
(Amersham Biosciences), and quantified as described
(26). Binding assays were
performed and analyzed as described previously
(26). Binding constants were
calculated as described previously
(24,
26) based on the known input
concentrations and a determination of the amount of complex formed, assuming a
simple bimolecular binding reaction. An additional assumption was that 100% of
the input molecules were active (i.e. capable of binding). For the
reaction A + B 
AB (where A is the 35S-labeled protein, and B
is the GST fusion protein), Kd 
([A]eq
x [B]eq)/[AB]eq, where [A]eq =
[A]0 – [AB]eq; [B]eq = [B]0
– [AB]eq; and [A]0 and [B]0 are the
input concentrations of A and B, respectively. [AB]eq is the
percentage of A that co-sediments with B multiplied by [A]0.
Kd estimates are calculated from multiple replicate
experiments; two representative examples are shown in
Table II.
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Peptides and Peptide Array—The soluble peptides used in this
study (see Fig. 6B)
were synthesized by United Biochemical Research, Inc. (Seattle, WA). Custom
synthesis of the peptide arrays used in this study was performed by Jerini AG
(Berlin, Germany). The peptides were N-terminally acetylated and anchored to
the membrane via a chemical linker at their C termini. Typically, an
5-cm2 membrane containing 50 peptide spots was used in an
experiment. The membrane was first equilibrated by incubation with binding
buffer A (20 mM Tris-HCl (pH 7.5), 125 mM potassium
acetate, 0.5 mM EDTA, 1 mM dithiothreitol, 0.1% (v/v)
Tween 20, and 12.5% (v/v) glycerol) for 15 min at room temperature and then
blocked by incubation with the same buffer containing 10 mg/ml bovine serum
albumin for 15 min at room temperature. The membrane was incubated with 10 ml
of the same buffer containing 1 mg/ml bovine serum albumin and 25 pmol of
in vitro translated, 35S-labeled JNK3 protein that had
been partially purified by ammonium sulfate precipitation from 500 µl of
reticulocyte lysate. This incubation was for 1 h at room temperature. The
membrane was washed for 1 min at room temperature in 100 ml of buffer A and
then for 1 min in 100 ml of ice-cold buffer A, allowed to dry, and quantified
on a PhosphorImagerTM (Amersham Biosciences). For quantification,
"-fold over background" was determined, i.e. the counts
emitted from the peptide spot divided by an average of several background
spots of the same area taken from other parts of the membrane where no
peptides were coupled.
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Protein Kinase Assays—The protein kinase assays for MEK2
phosphorylation of ERK2 and for ERK2 phosphorylation of Elk-1 were performed
as described (39). Kinase
reactions (20 µl) for MKK4 phosphorylation of JNK1 contained kinase assay
buffer (50 mM Tris-HCl (pH 7.5), 10 mM MgCl2,
1 mM EGTA, and 2 mM dithiothreitol), 2 µM
(1.8 µg) unactivated human JNK1, 0.02 units (15 ng) of active
full-length mouse MKK4, 50 µM ATP, 1 µCi of
[-32P]ATP, and the indicated concentration of peptide.
Reactions were for 20 min at 30 °C. JNK1 phosphorylation was quantified by
SDS-PAGE (12% gels), followed by analysis of relative incorporation using the
PhosphorImager.
Kinase reactions (20 µl) for JNK2 phosphorylation of c-Jun or ATF2
contained kinase assay buffer, 1 µM (740 ng) GST-c-Jun or 1
µM (700 ng) GST-ATF2, 0.8 milliunits (5 ng) of active human
JNK2, 50 µM ATP, 1 µCi of [-32P]ATP, and
the indicated concentration of peptide. Reactions were for 20 min at 30 °C
and were analyzed and quantified as described above.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-domain)
(31), ATF2
(34), Elk-1 (the D-domain)
(32), and the JIP-1 scaffold
protein (42)
(Fig. 1B). The term
D-site, an amalgam of -domain, D-domain, and docking site, will be
frequently used herein to refer to this class of related JNK-docking sites as
well as to similar docking sites for other MAPKs.
The D-site of MKK4 Is Necessary for JNK Binding—As an
initial test of the hypothesis that this region of MKK4 is a bona
fide JNK-docking site, the ability of MKK4 to bind to JNK3 was assessed
in an in vitro assay. Human JNK3 was fused at its N terminus to
Schistosoma japonicum GST, and the resulting fusion protein was
expressed in bacteria and purified by adsorption to glutathione-Sepharose
beads. GST-JNK3 (or GST alone as a control) was then incubated with either
full-length MKK4 or N-terminal truncation mutants of MKK4 that had been
produced in radiolabeled form by in vitro translation. Bead-bound
complexes were collected by sedimentation and analyzed by SDS-PAGE and
autoradiography. As shown in Fig.
2, full-length MKK4 bound efficiently to JNK3
(Kd 35 µM)
(Table II); this binding was
specific because precipitation of MKK4 protein did not occur when GST was used
instead of the GST-JNK3 fusion protein. MKK4-(37–399), which lacks the
first 36 residues of full-length MKK4, also bound to GST-JNK3. In contrast,
MKK4-(47–399), which lacks the first 46 residues, did not bind to JNK3.
Hence, residues 37–46, which contain the putative JNK-docking site and
the first anthrax lethal factor cleavage site, are necessary for high affinity
MKK4/JNK binding. Furthermore, the second anthrax lethal factor cleavage site
(approximately residues 54–59) is not sufficient for JNK3 binding.
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Localization of the MKK4 D-site—To confirm a previous report
that the N-terminal region of MKK4 is sufficient for binding to JNK
(20), a fusion of GST to the
first 94 residues of MKK4 was constructed (designated GST-MKK4-(1–94)).
Residues 1–94 comprise the entire portion of MKK4 that is N-terminal to
the kinase domain. To explore the specificity of binding, similar fusions of
GST to the N-terminal domains of MEK1 (residues 1–60) and MEK2 (residues
1–64) were also constructed. There are three JNK genes (JNK1,
JNK2, and JNK3) that are alternately spliced into at least 10
isoforms (43). The -
and -isoforms differ in their amino acid sequence between kinase
subdomains IX and X; the "2" isoforms have a mass of 54 kDa and
differ from the 46-kDa "1" isoforms in their C-terminal extension.
We assessed the binding of the JNK11, JNK11, JNK22, and
JNK31 isoforms to the N-terminal domains of MEK1, MEK2, and MKK4
(Fig. 3A). The binding
of ERK2 to all three MKKs was also examined. As shown in
Fig. 3B, all JNK
isoforms tested bound efficiently to GST-MKK4-(1–94), but did not bind
to GST-MEK1-(1–60) or GST-MEK2-(1–64). Conversely, ERK2 showed a
preference for binding to the N terminus of its cognate MKK MEK2. Hence, the
MKK N-terminal domains are selective for binding to their cognate MAPKs: the
MKK4 N-terminal domain prefers JNK, and the MEK2 N-terminal domain prefers
ERK2. However, this specificity is not absolute, as detectable binding of ERK2
to GST-MKK4-(1–94) was observed; indeed, MKK4-(1–94) bound to ERK2
just as well as, if not better, than MEK1-(1–60) did. This incomplete
specificity was also seen in experiments measuring the binding of full-length
MEK1, MEK2, and MKK4 to GST-ERK1, GST-ERK2, and GST-JNK3 (data not shown).
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The binding of in vitro translated, 35S-labeled JNK3 to
GST-MKK4-(1–94) was quantified (Kd 25
µM) (Table II).
This Kd agrees rather well with the value
(Kd 35 µM) obtained above for the
binding of 35S-labeled full-length MKK4 to GST-JNK3. In
vitro translated JNK3 migrated on SDS-polyacrylamide gels as a distinct
doublet (Fig. 3B); the
upper band presumably corresponded to full-length JNK3, whereas the lower
band, which was 4 kDa smaller, presumably represented the product of an
internal translation initiation or a premature termination. Whatever its
precise sequence, it bound to GST-MKK4-(1–94) with the same efficiency
as did full-length JNK3 (Fig.
3B).
To further localize the JNK-binding region of MKK4, residues 37–94 of
MKK4 were fused to GST and tested for binding to multiple MAPKs. Consistent
with the observations that both MKK4-(1–94)
(Fig. 3B) and
MKK4-(37–399) (Fig.
2B) were able to bind JNK, GST-MKK4-(37–94) was
also able to bind to all three JNK proteins
(Fig. 3C). The
affinity of this fusion for JNK3 (Kd 45
µM) was almost as strong as the affinity of
GST-MKK4-(1–94). Both JNK1 and JNK3 bound to the MKK4 fusions with
higher affinity than JNK2 did (compare the amount bound to the 10% input).
ERK2 also bound weakly to GST-MKK4-(37–94) (even more weakly than it did
to GST-MKK4-(1–94)). Finally, both the p38 and p38 MAPKs
bound weakly to GST-MKK4-(37–94) (Kd 200
µM) (Fig.
3C). This binding may be physiologically relevant, as
MKK4 can phosphorylate p38 in vitro
(4).
To delineate the contribution to the MKK4/MAPK interaction of the residues comprising the basic submotif of the putative D-site in MKK4, Lys40 and Lys42 were both changed to glutamate; Arg41 was changed to alanine; and this mutant, designated EAE, was fused to GST, yielding MKK4-(37–94)(EAE). As shown in Fig. 3C, this mutation abolished binding to all three JNK proteins and also to p38 and even to ERK2. Several conclusions can be drawn from these data. First, the basic submotif of the putative D-site in MKK4 is necessary for binding to its cognate MAPKs JNK and p38. Second, the weak non-cognate interaction of MKK4 with ERK2 is not a completely nonspecific interaction mediated by some "nonspecifically sticky" portions of each polypeptide, but is in fact dependent upon the same structural feature as the cognate interactions. In other words, ERK2 apparently binds weakly to the MKK4 D-site, indicating that this D-site has only limited ability to discriminate cognate MAPKs from non-cognate MAPKs (or that ERK2 has only a limited ability to discriminate cognate from non-cognate D-sites, or both). Finally, as the EAE mutation did not affect the integrity of the second lethal factor cleavage site (residues 54–59), yet completely abolished binding of JNK, ERK, and p38, it can be concluded that the second lethal factor cleavage site is not a high affinity MAPK-docking site. This is the first example of a lethal factor cleavage site that is not also a MAPK-docking site.
The D-site of MKK4 Is Sufficient for JNK Binding—To determine whether the D-site of MKK4 is sufficient for JNK binding, a peptide comprising residues 37–51 was synthesized and anchored to a cellulose membrane (44). A control peptide containing mutations of all three of the conserved basic residues, as well as of the second of the two conserved hydrophobic residues, was also synthesized and attached to the same membrane. These peptide spots were then incubated with radiolabeled JNK3 protein, and binding was quantified. As shown in Fig. 4, JNK3 bound very strongly to the wild-type MKK4 D-site, but exhibited no detectable binding to the mutant peptide. Hence, the D-site of MKK4 is sufficient for JNK binding.
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To investigate the N- and C-terminal borders of the MKK4 D-site, a series of shortened MKK4 peptides were assayed using the same peptide array method (data not shown). Removal of Met37 did not affect JNK3 binding; however, removal of Gln38 did somewhat diminish JNK3 binding. Similarly, removal of Pro51, Pro51 plus Asn50, or Ala49–Pro51 did not affect JNK3 binding; but removal of the next residue, Phe48, reduced JNK3 binding. These somewhat preliminary observations suggest that the minimal MKK4 D-site spans residues 38–48, comprising the sequence QGKRKALKLNF.
Analysis of Conserved Residues Required for Docking—To delineate the sequence features in the JNK-docking site of MKK4 that contribute to its stable and selective binding to JNK isoforms, amino acid substitutions were introduced into D-site consensus residues in this region (Fig. 5A). The D-site is a composite motif, composed of basic and hydrophobic submotifs (Fig. 1B). Single alanine substitution of either Lys residue in the Lys-Arg-Lys basic submotif of MKK4 had only moderate effects on JNK binding, reducing it to 60 and 90% of the wild-type level, respectively (Fig. 5B). In contrast, mutation of the Arg residue to alanine reduced JNK3 binding more substantially (37% of the wild-type level). Interestingly, double mutation of the Arg residue and either Lys residue almost completely abolished JNK3 binding, yet mutation of both lysines had much less of an effect (40% of the wild-type level). Substitution of all three basic residues with alanine eliminated JNK3 binding. These data suggest, first, that the basic submotif of the MKK4 D-site is critical for JNK binding and, second, that the three residues of the basic submotif bind in a partially cooperative manner to JNK3. Arg41 apparently participates in the most critical contact, but Lys40 and Lys42 are able to somewhat compensate for its loss.
Substitution of Leu44 or Leu46 of the conserved hydrophobic-X-hydrophobic submotif with alanine either alone or in combination also substantially reduced MKK4-JNK3 complex formation (Fig. 5, B and C). Hence, the hydrophobic submotif of the MKK4 D-site is also critical for JNK binding. These results collectively establish that residues 38–48 of MKK4 constitute a bona fide D-site.
D-site Peptides Inhibit MKK4 Binding to and Phosphorylation of JNK—To explore the role that MKK4/JNK docking plays in signal transmission from MKK4 to JNK, we tested whether synthetic peptides corresponding to D-sites are able to act as competitors of MKK4-mediated phosphorylation of JNK1 in vitro (Fig. 6A). Five different peptides were tested (Fig. 6B). The MKK4 D-site peptide corresponds to residues 37–52 of wild-type MKK4, and the MKK4(EAG) control peptide carries substitution mutations of three residues (K40E, R41A, and L46G) that are critical for JNK binding (Fig. 5). The JIP-1 peptide corresponds to residues 156–167 of the human JIP-1 scaffold protein (which is identical to residues 152–163 of mouse JIP-1); this region has been shown to bind to JNK (42). The MEK1 and MEK2 D-site peptides correspond to the MAPK-docking sites of the respective MKKs. Since the cognate MAPKs of MEK1 and MEK2 are ERK1 and ERK2, the MEK1 and MEK2 D-sites would not be expected to bind to JNK1. Indeed, this expected lack of interaction has been confirmed in GST co-sedimentation assays (Fig. 2B) (26), although it has not heretofore been examined in kinase inhibition assays, which can be more sensitive. Phosphorylation of unactivated JNK1 by limiting amounts of MKK4 was measured in the absence and presence of 200 µM peptide. As shown in Fig. 6C, the MKK4 D-site peptide was able to partially inhibit JNK1 phosphorylation, presumably because it was able to compete with full-length MKK4 for binding to JNK1 and thereby block MKK4/JNK docking. In contrast, the MKK4(EAG) mutant peptide had little or no inhibitory effect. In addition, the JIP-1 D-site peptide was also able to inhibit JNK1 phosphorylation, suggesting that full-length MKK4 and JIP-1 may compete for JNK binding. Notably, the MEK1 and MEK2 D-site peptides did not inhibit MKK4-mediated phosphorylation of JNK1. We have previously demonstrated that these two peptides can effectively inhibit the ability of both MEK1 (26) and MEK2 (39) (also see below) to phosphorylate ERK2. Hence, these two peptides exhibit good specificity for ERK2 versus JNK1.
To further examine the specificity of the D-site peptides, we performed the
reciprocal experiment and examined their ability to inhibit MEK2-mediated
phosphorylation of ERK2 (Fig.
6D). As we have previously shown
(39), the MEK2 D-site peptide
was a potent inhibitor of this reaction (IC50 15
µM), whereas the MEK1 D-site peptide was less effective
(IC50 100 µM). In contrast, a mutant version of
the MEK2 peptide, MEK2(EEAA) (Fig.
6B), had no inhibitory effect. Somewhat surprisingly, the
MKK4 and JIP-1 D-site peptides were almost as effective inhibitors of MEK2
phosphorylation of ERK2 as the MEK1 D-site peptide was. Hence, these
JNK-docking sites are not completely specific for their cognate MAPKs JNK1,
JNK2, and JNK3 (and p38); they are also able to bind with reasonable affinity
to ERK2, a non-cognate MAPK.
It is notable that the inhibition of MEK2 phosphorylation of ERK2 by D-site
peptides was considerably more extensive than the inhibition of MKK4
phosphorylation of JNK1 (Fig. 6, compare
C and D). Indeed, dose-response profiles (data
not shown) showed that D-site-mediated inhibition of MKK4 leveled out at
50% maximal inhibition. In contrast, D-site-mediated inhibition of MEK1
and MEK2 levels out at 75–80% inhibition
(26,
39). One possible explanation
for this difference is that relative to MEK2, MKK4 has a reduced dependence on
docking interactions with its cognate MAPKs. Another, more trivial possibility
is that a significant fraction of the particular preparation of active MKK4
used in Fig. 6 was in a lower
activity, uninhibitable state.
To demonstrate directly that the MKK4 and JIP-1 D-site peptides inhibit stable protein complex formation between MKK4 and JNK, a series of binding assays were performed. The ability of in vitro translated, radiolabeled full-length MKK4 protein to co-sediment with GST-JNK3 bound to glutathione-Sepharose beads was tested in the presence or absence of various D-site peptides. When the MKK4 and JIP-1 D-site peptides were added to the reactions, they inhibited the amount of MKK4 protein that co-sedimented with GST-JNK3 (Fig. 7). In contrast, the MKK4 mutant peptide did not appreciably inhibit MKK4 binding. Thus, the MKK4 and JIP-1 D-site peptides are both able to compete with MKK4 protein for binding to JNK3. In addition, the MEK2 D-site peptide did not appreciably inhibit MKK4 binding to JNK3 (Fig. 7), although it does substantially inhibit MEK2 binding to ERK2 (39), again demonstrating that these ERK-docking sites are specific for ERK versus JNK.
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D-site Peptides Inhibit JNK2 Phosphorylation of c-Jun and
ATF2—We have shown previously that peptides based on the D-sites of
MEK1 and MEK2 are able to inhibit not only MEK phosphorylation of ERK, but
also other ERK-dependent reactions as well, such as ERK2-mediated
phosphorylation of the Elk-1 transcription factor
(39). This cross-inhibition
strongly suggests that the D-sites in MEK1, MEK2, and Elk-1 all bind to the
same region of ERKs. Likewise, the JNK-docking site of MKK4 characterized in
this study displays noticeable sequence similarity to known JNK-docking sites
in transcription factors (Fig.
1B), suggesting that MKK4 and downstream transcription
factors may compete for binding to the same protein interaction site on JNKs.
To test this possibility, the MKK D-site peptides were assessed for their
ability to inhibit phosphorylation of GST-c-Jun by active JNK2
(Fig. 8, B and
C). JNK phosphorylates c-Jun at Ser63 and
Ser73 (31). The
MKK4 D-site potently inhibited JNK2 phosphorylation of c-Jun in a
dose-dependent manner (IC50 7.5 µM), whereas
the MKK4(EAG) control peptide showed no inhibitory activity whatsoever,
indicating that the MKK4 D-site and c-Jun compete for binding to JNK2. The
JIP-1 D-site peptide was also quite an effective inhibitor of JNK
phosphorylation of c-Jun, as has been previously reported
(45). In contrast, the MEK1
D-site peptide did not inhibit JNK2 (<20% inhibition at 100
µM), and the MEK2 D-site peptide displayed a weak inhibition
(IC50 105 µM).
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Like c-Jun, the ATF2 transcription factor is also a target of JNK
(46) and also contains a
JNK-docking site that displays some similarity to the MKK4 D-site
characterized in this study (Fig.
1B). JNK phosphorylates ATF2 at Thr69 and
Thr71 (46). When
the MKK4 D-site peptide was titrated into a JNK2 kinase assay using GST-ATF2
as a substrate, it inhibited JNK2 phosphorylation of ATF2 in a dose-dependent
manner (IC50 15 µM)
(Fig. 8, D and
E). Hence, the MKK4 D-site and ATF2 compete for binding
to JNK2. The JIP-1 D-site peptide was also a potent inhibitor of ATF2
phosphorylation. In contrast, neither the MKK4(EAG) mutant peptide nor the
MEK1 or MEK2 D-site peptide was able to inhibit ATF2 phosphorylation (<75%
inhibition at 100 µM).
The Elk-1 transcription factor contains a docking site for the ERK2 MAPK
(32), and this D-site also
exhibits similarity to the D-sites in MKKs
(Fig. 1B). To gain
further insight into the specificity of the MKK4 D-site for JNK
versus ERK, the panel of D-site peptides were also tested for their
ability to inhibit ERK2 phosphorylation of Elk-1. Elk-1 is phosphorylated by
ERK2 on at least six sites in its transcriptional activation domain
(47), resulting in a ladder of
bands displaying retarded electrophoretic mobility
(Fig. 8G);
peptide-dependent inhibition was revealed both by the progressive collapsing
of this ladder and by decreased phosphate incorporation overall
(Fig. 8F). As we have
previously shown (39), the
MEK1 and MEK2 D-site peptides were able to effectively inhibit ERK2
phosphorylation of Elk-1 (IC50 18.5 and 6.5 µM,
respectively) (Fig. 8, F and
G). Interestingly, the MKK4 and JIP-1 D-site peptides
were also reasonably effective inhibitors (IC50 50 and 65
µM, respectively). The ability of the MKK4 D-site to inhibit
ERK2 required conserved residues of the D-site consensus sequence, as the
MKK4(EAG) mutant peptide was not inhibitory. These data indicate that the MKK4
D-site does bind to ERK2, consistent with other results shown above (Figs.
3 and
6).
Selectivity of the MEK1, MEK2, and MKK4 D-sites—The above results suggest that the specificity of MKK/MAPK docking is somewhat limited, i.e. the specificity of binding between MAPKs and D-sites in their cognate MKKs is quantitative, but not qualitative. Typically, quantitative estimates of relative specificity are expressed as selectivity ratios. Hence, to see what insights could be gained from this approach, the selectivity of the D-sites in MEK1, MEK2, and MKK4 for their appropriate (cognate) versus inappropriate (non-cognate) MAPKs ERK2 versus JNK2 was estimated in two ways.
First, the selectivity of ERK and JNK for their cognate D-sites in a given reaction was expressed as the non-cognate D-site/cognate D-site IC50 ratio (Table III, part A). For example, the selectivity of the MKK4 D-site versus the MEK2 D-site for JNK2 was >10, estimated from the MEK2/MKK4 IC50 ratio in the c-Jun phosphorylation reaction. Indeed, selectivities of at least 10 were obtained comparing the MKK4/JNK2 interaction with the MEK1/JNK2 and MEK2/JNK2 interactions. These selectivities appear to be driven by the relatively strong binding of the MKK4 D-site to JNK2 and the minimal binding of the MEK1/2 D-sites to JNK2. In contrast, lower selectivities (between 6 and 10) were obtained comparing the MEK2/ERK2 and MKK4/ERK2 interactions. These lower selectivities are attributable to the moderate affinity binding of the MKK4 D-site to ERK2. Finally, the MEK1/ERK2 interaction displayed little or no selectivity compared with the MKK4/ERK2 interaction because the MEK1 D-site does not appear to bind to ERK2 that much better than the MKK4 D-site does.
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As a second means to estimate specificity, the selectivity of a given D-site for binding to its cognate MAPK, instead of to a non-cognate MAPK, was expressed as the non-cognate MAPK/cognate MAPK IC50 ratio. For example, the selectivity of the MKK4 D-site for JNK2 versus ERK2 was estimated by dividing the IC50 for its inhibition of ERK2 phosphorylation of Elk-1 (50 µM) by the IC50 for its inhibition of JNK2 phosphorylation of c-Jun (7.5 µM) or ATF2 (15 µM). The results of these calculations revealed that the MKK4 D-site had a selectivity of between 3 and 6 for JNK2 versus ERK2, whereas the MEK1 and MEK2 D-sites had a selectivity of at least 16 for ERK2 versus JNK2 (Table III, part B). Again, these numbers highlight the nontrivial propensity of the MKK4 D-site to bind to ERK2.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Ubiquity of MAPK-docking Sites in MKKs—Docking sites for MAPKs have now been identified in MEK1 (26, 30), MEK2 (26), MKK3 and MKK6 (27), and MKK4 (this work). Hence, of the seven MKKs in the human genome (48), MAPK-docking sites have so far been found in all but MEK5 and MKK7. The large N-terminal domain of MEK5 has been shown to bind to its cognate MAPK ERK52; however, this region does not contain a clear match to the D-site consensus sequence. The N-terminal domain of MKK7 has also been shown to bind to JNK (28), and a putative D-site in this domain has been predicted based on similarity to the consensus sequence (26, 40, 41). However, this region is not sufficient for JNK binding2; as such, it does not appear to be a bona fide JNK-docking site.
The characterized MAPK-docking sites in mammalian MKKs, including the
JNK-docking site in MKK4 characterized here, have the following properties in
common. 1) They mediate the formation of a relatively stable MKK-MAPK complex;
2) they are necessary and sufficient for the formation of this complex; and 3)
they share the core consensus sequence
(K/R)3-X1–5-(L/I)-X-(L/I). We
have estimated the affinities of the MEK1-ERK1, MEK1-ERK2, MEK2-ERK1, and
MEK2-ERK2 complexes (Kd 30, 15, 20, and 10
µM, respectively)
(26)3
and of the MKK4-JNK3 complex (Kd 30
µM) (this work). These estimates, obtained from binding assays,
are consistent with IC50 values obtained in peptide inhibition
assays (e.g. Table
III). Thus, human MKK-MAPK complexes appear to have affinities
with Kd values in the low micromolar range, consistent
with the affinities of protein/protein interactions mediated by certain other
modular domains or motifs involved in signaling, e.g. those mediated
by SH3 domains (49).
Relationship of MKK D-sites to Anthrax Lethal Factor Cleavage Sites—The MAPK-docking sites in MEK1, MEK2, MKK3, and MKK6 have been shown to be substrates for cleavage by the anthrax lethal factor protease (35). MKK4 is also cleaved by lethal factor at two sites in its N terminus (35); the first of these corresponds to the JNK-docking site identified herein. Thus, this study extends the correlation that all proven MAPK-docking sites found in the N termini of mammalian MKKs are also cleavage sites for anthrax lethal factor. However, we also demonstrated, by counterexample, that not all lethal factor cleavage sites are high affinity MAPK-docking sites. For instance, MKK4-(37–94) (EAE), which contains the second lethal factor cleavage site intact, did not bind with high affinity to the ERK, JNK, and p38 MAPKs.
Competition for JNK Docking—The D-sites in MKKs are related
to D-sites found in other MAPK-interacting proteins, including certain
transcription factors, MAPK phosphatases, and scaffolds
(34). This broader class of
D-sites fits the rough consensus sequence
(K/R)1–3-X1–5-
-X-,
where represents a hydrophobic residue, typically L or I, often V
(particularly in phosphatases), occasionally M. The similar primary structure
of D-sites suggests that they may bind to the same protein interaction site(s)
on their cognate MAPKs. Indeed, the MEK1 and MEK2 D-site peptides inhibit MEK
phosphorylation of ERK2, ERK2 phosphorylation of Elk-1, and dephosphorylation
of ERK2 by the MKP-1 phosphatase
(39); this extensive
cross-inhibition indicates that the D-sites in MEK1, MEK2, Elk-1, and MKPs
compete for binding to ERK. It was not clear, however, if the same situation
would apply to the JNK group of MAPKs because JNK2 mutations that reduce its
interaction with MKK4 lie on the opposite face of its tertiary structure from
mutations that reduce its interaction with c-Jun
(40,
50). Nevertheless, we found
that an MKK4 D-site peptide could inhibit JNK2-mediated phosphorylation of
c-Jun and ATF2. Almost certainly, this peptide inhibited these reactions by
binding to JNK2 and blocking it from docking with its transcription factor
substrates. Hence, it appears that the D-sites in MKK4 and c-Jun/ATF2 do
indeed compete for docking to JNK. Since MKK4 (like MKK7) is found in both the
nucleus and the cytoplasm of stimulated and unstimulated cells
(28), such competition
presumably occurs in vivo, at least to some extent. We have suggested
previously that the competitive docking of substrates and regulators to MAPKs
may play a role in enhancing signaling fidelity
(39). For example, ERK2
molecules that are docked to an appropriate physiological substrate, such as
Elk-1, may be protected from dephosphorylation by MAPK phosphatases as well as
from MEK1-mediated nuclear export. Similar mechanisms may also be relevant to
JNK signaling.
We also found that the D-site from the cytoplasmic protein JIP-1 could inhibit both MKK4/JNK binding and MKK4-mediated phosphorylation of JNK. These results suggest that, in the cell cytoplasm, JIP-1 competes with MKK4 for docking to JNK. JIP-1 is a putative scaffold protein that binds to JNK, MKK7, and certain upstream kinases (51). The mutually exclusive docking of JIP-1 and MKK4 to JNK may prevent the recruitment of MKK4 to the JIP-JNK complex and may also prevent MKK4-mediated activation of JIP-bound JNK. More generally, the competitive docking of scaffolds and MKKs to MAPKs would seem inconsistent with the proposed role of scaffold proteins as potentiators of MAPK activation (52). We speculate that under some circumstances, scaffold proteins may in fact function to inhibit MAPK activation. For example, JIP proteins and their cargo are transported by microtubule-based motors, and it may be useful to inhibit JNK activity while this is happening (53). We further speculate that some signal-regulated event, such as release from motor proteins or oligomerization, may switch the activity of scaffolds from inhibitors to activators of MAPK cascade signaling.
Specificity of D-site-mediated Interactions—To explore the
specificity of MKK D-sites for their cognate MAPKs, we tested the ability of
the MEK1 and MEK2 D-sites to inhibit JNK-dependent reactions and of the MKK4
and JIP-1 D-sites to inhibit ERK-dependent reactions. By and large, we found
that the MEK1 and MEK2 D-sites did not bind to JNK and did not appreciably
inhibit JNK-dependent reactions, although the MEK2 peptide did show some
ability to inhibit JNK2 phosphorylation of c-Jun. In contrast, the MKK4 and
JIP-1 D-site peptides were relatively good inhibitors of ERK2-dependent
reactions, and the MKK4 N terminus displayed significant, D-site-dependent
binding to ERK2. Hence, there appears to be appreciable, undiscriminating
binding of the MKK4 D-site to a non-cognate MAPK, ERK2. When the selectivity
of D-site/MKK interactions was quantified, it ranged from 20-fold or more
at best (MEK2 D-site for ERK2 versus JNK2) to 3-fold or less at
worst (MKK4 D-site for JNK2 versus ERK2). Ladbury and Arold
(49) have argued that
selectivities of this magnitude cannot, by themselves, account for the
requisite signaling specificity.
The limited specificity of D-site-mediated interactions is perhaps not surprising given the sequence similarity among D-sites. Indeed, the D-site of Elk-1 has apparently evolved to bind to both ERK and JNK so that Elk-1 can be regulated by both subfamilies of MAPKs (32). In this regard, it is notable that D-site-mediated interactions resemble interactions governed by SH3 domains in several respects. As summarized by Mayer (54), SH3 domains have "modest affinities" (Kd values in the low micromolar range) (49) and selectivities that are "generally marginal at best" (54). Nevertheless, the synthesis of selective peptide-like inhibitors of SH3 domain interactions has been achieved (55), and the same may ultimately prove true for certain D-site-mediated interactions.
What are the physiological implications of the relative lack of specificity in D-site-mediated interactions? These interactions are best viewed as one component of a "double selection" for fidelity in MAPK-dependent transactions (24, 26). For example, effective selectivity is presumably increased by the combined effects of multiple separate interactions between MKKs and their cognate MAPKs (56). Consequently, even though MKK4 docks to ERK2, it does not phosphorylate ERK2. Another mechanism that may enhance the specificity of relatively nonselective pairwise interactions is the assembly of multiprotein complexes (54, 57). The binding of both MKK and MAPK to a scaffold protein might function in such a manner, for instance (52). It is not clear, however, if this conception is entirely consistent with the observed competitive docking of MKKs and scaffolds to MAPKs. Such considerations highlight the theme that multiple, incompletely understood mechanisms contribute to specificity in cell signaling.
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FOOTNOTES |
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To whom correspondence should be addressed: Dept. of Developmental and Cell
Biology, 5205 McGaugh Hall, University of California, Irvine, CA 92697-2300.
Tel.: 949-824-6902; Fax: 949-824-4709; E-mail:
bardwell{at}uci.edu.
1 The abbreviations used are: MAPKs, mitogen-activated protein kinases; MKK,
MAPK kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal
kinase; MEK, MAPK/ERK kinase; JNKK, JNK kinase; SEK, SAPK/ERK kinase; SAPK,
stress-activated protein kinase; GST, glutathione S-transferase; SH3,
Src homology-3; MKP, MAPK phosphatase.
1 pmol (
