From the Department of Developmental and Cell
Biology, University of California, Irvine, California 92697 and the
§ Department of Molecular and Cell Biology, Division of
Biochemistry and Molecular Biology, University of California,
Berkeley, California 94720-3202
Received for publication, November 10, 2000, and in revised form, December 22, 2000
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ABSTRACT |
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The recognition of mitogen-activated protein
kinases (MAPKs) by their upstream activators, MAPK/ERK kinases (MEKs),
is crucial for the effective and accurate transmission of many signals.
We demonstrated previously that the yeast MAPKs Kss1 and Fus3 bind with
high affinity to the N terminus of the MEK Ste7, and proposed that a
conserved motif in Ste7, the MAPK-docking site, mediates this
interaction. Here we show that the corresponding sequences in human
MEK1 and MEK2 are necessary and sufficient for the direct binding of
the MAPKs ERK1 and ERK2. Mutations in MEK1, MEK2, or Ste7 that altered
conserved residues in the docking site diminished binding of the
cognate MAPKs. Furthermore, short peptides corresponding to the docking
sites in these MEKs inhibited MEK1-mediated phosphorylation of ERK2
in vitro. In yeast cells, docking-defective alleles of Ste7
were modestly compromised in their ability to transmit the mating
pheromone signal. This deficiency was dramatically enhanced when the
ability of the Ste5 scaffold protein to associate with components of
the MAPK cascade was also compromised. Thus, both the MEK-MAPK docking
interaction and binding to the Ste5 scaffold make mutually reinforcing
contributions to the efficiency of signaling by this MAPK cascade
in vivo.
Most cellular functions are regulated by protein phosphorylation,
and almost every known cellular signaling pathway utilizes one or more
protein kinases. Understanding how protein kinases recognize their
substrates with efficiency and fidelity is of great interest to
functional genomics and molecular pharmacology, as it could expedite
substrate prediction and drug target discovery, respectively. Perhaps
most importantly, it is key for understanding larger questions of
signal-response specificity (1, 2).
Kinase-substrate recognition and signal-response specificity are
particularly pressing issues in signaling networks that involve mitogen-activated protein kinase
(MAPK)1 cascades (3). MAPK
cascades contribute to the regulation of diverse responses, including
hormone and growth factor action, cell differentiation, cell cycle
progression, learning, inflammation, and stress responses, as well as
tumorigenesis and other pathological processes (4). At the core of
these cascades are a MAPK (also termed extracellular signal-regulated
kinase, or ERK), and a MAPK/ERK kinase (MEK or MKK) that phosphorylates
and thereby activates the MAPK. MEK activity can be regulated, in turn,
via phosphorylation by various classes of upstream kinase (5).
Activated MAPKs phosphorylate targets including transcription factors,
other kinases, and other enzymes. Some MAPKs also regulate some of
their targets by direct binding, rather than by (or in addition to)
phosphorylation (6, 7).
Both yeast and animal cells possess multiple parallel MAPK cascades.
Saccharomyces cerevisiae contains four fully elaborated MAPK
cascades, the mating pheromone response, filamentation invasion, cell
integrity, and high osmolarity response pathways (8). These pathways,
although distinct, share some components. Four mammalian MAPK pathways
have been well characterized, the ERK1/2 module, the JNK module, the
p38 module, and the ERK5 module (5). Other mammalian MEKs and MAPKs
have been identified as well, but await assignment to a pathway. It is
generally observed that MEKs in one pathway do not phosphorylate MAPKs
in other pathways, but how this selectivity is achieved is not well understood.
MAPK cascades have been highly conserved throughout eukaryotic
evolution. For example, human ERK1 and ERK2 are ~50% identical to
the yeast mating/invasive growth MAPKs, Fus3 and Kss1. Likewise, the
catalytic domains of human MEK1 and MEK2 are similar to that of the
yeast mating/invasive growth MEK, Ste7. Indeed, molecular evolutionary
analysis suggests that Fus3 and Kss1 are orthologs of ERK1 and ERK2,
and that the yeast high osmolarity response MAPK, Hog1, is the ortholog
of the mammalian JNK and p38 families (9). In fact, mammalian p38 can
functionally replace Hog1 in S. cerevisiae (10), and
ERK1 can substitute for Kss1 (11).
Given that MAPK cascades require the sequential action of at least
three tiers of protein kinases, properly directed kinase-substrate interactions are necessary to achieve efficiency and fidelity in these
signaling modules. Insights into kinase-substrate transactions have
come from studies of the phosphorylation of small peptides derived from
protein substrates (12, 13), or selected from combinatorial libraries
(14). Further understanding has come from crystal structures of such
peptides bound to the catalytic clefts of protein kinases (15).
However, peptides are often relatively poor substrates when compared
with native proteins. Furthermore, phosphorylation site consensus
sequences cannot fully account for the specificity observed in many
kinase-substrate relationships. For example, all MAPKs phosphorylate
substrates that have the core consensus, -(Ser/Thr)-Pro-. This
sequence, by itself, cannot be the sole determinant of MAPK specificity because it is found in more than 75% of all proteins and because it is
also at the core of phosphorylation sites recognized by cyclin-dependent kinases. Clearly, other factors must
contribute to MAPK recognition of unique substrates. In the case of MEK
recognition of MAPKs, altering the loop on MAPKs that is phosphorylated
by MEKs does not result in the predicted alterations in specificity (16-18). Also, unlike most protein kinases, MEKs do not recognize peptide versions of their target site (19).
Additional contributions to protein kinase target selection are
provided by "docking sites" on substrates that are separate from
the phosphoacceptor residues (reviewed in Ref. 20). Moreover, some
protein kinases use domains or subunits other than the catalytic domain
to aid in substrate recognition (21-23). Another means to influence
efficiency and fidelity of kinase-substrate transactions is via a
"scaffolding" or "anchoring" protein that binds both kinase and
substrate (24-26). The prototype MAPK-cascade scaffold is S. cerevisiae Ste5, which is essential for the mating pheromone response. Distinct regions of Ste5 bind the MEK Ste7 and the MAPKs Fus3/Kss1 (27). Ste5 also binds Ste11 (MEK kinase that phosphorylates and activates Ste7) (27-29) and Ste4 ( Current interest in docking and scaffolding stems not only from their
influence on signaling specificity and insulation, but also from their
role in shaping the kinetics, efficiency, and threshold properties of
within pathway signal transmission (42-44). Given that similar
functions have been attributed to kinase-substrate docking and to
scaffolding, it seems possible that these mechanisms may act
cooperatively within the cell. This has not been heretofore investigated, to our knowledge.
Here we present evidence that indicates that the MAPK-docking site of
MEKs has been structurally and functionally conserved from yeast to
humans. We also demonstrate that peptides corresponding to these
MAPK-docking sites inhibit MEK-mediated ERK phosphorylation. Finally,
we show that docking and scaffolding make mututally reinforcing, synergistic contributions to signal transmission in
vivo.
Yeast Strains and Media--
Standard yeast media and culture
conditions were as described previously (45). Yeast strain YLB105
(MATa ste5 Plasmids for in Vitro Transcription/Translation of Mammalian
Genes--
The following mammalian genes were used in this study:
human MEK1 (MAP2K1; GenBankTM
accession number NM_002755), human MEK2 (MAP2K2;
L11285), human ERK1 (MAPK3; X60188), and rat
ERK2 (M64300). To construct pGEM-MEK1, the MEK1 coding
sequence was amplified by high-fidelity polymerase chain reaction (PCR)
using Pfu DNA polymerase (Stratagene), primers LB111 (see Table
I for the sequence of all
oligonucleotides) and LB113, and template plasmid pBS-MEK1 (49). The
resulting PCR product was digested with BamHI and
SalI and inserted into the corresponding sites of pGEM4Z
(Promega). pGEM-MEK1 Plasmids for the Production of GST Fusion Proteins--
To
construct pGEX-STE71-25, oligonucleotide LB70 annealed to
LB71 was inserted into pGEX-4T-1 (Amersham Pharmacia Biotech) that had
been digested with EcoRI and SalI. To construct
pGEX-MEK11-19, oligonucleotide LB72 annealed to LB73 was
inserted into pGEX-4T-1 that had been digested with EcoRI
and SalI. pGEX-MEK11-19EE was constructed by
the same procedure, except that oligonucleotides encoding the indicated
amino acid substitutions were used. To construct
pGEX-MEK21-22, oligonucleotide LB74 annealed to LB75 was
inserted into pGEX-4T-1 that had been digested with EcoRI and SalI. pGEX-MEK21-22ATA was constructed by
the same procedure, except that oligonucleotides encoding the indicated
amino acid substitutions were used.
To construct pGEXLB, oligonucleotide LB101 annealed to LB102 was
inserted into pGEX-4T-1 that had been digested with BamHI and EcoRI. This results in the replacement of an encoded Pro
residue with a Gly-Gly-Gly-Gly-Gly-Ser-Gly sequence designed to
facilitate the independent functioning of the GST and fusion moieties.
The FUS3 coding sequence on an
EcoRI-SalI fragment was excised from pGEM4Z-meFUS3 (45) and inserted into the corresponding sites of pGEXLB,
yielding pGEXLB-FUS3. The ERK2 coding sequence was amplified by PCR
using as primers LB103 and LB104 and as the template plasmid pBS-ERK2
(45). The resulting PCR product was digested with BamHI and
SalI and inserted into the corresponding sites of pGEXLB,
yielding pGEX-GERK2. The plasmid encoding GST-ERK1 is described
elsewhere (50).
Yeast Expression Plasmids for STE7 and Alleles--
YCpT3-STE7
is a low copy (CEN), TRP1-containing vector, in which the
STE7 open reading frame, modified by silent
substitutions that introduce additional restriction sites, is expressed
from the STE7 promoter. This vector, which was used as the
starting point for construction of multiple alleles, was itself
constructed in several steps. First, the single AatII site
in plasmid YCpT-STE7 (45) was destroyed by digestion with
AatII, treatment of the resulting linearized DNA fragment
with the Klenow fragment of E. coli DNA polymerase I in the
presence of all 4 deoxyribonucleotide triphosphates, and
recircularization the resulting "filled-in" product, generating
YCpT2-STE7. Second, oligonucleotide LB88 annealed to LB89 was inserted
into YCpT2-STE7 that had been digested with BamHI and
MscI, yielding YCpT3-STE7. This resulted in the replacement of the sequence encoding residues 1-27 of Ste7 protein with a sequence
encoding the same amino acids but containing silent substitutions that
introduced a PstI site (spanning codons 7 and 8) and an
AatII site (spanning codons 22-23). YCpT3-STE7 was able to
fully complement the mating and invasive growth defects of a
ste7 Plasmids and Templates for in Vitro Transcription/Translation of
STE7 and Alleles--
The wild-type, AAAA, Other Yeast Expression Plasmids--
Low-copy (CEN4,
ARS1) plasmids YCpU (containing URA3) and YCpT
(containing TRP1) have been described elsewhere (48).
YCpH-STE11-4 (a gift of Deepak Voora, our (J. T.) laboratory)
contains the STE11-4 allele (51) inserted into the low-copy
plasmid pRS313 (46). The YCpU-based plasmids expressing STE5
alleles from the STE5 promoter are described elsewhere (28).
To construct the reporter plasmid YEpL-FUS1Z (a gift of Judy
Zhu-Shimoni, our (J. T.) laboratory), the URA3 gene was
excised from YEpU-FUS1Z (52) and replaced with the LEU2 gene
following a procedure described previously (52).
Transcription and Translation in Vitro--
Transcription and
translation reactions in vitro, the partial purification of
translation products by ammonium sulfate precipitation, and the
quantification of translation products were performed as described
previously elsewhere (45). In general, polypeptides encoded by
plasmid-borne alleles were produced using a coupled transcription/translation system (SPT3; Novagen); polypeptides encoded
by PCR products were produced by sequential transcription and translation.
Binding Assays--
GST fusion proteins were expressed and
purified from bacteria and bound to glutathione-Sepharose (Amersham
Pharmacia Biotech) as instructed by the manufacturer. GST fusion
proteins bound to glutathione-Sepharose beads were quantified by
comparison to known amounts of bovine serum albumin, following SDS-PAGE
and staining with Coomassie Blue. Binding assays were performed as
described (53). Binding reactions were analyzed by scintillation
counting and SDS-PAGE. SDS-PAGE gels were first Coomassie stained
(Gelcode blue, Pierce) and photographed, then treated with fluorography solution (Amplify; Amersham Pharmacia Biotech), dried down, and autoradiographed. For the experiments shown in Fig. 2, purified rat
ERK2 was purchased from Calbiochem, anti-ERK2 antiserum from New
England Biolabs, and purified human JNK1 ( Protein Kinase Assays--
Kinase reactions (20 µl) contained
20 mM MOPS (pH 7.2), 10 mM MgCl2, 1 mM EGTA, 25 mM Bioassays for Pheromone Response--
Mating assays were
performed as described (28). Pheromone-imposed cell cycle arrest and
recovery were measured in an agar diffusion (halo) bioassay as
described (45). For determination of reporter gene expression, strains
JCY107 or YLB105 were first transformed with the appropriate
STE5 and/or STE7 expression plasmids. Plasmid
YEpL-FUS1Z (containing the lacZ reporter gene
driven by the FUS1 promoter) was introduced in a second
transformation. Cells were grown in liquid culture, treated with 1 µM A Conserved Motif Near the N Termini of MEKs--
Our previous
study (45) demonstrated that the MAPKs Kss1 and Fus3 each bound to the
MEK Ste7 in the absence of any additional yeast proteins. Other yeast
MAPKs (Hog1 and Mpk1) did not bind to Ste7. The Ste7-MAPK complexes
displayed a higher affinity and stability than would be expected from a
prototypical enzyme-substrate interaction. Indeed, complex formation
did not require active site-phosphoacceptor residue contacts, and
apparently did not position the proteins in an enzyme-substrate
orientation (45).
Relatively stable, high-affinity associations have been documented for
other cognate MEK-MAPK pairs (54-57). For the yeast Ste7-Fus3 (or
Kss1) interaction, as well as for several mammalian MEK-MAPK
interactions, the region of the MEK responsible for MAPK binding has
been localized to the N-terminal, noncatalytic domain (45, 58-63). In
contrast to the catalytic domains, the noncatalytic domains of MEKs do
not exhibit extensive sequence conservation across widely divergent
species (Fig. 1A). However,
within this domain of Ste7, we noted a short sequence motif (which we
dubbed the "MAPK-docking site") that was conserved in seven other
MEKs across a broad range of species and positioned at or very near the
N termini of these (64). The more recent availability of sequence
information for many additional MEKs further strengthens our original
conclusion (Fig. 1B). Notable features of the MAPK-docking site motif are a cluster of at least two basic residues (Lys, Arg,
rarely His) separated by a spacer of 2-6 residues from a hydrophobic-X-hydrophobic (or
hydrophobic-X-hydrophobic-X-hydrophobic) sequence, where the hydrophobic residues are long-chain aliphatics (Leu, Ile, sometimes Val). Both in the spacer and in the sequence immediately C-terminal to the hydrophobic-X-hydrophobic
element, there is a high propensity for the presence of Pro, Asn,
and/or Gly, which are residues that are both turn-forming and
helix-breaking (65).
MAPK-docking Site of a MEK Is Sufficient for Binding of a Cognate
MAPK--
Our previous study indicated that residues 2-22 of Ste7
were necessary for the interaction of this MEK with its cognate MAPKs Kss1 and Fus3 (45). To determine whether this small region of Ste7 was
sufficient for MAPK binding, the first 25 residues of Ste7 were fused
to Schistosoma japonicum glutathione
S-transferase (GST), and the resulting fusion protein was
expressed in bacteria and purified by adsorption to glutathione-agarose
(GSH beads). GST-Ste71-25 (or GST alone as a control) was
then incubated with either Kss1 or Hog1 MAPKs 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. Kss1 bound to GST-Ste71-25, but not to
GST; Hog1 did not bind detectably to either (Fig.
2A). Fus3 also bound
specifically to GST-Ste71-25 (data not shown). Hence,
residues 1-25 of Ste7 are indeed sufficient for the specific binding
of its cognate MAPKs.
To ascertain whether the putative MAPK-docking sites in human MEK1 and
MEK2 were sufficient for binding to mammalian ERK1 and ERK2, the first
19 or 22 residues of MEK1 or MEK2, respectively, were also fused to
GST. The resulting polypeptides were then expressed in bacteria,
purified on GSH beads, and incubated with purified ERK2 or JNK1.
Bead-bound complexes were sedimented and analyzed by SDS-PAGE followed
by immunostaining with ERK2- or JNK1-specific antisera. Both
GST-MEK11-19 and GST-MEK21-22 bound ERK2;
this binding was specific because ERK2 did not bind to either GST alone
or empty beads (Fig. 2B). In contrast,
GST-MEK11-19 and GST-MEK21-22 did not bind
JNK1 above the nonspecific background displayed by either GST alone or
empty beads (Fig. 2B). ERK1 also bound specifically to
GST-MEK11-19 and GST-MEK21-22 (data not
shown). To achieve readily detectable ERK binding in these experiments,
a higher input of the bead-bound GST-docking site fusions was required
than was needed to observe specific Kss1 binding to
GST-Ste71-25 (compare Coomassie-stained panels in Fig. 2,
A and B), indicating that Kss1-Ste7 interaction is of higher affinity than the ERK2-MEK1 or ERK2-MEK2 interactions.
To delineate the contribution to the MEK-MAPK interaction of residues
that are highly conserved in the MAPK-docking site motif (Fig.
1B), Lys3 and Lys4 in the basic
cluster of MEK1 were both changed to glutamate, and this K3E/K4E
double mutant was fused to GST, yielding GST-MEK11-19EE. Similarly, a L12A/I14A double mutant of the
hydrophobic-X-hydrophobic motif of MEK2 was prepared,
yielding GST-MEK21-22ATA. Both mutant derivatives were
clearly defective in MAPK binding (Fig. 2C). ERK2 binding to
GST-MEK11-19EE was nearly abolished, and ERK2 binding to
GST-MEK21-22ATA was substantially reduced. Furthermore,
while GST-MEK11-19EE did not acquire the capacity to bind
JNK1 (Fig. 2C, lane 4), GST-MEK21-22ATA
exhibited trace binding to JNK1 (Fig. 2C, lane 6),
suggesting that this mutation led to a decrease in binding specificity.
The MAPK-docking Site Mediates ERK1 and ERK2 Binding to MEK1 and
MEK2--
Having demonstrated that the MAPK-docking sites in MEK1 and
MEK2 were sufficient for binding to ERK1 and ERK2, we asked if these
docking sites were necessary for the binding of full-length MEK1 or
MEK2 to ERK1 and ERK2. For this purpose, GST-ERK1 and GST-ERK2 were
expressed in and purified from bacteria, and then incubated either with
in vitro translated MEK1 or MEK2, or with MEK1 and MEK2
derivatives from which the MAPK-docking site had been deleted. MEK1
bound to GST-ERK1 and GST-ERK2 (but not to GST, as anticipated) whereas
binding of the mutant lacking the docking site
(MEK1
Since there are some similarities among the docking sites of MEK1,
MEK2, and Ste7 (Fig. 1B), we examined whether Ste7 could also associate with ERK1 and ERK2. Indeed, Ste7 bound to GST-ERK1 and
GST-ERK2; surprisingly, it did so with an apparent affinity (Kd Analysis of Conserved Residues Required for Docking--
To
delineate the sequence features in the MAPK-docking site of Ste7 that
contribute to its very high affinity binding to Kss1, Fus3, ERK1, and
ERK2, we introduced a number of substitution and deletion mutations
into the MAPK-docking site of Ste7 (Fig.
4A). Radiolabeled full-length
Ste7 bound with high affinity to GST-Fus3 (Kd
The roles of particular conserved residues in the MAPK-docking site
were evaluated by the use of substitution mutations. Three progressively more radical substitutions of the basic doublet of the
core docking site motif (R10A, R10E, and R9E/R10E) (Fig. 4A)
perturbed the interaction of Ste7N with GST-Fus3 with increasing severity. Ste7NR10A exhibited a modest, but readily
detectable, defect in binding (~50% of wild type);
Ste7NR10E showed an even more dramatic loss of binding
affinity (~25% of wild-type); and Ste7NR9E/R10E was as
severely defective in binding to GST-Fus3 (~5% of wild-type) as a
deletion of the entire docking site motif (Ste7N
To investigate sequence features of Ste7 that contribute to its high
affinity binding of MAPKs (relative to MEK1 and MEK2), we looked for
differences in the docking sites of these MEKs. Ste7 has a 7-residue
extension (FQRKTLQ) between its initiator methionine and the basic pair
in the core docking site motif, whereas MEK1 has only a single Pro and
MEK2 has just two residues (Leu/Ala). Deletion of six residues in this
N-terminal extension (Ste7N
To begin to explore the features of MAPKs that permit recognition by
the MAPK-docking sites of MEKs, binding of the series of Ste7N mutants
to GST-ERK2 was examined and compared with binding to GST-Fus3. The
spectrum of relative binding of the different Ste7N mutants to GST-ERK2
was quite similar to that observed for GST-Fus3 (Fig. 4B).
Mutants that bound relatively well to Fus3 (e.g.
Ste7NG14A, Ste7NR10A, and
Ste7N Docking Site Peptides Inhibit MEK1 Phosphorylation of ERK2--
To
explore the role that MEK-MAPK docking plays in signal transmission
from MEK to MAPK, we tested whether synthetic peptides corresponding to
the MAPK-docking site sequence were able to act as competitors of
MEK-dependent MAPK phosphorylation in vitro. Five different peptides were synthesized (Fig.
5A). Ste72-22, MEK11-17, and MEK21-20 correspond to the
MAPK-docking sites of the respective MEKs. Scram7 contains the same
residues as in Ste72-22, but in a randomized
("scrambled") order. MEK21-20EEAA is identical to
MEK21-20, except that it carries mutations in four
residues (R4E, R5E, L12A, and I14A) that are critical for MAPK binding
(see Fig. 2C). Phosphorylation of catalytically inactive
ERK2 by limiting amounts of MEK1 was measured in the absence and
presence of increasing concentrations of peptide. All three
MAPK-docking site peptides inhibited MEK1 phosphorylation of ERK2 and
did so in a concentration-dependent manner (Fig. 5,
B and C). Interestingly, these three peptides showed the same relative potency in inhibiting MEK1 phosphorylation of
ERK2 (Ste7>MEK2>MEK1) as they did in binding to ERK2, consistent with
the idea that the peptides inhibit by competing with MEK1 for ERK2
binding. The most potent peptide (Ste72-22) exhibited an
IC50 of ~10 µM. In contrast, the control
peptides (Scram7 and MEK21-20EEAA) had little or no effect
on ERK2 phosphorylation, even at a 10-fold higher concentration. To
assess the specificity of this inhibition, the peptides were also
tested for their ability to inhibit protein kinase A; none were able to
do so (IC50 Docking, by Itself, Plays a Minor Role in Transmission of the
Mating Pheromone Signal--
Analysis of the in vivo
function of MEK-MAPK docking in mammalian cells is complicated by the
fact that the expected phenotype of docking-defective mutants is
recessive, and no cell lines nullizygous for both MEK1 and MEK2 are
currently available. A further complicating factor is that MAPK cascade
components participate in multiple, potentially redundant interactions.
We chose, therefore, to examine the phenotype of MAPK-docking defective
mutants of Ste7, the MEK required for yeast mating pheromone response.
Key features of this differentiation-like process include
pheromone-imposed cell-cycle arrest, pheromone-induced gene
transcription, and a series of morphological and physiological changes
culminating in the fusion of two haploid cells of opposite mating type
to form a diploid (reviewed in Ref. 66).
To determine whether MEK-MAPK docking plays a positive role in
transmission of the mating pheromone signal, several MAPK-docking defective alleles of STE7 were individually expressed under
control of the endogenous STE7 promoter on a low-copy number
(CEN) plasmid in a MATa ste7
We previously reported that a Ste7 Docking and Scaffolding Have Overlapping Functions--
It seemed
possible that the ability of the Ste5 scaffold protein to bind both
Ste7 and Fus3 might mask any defect in signal transmission caused by
ablation of the MEK-MAPK interaction mediated by the docking site. To
address whether scaffolding by Ste5 might serve a partially redundant
function with direct MEK-MAPK docking, it was necessary to devise an
experimental strategy that would separate the scaffold function of Ste5
from its crucial role in Ste11 activation. Cytoplasmic proteins (like
Ste5) that bind both a membrane-bound protein (such as Ste4-Ste18 = G
To test whether there is functional overlap between scaffolding and
docking, MAPK-docking defective alleles of STE7, or
scaffolding-defective alleles of STE5, or both, were
expressed from their own promoters on low-copy plasmids in a
MATa ste5
To determine whether docking and scaffolding likewise have mutually
reinforcing functions in determining overall mating proficiency, quantitative mating assays were performed. Because mating efficiency can be measured over several orders of magnitude using this extremely sensitive assay, the Ste5V763A/S861P mutant, which exhibits
an essentially complete defect in Ste7 binding, was utilized. As demonstrated previously for ste5 and ste7 single
mutants (67), a ste5
A way to bypass the adapter function of Ste5 for Ste11 activation is by
using a constitutively hyperactive Ste11 variant (30, 51). The Ste11-4
mutant contains a substitution (T596I) in the catalytic domain of Ste11
that substantially increases pheromone-independent transcription of
FUS1 and other pheromone-responsive genes (51). Deletion of
Ste4 (loss of G
A plasmid expressing Ste11-4 was transformed into cells containing wild
type Ste7, or MAPK docking-site mutants of Ste7, and either containing
or lacking Ste5. Expression of a FUS1-lacZ reporter gene was
monitored in a quantitative assay. When both wild-type Ste5 and
wild-type Ste7 were present, the presence of Ste11-4 resulted in a high
level of reporter gene expression (Fig. 6D). Reporter gene
expression was abolished in the absence of Ste7, and was reduced by
3-fold in the absence of Ste5, consistent with previous results (51).
Ste7 mutants with alterations of the MAPK-docking site displayed a
reduced ability to transmit the Ste11-4 signal in the presence of Ste5;
reporter gene expression was about 30% of that observed for wild-type
Ste7. However, these mutants exhibited a much more substantial defect,
relative to wild-type Ste7, in the absence of Ste5, an additional
5-20-fold reduction in reporter gene expression (Fig. 6D
and data not shown). In contrast, the
Ste7 Conservation of the MEK-MAPK Docking Interaction--
This study
investigated stable, high affinity complex formation between mammalian
MEK1 and MEK2 and their MAPK targets ERK1 and ERK2, as well as between
the yeast MEK Ste7 and its targets Fus3 and Kss1. For all of these
interactions, a short N-terminal region (the MAPK-docking site)
in these MEKs was found to be both necessary and sufficient for the
formation of stable MEK-MAPK complexes. The MAPK-docking site is
conserved between yeast Ste7 and human MEK1 and MEK2, and putative
docking sites are also found in MEKs from organisms representative of
many different phyla and even across kingdoms (Fig. 1B).
Mutation or deletion of conserved residues in the docking site
substantially reduced complex formation between cognate yeast and
mammalian MEK-MAPK pairs. These same mutations significantly reduced
the ability of Ste7 to transmit the mating pheromone signal in
vivo, as revealed when the contribution of the Ste5 scaffold
protein was compromised or eliminated. In addition, peptides
corresponding to the MAPK-docking sites (but not mutant versions
thereof) inhibited MEK1-mediated phosphorylation of ERK2 in
vitro. Moreover, the Ste7-docking site was able to bind to and
inhibit mammalian ERKs, and yeast Fus3 and mammalian ERK2 displayed the
same relative binding affinities to a series of Ste7-docking site
mutants. We conclude that MEK-MAPK docking has been structurally and
functionally conserved from yeast to humans.
Function of MEK-MAPK Docking--
MEKs phosphorylate their target
MAPKs on a Thr and a Tyr located on a surface loop (the "activation
loop") situated between conserved protein kinase subdomains VII and
VIII. The MEK-MAPK docking interaction is distinct from this active
site/target peptide interaction. Our previous study demonstrated that
the activation loop of Kss1 was not required for Ste7 binding, and that
the C-terminal catalytic domain of Ste7 was also dispensable for
high-affinity Ste7-MAPK association (45). Thus, neither the active site
of the MEK, nor the target loop on the MAPK, are required for the docking interaction. As such, the docking interaction and the active
site-target peptide interaction could constitute a double selection for
fidelity in MEK-MAPK transactions, analogous to that proposed for some
tyrosine kinase-substrate interactions (Ref. 23 and see Fig.
7A). Furthermore, the docking
interaction represents an independent target site for drug development.
In this regard, our finding that MAPK-docking site peptides can inhibit MEK1 phosphorylation of ERK2 in vitro is notable as a
"proof of concept," because demonstrating that peptides serve as
effective inhibitors often indicates the possibility for designing more pharmacologically useful, small-molecule mimetics (68, 69). Surprisingly, we found that a peptide based upon the docking site of a
yeast MEK (Ste7) was better at binding and inhibiting the interaction
of a mammalian MEK with its target MAPK (ERK2) than were peptides from
the cognate human MEKs. Thus, comparative studies of evolutionarily
conserved protein-protein interactions of this sort may provide
additional insights for the development of more effective drug
leads.
The peptide inhibition experiments presented here support and extend
previous observations that had suggested that the MEK-MAPK docking
interaction increases the efficiency of MAPK activation. One line of
evidence came from studies of the lethal factor (LF) of Bacillus
anthracis (anthrax). LF is a protease that cleaves the
MAPK-docking site sequence in both MEK1 and MEK2 between the doublet of
basic residues and the hydrophobic-X-hydrophobic element (70, 71), and hence would be predicted to substantially reduce the
MEK-MAPK docking interaction, based on our results. Indeed, LF
treatment inhibits MEK activation of MAPK in an in vitro
kinase assay, and also inhibits ras-stimulated MAPK
phosphorylation in NIH-3T3 cells (70). A positive role for the MEK N
terminus in MAPK activation was also indicated by analysis of
N-terminal truncation and substitution mutants in MEK1 (63, 72, 73). In
many cases, however, the reported effect of these alterations on MAPK
activation was not dramatic, as is discussed further below. An
additional function for high-affinity MEK-MAPK binding has been
demonstrated by Nishida and colleagues (58, 74), who found that an
N-terminal region of MEK1 that contains the MAPK-docking site, in
combination with an immediately adjacent nuclear export signal
(residues 33-44), functions to retain unphosphorylated ERK in the cytoplasm.
MAPK-docking Sites in Other Proteins--
After our description of
the nature of the MAPK-docking site motif in MEKs (64), we and others
noted sequence similarity between MAPK-docking sites in MEKs and
putative or identified MAPK-docking sites in MAPK substrates,
phosphatases, and scaffolds2
(20, 73, 75). Indeed, mutagenesis studies of MAPK-docking sites found
in MEKs (this study), MAPK phosphatases (76), and transcription factors
(77) have all demonstrated a requirement for a cluster of basic
residues and a nearby hydrophobic-X-hydrophobic element.
This situation suggests that these proteins could compete for MAPK
binding, and that any individual complex may be dynamic in the cell. It
further suggests that a peptide or appropriately engineered drug that
disrupts the MEK-MAPK docking interaction might also block the
interaction of MAPKs with downstream substrates and other partners. On
the other hand, MAPKs do not recognize all their high-affinity targets
in the same way. For example, Kss1 binds with high-affinity not only to
Ste7 (45), but also to its target transcription factor, Ste12 (52).
Mutations in Kss1 that impair binding to Ste12 have no effect on
docking to Ste7 (6), demonstrating that the Ste7- and Ste12-binding
sites on Kss1 are distinct. In addition, it has been demonstrated that at least one other type of independent ERK-docking site motif (FXFP) exists (75), which presumably associates with a
structural element in MAPKs that is distinct from that mediating
binding to the MAPK-docking site motif in MEKs. Thus, it should be
possible to develop drugs that specifically block interaction of a
kinase with a subset of its activators or targets. Such agents might provide more specific and less toxic means for therapeutic intervention than kinase inhibitors that target the active site. MAPK inhibitors could be efficacious in treating several pathological conditions, including inflammation, cancer, and myocardial injury (68, 78).
Although the MAPK-docking site of MEKs is found near the N terminus of
these enzymes, similar sequences in MAPK substrates are often found
elsewhere within these polypeptides. However, our results support the
idea that the class of MAPK-docking site that exists in MEKs can
function modularly, because it is able to bind its cognate MAPK when
the site is positioned at its native location at the N terminus (45),
or at the C terminus, as we have shown here (e.g.
GST-Ste71-25), or even in the middle of a polypeptide, as
we have shown elsewhere (e.g. GST-Ste71-172 (6,
45)).
The complementary site(s) on MAPKs that interact with the MAPK-docking
motif of MEKs and related sequences are not completely clear. There is
good evidence for the involvement of an acidic patch that is the site
of the sevenmaker mutation in the Drosophila MAPK
rolled (73, 79). This acidic patch probably interacts with
the basic cluster of the MAPK-docking site (73). Other studies,
however, have implicated the N terminus of a MAPK (63), or a surface
loop lying below the catalytic pocket (80). The MEK-MAPK
enzyme-substrate interaction may also involve multiple regions of MAP
kinases (81). Whatever region(s) of the MAPKs interact with the
MAPK-docking site in MEKs, our results suggest that its properties have
been well conserved, as both yeast and mammalian MAPKs exhibited the
same spectrum of binding affinities when tested against various docking
site mutants.
Variation in the Strength of MEK-MAPK Docking--
One aspect of
MEK-MAPK docking that has not been well conserved between yeast and
humans is the strength of this interaction. We first characterized the
Ste7-Kss1 and Ste7-Fus3 interaction by means of a
co-immunoprecipitation assay using in vitro translated proteins (45). Using this assay, Ste7-ERK2 and MEK1-ERK interactions could not be detected (45). However, those experiments were performed
using dilute concentrations of the interacting partners, such that
associations with Kd > 0.5 µM could
not be detected. Indeed, as we have estimated here, the
Kd for interaction of full-length Ste7 with GST-ERK2
is about 1 µM, whereas binding of Ste7 to GST-Fus3 is
about 10-fold stronger. Of course, the assay method used also
influences these values. For example, previously we calculated the
Kd for Ste7-Fus3 interaction to be about 5 nM, when we used radiolabeled native (untagged) molecules,
free in solution, which were then immunoprecipitated (45); however,
using the cosedimentation assay described here, radiolabeled Ste7
associated with bead-bound GST-Fus3 with a Kd of
about 100 nM. This difference suggests that the structure
of Fus3 in the GST fusion may be suboptimal for Ste7 binding (compared with native Fus3) and/or that in vitro translation of Fus3
yields a higher fraction of properly folded molecules (compared with expression and purification of the GST-Fus3 chimera from bacteria). Nonetheless, as assessed by either assay method, Ste7-Fus3 interaction was ~100-fold stronger than that of the tightest mammalian MEK-ERK complex (MEK2-ERK2). This situation is likely counterbalanced by the
fact that MEK1, MEK2, ERK1, and ERK2 are present in mammalian cells at
much higher concentrations than the corresponding levels of Ste7, Fus3,
and Kss1 in yeast cells (82). We showed here that these
species-specific differences in binding affinity seem to be due largely
to changes in the MEKs, rather than in the MAPKs. In fact, the
MAPK-docking site of Ste7 bound to mammalian ERK1 and ERK2 with a
significantly higher affinity than did the docking sites of human MEK1
and MEK2. Presumably, therefore, differences in the precise nature of,
and spacing between, the core elements in the MAPK-docking motif, and
differences in the surrounding residues, can fine tune the affinity of
these sites to adjust the strength of MEK-MAPK association so that it
is appropriate to its physiological setting. Ste7-Fus3 complexes appear
to represent a particularly high-affinity version of the MEK-MAPK
docking interaction.
Functional Overlap between Docking and Scaffolding in Signal
Transmission--
Interaction between the active site of a protein
kinase and the target phosphoacceptor residues on its substrate (Site
1, Fig. 7A) can be enhanced either by docking between other
regions of the kinase and substrate (Site 2, Fig. 7A) or by
the binding of both to a scaffold protein (Fig. 7B). This
logic suggested that scaffolding and docking might have similar,
mutually reinforcing roles in achieving efficient signal transmission
(Fig. 7C). Indeed, by combining scaffolding-defective
alleles of STE5 with docking-defective alleles of
STE7, we have demonstrated here that scaffolding and docking
do in fact have overlapping functions in vivo. We used three
different means to specifically compromise the scaffold function of
Ste5, and to isolate the scaffold function of Ste5 from its role as an
adapter at the Ste11 (MEKK) activation step of the mating pheromone
response pathway. First, we used a Ste5 point mutant with a reduced,
but measurable, binding affinity for Ste7. Strains expressing this
mutant alone, or MAPK-docking site mutants of Ste7 alone, showed only
modest defects in pheromone response, but the double mutants exhibited
a strikingly synergistic defect. Second, we used a strain expressing a
double-point mutant of Ste5 that is unable to bind Ste7 and, hence,
completely crippled in its scaffolding function, as we demonstrated.
This mutant had a significant signaling defect, that was dramatically
enhanced when combined with MAPK-docking site mutants of Ste7. Third,
the scaffolding function of Ste5 was ablated by deleting the
STE5 gene, and the requirement for Ste5 as an adapter in
Ste11 activation was bypassed by using a constitutively active allele
of Ste11. Once again, the diminished signaling caused by the absence of scaffolding or docking was substantially enhanced by the combination of
both defects. Therefore, we conclude that docking and scaffolding share
mutually reinforcing roles in promoting signal transmission.
The minor signaling defects we observed in yeast expressing
MAPK-docking site mutants of Ste7 in an otherwise wild-type background are similar to the very modest effects of MEK1 N-terminal mutations on
ERK2 activation seen in cell-based assays. For example, Tanoue et
al. (73) observed less than 50% reduction in ERK2 activity in
such circumstances. Hence, in mammalian cells, as in yeast, the binding
of MEK and MAPK to a scaffold, such as MP1, KSR, or JIP, may compensate
for loss of direct MEK-MAPK docking.
Implications of Cooperative Docking and Scaffolding--
The
functional overlap we observed between scaffolding and docking suggests
that these two means of bringing kinase and substrate together may
share common properties. First, based on our results in yeast, both
clearly play a positive role in signal transmission. In this capacity,
each helps to facilitate signaling, but neither is absolutely essential
for achieving at least some signal throughput. Second, both may
participate in insulating a given pathway from parallel pathways that
share similar or identical components. Thus, the MAPK-docking site of
Ste7 bound to Fus3 (and Kss1), but not Hog1, and the MAPK-docking sites
in MEK1 and MEK2 bind ERK1 and ERK2, but not JNK1. Loss of Ste5
drastically reduces pheromone response, but has no effect on any other
yeast MAPK pathway (8); similarly, JIP-family scaffolds bind JNKs, but not ERK2 or p38 (40). Third, both scaffolding and docking proteins can
dictate subcellular localization of their associated components (25,
74, 83), although in some cases (e.g. Ste5) this
localization function may be independent of docking or scaffolding
per se (30, 37). Finally, in some cases, docking and/or
scaffolding could hinder signal amplification (42). Our results
indicate that, in the yeast mating MAPK cascade, the net effect of
docking and scaffolding is to augment signal propagation. Similarly,
Scott and Zuker (84) have proposed that the InaD scaffold protein limits signal amplification in Drosophila photoreceptor
cells, yet acts overall as a positive effector of phototransduction.
It is possible to conceptualize docking and scaffolding such that one
may be considered a special case of the other (compare Fig. 7,
A and B). One popular model suggests that
MAPK-cascade scaffolds function by making the dual phosphorylation of
MAPKs by MEKs processive rather than distributive (42, 43). Such a
situation would have the effect of making the stimulus-response properties of the MAPK cascade less switch-like (82). Like a scaffold,
it is conceivable that the MEK-MAPK docking interaction also increases
the likelihood of efficient dual phosphorylation. Yet, our evidence
suggests that dual phosphorylation cannot occur without prior
dissociation of the high-affinity Ste7-MAPK complex, indicating
nonprocessivity (45). Hence, docking and scaffolding may function to
increase the local effective concentrations of kinase and substrate
(85), or to hold the proteins in an enzyme-substrate orientation long
enough for a relatively slow catalytic step to occur (86). If so,
docking and scaffolding may not be incompatible with switch-like responses.
A strong argument could have been made that docking and scaffolding
function to ensure signaling specificity and to insulate against
adventitious activation, at the expense of signal flux. That is,
selectivity gained by stable complex formation might need to be "paid
for" by kinetics limited by the dissociation of the stable complexes
and by a reduced capacity for signal amplification. It is remarkable
then that most of the extant experimental evidence indicates that
docking and scaffolding potentiate signal transmission. Whether docking
and scaffolding also function to increase signaling specificity
in vivo remains a largely untested hypothesis, although there is some evidence that Ste5 may play such a role (87). No
mechanistic model of scaffolding and/or docking has received both
theoretical and experimental support. Appreciation of their overlapping
roles may facilitate the development of such a model.
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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subunit of the
membrane-bound G
complex of the pheromone receptor-coupled G
protein) (30-32). Hence, Ste5 is thought to function, first, by
recruiting Ste11 to the plasma membrane, where it can be activated by
the Ste20 protein kinase (which also binds G
) (33-35), and second,
by co-localizing, sequestering, and properly organizing the component
protein kinases of the mating MAPK cascade (36, 37). In this second
role, Ste5 is thought to enhance signal transmission from MEK kinase to
MEK to MAPK, and to insulate the mating pheromone response pathway from
other pathways that use some of the same components. Despite initial
expectations, no metazoan homologues of Ste5 have been identified.
However, analogous scaffolding functions have been ascribed to
mammalian MP1 and KSR (which bind both MEK and ERK) (38, 39), and to
JIP1-JIP3 (which bind members of the JNK cascade) (40, 41).
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ste7
) was derived
from YPH499 (46) by replacing the endogenous STE5 and
STE7 alleles with the
ste5
::LYS2 (47) and
ste7
::ADE2 (29) alleles,
respectively. Strain JCY107 (MATa
ste7
) has been described (48). Strain DC17
(MAT
his1) was used as a mating tester.
3-11 was constructed
using the same procedure except that primer LB112 was substituted for
LB111. To construct pGEM-MEK2, the MEK2 coding sequence was amplified
by PCR using primers LB114 and LB116 and template plasmid pBS-MEK2
(49). The resulting PCR product was digested with EcoRI and
XhoI and inserted into pGEM4Z that had been digested with
EcoRI and SalI.
pGEM-MEK2
4-16 was constructed using the
same procedure except that primer LB115 was substituted for LB114.
Oligonucleotides used in this study
strain, and was used as the wild-type control for
the experiments in this study. To construct
YCpT3-STE79E10E, oligonucleotide LB105 annealed to LB106B
was used to replace the corresponding PstI-AatII
fragment of YCpT3-STE7. YCpT3-STE7R10A,
YCpT3-STE715A17A, and YCpT3-STE7AAAA were
constructed by the same procedure, except that oligonucleotides encoding the indicated substitutions were employed. The
BamHI-PstI fragment of YCpT3-STE7 was replaced by
oligonucleotide LB127 annealed to LB128, yielding
YCpT3-STE7
2-7. The
BamHI-AatII fragment of YCpT3-STE7 was replaced
by oligonucleotide LB131 annealed to LB132, yielding
YCpT3-STE7
2-19. The
AatII-MscI fragment of YCpT3-STE7 was replaced by
oligonucleotide LB133 annealed to LB134, yielding
YCpT3-STE7
20-26. The
BamHI-MscI fragment of YCpT3-STE7 was replaced by
oligonucleotide LF4 annealed to LF5, yielding
YCpT3-STE7
2-22.
2-7,
2-19, and
20-26 alleles of STE7 were subcloned from their
YCpT3-based vectors into pGEM4Z as BamHI-HinDIII
fragments. Fragments encoding the N-terminal third of these alleles
(through residue 172 of wild-type STE7) were then amplified
by PCR using primers SP6PL and STE7-X-172. The wild-type allele
was produced as a fragment encoding the first 98 residues by PCR using
primers STE7-1-X and STE7-X-98 (45). An SP6 promoter was added to this
fragment in a second round of PCR using primers SP6EP and STE7-X-98, as
described (45). The same procedure was used to produce N-terminal
fragments of the
9-19, G14A, R10A, R10E, 9E10E, 15A17A, and 4E5E
alleles, except that oligonucleotides encoding the indicated amino acid
changes were used in place of STE7-1-X.
1 isoform,
GenBankTM L26318) and anti-JNK1 antiserum from Santa Cruz
Biotechnology. Binding constants were calculated as described (45),
based on the known input concentrations and a determination of the
amount complex formed, assuming a simple bimolecular binding reaction. A further assumption was that 100% of input molecules were active (i.e. capable of binding). We find Kd
values calculated in this manner to be useful for relative comparisons
of the affinities of different protein-protein interactions measured
using the same assay. We have noted, however, that the
Kd for (essentially) the same interaction,
determined in two different assays (e.g. GST cosedimentation
versus coimmunoprecipitation), can vary up to 20-fold.
-glycerophosphate, 1 mM sodium orthovanadate, 1 mM dithiothreitol, 1 µg (1.25 µM) of inactive mouse ERK2 (K52R mutation; New
England Biolabs), 0.1 unit of active human MEK1 (Upstate
Biotechnology), 50 µM ATP, 1 µCi of
[
-32P]ATP, and the indicated concentration of peptide.
ERK2 and peptide were preincubated in buffer for 10 min at 37 °C,
then returned to ice prior to the addition of ATP and MEK1. Reactions
were for 20 min at 30 °C. Reactions were analyzed by SDS-PAGE and
quantified on a PhosphorImager. The results were also verified
qualitatively by immunostaining with antisera specific for the dually
phosphorylated ERK isoform (New England Biolabs).
F mating pheromone for 90 min, and harvested as
described (45). For analysis of expression stimulated by the
STE11-4 allele, plasmid YCpH-STE11-4 was
introduced in a third transformation. Transformant colonies were
harvested directly from plates after 3-4 days growth. Cell extracts
were prepared and
-galactosidase activity quantified as described
(45).
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Fig. 1.
A MAPK-docking site near the N terminus of
MEKs. A, schematic representation of S. cerevisiae Ste7, and human MEK1 and MEK2, showing the positions of
the MAPK-docking site (solid bar) and the protein kinase
catalytic core (hatched region). The percent amino acid
identity between Ste7 and MEK1 and between MEK1 and MEK2 is indicated
for the N-terminal and catalytic domains. Relatedness between Ste7 and
MEK2 (<20 and 36% identity, respectively, in the N-terminal and
catalytic domains) is similar to that between Ste7 and MEK1. Length of
each protein is indicated on the right. B,
alignment of the N termini of representatives of the MEK/MKK/MAPKK
family of dual-specificity protein kinases. All sequences start from
the initiator methionine. Positions of the doublet of basic residues
and the nonpolar residues of the hydrophobic-X-hydrophobic
element of the core MAPK-docking site motif are indicated by
asterisks. One-residue gaps are indicated by
hyphens ( ); spaces are for visual clarity. Source of the
sequence shown is given on the right. Accession numbers for
the NCBI Nucleotide or Protein data bases are given. In most cases,
rodent and other mammalian MEKs are identical to their human orthologs
in the docking-site region; hence they are not shown to reduce
redundancy. MEKs containing an N-terminal sequence that nearly fits the
above consensus (e.g. Drosophila Dsor1 (Q24324)
and p38 MAPKK (CAB45101), and Leishmania MAPKK (CAB45417))
are also not shown.
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Fig. 2.
The MAPK-docking site is sufficient for MAPK
binding. A, Kss1, but not Hog1, binds to the
MAPK-docking site of Ste7. Radiolabeled (35S) Kss1 and Hog1
proteins were prepared by in vitro translation, partially
purified by ammonium sulfate precipitation, and portions (5% of the
amount added in the binding reactions; input) were resolved on a 10%
SDS-polyacrylamide gel (lanes 1 and 2). Samples
(1 pmol) of the same proteins were incubated with 1 or 10 µg of GST
(lanes 3-6) or GST-Ste71-25 (lanes
7-10), bound to glutathione-Sepharose (GSH) beads, and the
resulting bead-bound protein complexes were isolated by sedimentation
and resolved by 10% SDS-PAGE on the same gel. The gel was analyzed by
staining with Coomassie Blue for visualization of the bound GST fusion
protein (lower panel), and by autoradiography for
visualization of the bound radiolabeled protein (upper
panel). B, ERK2, but not JNK1, binds to the
MAPK-docking sites of MEK1 and MEK2. Purified ERK2 or JNK1 1 proteins
(1 pmol) were incubated with ~100 µg of GST,
GST-MEK11-19, or GST-MEK21-22 that were
pre-bound to GSH beads (lanes 2-4), or with empty GSH beads
only (lane 5). Bead-bound complexes were isolated, resolved
by 10% SDS-PAGE, and analyzed by staining with Coomassie Blue for
visualization of the bound GST fusion protein (lower panel),
and by immunostaining with anti-ERK2 (upper panel) or
anti-JNK1 (middle panel) antisera. Input (10% of the amount
added in the binding reactions) is shown in lane 1. The
middle panel is overexposed to highlight that there was no
binding of JNK1 above background. C, alteration of conserved
residues in the MAPK-docking sites of MEK1 and MEK2 result in
diminished MAPK binding. Experimental design as in B, except
that the GST fusion proteins were GST, GST-MEK11-19,
GST-MEK11-19EE, GST-MEK21-22, and
GST-MEK21-22ATA (lanes 2-6,
respectively).
3-11) was markedly reduced (Fig.
3A). Similarly, MEK2 bound to
GST-ERK1 and GST-ERK2, but binding of
MEK2
4-16 was greatly diminished (Fig.
3B). We quantified formation of these complexes using a
method described in detail elsewhere (Ref. 45, see also "Experimental
Procedures"). MEK2-ERK2 interaction had the highest apparent affinity
(Kd
9 µM); and MEK1-ERK1
interaction had a somewhat lower affinity (Kd
29 µM). For all MEK-ERK pairings, deletion of the
MAPK-docking site decreased binding affinity in the range 5-10-fold.
Interestingly, it has been reported that the MP1 scaffold protein shows
some specificity for MEK1-ERK1 over MEK2-ERK2 (38), and may thus
compensate for the weaker intrinsic binding affinity of the MEK1-ERK1
pair.
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Fig. 3.
High-affinity interaction between full-length
MEK and ERK requires the MAPK-docking site. A, MEK1,
but not MEK1 3-11, binds to ERK1 and ERK2.
Radiolabeled (35S) MEK1 and
MEK1
3-11 proteins (~1 pmol) were
incubated with 10 µg of purified GST, GST-ERK1, or GST-ERK2 that were
pre-bound to GSH beads, and bead-bound protein complexes were isolated
and analyzed as in Fig. 2A. Inputs (5% of the amount added
in the binding reactions) are shown in lanes 1 and
2. B, MEK2, but not
MEK2
4-16, binds to ERK1 and ERK2.
Experimental design as in A, except that the radiolabeled
proteins were MEK2 and MEK2
4-16, as
indicated.
1 µM) higher than that of
either MEK1 or MEK2 (data not shown). To confirm this result and to
determine whether the differences in affinity were due mainly to
differences in the MAPK-docking site sequences per se, we
assessed the ability of GST-Ste71-25,
GST-MEK11-19, and GST-MEK21-22 to bind
purified ERK1 and ERK2. Indeed, the Ste7-docking site bound both
mammalian MAPKs and did so with apparent affinities at least 10-fold
higher than those displayed by the human MEK1 and MEK2-docking sites
(data not shown).
0.1 µM), whereas a mutant lacking the entire MAPK-docking site (Ste7
2-19), bound with reduced, but
still readily detectable, affinity (Kd
1.4 µM). This result is consistent with our previous
observations suggesting that, in addition to MEK-MAPK binding mediated
by the docking site, there is also a detectable interaction between the
catalytic domain of Ste7 and the MAPKs, possibly mediated by active
site-phosphoacceptor loop contacts (45). Therefore, to probe the
specific contributions of the docking interaction, binding of the
N-terminal noncatalytic domain of Ste7 (Ste7N) to GST-Fus3 was examined
(Fig. 4B). Ste7N corresponded to residues 1-172 or residues
1-98, depending on the experiment (see "Experimental Procedures"
for details). The results obtained were virtually indistinguishable
when the longer or shorter construct (and mutant versions thereof) were
compared. Like full-length Ste7, Ste7N bound with high affinity to
GST-Fus3 (Kd
0.2 µM), whereas
deletion of the core of the MAPK-docking site (residues 9-19) greatly
reduced the ability of Ste7N to bind to GST-Fus3 (Fig. 4B).
A somewhat larger deletion (residues 2-19), that removed an upstream
pair of basic residues in Ste7N (see Fig. 1B), eliminated
essentially all of the residual binding to GST-Fus3. In contrast, a
deletion (residues 20-26) that removed seven residues immediately
C-terminal to the proposed MAPK-docking site caused only a slight
reduction in binding affinity (Fig. 4B). This deletion
removed a Pro, a Gly, and two Asn, which are residues frequently
represented in the sequences in MEKs immediately C-terminal to the core
docking site motif (see Fig. 1B). It is possible, however,
that the next residue (Gly27) partially compensated for the
loss of the other turn forming, helix-breaking residues.
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Fig. 4.
Conserved residues in the MAPK-docking site
of Ste7 are required for high-affinity MAPK binding. A,
Ste7 MAPK-docking site mutants analyzed. The first 27 residues of Ste7
are shown. The positions of substitution mutations are shown in
bold and underlined. Residues removed in deletion
mutations are indicated by hyphens ( ); spaces are for
visual clarity. The name of the mutant allele is on the
left; a summary of the binding data, shown in B
is on the right. B, relative binding of
radiolabeled Ste7N (either Ste71-172 or
Ste71-98) and mutant derivatives thereof to GST-Fus3
(solid bars) or to GST-ERK2 (open bars).
35S-Ste7N and mutants thereof (2 pmol) were incubated with
20 pmol of GST, 2 pmol of GST-Fus3, or 2 pmol of GST-ERK2 bound to GSH
beads, and the resulting bead-bound protein complexes were quantified
in a scintillation counter. The nonspecific background adsorption of
each radiolabel protein to GST alone was subtracted. Under these
conditions, 8.1% of the input wild-type Ste7N bound to GST-Fus3, and
4.7% bound to GST-ERK2. Results were normalized by setting these
values as 100%. Data shown are the average of at least three
experiments; error bars indicate standard deviations. The
relative affinity of the weak-binding derivatives was confirmed in
experiments using 20 pmol of GST-Fus3 (not shown). C,
representative data for experiments shown above. Radiolabeled wild-type
Ste7N, or the AAAA or
2-7 derivatives, as indicated, was incubated
with 20 pmol of GST (G; lane 2), or with 2 or 20 pmol of
GST-Fus3 (lanes 3 and 4, respectively) that had
been pre-bound to GSH beads. Bead-bound complexes were isolated,
resolved by 12% SDS-PAGE, and visualized by autoradiography. Input
(10% of the amount used in the binding reactions) is shown in
lane 1.
9-19) (Fig. 4B).
Substitution of Leu15 and Leu17 of the
hydrophobic-X-hydrophobic element of the motif with Ala also
substantially reduced formation of the GST-Fus3-Ste7N complex (Fig.
4B); substitution of these residues in conjunction with the
basic doublet (Ste7NAAAA) had an even more pronounced
effect (Fig. 4, B and C).
2-7) greatly
reduced binding to Fus3 (Fig. 4B), as did substitution of
residues 2-8 with a single Pro (data not shown). We noted that this
stretch of sequence, like the core docking site motif, contains a basic
pair. Indeed, charge-reversal mutations at this site
(Ste7NR4E/R5E) resulted in a binding defect comparable to
that observed for Ste7N
2-7 (Fig.
4B). However, Ste7N
2-7 still bound to GST-ERK2 with a higher affinity than did either MEK1 or MEK2
(data not shown), suggesting that additional variation among the
MAPK-docking sites of Ste7, MEK1, and MEK2 also contribute to the
observed differences in affinity.
20-26) also bound relatively well to
ERK2, and mutants that bound poorly to Fus3 (e.g.
Ste7NL15A/L17A, Ste7N
9-19) also
bound poorly to ERK2. Moreover, the series of mutants in the basic
cluster that showed a graded decrease in binding affinity to Fus3
(Ste7NR10A, Ste7NR10E, and
Ste7NR9E/R10E) showed the same pattern of binding to ERK2.
These data indicate that the structural and chemical features of the
region(s) of Fus3 and ERK2 that interact with the MAPK-docking site on
MEKs must share substantial similarity.
100 µM; data not
shown).
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Fig. 5.
Inhibition of MEK1-dependent
phosphorylation of ERK2 by MAPK-docking site peptides.
A, peptides used. Sequences of the synthetic peptides
prepared for this study are shown; substitution mutations are indicated
in bold and underlined. The corresponding
residues of the full-length proteins are given on the right.
B and C, effect of peptides on phosphorylation of
ERK2 by MEK1. Purified, full-length, catalytically inactive ERK2 (1.25 µM) was incubated with 1 unit of purified active MEK1 and
[ -32P]ATP for 20 min, in the absence or presence of
the specified concentrations of the indicated peptides. ERK2
phosphorylation was quantified by SDS-PAGE followed by analysis of
relative incorporation using a PhosphorImagerTM (Molecular
Dynamics, Inc.). B, autoradiogram of a representative
experiment. C, results plotted as percent phosphorylation
relative to that observed in the absence of any added peptide. Data are
the average of at least three experiments.
strain. Under these conditions, all of the mutant Ste7 derivatives
exhibited only a modest defect in mating pheromone response, as
assessed by several types of assays. To obtain a quantitative measure
of signal throughput, we monitored expression of a pheromone-inducible
reporter gene (FUS1-lacZ), which is expressed at a low, but
detectable, level in the absence of pheromone stimulation, but is
induced about 100-fold after addition of a saturating concentration of
pheromone. Docking-defective mutants of Ste7 supported basal expression
at 60% of the wild-type level. Following pheromone stimulation,
docking-defective mutants of Ste7 supported induced expression at 85%
of the level of expression observed for induced wild-type cells (data
not shown). The docking-defective Ste7 mutants also exhibited only a
slight defect in pheromone-imposed cell-cycle arrest (see below), and
no significant defect in mating proficiency (see below).
2-22
mutant had a moderate defect in pheromone response (45), in contrast to
the rather minor defect we observed in this present study for
Ste7
2-19 and other docking site-defective
mutants expressed in an otherwise wild-type background. Sequencing of
the complete open reading frame of the
Ste7
2-22 construct used in our previous
study (45) revealed a cloning artifact that resulted in truncation of
the last three residues of the STE7 coding sequence (data
not shown). We therefore reconstructed Ste7
2-22; this new construct behaved identically to Ste7
2-19 in all assays tested (data not shown).
) and another cytoplasmic protein (such as Ste11 = MEKK),
and thereby recruit the latter protein to the membrane, are often
referred to as adapters (25). To separate the adapter function of Ste5
from its subsequent role as a scaffold for the MAPK cascade components,
we made use, first, of Ste5 point mutants with specific defects in Ste7
binding (28). It has been shown that Ste5D746G greatly
reduces, but does not completely prevent, Ste7 binding to Ste5, whereas
Ste5V763A/S861P eliminates detectable Ste7 binding to Ste5.
Both Ste5D746G and Ste5V763A/S861P, however,
exhibit undiminished binding to Ste4 and Ste11 (and to Fus3),
consistent with the idea that they are specifically defective in the
scaffold function of Ste5 (28).
ste7
double-delete strain. These cells were then assayed for mating
pheromone response. First, as a measure of the long-term effects of
pheromone action, pheromone-imposed cell-cycle arrest and recovery from
arrest were monitored using the standard halo bioassay (Fig.
6A). To obtain the optimal
window for detecting a genetic interaction in this assay, the
Ste5D746G mutant was used because, like the Ste7
MAPK-docking site mutants, it displays only a modest signaling defect
when expressed in place of Ste5 in an otherwise wild-type background. Indeed, when Ste5D746G was coexpressed with wild-type Ste7,
only a minor reduction in pheromone-imposed G1 arrest was
observed, as indicated by the roughened edges and slightly smaller
diameter of the haloes (Fig. 6A). A comparable modest defect
was observed in cells expressing wild-type Ste5 in combination with the
docking-defective mutant Ste7
2-19.
Strikingly, however, cells expressing both Ste5D746G and
Ste7
2-19 displayed smaller and much more
turbid haloes (Fig. 6A), indicating a marked reduction in
the initial strength of pheromone-imposed arrest and/or a substantially
accelerated rate of recovery (or both). Other Ste7 docking-defective
mutants behaved comparably; in contrast, a control
(Ste7
20-26) behaved like wild-type Ste7
(data not shown). Thus, the scaffolding function of Ste5 acts
synergistically in concert with the docking function of Ste7 (and vice
versa) to achieve efficient and sustained pheromone-imposed
G1 arrest.
View larger version (75K):
[in a new window]
Fig. 6.
Overlap between the MAPK docking function of
Ste7 and the scaffold function of Ste5. A, synergistic
effect of scaffolding-deficient Ste5 and docking-defective Ste7 on
pheromone-imposed cell-cycle arrest. Yeast strain YLB105
(MATa ste5
ste7
) was transformed with a plasmid encoding
wild-type Ste5, or Ste5D746G, or an "empty" vector, in
combination with a plasmid encoding wild-type Ste7,
Ste7
2-19, or an empty vector. The resulting
transformants were plated as a lawn, onto which were placed discs
containing 12 µg (left) or 3 µg (right) of
-factor mating pheromone. The plates were photographed 48 h
later. Pheromone-imposed cell-cycle arrest is indicated by the zone of
growth inhibition ("halo") surrounding a disc. B and
C, scaffolding-defective Ste5 unmasks a role for the
Ste7-MAPK docking interaction in mating proficiency. Strain YLB105 was
transformed with a plasmid encoding wild-type Ste5 or
Ste5V763A/S861P (denoted VASP in B), in
combination with a plasmid encoding wild-type Ste7, or mutant
derivatives thereof. B, the resulting transformants were
streaked onto a plate selective for plasmid maintenance
(top), then scored for mating to an appropriate tester
strain using a qualitative assay (bottom). C, the
resulting transformants were scored for mating to an appropriate tester
strain using a quantitative assay. Mating efficiencies (diploids formed
per input MATa haploid) are normalized to the value (0.51)
obtained for cells expressing wild-type Ste5 and wild-type Ste7.
Error bars indicate standard deviations of duplicate
experiments. D and E, partially redundant roles
for scaffolding and docking in transmitting a signal from
constitutively active MEKK (Ste11). Strain YLB105 was transformed with
a plasmid encoding wild-type Ste5 or an empty vector (D and
E), or Ste5V763A/S861P (E), in
combination with a plasmid encoding wild-type Ste7, or mutant
derivatives thereof. The resulting transformants were then further
transformed with a plasmid containing a reporter gene regulated by the
mating MAPK cascade (FUS1-lacZ), and a plasmid carrying
dominant, constitutively active STE11-4. After 4 days, the
resulting colonies were pooled, and expression of the reporter gene was
assessed by determination of
-galactosidase specific activity.
Values are normalized to that obtained for cells expressing wild-type
Ste5 and wild-type Ste7, and represent the average of at least three
experiments. Error bars indicate standard deviations.
ste7
double mutant is
completely sterile, and remained sterile (frequency of diploid
formation <10
7) when transformed with either a plasmid
expressing STE5 alone (making the resulting strain
functionally ste7
) or a plasmid expressing
STE7 alone (making the resulting strain functionally ste5
) (data not shown). When transformed with both
plasmids, however, these functionally wild-type cells mated with high
efficiency, as judged by either a plate mating assay (Fig.
6B) or a quantitative mating assay (~50% diploid
formation). Relative to this reconstructed wild-type situation, cells
coexpressing Ste5V763A/S861P and wild-type Ste7 mated with
a reproducibly reduced efficiency, as assessed either on plates (Fig.
6B) or using the quantitative method (~12% of wild-type;
Fig. 6C). In contrast, there was no statistically significant difference in mating efficiency between the reconstructed wild-type control and cells coexpressing wild-type Ste5 and any of the
docking site-defective Ste7 mutants (Fig. 6C). However, all
of the docking site-defective Ste7 mutants showed a strong effect when
each was coexpressed with Ste5V763A/S861P: mating efficiency was typically reduced a further 10-20-fold over the impairment caused by Ste5V763A/S861P alone, as judged
either on plates (Fig. 6B) or using the quantitative test
(Fig. 6C). Only Ste7
20-26, a
mutant that has no MAPK-binding defect (Fig. 4B), behaved
like wild-type Ste7 when combined with Ste5V763A/S861P
(Fig. 6B).
) has no effect on the degree of FUS1
expression elicited by Ste11-4; however, deletion of Ste5 reduces
Ste11-4-dependent FUS1 expression severalfold
(Ref. 51, see also below). The fact that loss of Ste5 reduces signaling by constitutively active Ste11 has been interpreted to reflect the role
of the scaffold function of Ste5 in potentiating MEKK (Ste11)
phosphorylation of MEK (Ste7), and/or MEK phosphorylation of MAPK
(30).
20-26 mutant, which has no MAPK-docking
defect in vitro, behaved just like wild-type Ste7 in either
the presence or absence of Ste5. Moreover, we found that cells
containing Ste5V763A/S861P behaved identically to cells lacking Ste5 entirely (Fig. 6E), supporting the contention
that Ste5V763A/S861P is ineffective as a scaffold.
Consistent with the results seen in cells entirely lacking Ste5, cells
expressing both Ste5V763A/S861P and
Ste7
2-19 displayed a markedly synergistic
defect in transmitting the Ste11-4-dependent signal (Fig.
6E). Hence, as in the previous two experiments, the phenotype of the docking defect was enhanced by the scaffolding defect. Thus, as judged by three separate experimental
approaches and using three different biological end points (cell-cycle
arrest, mating, and transcriptional induction), mutants of Ste7
defective in MAPK docking displayed a substantial reduction in
pheromone response only when the scaffold function of Ste5 was compromised.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (26K):
[in a new window]
Fig. 7.
Diagrammatic representation of interactions
between MEKs, MAPKs and scaffold proteins. A, the
ability of a MEK to interact with its cognate MAPK both by an active
site-target residue interaction (1) and by a docking
interaction (2) comprises, in principle, a "double
selection" that has the potential to promote the efficiency and
fidelity of MAPK activation. To aid conceptualization, these two
interactions are shown as occurring simultaneously. Alternatively, they
may occur in kinetically distinct steps (45, 85). The region of the
MAPK that is phosphorylated by the MEK is represented by a chain of
five circles. B, view of scaffolding as special
case of docking (or visa versa). Compare with A, the
scaffold protein is indicated by an S. C,
overlapping roles of docking and scaffolding in promoting MAPK cascade
signaling. MAPK activation is promoted by direct MEK-MAPK binding via
the active site (1) and the docking site (2), and
by binding of both MEK and MAPK to a scaffold (S) protein
(3). In support of the scheme shown, it is known that the
catalytic domain of Ste7 binds to Ste5 (27, 29), whereas the N terminus
of Ste7 binds to Fus3 (this study and Refs. 29 and 45). It is likely,
therefore, that Ste7 can bind to both Ste5 and Fus3 simultaneously. We
have shown here that mutations in the MAPK-docking site of MEK
(2), combined with mutations in the MEK-binding site
(3) of the scaffold Ste5, or with deletion of the scaffold
entirely, leads to a synergistic reduction in MAPK activation in the
yeast mating pheromone response pathway.
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ACKNOWLEDGEMENTS |
---|
We thank Natalie Ahn, Melanie Cobb, Kun-Liang Guan, Steven Pelech, George Sprague, and Mike Weber for generous gifts of reagents.
![]() |
FOOTNOTES |
---|
* This work was supported by a Special Fellow Award from the Leukemia and Lymphoma Society (to L. B.) (work done at Berkeley), National Institutes of Health Research Grant GM21841 (to J. T.), resources provided by the Berkeley Campus Cancer Research Laboratory; and a Burroughs Wellcome Foundation New Investigator Award, Beckman Foundation Young Investigator Award, by seed money provided by Grant IRG 98-279 from the American Cancer Society, and National Institutes of Health Research Grant GM60366 (all to L. B.) (work done at Irvine).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.
¶ To whom correspondence should be addressed: Dept. of Developmental and Cell Biology, 5207 Biological Sciences II, University of California, Irvine, CA 92697-2300. Tel.: 949-824-6902; Fax: 949-824-4709; E-mail: bardwell@uci.edu.
Published, JBC Papers in Press, December 29, 2000, DOI 10.1074/jbc.M010271200
2 J. X. Zhu-Shimoni and J. Thorner, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are: MAPK, mitogen-activated protein kinase: ERK, entracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; MOPS, 4-morpholinepropanesulfonic acid; LF, lethal factor; JNK, c-Jun N-terminal kinase.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Graves, J. D., and Krebs, E. G. (1999) Pharmacol. Ther. 82, 111-21[CrossRef][Medline] [Order article via Infotrieve] |
2. | Hunter, T. (2000) Cell 100, 113-127[Medline] [Order article via Infotrieve] |
3. |
Schaeffer, H. J.,
and Weber, M. J.
(1999)
Mol. Cell. Biol.
19,
2435-2444 |
4. | Lewis, T. S., Shapiro, P. S., and Ahn, N. G. (1998) Adv. Cancer Res. 74, 49-139[Medline] [Order article via Infotrieve] |
5. | Garrington, T., and Johnson, G. (1999) Curr. Opin. Cell Biol. 11, 211-218[CrossRef][Medline] [Order article via Infotrieve] |
6. |
Bardwell, L.,
Cook, J. G.,
Voora, D.,
Baggott, D. M.,
Martinez, A. R.,
and Thorner, J.
(1998)
Genes Dev.
12,
2887-2898 |
7. |
Shapiro, P. S.,
Whalen, A. M.,
Tolwinski, N. S.,
Wilsbacher, J.,
Froelich-Ammon, S. J.,
Garcia, M.,
Osheroff, N.,
and Ahn, N. G.
(1999)
Mol. Cell. Biol.
19,
3551-3560 |
8. |
Gustin, M. C.,
Albertyn, J.,
Alexander, M.,
and Davenport, K.
(1998)
Microbiol. Mol. Biol. Rev.
62,
1264-1300 |
9. | Caffrey, D. R., O'Neill, L. A., and Shields, D. C. (1999) J. Mol. Evol. 49, 567-582[Medline] [Order article via Infotrieve] |
10. | Galcheva-Gargova, Z. B., Derijard, B., Wu, I. H., and Davis, R. J. (1994) Science 265, 806-808[Medline] [Order article via Infotrieve] |
11. |
Atienza, J. M.,
Suh, M.,
Xenarios, I.,
Landgraf, R.,
and Colicelli, J.
(2000)
J. Biol. Chem.
275,
20638-20646 |
12. | Kemp, B. E., Parker, M. W., Hu, S., Tiganis, T., and House, C. (1994) Trends Biochem. Sci. 19, 440-444[CrossRef][Medline] [Order article via Infotrieve] |
13. | Pearson, R. B., and Kemp, B. E. (1991) Methods Enzymol. 200, 62-81[Medline] [Order article via Infotrieve] |
14. | Songyang, Z., Lu, K. P., Kwon, Y. T., Tsai, L. H., Filhol, O., Cochet, C., Brickey, D. A., Soderling, T. R., Bartleson, C., Graves, D. J., DeMaggio, A. J., Hoekstra, M. F., Blenis, J., Hunter, T., and Cantley, L. C. (1996) Mol. Cell. Biol. 16, 6486-6493[Abstract] |
15. | Johnson, L. N., Lowe, E. D., Noble, M. E., and Owen, D. J. (1998) FEBS Lett. 430, 1-11[CrossRef][Medline] [Order article via Infotrieve] |
16. | Brunet, A., and Pouyssegur, J. (1996) Science 272, 1652-1655[Abstract] |
17. |
Jiang, Y.,
Li, Z.,
Schwarz, E.,
Lin, A.,
Guan, K.,
Ulevitch, R.,
and Han, J.
(1997)
J. Biol. Chem.
272,
11096-11102 |
18. |
Robinson, M. J.,
Cheng, M.,
Khokhlatchev, A.,
Ebert, D.,
Ahn, N.,
Guan, K. L.,
Stein, B.,
Goldsmith, E.,
and Cobb, M. H.
(1996)
J. Biol. Chem.
271,
29734-29739 |
19. |
Seger, R.,
Ahn, N. G.,
Posada, J.,
Munar, E. S.,
Jensen, A. M.,
Cooper, J. A.,
Cobb, M. H.,
and Krebs, E. G.
(1992)
J. Biol. Chem.
267,
14373-14381 |
20. | Holland, P. M., and Cooper, J. A. (1999) Curr. Biol. 9, R329-R331[CrossRef][Medline] [Order article via Infotrieve] |
21. |
Kolman, M. F.,
and Egelhoff, T. T.
(1997)
J. Biol. Chem.
272,
16904-16910 |
22. |
Schulman, B. A.,
Lindstrom, D. L.,
and Harlow, E.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
10453-10458 |
23. | Songyang, Z., Carraway, K. L., III, Eck, M. J., Harrison, S. C., Feldman, R. A., Mohammadi, M., Schlessinger, J., Hubbard, S. R., Smith, D. P., Eng, C., Lorenzo, M. J., Ponder, B. A. J., Mayer, B. J., and Cantley, L. C. (1995) Nature 373, 536-539[CrossRef][Medline] [Order article via Infotrieve] |
24. | Karandikar, M., and Cobb, M. H. (1999) Cell Calcium 26, 219-226[CrossRef][Medline] [Order article via Infotrieve] |
25. |
Pawson, T.,
and Scott, J. D.
(1997)
Science
278,
2075-2080 |
26. | Whitmarsh, A. J., and Davis, R. J. (1998) Trends Biochem. Sci. 23, 481-485[CrossRef][Medline] [Order article via Infotrieve] |
27. | Choi, K.-Y., Satterberg, B., Lyons, D. M., and Elion, E. A. (1994) Cell 78, 499-512[Medline] [Order article via Infotrieve] |
28. |
Inouye, C.,
Dhillon, N.,
Durfee, T.,
Zambryski, P. C.,
and Thorner, J.
(1997)
Genetics
147,
479-492 |
29. |
Printen, J. A.,
and Sprague, G. F., Jr.
(1994)
Genetics
138,
609-619 |
30. | Feng, Y., Song, L., Kincaid, E., Mahanty, S., and Elion, E. (1998) Curr. Biol. 8, 267-278[Medline] [Order article via Infotrieve] |
31. |
Inouye, C.,
Dhillon, N.,
and Thorner, J.
(1997)
Science
278,
103-106 |
32. | Whiteway, M. S., Wu, C., Leeuw, T., Clark, K., Fourest-Lieuvin, A., Thomas, D. Y., and Leberer, E. (1995) Science 269, 1572-1575[Medline] [Order article via Infotrieve] |
33. | Drogen, F., O'Rourke, S., Stucke, V., Jaquenoud, M., Neiman, A., and Peter, M. (2000) Curr. Biol. 10, 630-639[CrossRef][Medline] [Order article via Infotrieve] |
34. | Leeuw, T., Wu, C., Schrag, J., Whiteway, M., Thomas, D., and Leberer, E. (1998) Nature 391, 191-195[CrossRef][Medline] [Order article via Infotrieve] |
35. |
Pryciak, P. M.,
and Huntress, F. A.
(1998)
Genes Dev.
12,
2684-2697 |
36. |
Elion, E. A.
(1998)
Science
281,
1625-1626 |
37. |
Sette, C.,
Inouye, C. J.,
Stroschein, S. L.,
Iaquinta, P. J.,
and Thorner, J.
(2000)
Mol. Biol. Cell
11,
4033-4049 |
38. |
Schaeffer, H. J.,
Catling, A. D.,
Eblen, S. T.,
Collier, L. S.,
Krauss, A.,
and Weber, M. J.
(1998)
Science
281,
1668-1671 |
39. | Therrien, M., Michaud, N. R., Rubin, G. M., and Morrison, D. K. (1996) Genes Dev. 10, 2684-2695[Abstract] |
40. |
Kelkar, N.,
Gupta, S.,
Dickens, M.,
and Davis, R.
(2000)
Mol. Cell. Biol.
20,
1030-1043 |
41. |
Whitmarsh, A. J.,
Cavanagh, J.,
Tournier, C.,
Yasuda, J.,
and Davis, R. J.
(1998)
Science
281,
1671-1674 |
42. | Burack, W. R., and Shaw, A. S. (2000) Curr. Opin. Cell Biol. 12, 211-216[CrossRef][Medline] [Order article via Infotrieve] |
43. |
Levchenko, A.,
Bruck, J.,
and Sternberg, P.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
5818-5823 |
44. | Tsunoda, S., Sierralta, J., and Zuker, C. S. (1998) Curr. Opin. Genet. Dev. 8, 419-422[CrossRef][Medline] [Order article via Infotrieve] |
45. | Bardwell, L., Cook, J. G., Chang, E. C., Cairns, B. R., and Thorner, J. (1996) Mol. Cell. Biol. 16, 3637-3650[Abstract] |
46. |
Sikorski, R. S.,
and Hieter, P.
(1989)
Genetics
122,
19-27 |
47. | Hasson, M. S., Blinder, D., Thorner, J., and Jenness, D. D. (1994) Mol. Cell. Biol. 14, 1054-1065[Abstract] |
48. | Cook, J. G., Bardwell, L., and Thorner, J. (1997) Nature 390, 85-88[CrossRef][Medline] [Order article via Infotrieve] |
49. |
Zheng, C.-F.,
and Guan, K.-L.
(1993)
J. Biol. Chem.
268,
11435-11439 |
50. | Charest, D. L., Mordret, G., Harder, K. W., Jirik, F., and Pelech, S. L. (1993) Mol. Cell. Biol. 13, 4679-4690[Abstract] |
51. | Stevenson, B. J., Rhodes, N., Errede, B., and Sprague, G. F., Jr. (1992) Genes Dev. 6, 1293-1304[Abstract] |
52. |
Bardwell, L.,
Cook, J. G.,
Zhu-Shimoni, J. X.,
Voora, D.,
and Thorner, J.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
15400-15405 |
53. | Cook, J. G., Bardwell, L., Kron, S. J., and Thorner, J. (1996) Genes Dev. 10, 2831-2848[Abstract] |
54. |
Posas, F.,
and Saito, H.
(1997)
Science
276,
1702-1705 |
55. |
Zanke, B. W.,
Rubie, E. A.,
Winnett, E.,
Chan, J.,
Randall, S.,
Parsons, M.,
Boudreau, K.,
McInnis, M.,
Yan, M.,
Templeton, D. J.,
and Woodgett, J. R.
(1996)
J. Biol. Chem.
271,
29876-29881 |
56. |
Zheng, C.-F.,
and Guan, K.-L.
(1993)
J. Biol. Chem.
268,
23933-23939 |
57. |
Zhou, G.,
Bao, Z. Q.,
and Dixon, J. E.
(1995)
J. Biol. Chem.
270,
12665-12669 |
58. |
Fukuda, M.,
Gotoh, Y.,
and Nishida, E.
(1997)
EMBO J.
16,
1901-1908 |
59. | Kieran, M. W., Katz, S., Vail, B., Zon, L. I., and Mayer, B. J. (1999) Oncogene 18, 6647-6657[CrossRef][Medline] [Order article via Infotrieve] |
60. |
Enslen, H.,
Brancho, D. M.,
and Davis, R. J.
(2000)
EMBO J.
19,
1301-1311 |
61. |
Tournier, C.,
Whitmarsh, A. J.,
Cavanagh, J.,
Barrett, T.,
and Davis, R. J.
(1999)
Mol. Cell. Biol.
19,
1569-1581 |
62. |
Xia, Y.,
Wu, Z.,
Su, B.,
Murray, B.,
and Karin, M.
(1998)
Genes Dev.
12,
3369-3381 |
63. |
Xu, B.,
Wilsbacher, J. L.,
Collisson, T.,
and Cobb, M. H.
(1999)
J. Biol. Chem.
274,
34029-34035 |
64. | Bardwell, L., and Thorner, J. (1996) Trends Biochem. Sci. 21, 373-374[CrossRef][Medline] [Order article via Infotrieve] |
65. | Creighton, T. E. (1993) Proteins: Structures and Molecular Properties , 2nd Ed. , W. H. Freeman & Co., New York |
66. | Bardwell, L., Cook, J. G., Inouye, C. J., and Thorner, J. (1994) Dev. Biol. 166, 363-379[CrossRef][Medline] [Order article via Infotrieve] |
67. | Hartwell, L. H. (1980) J. Cell Biol. 85, 811-822[Abstract] |
68. | Gibbs, J., and Oliff, A. (1994) Cell 79, 193-198[Medline] [Order article via Infotrieve] |
69. | Lowman, H. B. (1997) Annu. Rev. Biophys. Biomol. Struct. 26, 401-424[CrossRef][Medline] [Order article via Infotrieve] |
70. |
Duesbery, N. S.,
Webb, C. P.,
Leppla, S. H.,
Gordon, V. M.,
Klimpel, K. R.,
Copeland, T. D.,
Ahn, N. G.,
Oskarsson, M. K.,
Fukasawa, K.,
Paull, K. D.,
and Vande Woude, G. F.
(1998)
Science
280,
734-737 |
71. | Vitale, G., Pellizzari, R., Recchi, C., Napolitani, G., Mock, M., and Montecucco, C. (1998) Biochem. Biophys. Res. Commun. 248, 706-711[CrossRef][Medline] [Order article via Infotrieve] |
72. | Mansour, S. J., Candia, J. M., Matsuura, J. E., Manning, M. C., and Ahn, N. G. (1996) Biochemistry 35, 15529-15536[CrossRef][Medline] [Order article via Infotrieve] |
73. | Tanoue, T., Adachi, M., Moriguchi, T., and Nishida, E. (2000) Nat Cell Biol 2, 110-116[CrossRef][Medline] [Order article via Infotrieve] |
74. |
Adachi, M.,
Fukuda, M.,
and Nishida, E.
(2000)
J. Cell Biol.
148,
849-856 |
75. |
Jacobs, D.,
Glossip, D.,
Xing, H.,
Muslin, A. J.,
and Kornfeld, K.
(1999)
Genes Dev.
13,
163-175 |
76. |
Zúñiga, A.,
Torres, J.,
Ubeda, J.,
and Pulido, R.
(1999)
J. Biol. Chem.
274,
21900-21907 |
77. |
Yang, S. H.,
Whitmarsh, A. J.,
Davis, R. J.,
and Sharrocks, A. D.
(1998)
EMBO J.
17,
1740-1749 |
78. | Lee, J. C., Kumar, S., Griswold, D. E., Underwood, D. C., Votta, B. J., and Adams, J. L. (2000) Immunopharmacology 47, 185-201[CrossRef][Medline] [Order article via Infotrieve] |
79. |
Rubinfeld, H.,
Hanoch, T.,
and Seger, R.
(1999)
J. Biol. Chem.
274,
30349-30352 |
80. | Kallunki, T., Su, B., Tsigelny, I., Sluss, H. K., Dérijard, B., Moore, G., Davis, R., and Karin, M. (1994) Genes Dev. 8, 2996-3007[Abstract] |
81. |
Wilsbacher, J. L.,
Goldsmith, E. J.,
and Cobb, M. H.
(1999)
J. Biol. Chem.
274,
16988-16994 |
82. | Ferrell, J. E., Jr. (1996) Trends Biochem. Sci. 21, 460-466[CrossRef][Medline] [Order article via Infotrieve] |
83. |
Stewart, S.,
Sundaram, M.,
Zhang, Y.,
Lee, J.,
Han, M.,
and Guan, K. L.
(1999)
Mol. Cell. Biol.
19,
5523-5534 |
84. | Scott, K., and Zuker, C. S. (1998) Nature 395, 805-808[CrossRef][Medline] [Order article via Infotrieve] |
85. | Kallunki, T., Deng, T., Hibi, M., and Karin, M. (1996) Cell 87, 929-939[Medline] [Order article via Infotrieve] |
86. | Prowse, C. N., Hagopian, J. C., Cobb, M. H., Ahn, N. G., and Lew, J. (2000) Biochemistry 39, 6258-6266[CrossRef][Medline] [Order article via Infotrieve] |
87. | Yashar, B., Irie, K., Printen, J. A., Stevenson, B. J., Sprague, G. F., Jr., Matsumoto, K., and Errede, B. (1995) Mol. Cell. Biol. 15, 6545-6553[Abstract] |