From the
Raf-1 is a serine/threonine protein kinase positioned downstream
of Ras in the mitogen-activated protein kinase cascade. Using a yeast
two-hybrid strategy to identify other proteins that interact with and
potentially regulate Raf-1, we isolated a clone encoding the
carboxyl-terminal half of the G
Raf-1 is a serine-threonine protein kinase critically positioned
in a kinase cascade linking activated growth factor receptors with the
nucleus and ultimately regulating the mitogenic response. Structurally,
Raf-1 can be subdivided into three regions, CR1, CR2, and CR3, which
are conserved through evolution and among the various isoforms of Raf-1
(1). CR1 consists of a Ras binding domain (amino acids
53-132)(2, 3) and a zinc finger similar to those
found in protein kinase C. A serine/threonine-rich region with multiple
phosphorylation sites, including an autophosphorylation site
(Thr
Raf-1 has been
shown to bind Ras, in a GTP-dependent
fashion(2, 7, 8, 9) . This binding event
appears to be a critical step in the activation of
Raf-1(3, 10) . Current models of activation suggest that
Raf-1 is bound in a native complex with 14-3-3 protein(s)(11, 12) and hsp50 and hsp90 proteins(13, 14) .
Activated Ras is thought to recruit Raf-1 to the plasma membrane, where
it interacts with other modulators of its activity and with relevant
substrates(15, 16) . To identify other proteins that
interact with and potentially regulate Raf-1, we utilized two-hybrid
interaction screening with the amino-terminal regulatory domain of
Raf-1 as the target protein. An interacting clone was identified which
encodes the carboxyl-terminal half of the G
The pAS1-
In experiments
where binding affinities were estimated, various concentrations of
G
In experiments
where proteins were added to compete with binding of G
Two-hybrid screening in yeast was used to identify cDNAs
coding for proteins which bind the regulatory region of Raf-1 protein
kinase. One such cDNA encoded amino acids 194-340 of the
G
To
further characterize the interaction between Raf-1 and
G
c-H-Ras (2, 7, 9) and 14-3-3 proteins (11, 12) have also been reported to bind to the amino
terminus of Raf-1. The carboxyl terminus of
We next compared the relative
affinities of Raf/330 and the carboxyl terminus of
These experiments provide evidence for a direct and specific
interaction between the Raf-1 protein kinase and the G
A similar
G
A third avenue through which Raf-1 could influence
G-protein receptor signaling is provided by our finding that Raf/330
can inhibit association of G
Another
consequence of Raf/G
In conclusion, we have identified and characterized a novel
interaction between the serine-threonine protein kinase Raf-1 and the
G
Constructs encoding either
the NH
Roman Herrera for the generous gift of the
pAS1-
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
subunit of
heterotrimeric G-proteins. In vitro, purified G
subunits specifically bound to a GST fusion protein encoding
amino acids 1-330 of Raf-1 (Raf/330). Binding assays with
truncation mutants of GST-Raf indicate that the region located between
amino acids 136 and 239 is a primary determinant for interaction with
G
. In competition experiments, the carboxyl
terminus of
-adrenergic receptor kinase (
ARK) blocked the
binding of G
to Raf/330; however, the
Raf-1-binding proteins, Ras and 14-3-3, had no effect. Scatchard
analysis of in vitro binding between Raf/330 and
G
revealed an affinity of interaction (K
= 163 ± 36 nM),
similar to that seen between G
and
ARK (K
= 87 ± 24 nM).
The formation of native heterotrimeric G
complexes, as measured by pertussis toxin ADP-ribosylation of
G
, could be disrupted by increasing amounts of Raf/330,
with an EC
of approximately 200 nM, in close
agreement with the estimated binding affinity. In vivo complexes of Raf-1 and G
were isolated from
human embryonic kidney 293-T cells transfected with epitope-tagged
G
The identification and characterization of this
novel interaction raises several possibilities for signaling cross-talk
between growth factor receptors and those receptors coupled to
heterotrimeric G-proteins.
)(4) , comprises CR2. The catalytic region of
the protein, CR3, makes up most of the COOH-terminal half of the
molecule. Previous studies have demonstrated that truncations or
fusions which result in the loss of the amino-terminal portion of Raf-1
are oncogenically active(5, 6) . Thus, regions contained
in the amino-terminal portion of Raf-1 are presumed to be critical for
regulating the biological activity of this kinase.
subunit of
heterotrimeric G-proteins.
(
)In this report, we
have characterized this interaction, raising the possibility that the
binding of G
to Raf-1 could play a role in
regulation of the mitogen-activated protein kinase pathway by
G-protein-coupled receptors and/or potentially provide a junction for
integration of signals generated by tyrosine kinase growth factor
receptors and G-protein-coupled receptors.
Constructs
GST-Raf fusion proteins were made by
PCR amplification of sequences encoding amino acids 1-135
(Raf/135), 1-239 (Raf/239), 136-239 (Raf/136-239),
and 1-330 (Raf/330) of human Raf-1. Amplified fragments were
cloned into pCR-script (Stratagene) and subcloned into
pGEX-KG(17) . Full-length Raf-1 was expressed as a MAL fusion
protein using the pMALc2 vector (New England Biolabs). GST-14-3-3 was
made by amplifying the complete coding sequence of human 14-3-3 .
GST-
-adrenergic receptor kinase (
ARK) contains amino acids
467-689 of rat
ARK cloned into pGEX-KT (prepared from a cDNA
clone kindly provided by Antonio DeBlasi, Instituto di Ricerche
Farmalogiche Mario Negri). GST-Ras and GST-PEST tyrosine phosphatase
(PTP-PEST) have been described previously(3, 18) .
pc
was generated by PCR using custom primers
coding for a FLAG epitope tag (DYKDDDDK) in frame with the carboxyl
terminus of G
. The amplified fragment was
cloned into the BamHI/EcoRI sites of the expression
vector pcDNA3 (Invitrogen). The G
subunit was also
amplified by PCR and cloned into pcDNA3 at the EcoRI and XbaI sites. The expression plasmid pcRaf-KT3 codes for the
full-length Raf-1 protein in pcDNA3 and was generously provided by
Angus MacNicol (University of Chicago).
Yeast Two-hybrid Cloning
The plasmid pAY-Raf was
constructed by amplifying the sequence coding for amino acids
1-320 by PCR and cloning into pAS1-CYH2 vector originally
described by Durfee et al.(19) . This plasmid was
co-transformed with a HeLa cell cDNA library, constructed in
pACT(19) , into the Y190 lacZ/HIS3 reporter strain of
yeast (20) using the lithium acetate procedure(21) . The
transformation mix was plated onto dishes containing synthetic complete
media lacking tryptophan, leucine, and histidine, in the presence of
3-aminotriazole, and incubated for 7-10 days at 30 °C.
His colonies were analyzed for
-galactosidase
activity using a filter lift procedure(22) . Library-derived
plasmids from His+/
- gal+ clones were rescued and
transformed into Escherichia coli for plasmid preparation and
DNA sequencing.
-integrin construct encodes the
COOH-terminal 20 amino acids of the
-integrin receptor
as a fusion with the Gal-4 DNA binding domain in the pAS1-CYH2 vector.
The pActPTP-PEST construct has been described previously(18) .
Purification of G
The GSubunits and
ADP-ribosylation Assay
subunits
of heterotrimeric G-proteins were purified from bovine brain as
described by Sternweis and Pang(22) . Proteins were stored in
elution buffer at -70 °C at a protein concentration of
0.5-3.0 mg/ml. ADP-ribosylation reactions were carried out
essentially as described previously(23) . Pertussis toxin was
from List Biochemicals.
In Vitro Binding of Raf and
G
The various fusion proteins were purified
as described previously (17) on glutathione-Sepharose (Pharmacia
Biotech Inc.) or amylose resin (New England Biolabs). Immobilized
fusion protein (0.2-0.5 µg) was mixed with purified
G subunits (300 nM final concentration)
in phosphate-buffered saline containing 0.1% Lubrol. After a 60-min
incubation, beads were washed three times with the same buffer followed
by separation of bound proteins by SDS-polyacrylamide gel
electrophoresis. Proteins were transferred to nitrocellulose and
immunoblotting was performed with anti-G
antibodies
(catalog number 261, Santa Cruz Biotechnology) and
I-labeled protein A (Amersham Corp).
were used in the in vitro binding
assay. The resulting immunoblots were quantified by exposure on a
PhosphorImager (Molecular Dynamics) with known quantities of pure
G
subunits run as standards.
to Raf-1, G
subunits were added at a
concentration of 30 nM, and a 10-fold excess (mole/mole) of
the competitor protein was used.
In Vivo Association
Human embryonic kidney 293-T
cells (donated by Akilesh Pandey, University of Michigan) were
transfected with expression plasmids encoding Raf-1 (pcRaf-KT3),
epitope-tagged G (pc
), and
G
(pc
) proteins, either alone or in
combination using a calcium phosphate procedure (Stratagene). At 24 h
after transfection, cells were washed twice with ice-cold
phosphate-buffered saline prior to lysis in buffer A (50 mM HEPES [pH 7.5], 1% Nonidet P-40, 0.5% sodium
deoxycholate, 150 mM NaCl, 50 mM NaF, 1 mMpara-nitrophenyl phosphate, 1 mM orthovanadate, 20
nM calyculin-A, 1 mM phenylmethylsulfonyl fluoride, 1
µM leupeptin, 1 µM antipain, and 0.1
µM aprotinin). Lysates were clarified by centrifugation
and incubated for 1.5 h with 25 µl of FLAG-specific monoclonal
antibody M2 immobilized on beads (Kodak). After washing three times
with lysis buffer, precipitates were analyzed by immunoblotting with
anti-Raf (catalog number 227; Santa Cruz) or anti-
(catalog number
378; Santa Cruz) antibodies as outlined above, except enhanced
chemiluminescence (Amersham Corp.) was used for detection(4) .
Aliquots of the clarified extracts were also routinely analyzed to
monitor expression levels.
subunit of heterotrimeric G-proteins. Specificity of
this interaction was tested by measuring the ability of the two-hybrid
constructs, pAY-Raf and pAct-
to support
His
prototropy and lacZ expression when expressed
either with the corresponding Gal-4 domain lacking a fusion, or with
irrelevant proteins. summarizes these data and
demonstrates a specific interaction between the amino terminus of Raf-1
and the carboxyl terminus of the G
subunit.
, we tested the ability of purified G
subunits to bind to Raf/330, to GST alone, or other GST-fusion
proteins, in an in vitro binding assay (Fig. 1A). Raf/330 and GST-
ARK (which has
previously been shown to bind
G
(24, 25) ) bound G
in this assay. Little or no binding, however, was detected
between G
subunits and GST alone, GST-Ras or
GST-PTP-PEST, suggesting that the Raf/G
interaction is specific.
Figure 1:
In vitro binding of GST-Raf
fusion proteins to purified G subunits. A, purified G
subunits were incubated
with GST alone or various GST-fusion proteins immobilized on
glutathione-Sepharose. After washing, bound G
subunits were detected by immunoblotting. A small aliquot of
purified G
protein was run (Std) to
confirm the migration of the G
subunit. B,
glutathione beads containing either GST alone or various GST-Raf fusion
proteins were incubated with purified G
subunits
in an in vitro binding assay. Binding of the bound
G
was assessed as described above. Quantitation of
the immunoblot by PhosphorImager analysis provided values of: GST, 100
counts; GST-Raf 1-(1-330), 14,145 counts; GST-Raf-(1-135), 2,682
counts; GST-Raf-(1-239), 13,927 counts. C, amylose resin
preincubated with either maltose-binding protein alone (Mal)
or maltose-binding protein as a fusion with full-length Raf-1
(pMAL-Raf-1) was incubated with purified G
subunits in the absence (-) or presence of potential
competitors as described under ``Experimental Procedures.''
Bound proteins were detected by immunoblotting with anti-G
antibodies. Figures are typical results from two or three
independent experiments.
To better define the region of the
Raf-1 amino terminus responsible for the binding of G subunits, we constructed additional GST-Raf proteins: one encodes
amino acids 1-135, which includes the Ras binding domain, but
excludes the cysteine rich region and CR2; the other codes for amino
acids 1-239, which includes all of CR1 but stops just prior to
the beginning of CR2. These constructs were compared with the full
amino terminus (Raf/330) for their ability to bind G
in vitro. As shown in Fig. 1B, deletion
of the CR2 region has no effect on the binding of G
subunits to GST-Raf/259. In contrast, the additional removal of
amino acids 136-239 results in over 80% loss of G
binding. A fusion protein coding for amino acids 136 through 239
(Raf/136-239) bound G
to the same extent as
Raf/330 or Raf/239 (not shown), further demonstrating the importance of
this region in binding G
. We cannot exclude,
however, the possibility that other sequences in Raf-1 (e.g. 1-135) may participate in and contribute to the stable
interaction of Raf-1 and G
.
ARK, which contains a
PH (pleckstrin homology) domain, is known to bind to
G
. We tested whether these proteins could effect
the interaction between Raf/330 and G
in the in vitro binding assay (Fig. 1C). GST-
ARK,
but not GST-Ras or GST-14-3-3, strongly inhibited binding of
G
to Raf/330.
ARK (a protein
dependent upon binding to G
(24) for
carrying out its physiological function) for binding to
G
. Scatchard analysis revealed a K
of approximately 163 (±36; n = 3) nM for binding of G
to
Raf/330 compared to a K
of approximately
87 (±24; n = 3) nM for G
binding to
ARK (Fig. 2).
Figure 2:
Scatchard analysis of G binding to Raf/330 and
ARK-CT. Increasing amounts of
G
were incubated with beads containing immobilized
GST-Raf-(1-330) or GST-
ARK-CT in an in vitro binding assay. Bound G
was detected
by immunoblotting with anti-G
antibodies and quantified
by PhosphorImager analysis using comparison with known amounts of
purified G
as described under ``Experimental
Procedures.'' Constants were derived from three independent
experiments. A representative experiment is
shown.
ADP-ribosylation of
G by pertussis toxin requires the intact heterocomplex
between G
and G
subunits(26) . To determine if Raf/330 could inhibit
association of G
and G
, we
investigated the effects of increasing amounts of Raf/330 on the
pertussis toxin-catalyzed ADP-ribosylation of G
in the
presence of G
(Fig. 3). We found that the
Raf/330 inhibited the ADP-ribosylation of G
with an
EC
of approximately 200 nM. GST alone had no
effect on ADP-ribosylation (not shown).
Figure 3:
Inhibition of pertussis toxin-mediated
ADP-ribosylation of G. Increasing amounts of GST-Raf-(1-330)
were added to an ADP-ribosylation reaction containing G
and G
subunits purified from bovine brain,
P-NAD, and the active subunit of pertussis toxin.
Reactions were carried out as described under ``Experimental
Procedures.'' Following separation by SDS-polyacrylamide gel
electrophoresis and autoradiography, the band corresponding to
G
and [
P]ADP-ribose was
quantified by densitometry on a Bioimager (Millipore). Results
represent duplicate determinations
(±range).
The ability of
G subunits to form complexes with Raf-1 in
vivo was examined in human embryonic kidney 293-T cells. In order
to facilitate the efficient and specific precipitation of
G
subunits, we engineered a FLAG epitope tag onto
the carboxyl terminus of G
293-T cells were
transfected with expression plasmids coding for Raf-1, G
and G
subunits, or combinations of these
proteins. When equal amounts of Raf-1 protein are overexpressed (Fig. 4A), Raf-1 was specifically co-precipitated with
anti-FLAG antibody only with concomitant expression of
G
. Significantly, the co-precipitation of
endogenous Raf-1 protein with anti-FLAG antibody is detected in 293-T
cells transfected with G
and G
alone (Fig. 4B). Thus, as the levels of
G
expression are quite modest (2-3-fold
over endogenous), the formation of in vivo complexes between
Raf-1 and G
does not require significant
overexpression of the proteins. Under the conditions employed we
estimate that between 2 and 4% of the Raf-1 protein is co-precipitated
with G
, similar to the values previously reported
for in vivo complexes between Ras and Raf-1(27) .
Figure 4:
Co-immunoprecipitation of Raf-1 with
G. 293-T cells were transfected, either alone or
in combination, with expression plasmids coding for Raf-1,
G
, or G
or with vector alone as
a control. A, top: anti-FLAG immunoprecipitates from 293-T
cells transfected as shown were analyzed for the presence of
G
and Raf-1 proteins by simultaneous immunoblotting
with anti-G
and anti-Raf antibodies. A,
bottom: aliquots of the clarified whole cell extracts (WCL) were monitored for the expression levels of Raf-1 and
G
. B, top: anti-FLAG immunoprecipitates from
cells transfected either with vector or a combination of
G
and G
were analyzed for the
co-precipitation of G
and Raf-1 proteins by
simultaneous immunoblotting with anti-G
and anti-Raf
antibodies. B, bottom: portions of the clarified whole cell
lysates (WCL) were immunoblotted to determine the expression
levels of Raf-1 and G
Similar results were obtained in
three other experiments.
subunits of heterotrimeric G-proteins. The affinity of this
interaction is similar to that of G
for
ARK
in the in vitro binding studies. There are several conceivable
physiological ramifications of such an interaction. One possibility is
that G
is directly involved in the activation of
Raf-1. G-protein receptor-coupled agonists, such as platelet-activating
factor (28) and lipopolysaccharide(29) , have been shown
to stimulate Raf-1 and the mitogen-activated protein kinase pathway in
a Ras-independent manner. Robbins et al.(30) have
shown that AlF
-induced mitogen-activated
protein kinase activation was only minimally inhibited by dominant
negative Ras. In these cases, free G
could act
analogously to Ras, functioning to recruit Raf-1 to the plasma membrane
where it could interact with other regulators and with substrates. A
parallel interaction between certain protein kinase C isoforms and a
G
-related protein termed RACK1 has been reported by Ron et al.(31) .
-dependent mechanism has been proposed for the
recruitment of
ARK to the membrane. Once translocated by
G
,
ARK phosphorylates agonist-bound G-protein
receptor, resulting in receptor desensitization (24). Here we show that
Raf/330 and
ARK can compete for binding to G
.
This result implies that Raf-1 could potentiate receptor signaling by
interfering with the capacity of
ARK to desensitize activated
receptor. In this way activation of a tyrosine kinase growth factor
receptor pathway could impinge upon G-protein receptor signaling. For
example, Raf-1 could be involved in regulation of cAMP levels by
binding to G
subunits and inhibiting
ARK-induced down-regulation of G
-linked
receptors.
and G
subunits. By influencing the ratio of free G
and
G
subunits, Raf-1 might exert either a positive or
a negative effect on signaling. G
and G
can act in concert to potentiate effector function as in the case
of adenyl cyclase type II and IV(32) . In other systems,
G
can be either directly stimulatory or inhibitory
by interaction with effectors such as adenyl cyclase, phospholipase
C-
, or potassium channels(33) . In addition,
G
retains the potential to indirectly dampen
signals by sequestering G
(33) .
interaction might be negative
regulation of Raf-1. The recruitment of Raf-1 to the membrane by free
G
subunits might prevent it from interacting with
GTP-Ras and subsequently activated. Although our data do not support a
direct competition between Ras and G
, they do
support a high affinity interaction between Raf-1 and
G
. As the intracellular concentrations of
G
can be as high as 500 µM in some
tissues(33) , and the percentage of Ras that is GTP bound is
typically low (10-25%), it is possible that liberated
G
subunits could effectively recruit and sequester
Raf-1. This mechanism would allow G-protein agonists which stimulate
cAMP production to dampen growth factor-mediated signals through
parallel and potentially synergistic pathways(34, 35) .
subunits of heterotrimeric G-proteins. This
interaction represents a potentially important point of cross-talk and
regulation between the growth factor receptor pathway and signals
emerging from G-protein receptors. Given the diversity of cellular
effects in which G
subunits have been implicated,
the functional relationship between these proteins may be quite
complex. The identification and characterization of this interaction
allows the functional significance of this interaction to be tested in
the various model systems.
Table: Yeast two-hybrid interactions between Raf,
G, and control constructs
terminus of Raf (pAY-Raf), the fragment of
G
isolated in a library screen
(pACT-G
), irrelevant proteins (PTP-PEST;
pAS1-
-integrin), or fusion vector alone (pACT) were co-transformed
into yeast, and co-transformants were selected by plating on media
lacking leucine and tryptophan. Two independent co-transformants were
evaluated for the ability to support growth on media lacking histidine
(His3) and to induce the expression of
-galactosidase (
-gal).
SNF1 and SNF4 plasmids were included as a positive control.
ARK,
-adrenergic receptor
kinase.
-integrin construct, Stanley Fields for the yeast two-hybrid
reagents, and Alan Saltiel for critical reading of the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.