(Received for publication, June 23, 1995)
From the
We have purified 3500-fold from rabbit skeletal muscle a 12,020-Da mitogen-activated protein kinase kinase (MEK)-enhancing factor (MEF) that stimulates both mitogen-activated protein kinase (MAPK) autophosphorylation and the rate (24-fold) at which the enzyme is phosphorylated by MEK in vitro. This was manifest by the finding that in the presence of MEF, molar equivalents of MEK to MAPK were sufficient to produce fully phosphorylated (2.1 ± 0.4 mol/mol; S.D., n = 3) and activated MAPK. This contrasted with the 40:1 molar excess ratio of MEK to MAPK required to produce fully phosphorylated and activated MAPK in the absence of MEF. Phosphoamino acid analysis revealed that in the presence of MEF, phosphorylation of MAPK by MEK was ordered, with Tyr-185 phosphorylation preceding Thr-183 phosphorylation. However, the rate at which Thr-183 was phosphorylated relative to Tyr-185 was greatly increased. The finding that MEF stimulated MAPK autophosphorylation and increased its ability to be phosphorylated by MEK suggests a mechanism of action in which MEF interacts with MAPK to alter its conformation.
The mitogen-activated protein kinases (MAPKs) ()p42
(ERK2) and p44
(ERK1)
are thought to mediate the actions of numerous hormones controlling
cellular events as diverse as growth, division, and metabolism (for
reviews, see (1, 2, 3, 4) ).
p42
and p44
belong to a conserved family
of protein kinases that also includes p54
(5, 6) and the stress-activated protein kinases JNKs/SAPKs and
p38
/RKs(7, 8, 9, 10, 11) .
All MAPKs in the family are activated uniquely by dual phosphorylation
at sites that contain the sequence TXY, seven residues
N-terminal to the conserved APE motif found in most protein
kinases(5, 7, 8, 12, 13) .
The MAP kinase kinases (MEKs) also comprise a related gene family and
appear to function exclusively to activate their respective
MAPKs(7, 9, 14, 15, 16, 17, 18) .
Significantly, many features of the MAPK pathway in higher eukaryotes
are conserved in the pheromone and stress response signal transduction
pathways of fission and budding
yeast(19, 20, 21, 22) .
Much
attention in recent years has focused on delineating the immediate
steps from cell-surface receptors to the activation of p42 and p44
. To date, two tentative connections have
been made. In one pathway, several laboratories, utilizing a
combination of overexpression and antisense techniques, have identified
p74
and possibly its homologs A-Raf and B-Raf
as activators of MEK in
vitro(23, 24, 25, 26, 27, 28, 29) .
Indeed, raf has been demonstrated to phosphorylate MEK on two
serine residues, 218 and 222, and mutation of these residues to
glutamic or aspartic acid results in constitutive
activation(30, 31, 32, 33, 34, 35) .
The mechanism(s) by which p74
is activated by
hormone receptors is not completely understood, but appears to involve
an interaction with GTP-bound
p21
(29, 36, 37, 38, 39, 40, 41, 42, 43) .
In yeast, an alternative pathway suggests that MEKs are activated by
heterotrimeric G protein-coupled receptors via the activation of STE-11
homologs of MEK kinases
(MEKKs)(19, 20, 21, 22, 44) .
Other less well characterized activators of MEK have also been
described, including c-mos(45, 46, 47) , a 400-kDa MEK-activating
factor in Xenopus(48) , and a 50-60-kDa
insulin-stimulated MEK kinase (I-MEKK) in isolated adipocytes (49) and NIH 3T3 cells(29) . The existence of multiple
MEK-activating pathways has led to the hypothesis that MEK acts as a
signaling convergence point, explaining how so many diverse cellular
agonists can activate MAPKs.
Our laboratory has had a long standing
interest in the mechanism by which insulin both activates and then
inactivates MAPK in vivo. We recently identified in isolated
adipocytes a MEK kinase (I-MEKK) that, like MEK and MAPK, showed acute
phasic activity kinetics in response to the hormone(50) . Since
this initial report, we have made several unsuccessful attempts to
purify I-MEKK from insulin-treated adipocytes. Although I-MEKK remains
stable over anion-exchange and gel filtration chromatography, further
purification results in immediate loss of all activity. These findings
led us to conclude that activation of MEK by MEK kinases in vitro requires additional factors that are lost in subsequent
purification steps. Accumulating genetic evidence in yeast may support
this hypothesis. In Saccharomyces cerevisiae, the conserved
MAPK module, comprising STE-11 (MEKK), STE-7 (MEK), and FUS-3 (MAPK),
requires a fourth protein, STE-5, to mediate pheromone responses ((51) ; see also (52) ). In the module, STE-5 has been
proposed to tether these protein kinases together in order to bring
about their sequential activation. If an STE-5-like protein exits to
activate MEK in higher eukaryotes, this might also explain
discrepancies we have observed in the mechanism by which MEK activates
MAPK in vitro. We had reported earlier that in the presence of
catalytic amounts of purified MEK, the Tyr-185 phosphorylated form of
MAPK accumulated. To produce the fully activated and phosphorylated
form of MAPK, repeated additions of purified MEK were
required(49) . From this study, we concluded that
phosphorylation of MAPK by MEK was ordered, with Tyr-185
phosphorylation preceding Thr-183 phosphorylation. Recently, Goldsmith
and co-workers (53) solved the apo structure of p42 to 2.3-Å resolution. In the apo structure, Tyr-185 is
buried in a hydrophobic pocket near the ATP-binding cleft, whereas
Thr-183 is exposed on the surface of the enzyme. These findings are
paradoxical since the apo structure would indicate that MEK is unlikely
to phosphorylate MAPK on Tyr-185 first. Taken together, these
observations strongly support the hypothesis that additional factors
may be required for the activation of MAPK and possibly MEK. In this
paper, we report the purification of a 12,020-Da protein that potently
enhances the activation of MAPK by MEK. We propose that our findings
have general implications for some of the models currently proposed for
MAPK activation in vivo.
Figure 2:
Anion-exchange chromatography of rabbit
muscle extract showing elution of MEF activity. Rabbit skeletal muscle
extract (800 ml) was prepared as described under ``Methods''
and applied to the anion-exchange column. The column was washed with 2
liters of buffer B and then developed with a 2-liter salt gradient to 1 M in buffer B. Column fractions (15 ml) were assayed for MAPK
activity in the presence () or absence (
) of S218D/S222D
MEK1. The data shown for activity in the presence of S218D/S222D MEK1
were derived following subtraction of the activity measured in the
absence of mutant MEK. Data shown are from a single representative
experiment.
Figure 3:
Gel filtration analysis of MEF activity. A
concentrated sample (200 µl) of the column wash from anion-exchange
chromatography containing MEF activity was applied to a Waters SW300
gel filtration column (8.0 300 mm), and the column was
developed at 1.0 ml/min. Column fractions (200 µl) were assayed for
MAPK activity in the presence (
) or absence (
) of
S218D/S222D MEK1. Molecular mass markers were as follows: 150 kDa,
aldolase; 69 kDa, bovine serum albumin; 42 kDa, MAPK; 12 kDa, MEF; and
8 kDa, aprotinin. Data shown are from a single experiment; however,
this was repeated with identical results.
Figure 4:
Reverse-phase chromatography of MEF.
Heat-treated MEF was purified to homogeneity over C (A) and then C
(B) reverse-phase
columns developed (1.0 ml/min) with the indicated gradients of O.1%
trifluoroacetic acid/water and acetonitrile. In A, MEF
activity was measured on column fractions following microdialysis
against buffer C. In B, peaks of absorbance were collected by
hand, and MEF activity was measured following microdialysis against
buffer C. Absorbance was measured by an on-line photodiode array
detector at 214 nm.
Figure 1:
Phosphorylation of MAPK by native MEK.
Purified recombinant p42 (0.5 µM) was
incubated for 30 min in a 30-µl assay containing 300 µM ATP, 7.5 mM MgCl
, 25 mM Hepes, pH
7.4, 1 mM dithiothreitol, 1 mM EDTA, and the
indicated concentrations of purified rabbit skeletal muscle MEK1.
Reactions were terminated by the addition of ice-cold 25%
trichloroacetic acid. Following centrifugation (14,000
g for 5 min) and washing, the radioactivity in the precipitated
protein was determined. Insets are the results of phosphoamino
acid analysis (62) of trichloroacetic acid-precipitated MAPK
from reactions containing 1 or 10 µM MEK. Results are
means ± S.D. of three separate
experiments.
Fig. 2shows an anion-exchange chromatography column profile of muscle extract in which fractions were assayed for their ability to activate MAPK in the presence and absence of S218D/S222D MEK1. In the absence of the mutant kinase, four distinct peaks of MAPK activity were observed, a broad peak eluting in the column wash (I) and three other sharper peaks (II-IV) eluting with the salt gradient. When column fractions were assayed in the presence of S218D/S222D MEK1, four peaks were also observed; however, peak I (designated MEF) was more striking. Indeed, the activity of the mutant kinase in this region was 5-6 times greater than that measured in the absence of column fraction or in other regions of the column profile. The enhanced activity in the MEF peak area could not be accounted for by simply adding the constitutive basal activity of S218D/S222D MEK1 to the activity of MAPK measured in the absence of the mutant kinase. Indeed, the data shown in Fig. 2(and subsequently Fig. 3) were derived following subtraction of the constitutive basal activity of S218D/S222D MEK1 from the data. To test whether the MEF peak contained any MEK kinase activity, the anion-exchange column profile was also assayed with wild-type recombinant MEK1. However, in these experiments, no evidence of MEK kinase activity was found in this region (data not shown). In contrast to the MEF peak, peaks II-IV do not change significantly in the presence or absence of S218D/S222D MEK1 once the correction is made for constitutive activity. These later eluting peaks were identified by Western blotting as endogenous MEK (peak II) and MAPK (peaks III and IV) (data not shown).
Further purification of MEF was
carried out by gel filtration (Fig. 3). Fig. 3shows that
the majority of MEF activity (>90%) eluted on gel filtration with a
putative molecular mass of 12 kDa. At its apex, MEF stimulated
S218D/S222D MEK1 7-8-fold over its basal constitutive activity.
Two minor peaks of MEF activity, of 80 and >150 kDa, respectively,
were also recovered from the column. However, because both peaks were
less well defined and eluted with the majority of the protein, further
purification was not carried out at this stage. All subsequent studies
were performed on the 12-kDa MEF fraction. As observed on
anion-exchange chromatography, when the gel filtration profile was
assayed in the absence of S218D/S222D MEK1, MEF also stimulated MAPK
autophosphorylation (see Fig. 6) and activation (Fig. 3).
These findings suggest that either MEF is a MEK or it stimulates MAPK
autophosphorylation. However, since the molecule is not of sufficient
molecular size to accommodate the minimum consensus sequence required
for a protein kinase, this suggests that MEF is unlikely to be a
MEK(13) . Consequently, it is more likely that MEF interacts
with MAPK to stimulate autophosphorylation.
Figure 6:
Effects of MEF on phosphorylation of MAPK.
MAPK (1 µM) was incubated at 30 °C in assays (500
µl) containing buffer C, 300 µM ATP (2500 cpm/nmol),
7.5 mM MgCl, and the following combinations:
, without MEF;
, 10 units of MEF;
, 1 µM S218D/S222D MEK1;
, 1 µM S218D/S222D MEK1 and 10
units of MEF;
, 1 µM S218D/S222D MEK1 and 10 units
of heat-treated MEF. At the indicated times, 50 µl of the reaction
mixtures was removed and added to 0.5 ml of ice-cold trichloroacetic
acid (25%, w/v). A, stoichiometry of MAPK; B,
autoradiograms following SDS-PAGE of the trichloroacetic
acid-precipitated proteins phosphorylated with the indicated
combinations of MEK, MEF, and MAPK. Experiments shown were conducted on
partially purified MEF isolated from the SW300 column. Similar data
were also obtained when purified rabbit skeletal MEK1 was used instead
of S218D/S222D MEK1.
Gel filtration fractions
were also assayed for their ability to phosphorylate and activate
wild-type recombinant MEK1. However, as observed on anion-exchange
chromatography, no phosphorylation or activation of wild-type MEK was
observed across the column profile (data not shown), further ruling out
the possibility that MEF is a MEK kinase that phosphorylates MEK at
novel activating sites other than Ser-218 and Ser-222. The effect of
MEF on stimulating MAPK autophosphorylation appears to be selective for
MAPK since when column fractions were assayed with four other distinct
protein kinases, including casein kinase II, p70 ribosomal protein S6
kinase, cyclic AMP-dependent protein kinase, or p60,
no effect on the activity of these enzymes was observed (data not
shown).
Following gel filtration, MEF was purified to homogeneity
over two reverse-phase HPLC columns, C and then C
(Fig. 4, A and B, respectively). Fig. 4A shows that two major peaks of MEF activity,
eluting at 4 and 57 min, respectively, were recovered from the C
reverse-phase column. Approximately 50% of the activity eluting
at 4 min re-eluted at 57 min when this material was reapplied to the
column, suggesting that this was merely unbound MEF. MEF activity
eluting at 57 min was pooled and applied to the C
column.
When the column was developed, MEF was eluted at 54 min as a single
homogeneous protein as judged by SDS-PAGE and silver staining (Fig. 5). Analysis of the purified protein by time-of-flight
laser desorption mass spectrometry gave the molecular mass of MEF as
12,020 Da, consistent with gel filtration (Fig. 3) and SDS-PAGE (Fig. 5) data. Starting with 300 g of rabbit skeletal muscle,
MEF was purified to homogeneity 3500-fold (estimated from the
FFQ-Sepharose step) with a recovery of 2.7% (Table 1). The
relatively poor recovery appears to be due to significant loss of
activity during the reverse-phase chromatography steps.
Figure 5:
SDS-PAGE and silver stain of purified MEF.
MEF isolated from the C reverse-phase column was taken to
dryness by vacuum concentration and resuspended in water (100 µl).
The purified protein (25 µl) was applied to an SDS-20% (w/v)
polyacrylamide gel.
The most striking effects on MAPK phosphorylation were
observed when MEF (heat-treated or non-heat-treated) was included in
the assay with MEK (Fig. 6, A (,
) and B (panel iv)). Within 5 min, MAPK was phosphorylated to a
stoichiometry of 0.78 mol/mol, reflecting a 24-fold increase in the
rate of phosphorylation measured at this time point in the absence of
MEF. By the 20-min time point, MAPK was phosphorylated to close to 2.0
mol/mol (2.1 ± 0.4 mol/mol; S.D., n = 3) and had
a specific activity toward myelin basic protein of 0.35 ± 0.01
µmol/min/mg (S.D., n = 3). This compared with the
0.084 mol/mol and a specific activity of 0.01 µmol/min/mg achieved
at this same time point in assays containing MEK, but no MEF. MEF also
enhanced the ability of MEK to phosphorylate KR52 MAPK, achieving a
stoichiometry of 1.86 ± 0.4 mol/mol (S.D., n =
3) by 20 min. This suggests that although KR52 is unable to
autophosphorylate, it can interact with MEF and be phosphorylated by
MEK to a stoichiometry close to that achieved with wild-type MAPK.
Significantly, the data shown in Fig. 6contrast dramatically
with those shown in Fig. 1in that a molar excess of MEK over
MAPK was no longer required to produce fully phosphorylated and
activated MAPK. In Fig. 1, a MEK/MAPK molar ratio of 40:1 was
required to produce stoichiometrically phosphorylated MAPK within a
30-min period. However, as shown in Fig. 6, in the presence of
MEF, molar equivalents of MEK to MAPK were now sufficient to produce
fully phosphorylated and activated enzyme within this time frame.
Preliminary findings indicate that in the presence of MEF and native
MEK, the ratio of MEK to MAPK can be titrated down still further while
retaining reasonable rates of MAPK phosphorylation, although at the
time of writing, it is not clear whether molar equivalents of MEF to
MAPK are required to fully phosphorylate and activate the enzyme in the
presence of MEK. (
)
In assays that contained MAPK, a low
level of GST-S218D/S222D MEK1 phosphorylation was also observed (Fig. 6B, panelsiii and iv). This finding is consistent with data of others showing
phosphorylation of MEK by MAPK at sites other than Ser-218 and
Ser-222(55) . However, in our hands, phosphorylation of MAPK by
MEK does not appear to affect activity. ()As shown in the
autoradiogram in Fig. 6B (panelii)
and also as indicated during column chromatography, purified MEF did
not bring about phosphorylation of GST-MEK, further ruling out the
possibility that the protein stimulates MEK autophosphorylation or that
it is a MEK kinase.
The effect of MEF on MAPK autophosphorylation
and phosphorylation by MEK suggests a mechanism of action in which MEF
interacts with MAPK to alter its conformation. To explore this
hypothesis, the relative rates of Tyr-185 and Thr-183 were measured (Fig. 7). As shown earlier(49) , when MAPK is
phosphorylated by MEK alone, Tyr-185 phosphorylation proceeds at a
significantly greater rate than Thr-183 phosphorylation (Fig. 7A). Quantitation of the two amino acids by
PhosphorImager analysis reveals the relative rate of tyrosine
phosphorylation to be 20-fold greater than that of threonine
phosphorylation at the 10-min time point and
5-fold greater by 90
min (Fig. 7A). However, when MAPK is phosphorylated by
MEK in the presence of MEF, a significant increase in the rate of
threonine phosphorylation relative to tyrosine is observed (Fig. 7B). Within 5 min, threonine phosphorylation was
45% of that measured for tyrosine, and by 20 min, both residues
were equally phosphorylated. These data suggest that in the presence of
MEF, phosphorylation of MAPK is still ordered (Tyr-185 precedes
Thr-183), but the rate and efficiency of phosphorylation of threonine
relative to tyrosine is greatly enhanced.
Figure 7:
Effects of MEF on kinetics of Tyr-185 and
Thr-183 phosphorylation by MEK. Phosphoamino acid analysis was carried
out on trichloroacetic acid precipitates of MAPK phosphorylated by
GST-S218D/S222D MEK1 in the presence (A) or absence (B) of MEF. To detect the presence of phosphothreonine, B was exposed for 48 h compared with a 12-h exposure in A.
Quantitation of the P-labeled amino acids was carried out
on a Molecular Dynamics PhosphorImager.
Data demonstrating that
MEF did not cause phosphorylation of MAPK at additional sites other
than Tyr-185 and Thr-183 were obtained after tryptic peptide mapping of
the P-labeled protein by reverse-phase HPLC (Fig. 8). Comparison of the peptide maps obtained from MAPK
phosphorylated in the presence or absence of MEF revealed similar
patterns. In both cases,
P-labeled tryptic peptides were
recovered eluting at 28.00 and 38.00 min, respectively. Although the
amount of radioactivity in the 38.00 min peptide was greater in the
MAPK sample phosphorylated by MEK in the presence of MEF (Fig. 8A), phosphoamino acid analysis of the
phosphopeptides revealed the 28.00 min peptide to contain
phosphotyrosine and the 38.00 min peptide to contain equal proportions
of phosphotyrosine and phosphothreonine (Fig. 8A, inset). These findings are consistent with phosphorylation of
MAPK by MEK at its activating site,
VADPDHDHTGFLTEYVATR(12) , and no other.
Figure 8:
Reverse-phase HPLC following tryptic
digestion of P-labeled MAPK. MAPK was phosphorylated by
S218D/S222D MEK1 in the presence (A) and absence (B)
of MEF and digested with trypsin as described previously(17) .
The digests were applied to a Waters Nova-Pak C
reverse-phase column (3.9
150 mm) equilibrated in 0.1%
trifluoroacetic acid, and the column was developed (1.0 ml/min) with
acetonitrile in 0.1% trifluoroacetic acid as indicated. Absorbance was
monitored using an on-line photodiode array detector at 214 nm, and
radioactivity (
) was determined in fractions (1.0 ml) by
Cerenkov counting. Insets in A are results of
phosphoamino acid analysis of the
P-labeled peptides. The
radioactivity eluting in the column breakthrough fraction was found to
be due to free
[
-
P]ATP.
In this paper, we have purified to homogeneity a 12,020-Da protein (MEF) that enhances the activation of MAPK by MEK. Three lines of evidence indicate that MEF is not a MEK kinase or a MEK. First, MEF is not of sufficient molecular size to accommodate the minimum consensus sequence required for protein kinases(13) . Second, MEF did not phosphorylate wild-type MEK in the absence of active MAPK. Third, MEF did not phosphorylate catalytically inactive KR52 MAPK, but like wild-type MAPK, did enhance the ability of the mutant to be phosphorylated by MEK. Phosphoamino acid analysis revealed that in the presence of MEF, phosphorylation of MAPK by MEK was ordered, with Tyr-185 phosphorylation preceding Thr-183 phosphorylation. However, the rate at which Thr-183 was phosphorylated relative to Tyr-185 was greatly increased. This was manifest by the finding that in the presence of MEF, molar equivalents of MEK to MAPK were now sufficient to produce fully phosphorylated and activated MAPK. This contrasts with the 40:1 molar excess ratio of MEK to MAPK required to produce fully phosphorylated and activated MAPK in the absence of MEF. The finding that MEF stimulated MAPK autophosphorylation and increased its ability to be phosphorylated by MEK 24-fold suggests a mechanism of action in which MEF interacts with MAPK to alter its conformation (Fig. 9).
Figure 9: Schematic diagram showing mechanism of MEF action. MEF is envisaged to interact with the lower lobe of MAPK since this region contains the substrate recognition domain and the larger part of nonconserved amino acid sequences(53, 61) . Upon binding, MEF alters the conformation of MAPK, exposing Tyr-185 to the surface and enhancing its rate of autophosphorylation. In this transitional conformation, MAPK is more readily phosphorylated at Tyr-185 and Thr-183 by MEK.
The proposed mechanism of action of MEF shown in Fig. 9is reminiscent of the regulation of cdc2 kinase by cyclins (for reviews, see (56, 57, 58) ). Prior to entry into mitosis, cyclin B accumulates and complexes with cdc2 kinase, forming maturation promoting factor. As the maturation promoting factor complex, cdc2 is phosphorylated at three separate regulatory sites, one of which is necessary for subsequent activation (Thr-181) and the other two of which are inhibitory sites (Thr-14 and Tyr-15). Significantly, these phosphorylations are dependent upon the presence of cyclin B. At the onset of mitosis, cdc2 is activated by dephosphorylation at Thr-14 and Tyr-15 by the phosphatase cdc25, a process that requires the presence of phosphate at Thr-181 and cyclin B(59, 60) . Cyclin B degradation at the metaphase-anaphase transition marks the end of division with concomitant inactivation of cdc2. This elaborate mechanism of regulation is thought to act as an important mitotic checkpoint since activation of cdc2 is a critical step that commits the cell to enter mitosis. The findings presented herein suggest that MAPK has more in common with cdc2 than simply regulation by dual phosphorylation of two closely spaced tyrosine and threonine residues. Indeed, the finding that MEF may be necessary to bring about full activation of MAPK in vivo tempts one to speculate that it may act in a capacity that renders it equivalent to cyclin B. Although we have yet to carry out studies to determine whether MEF itself is a target for hormonal regulation, by analogy with cyclin B, this possibility would add another level of control to prevent unwanted MAPK activation. Although no natural oncogenes of MEK or MAPK have been discovered, several laboratories have demonstrated that transfection of fibroblasts with the constitutively active truncated S118D/S122D MEK mutant is highly transforming(33, 34) .
Identification of MEF is
critical for determining its role in the regulation of the MAPK
pathway. Preliminary analysis of tryptic fragments of MEF by mass
spectrometry indicates that it is unrelated to any known protein in the
current data base. MEF may be a fragment of a larger protein; indeed,
two other less well defined MEF peaks of 50 and >150 kDa were
detected upon gel filtration (Fig. 3). We are currently
investigating whether these are complexes of MEF or MEF combined with
other proteins, such as MEK or MAPK. We originally hypothesized that
MEF might be a mammalian homolog of STE-5 (100,000 kDa), although
its small molecular size and lack of effect on MEK autophosphorylation
may suggest other wise. However, the findings presented in this paper,
demonstrating that additional factors exist in the regulation of MAPK,
lead one to speculate that proteins with MEF-like function may be
necessary for activation of other members of the MAPK pathway, such as
MEK. In yeast, STE-5 appears to fulfill such a role. As discussed
earlier, STE-5 is thought to tether STE-11, STE-7, and FUS-3 in a large
complex that is necessary for the transmission of pheromone responses.
In S. cerevisiae, transmission of this signal is thought to
begin with activation of the G protein-coupled pheromone receptor that
activates the protein kinase STE-20. STE-20 is subsequently thought to
activate STE-11 while it is tethered to STE-5. Activation of STE-7 and
FUS-3 then ensues. The yeast paradigm has been proposed by several
groups to be the model for MAPK activation in mammalian cells
responding to hormones that act through G protein-coupled receptors,
such as thrombin(21) .
Our original motivation of searching for MEK and MAPK activators was driven by the observation that MAPK was a relatively poor substrate for MEK in vitro, an observation that was counter-intuitive to the role of MEK in vivo. The finding that MEF improves the ability of MAPK to be phosphorylated by MEK significantly addresses this discrepancy. However, this finding, coupled with the yeast paradigm, leads one to speculate that similar effectors may also be required to activate MEK. If such factors exist, it will be necessary to re-evaluate c-Raf and c-Mos as MEKKs. In the case of I-MEKK, it has been our experience that inclusion of recombinant or purified native MEK alone in assays is insufficient to isolate the enzyme beyond the crudest of fractionation steps. In this case, it appears that other factors are necessary and that these must be identified before I-MEKK can be isolated and studied.