From the
Mitogen-activated protein kinase kinase kinase (MEKK1) is a
serine-threonine kinase that regulates sequential protein kinase
pathways involving stress-activated protein kinases and
mitogen-activated protein kinases. MEKK1 is activated in response to
growth factor stimulation of cells and by expression of activated Ras.
We demonstrate that the kinase domain of MEKK1 (MEKK
Ras plays a critical role in multiple signal transduction
pathways. Ras is a guanine nucleotide-binding protein, which cycles
between an active GTP-bound form and an inactive GDP-bound
form
(1, 2) . Due to its importance as a mediator of
mitogenic stimuli, direct effectors of Ras
How the Ras
An endogenous MEKK1 activity
immunoprecipitated from PC12 cells was recently found to be growth
factor-regulated in a Ras-dependent manner
(16) . The study
showed that expression of dominant negative N17Ras inhibited epidermal
growth factor-stimulated MEKK1 activity, and that an oncogenic Ras
mutant stimulated MEKK1 activity. We were interested to understand if
MEKK1 interacts directly with Ras and, if so, which region of MEKK1 is
necessary for that interaction. To address these questions, we examined
the ability of COS cell-expressed MEKK1 proteins and purified
recombinant MEKK1 proteins to bind to GST-Ras
In all of the binding
studies using COS cell lysates, a significant fraction of the expressed
MEKK1 and C4Raf remained in the unbound fraction (data not shown). This
was due to a very abundant expression of MEKK1 and C4Raf proteins and
the use of limiting GST-Ras
At present, five different
proteins have been shown to interact with Ras in a GTP-dependent
manner. Two of these proteins (Raf-1 and MEKK1) are protein
serine-threonine kinases, one is a lipid kinase (PI 3-kinase), and two
additional regulatory proteins (Ras-GAP and neurofibromin) function to
regulate Ras GTPase
activity
(3, 4, 5, 6, 25, 26) .
Raf-1, MEKK1, and PI 3-kinase have each been shown to have increased
activity in cells expressing GTPase-deficient
Ras
(7, 16) , consistent with their interaction with
Ras
A consensus sequence for effector binding to
Ras has not been identified. It is also apparent that different
functional domains of Ras effectors bind to Ras in a GTP-dependent
manner. The Ras binding domain for Raf-1 is encoded in the extreme
NH
)
binds to GST-Ras
in a GTP-dependent manner. Purified
bacterially expressed MEKK
binds to
GST-Ras
(GTP
S) (GTP
S is guanosine
5`-3-O-(thio)triphosphate), demonstrating a direct interaction
of the two proteins. A Ras effector domain peptide blocks the binding
of MEKK
to GST-Ras
(GTP
S).
MEKK
complexed with GST-Ras
(GTP
S) is
capable of phosphorylating MEK1. These findings indicate that MEKK1
directly binds Ras
GTP. Thus, Ras interacts with protein kinases
of both the Raf and MEKK families.
GTP have been searched
for other than the GTPase-activating protein (GAP)
(
)
that would regulate signal transduction in a Ras-dependent
manner. Recently, several groups elegantly demonstrated that the
serine-threonine kinase Raf-1 interacts directly with Ha-Ras both
in vivo and in vitro. They have mapped the region of
interaction to be a portion of the amino-terminal domain of Raf-1
(3-6). An additional effector for Ras
GTP was recently
identified as the catalytic subunit of phosphatidylinositol 3-kinase
(PI 3-kinase) (7).
GTP/Raf-1 interaction
participates in the activation of Raf-1 is not clear at this time, but
a second regulatory event appears to be required for increased Raf
activity. Activated Raf-1 phosphorylates and activates
mitogen-activated protein kinase kinase (MEK), which in turn activates
mitogen-activated protein kinase (MAPK). Similar to the multiple
discreet MAPK pathways that have been identified in yeast
(8) ,
there are also multiple MAPK pathways characterized in mammalian
cells
(9, 10, 11) . It was due to the homology of
several yeast MAPK pathways that the serine/threonine kinase MEK kinase
(MEKK1) was originally cloned. This kinase is parallel to Raf-1 in the
MAPK cascade in that it acts directly upstream of MEK and is capable of
phosphorylating and activating MEK
(12) . MEKK1 has also been
shown to be the upstream activator of stress-activated protein kinase
kinase (SEK), which then phosphorylates and activates stress-activated
protein kinase/Jun kinase (SAPK/JNK) family
members
(13, 14) . SAPK/JNKs are MAPK homologs that have
been shown to be activated in response to cellular stress such as heat
shock or UV irradiation
(15) . While both Raf-1 and MEKK1 are
upstream activators of MEK, only MEKK1 has been shown to be an upstream
activator of SEK
(14) .
.
COS Cell Expression of MEKK1 Constructs
COS
cells were transiently transfected by the DEAE-dextran protocol as
described previously
(17) . MEKK1 encodes the original
full-length MEKK1 described previously
(12) , which is actually a
partial cDNA of the full-length MEKK1 gene.()
MEKK
encodes a 1270-base pair fragment of the amino
terminus constructed by an EcoRI(322)-PstI(1592)
restriction digest. MEKK
encodes a 1435-base pair
fragment encoding the entire kinase domain constructed by an
NcoI(1541)-SspI(2976) restriction digest. The
different MEKK1 constructs were ligated into the plasmid pCMV5 for
expression studies.
Binding of Expressed MEKK1 to
GST-Ras
COS cells expressing various MEKK1
proteins were lysed in EB (1% Triton X-100, 10 mM Tris HCl, pH
7.4, 5 mM EDTA, 50 mM NaCl, 50 mM NaF, 0.1%
bovine serum albumin, 0.2 unit/ml aprotinin, 1 mM
phenylmethylsulfonyl fluoride, 2 mM
NaVO
). Lysates were separated into two equal
parts for separate binding reactions. Half of the lysate was incubated
with GST-agarose (1.5 µg), while half of the lysate was incubated
with GST-Ras
-agarose (1.5 µg, Upstate Biotechnology
Inc.) for 1 h at 4 °C. The GST-Ras
was preincubated
at 30 °C for 30 min with 1 mM nucleotide (GDP or
GTP
S). The nucleotide binding reaction was stopped by adding
MgCl
to a final concentration of 20 mM. After the
1-h binding reaction, the agarose beads were pelleted at 2000 rpm for 2
min and washed three times with phosphate-buffered saline + 1.0%
Triton X-100. The washed agarose beads were boiled in Laemmli SDS
sample buffer and the proteins resolved by SDS-polyacrylamide gel
electrophoresis. Proteins were transferred onto nitrocellulose for
immunoblotting with MEKK1 antibodies. Both MEKK
antibody
raised against an NH
-terminal fusion protein
(16) and MEKK
antibody raised against a
COOH-terminal peptide were used
(12) .
-agarose per binding reaction.
Binding of recombinant MEKK
A construct encoding the kinase domain of
a Rat MEKK1 cDNA (95% identical to mouse MEKK1) with a
NHto
GST-Ras
-terminal hexahistidine tag was kindly provided by Dr.
Melanie Cobb. MEKK
was expressed in bacteria, and
soluble active enzyme was purified on Ni
-NTA-agarose
(18). Purified recombinant MEKK
was incubated with
either GST or GST-Ras
in PAN (10 mM PIPES, pH
7.0, 100 mM NaCl, 0.2 unit/ml aprotinin) for 1 h at 4 °C.
The agarose beads were pelleted and washed three times in PAN. The
washed agarose beads were then incubated in kinase buffer (20
mM PIPES, pH 7.0, 10 mM MnCl
, 40 µCi
of [
-
P]ATP, 20 µg/ml aprotinin, and 100
ng of recombinant kinase-inactive MEK1 (KMMEK1) as substrate in a final
volume of 150 µl at 30 °C for 20 min. Reactions were terminated
by addition of 5
Laemmli SDS sample buffer, boiled, and
resolved by SDS-PAGE.
RESULTS
To validate our Ras binding assay, we tested the ability of
GST-Ras to bind to expressed C4Raf protein
(19) .
C4Raf encodes the amino terminus of Raf-1, which has been shown to
interact with Ras
GTP
(3, 4, 5, 6) .
As seen in Fig. 1A, C4Raf bound to
GST-Ras
(GTP
S)-agarose but not to the GST-agarose
control. Additionally, no Raf immunoreactive proteins were detected
bound to Ras from control transfected cells (pCMV5).
Figure 1:
A,
C4Raf binds GST-Ras(GTP
S). Lysates of either empty
vector (pCMV5) or C4Raf transiently expressed in COS cells, as
described under ``Materials and Methods,'' were incubated
with GST-agarose (1.5 µg) or
GST-Ras
(GTP
S)-agarose (1.5 µg) for 1 h at 4
°C. The agarose was pelleted and washed three times with lysis
buffer and resuspended in sample buffer, and proteins were resolved by
SDS-PAGE. Proteins were transferred onto nitrocellulose and
immunoblotted with Raf-1 antibody. Similar results were obtained in
three independent experiments. B, MEKK1 binds GST-Ras
in a GTP-dependent manner. Lysates of either empty vector (pCMV5)
or MEKK1 transiently expressed in COS cells, as described under
``Materials and Methods,'' were incubated with GST-agarose
(1.5 µg), GST-Ras
(GDP)-agarose (1.5 µg), or
GST-Ras
(GTP
S)-agarose (1.5 µg) for 1 h at 4
°C. The agarose was pelleted, washed three times with lysis buffer,
and resuspended in sample buffer, and proteins were resolved by
SDS-PAGE. Proteins were transferred onto nitrocellulose and
immunoblotted with MEKK
antibody. A triplet of MEKK1
immunoreactive bands is created by phosphorylation of MEKK1 at multiple
sites within the NH
terminus (20). The faster migrating
MEKK1 immunoreactive species is derived from MEKK1 (12) and is created
by initiation of translation at an internal methionine within the MEKK1
open reading frame (C. Lange-Carter, unpublished results). Similar
results were obtained in three independent
experiments.
MEKK1 Binds GST-Ras in a GTP-dependent Manner
An
endogenous MEKK1 activity immunoprecipitated from PC12 cells was
recently shown to be growth factor-regulated in a Ras-dependent
manner
(16) . In order to determine if MEKK1 could bind directly
to Ras in vitro, we designed experiments in which various
MEKK1 proteins were expressed in COS cells and then incubated with
GST-Ras-agarose beads that had been preloaded with GDP or
GTP
S. MEKK1 transiently expressed in COS cells was capable of
binding GST-Ras
in a GTP-stimulated manner
(Fig. 1B). The predominant triplet of MEKK1
immunoreactive bands is created by phosphorylation of MEKK1 at multiple
sites within the NH
terminus
(20) . The faster
migrating MEKK1 immunoreactive species is derived from MEKK1
(12) and is created by initiation of translation at an internal
methionine within the MEKK1 open reading frame.
MEKK1 from
COS cell lysates bound to GST-Ras
(GTP
S), while very
little binding to GST-Ras
(GDP) was detectable. With the
conditions used MEKK1 binding to GST-Ras
(GTP
S) was
at least 5-fold greater than the binding to GST-Ras
(GDP).
No detectable MEKK1 was bound to GST.
MEKK1 Binds GST-Ras through Its Kinase Domain
In
order to map which domain of MEKK1 is critical for binding to Ras, we
expressed different domains of the MEKK1 protein in COS cells. The
COOH-terminal kinase domain, MEKK, of MEKK1 binds Ras.
MEKK
bound to GST-Ras
in a GTP-stimulated
manner (Fig. 2). Little MEKK
bound to
GST-Ras
(GDP). Thus the GTP
S-dependent binding to
GST-Ras
is encoded within the COOH-terminal catalytic
domain of MEKK1. No detectable MEKK
was bound to GST.
Interestingly, when a MEKK
protein was expressed that
encodes a 858-base pair fragment of the amino terminus no binding to
Ras was detected. Fig. 3shows that in contrast to the ability of
Raf-1 to bind to Ras through its amino terminus
(Fig. 1A), MEKK
failed to bind
GST-Ras
(GTP
S) even though the protein was expressed
to levels similar to that of MEKK1 in the same experiment.
Figure 2:
MEKK binds
GST-Ras
(GTP
S). Lysates of either empty vector
(pCMV5) or MEKK
transiently expressed in COS cells, as
described under ``Materials and Methods,'' were incubated
with GST-agarose (1.5 µg), GST-Ras
(GDP)-agarose (1.5
µg), or GST-Ras
(GTP
S)-agarose (1.5 µg) for 1
h at 4 °C. The agarose was pelleted, washed three times with lysis
buffer, and resuspended in sample buffer, and proteins were resolved by
SDS-PAGE. Proteins were transferred onto nitrocellulose and
immunoblotted with MEKK
antibody. Similar results were
obtained in three independent experiments.
Figure 3:
MEKK does not bind
GST-Ras
(GTP
S). Lysates of empty vector (pCMV5),
MEKK
, or MEKK1 transiently expressed in COS cells, as
described under ``Materials and Methods,'' were incubated
with GST-agarose (1.5 µg) or
GST-Ras
(GTP
S)-agarose (1.5 µg) for 1 h at 4
°C. The agarose was pelleted, washed three times with lysis buffer,
and resuspended in sample buffer, and proteins were resolved by
SDS-PAGE. Proteins were transferred onto nitrocellulose and
immunoblotted with MEKK
antibody. Similar results were
obtained in three independent experiments.
Direct Interaction between Ras and the Kinase Domain of
MEKK1
A question that cannot be addressed by the COS cell
expression binding studies was: is the interaction between MEKK1 and
Ras direct or mediated through other MEKK1-associated proteins present
in the COS cell lysate? In order to address this question, we
bacterially expressed recombinant MEKK protein. This
protein was isolated from bacterial lysates using
Ni
-NTA-agarose. The soluble protein was an active
kinase capable of phosphorylating one of its potential substrates,
kinase-inactive MEK1 (KMMEK1) (Fig. 4A). MEKK1 has been
shown to phosphorylate and activate either MEK1 or SEK in
vitro(12, 14) . In addition the purified
MEKK
protein was recognized by MEKK
antibody when immunoblotted (data not shown). We used the
recombinant MEKK
protein to determine whether the
interaction between MEKK
and Ras was direct. Binding of
MEKK
recombinant protein to
GST-Ras
(GTP
S) was assayed by an in vitro kinase assay on washed beads with kinase-inactive MEK1 as
substrate. Fig. 4A demonstrates that there was indeed
direct binding of Ras
GTP
S to purified MEKK
.
The interaction between Ras and MEKK
was GTP-stimulated
as measured by the increased phosphorylation of KMMEK1 using
GST-Ras
(GTP
S) beads incubated with recombinant
MEKK
. Little or no KMMEK1 phosphorylation could be
detected with GST-Ras
(GDP) beads incubated with
recombinant MEKK
.
Figure 4:
A, recombinant
MEKK binds GST-Ras
(GTP
S). Purified
recombinant MEKK
was incubated with GST-agarose (1.5
µg), GST-Ras
(GDP)-agarose (1.5 µg), or
GST-Ras
(GTP
S) (1.5 µg) for 1 h at 4 °C in
PAN as described under ``Materials and Methods.'' The agarose
beads were pelleted and washed three times in PAN. The washed agarose
beads were then incubated in kinase buffer with 100 ng of recombinant
kinase-inactive MEK1 (KMMEK1) as substrate in a final volume of 150
µl at 30 °C for 20 min. A control reaction containing wild-type
MEK1 (WTMEK1), which autophosphorylates, serves as a marker
for the KMMEK1 substrate. Reactions were terminated by addition of 5
Laemmli SDS sample buffer, boiled, and resolved by SDS-PAGE.
The autoradiogram is shown. Similar results were obtained in three
independent experiments. B, Ras effector domain peptide blocks
binding of MEKK
to GST-Ras
(GTP
S).
GST-Ras
(GTP
S)-agarose was preincubated with a Ras
effector domain peptide (peptide encodes residues 17-42 of
Ha-Ras) or a control peptide
([D-Arg
,D-Phe
,D-Trp
,Leu
]substance
P peptide) (21) 100 µM of each for 1 h at 4 °C. The
agarose was then incubated with MEKK
and assayed as
described for panelA. The autoradiogram is shown.
Similar results were obtained in three independent
experiments.
Ras Effector Domain Peptide Inhibits Binding of
MEKK
We also utilized this
recombinant binding system to determine which region of Ras was
involved in binding MEKKto GST-Ras
. The binding of MEKK
to GST-Ras
was blocked by preincubating the
GST-Ras
(GTP
S) beads with a Ras effector domain
peptide. The Ras effector peptide encodes residues 17-42 of
Ha-Ras and has been shown to block the binding of Ras to other
effectors including GAP, Raf-1, and PI
3-kinase
(5, 7, 21) .
GST-Ras
(GTP
S)-agarose, preincubated with Ras
effector peptide for 1 h prior to incubation with MEKK
,
was unable to bind MEKK
(Fig. 4B);
GST-Ras
(GTP
S) incubated with buffer alone or in the
presence of a control peptide
([D-Arg
,D-Phe
,D-Trp
,Leu
]substance
P peptide)
(22) , which does not encode the Ras effector domain,
bound MEKK
(Fig. 4B). These studies
clearly show that MEKK1 interacts directly with Ras in vitro in a GTP-dependent manner via the COOH-terminal region of MEKK1
that encodes the catalytic kinase domain. MEKK1, Raf-1, PI 3-kinase,
and GAP all bind to the Ras effector domain.
DISCUSSION
Ras is a critical component of tyrosine kinase growth factor
receptor and G-protein-coupled receptor regulation of signal
transduction pathways controlling mitogenesis and
differentiation
(23, 24) . Raf-1 and the p110 catalytic
subunit of PI 3-kinase have been shown to directly interact with Ras in
a GTP-dependent
manner
(3, 4, 5, 6, 7) . In this
report we have demonstrated that MEKK1 is also a potential Ras effector
and selectively binds to Ras in a GTP-stimulated manner. This finding
supports at a biochemical level the observation that MEKK1 is activated
in a Ras-dependent manner in response to growth factors in PC12
pheochromocytoma cells
(16) .
GTP being involved in their regulation. The ability of Ras to
regulate multiple effector proteins is consistent with the diversity of
signal transduction pathways controlled by cell surface receptors in a
Ras-dependent manner.
-terminal regulatory domain of
Raf-1
(3, 4, 5, 6) , while the Ras
binding domain is encoded within the catalytic domain of MEKK1. Both
Raf-1 and MEKK1 binding to Ras is blocked by a Ras effector domain
peptide. The prediction from these findings is that Raf-1, MEKK1, and
other Ras effectors would compete for interaction with Ras
GTP
presumably at the Ras effector domain. The relative abundance and
affinity of each Ras effector in different cells may influence the
magnitude, onset, and duration of each effector response. Secondary
inputs such as phosphorylation of the different Ras effectors may also
influence their interaction with Ras
GTP. It is now possible to
begin to define the kinetic properties of Ras effector activation in
cells relative to effector affinity for Ras
GTP. In this regard
MEKK1 has been shown to preferentially regulate the SEK/Jun kinase
pathways relative to MAPK
(14) . Activation of the SEK/Jun kinase
pathway is generally slower in onset and maintained at maximal activity
longer than the activation of MAPK, consistent with the finding that
MEKK1 is persistently activated compared to Raf in PC12
cells
(16) . As additional MEKKs are characterized, it will be
important to characterize their regulation and interaction with
Ras
GTP. Undoubtedly additional Ras effectors will be identified
in the near future.
S, guanosine
5`-3-O-(thio)triphosphate; PIPES,
1,4-piperazinediethanesulfonic acid; KMMEK, kinase-inactive MEK; PAGE,
polyacrylamide gel electrophoresis; GST, glutathione
S-transferase.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.