(Received for publication, May 3, 1995; and in revised form, June 13, 1995)
From the
The guanine nucleotide exchange factor Son of sevenless (Sos)
performs a crucial step in the coupling of receptor tyrosine kinases to
Ras activation. Mammalian cells contain two related but distinct Sos
proteins, Sos1 and Sos2. Although they share a high degree of overall
similarity, it is not known to what extent their biological and
biochemical properties overlap. In the present study, we have compared
the interactions of the two human homologues of Sos, hSos1 and hSos2,
with the adaptor protein Grb2. We show that hSos2 interacts with Grb2
via its proline-rich COOH-terminal domain and that this interaction is
dependent on the SH3 domains of Grb2. In general, these characteristics
are similar to the ones reported previously for the interaction of
hSos1 with Grb2. However, the apparent binding affinity of hSos2 for
Grb2 is significantly higher relative to that of hSos1 both in
vitro and in vivo. The region conferring this higher
binding affinity has been mapped to residues 1126-1242 of the
hSos2 COOH-terminal domain. These results suggest that Sos1 and Sos2
may differentially contribute to receptor-mediated Ras activation.
Ras proteins are small membrane-bound GTPases that play a
central role in signal transduction pathways initiated by many receptor
tyrosine kinases(1) . Activation of these receptor tyrosine
kinases promotes the accumulation of the active GTP-bound form of Ras
proteins. To reach the active GTP-bound state, Ras proteins must first
release bound GDP. This rate-limiting step in GTP binding is catalyzed
by guanine nucleotide exchange factors
(GNEFs)( Members of the Sos family of Ras
GNEFs contain a proline-rich sequence at their carboxyl terminus that
binds the SH3 domains of the adaptor protein Grb2/S.E.5/Drk. Recently,
the three-dimensional structures of both the amino- and
carboxyl-terminal SH3 domains of Grb2 bound to a proline-rich peptide
from Sos1 have been
elucidated(6, 7, 8, 9) . The salient
feature of these structures is that the peptide adopts the conformation
of a left-handed polyproline type II helix and interacts with three
major sites on the SH3 domain in an orientation opposite to that of the
proline-rich peptide binding to SH3 domains of the p85, Fyn, and Abl
proteins(10, 11) . In virtually all cells examined
thus far, Sos is found in complex with Grb2. Upon growth factor
stimulation, the Grb2-Sos complex binds directly to the activated
receptors or to an intermediate protein, e.g. SHC, through the
SH2 domain of Grb2. However, the guanine nucleotide exchange activity
of Sos is not measurably affected by growth factor
stimulation(12, 13) . Thus, it has been postulated
that stimulation of the receptors serves to translocate the Grb2-Sos
complex into proximity with its target, membrane-bound Ras. In support
of this mechanism is the observation that the targeting of Sos1 to the
plasma membrane is sufficient for the activation of the Ras signaling
pathway(14, 15) . Mammalian cells contain two
related but distinct Sos proteins designated Sos1 and
Sos2(4, 5) . Both Sos1 and Sos2 are widely expressed
during development and in adult tissues. Alignment of the two murine or
the two human Sos proteins shows that they share a high degree of
similarity (65% overall amino acid identity). However, at their
carboxyl-terminal region the homology between Sos1 and Sos2 is
scattered and the conserved regions are mostly restricted to short
proline-rich motifs. In the present study, we have compared the
interactions of the two human homologues of Sos, hSos1 and hSos2, with
Grb2. Both Sos proteins interact with the SH3 domains of Grb2 via their
proline-rich carboxyl-terminal domain. However, the apparent binding
affinity of hSos2 for Grb2 is significantly higher relative to that of
hSos1 both in vitro and in vivo. These findings
indicate that Sos1 and Sos2 may play differential roles in
receptor-mediated Ras-activation.
The biochemical features and physiological significance of
the interaction between Grb2 and Sos1 have been extensively
documented(5, 12, 13, 25, 26, 27) .
In contrast, very little is known about the interaction of Sos2 with
Grb2. In order to characterize the interactions of hSos2 with Grb2, we
first used the two-hybrid system. Initially, we examined the ability of
Grb2 to interact with various regions of hSos2. Pairwise combinations
of Grb2 fused to the GAL4 transcriptional activation domain (GAD) and
hSos2 fused to the GAL4 DNA binding domain (GBD) were coexpressed in
the yeast reporter strain YPB2, and their ability to interact was
tested by the induction of
Figure 1:
Two-hybrid analysis of the
interaction of Grb2 with hSos2. A, various regions of hSos2
fused to the GAL4 activation domain were cotransformed with Grb2 fused
to the GAL4 binding domain into yeast YPB2 strain. The interaction
between the two hybrid proteins is indicated by the induction of LacZ
expression (darkcolor). Each patch represents an independent transformant. The S. cerevisiae SNF4 fusion protein was used as a negative control. The regions of
hSos2 corresponding to each hSos2 construct are shown schematically on
the rightside. B, pairwise transformations
of COOH-terminal domain of hSos2 fused to the GAL4 activation domain
(hSos2 IV) and various Grb2 mutants fused to the GAL4 binding domain
were performed as indicated in the graph. Cell lysates from
each transformation were then prepared and assayed for
To further analyze the interaction of Grb2-Sos2, we examined the
ability of the COOH-terminal domain of hSos2 (hSosIV) to interact with
Grb2 proteins containing point mutations in the SH2 (E89K, S90N) or SH3
(P49L, G203R) domains. These mutations correspond to the mutations
originally identified in the loss of function alleles of sem-5 in Caenorhabditis elegans(28, 29) .
Biochemical and genetic characterization of Grb2 proteins containing
these point mutations indicated that mutations in the amino- or
carboxyl-terminal SH3 domains of Grb2 (P49L and G203R, respectively)
impair its ability to interact with Sos1, whereas mutations in the SH2
domain have no effect on Grb2-Sos1
interaction(5, 25, 27) . The interaction
between these Grb2 mutants expressed as GBD fusions and the hSos2IV-GAD
fusion was assessed using a quantitative assay for Because the primary sequences of the
COOH-terminal domains of hSos1 and hSos2 show a significant divergence,
we sought to determine whether these differences might influence the
relative binding affinities of hSos1 and hSos2 for Grb2. It has been
reported that the level of induction of
Figure 2:
Comparison between the interactions of
hSos1 and hSos2 with Grb2. A, Grb2 fused to the GAL4 binding
domain was cotransformed into yeast YPB2 strain with GAL4 activation
domain fusions of either hSos1 COOH-terminal domain (hSos1 IV, residues
1019-1333) or hSos2 COOH-terminal domain (hSos2 IV, residues
1017-1332). Cell lysates from each transformation were then
prepared and assayed for
To
test whether the apparent affinity difference between hSos1 and hSos2
detected in the two-hybrid system can be confirmed by an independent
assay, the binding of hSos1 or hSos2 to Grb2 was compared using
immobilized GST-Grb2 fusion protein. COS-1 cells were transfected with
cytomegalovirus-based mammalian expression constructs encoding
full-length hSos1 or hSos2 tagged with the HA epitope at the amino
terminus. Increasing amounts of lysates prepared from
[ We next wished to determine
whether the difference between the apparent affinities of Sos1 and Sos2
for Grb2 detected in vitro can also be observed in
vivo. COS-1 cells were transfected with HA-hSos1 or HA-hSos2
expression plasmids. Lysates were immunoprecipitated with anti-HA
antibodies, and the immunoprecipitates were blotted with anti-Grb2 or
anti-HA antibodies. As can be seen, the amount of Grb2
coimmunoprecipitated with hSos was significantly higher than that
coimmunoprecipitated with hSos1 (Fig. 2C).
Immunoblotting with anti-Grb2 antibodies of supernatant from hSos2
immunoprecipitates revealed that essentially the total pool of Grb2
molecules is associated with hSos2 (not shown). To rule out the
possibility that the observed differences in the apparent affinities of
hSos1 and hSos2 for Grb2 are unique to the COS-1 cell system, the same
analysis was carried out using human 293 cells. Consistent with the
results obtained with the COS-1 cells, the hSos2 immunoprecipitate from
the 293 cells contained a significantly higher amount of Grb2 relative
to the amount of Grb2 present in the hSos1 immunoprecipitate (Fig. 2C). These data demonstrate that the preferential
interaction of Grb2 with hSos2 occurs also in vivo. To gain
an insight into the molecular basis underlying the differential
interactions of hSos1 and hSos2 with Grb2, experiments were performed
to define the region within the COOH terminus of hSos2 that confers the
apparent higher binding affinity for Grb2. Chimeric HA-hSos1/Sos2
constructs were generated by substituting various segments from the
COOH-terminal region of hSos1 for the corresponding segments from the
COOH-terminal regions of hSos2 (Fig. 3A). Each of these
constructs was transfected into COS-1 cells. Lysates prepared from the
transfected cells were immunoprecipitated with anti-HA antibodies, and
the association of the chimeric hSos1/hSos2 proteins with Grb2 was
determined by immunoblotting of the anti-HA immunoprecipitates with
anti-Grb2 antibodies. Fig. 3B shows that a region of
117 amino acids corresponding to residues 1126-1242 of hSos2
imparts the increase in the apparent affinity of hSos2 toward Grb2.
Significantly, this region represents the most divergent region between
hSos1 and hSos2 suggesting that sequence differences between hSos1 and
hSos2 within this region may specify their differential interaction
with Grb2.
Figure 3:
Identification of residues within hSos2
that confer the increase in the apparent affinity of hSos2 toward Grb2. A, a schematic representation of the various hSos1/Sos2
chimeric constructs. The residues defining the site of insertion of the
hSos2 fragments were chosen based on sequence alignment of hSos1 and
hSos2. B, HA-tagged chimeric constructs were transfected into
COS-1 cells, and chimeric proteins were immunoprecipitated by anti-HA
antibody. Immunoblotting of the immunoprecipitates (IP) with
anti-Grb2 and anti-HA antibodies was performed as described in the
legend to Fig. 2. C, sequence of the proline-rich
SH3-binding motifs in the COOH-terminal domains of hSos1 and hSos2. P and R denote the highly conserved proline and
arginine residue. X tends to be an aliphatic residue. Residues
enclosed in the shadedboxes are crucial for high
affinity SH3 binding. The intervening scaffolding residues (p)
also tend to be prolines. Underlinedsequences represents perfect matches with the consensus Grb2 SH3 domain
binding motif XPpXP.
Recent structural studies of the interactions of various
SH3 domains with proline-rich peptides have indicated that proline-rich
sequences bind to SH3 domains in a PPII helix formation that can adopt
either a plus or minus orientation(31) . An XPpXP motif has been identified as critical for
binding in either orientation. All the proline-rich SH3 binding motifs
of hSos1 and hSos2 (Fig. 3C) can adopt only the minus
binding orientation. It is of interest to note that the region within
the COOH-terminal domain of hSos2 that confers the increased affinity
toward Grb2 contains three perfectly matched XPpXP
motifs whereas the corresponding region in hSos1 contains only one such
motif (Fig. 3C). Thus the higher affinity of hSos2 for
Grb2 relative to hSos1 could be due to the increased number of the high
affinity SH3 binding sites and/or their relative position within the
COOH-terminal domain of hSos2. Characterization of the structural basis
for the preferential interaction of Sos2 with Grb2 should provide
insight into the molecular determinants that specify SH3
domain-mediated interactions.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)(2) . GNEFs are highly conserved in
evolution. They were first identified through genetic studies in lower
eukaryotes (reviewed in (3) ). In Saccharomyces
cerevisiae, CDC25 and SCD25 are GNEFs for Ras, and in Schizosaccharomyces pombe Ste6 is the homologue of CDC25. In Drosophila, the GNEF for Ras is encoded by the son of
sevenless gene (sos). Two mammalian homologues of Drosophila Sos have been identified, Sos1 and
Sos2(4, 5) . All Ras GNEFs share a region of homology
with the COOH-terminal 450 amino acids of CDC25, which constitutes the
catalytic domain of these proteins.
Antibodies
The anti-HA mouse monoclonal
antibody, 12CA5, was from Babco. The anti-Grb2 rabbit polyclonal
antibody 86 was a gift from Dr. Joseph Schlessinger (New York
University, New York).The Two-hybrid System
Plasmids PGADGH, PGBT10,
PGADGH-SNF1, PGBT9-SNF4, and the host yeast strain YPB2 were described
previously (5) . Replicative plasmid PGAD1318 is a PGADGH
plasmid in which the frame of the cloning sites has been changed. The
LacZ filter assay was carried out according to published
procedures(16) . Quantitative assays for -galactosidase
activity were performed as described(17) , using O-nitrophenolgalactosidase (Boehringer Mannheim) as an
indicator. Protein concentration was determined using Blue G-250-based
reagent (Pierce).
Plasmid Construction
hsos1 and hsos2 cDNAs were provided by Pierre Chardin (Institut de Pharmacologie
Moleculaire et Cellulaire, Valbonne, France). All molecular cloning
techniques were performed as described(18) . DNA restriction
endonucleases, DNA polymerase I Klenow fragment, T4 DNA ligase (New
England Biolabs Inc.), shrimp alkaline phosphatase (Amersham-USB) and
Pfu polymerase (Stratagene) were used as recommended by the suppliers.
All DNA fragments were purified using Geneclean II kits (Bio101), and
all polymerase chain reaction fragments were verified using Sequenase
2.0 kits (Amersham-USB).Constructs Created for the Two-hybrid
Assay
Wild-type and mutant grb2 sequences were
subcloned into the PGBT10 vector as BamHI-SalI
fragments. Full-length hSos2 was cloned into the BamHI site of
PGAD1318. The region corresponding to amino acids 1017-1332 of
hSos2 was amplified by primers S1
(5`-cgtaggatccaactgcaaacagccacctcgatttcc-3`) and R
(5`-ccttgtcgactcattggggagtttctgcattttctag-3`) and then
subcloned into PGAD1318 to create PGAD1318-hSos2IV. PGAD1318-hSos1IV
encoding an identical region(1019-1333) from hSos1 was a gift
from Dr. J. Camonis (INSERM, Valbonne, France). The BamHI/NcoI, BamHI/NdeI, or NdeI/NcoI fragments of hsos2 were subcloned
into PGAD1318 to create PGAD-hSos2I, PGAD-hSos2II, or PGAD-hSos2III,
respectively.Mammalian Expression Constructs
The mammalian
expression vector PCGN used in this study was a gift from Dr. M. Tanaka
(Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). This vector
contains the cytomegalovirus promoter and multicloning sites that allow
the expression of genes fused 3` to the hemagglutinin (HA) epitope (19) . To create various hSos1/2 hybrids, the full-length hsos1 gene was first subcloned into SmaI and HindIII sites of Bluescript (pSK,
Stratagene). The various length fragments of hsos2 COOH-terminal region were amplified by polymerase chain reaction
using Pfu DNA polymerase, and all products were named according to the
primers used. Primers P1 (5`-tatggatccctgaaactgagctgg-3`) and
R1(5`-cacaagctttcattggggagtttc-3`) were used to amplify the
region corresponding to amino acids 1073-1332 of hSos2. The
product, P1R1, was subcloned into BamHI/HindIII sites
of SK-hsos1. A fragment corresponding to amino acids
1126-1332 of hSos2 was amplified by primers P2
(5`-ggccatggctcaaagtctttcttt-3`) and R1, and the product,
P2R1, was then subcloned into NcoI/HindIII sites of
SK-hsos1. The region corresponding to residues 1126-1242
was amplified by primers P2 and R2
(5`-gatccatggtcgactctgtgaagatgc-3`), and the product, P2R2,
was subcloned into NcoI sites of SK-hsos1. All
resulting products and full-length hSos1 were then subcloned into XbaI/SmaI sites of PCGN vector. Full-length hsos2 was first cloned into PGADGH vector as a BamHI fragment
and then isolated as a SpeI/SmaI and inserted into XbaI/SmaI sites of PCGN vector.
Transfection
Transfections of COS-1 and 293 cells
were performed using Lipofectin (Life Technologies, Inc.), according to
the manufacturer's instructions. Briefly cells were cultured on
10-cm plates in Dulbecco's modified Eagle's medium (DMEM,
Life Technologies, Inc.) supplemented with 5% fetal calf serum for
COS-1 or 10% calf serum for 293, 100 units/ml penicillin, and 100
µg/ml streptomycin until 70% confluent. The growth medium was then
aspirated, and the cells were rinsed one time with Opti-MEM medium
(Life Technologies, Inc.). 4 ml of Opti-MEM medium were added to the
rinsed cells. A mixture was made in which 1 µg of plasmid DNA
diluted in 500 µl of Opti-MEM was mixed with 20 µl of
Lipofectin diluted in 500 µl of Opti-MEM medium. The mixtures were
incubated 10-15 min at room temperature and added to the cells.
Following an overnight incubation, the transfected medium were
supplemented with 5 ml of DMEM plus 10% fetal calf serum. The
transfected cells were harvested 2 days after transfection.Cell Labeling, Immunoprecipitation, and
Immunoblotting
Metabolic labeling was carried out by incubating
cells in methionine-free DMEM containing 5% dialyzed fetal calf serum
and 125 µCi/ml medium of ExpressS-methionine label
(DuPont NEN) for 14 h. At the end of labeling period, the cells (5
10
) were washed two times with ice-cold
phosphate-buffered saline solution and lysed in 1 ml of cold co-IP
buffer (150 mM NaCl, 10 mM Tris/HCl, pH 7.4, 1%
Triton X-100, 10% glycerol, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 mg/ml pepstatin, 1% aprotinin,
10 µg/ml leupeptin, 1 mM sodium vanadate, 10 µg/ml
soybean trypsin inhibitor, and 1 µM okadaic acid). The
lysates were clarified by centrifugation at 4 °C for 15 min at
14,000
g. The clarified lysates were preincubated with
50 µl of a 50% protein A-Sepharose (Pharmacia Biotech Inc.)
solution for 30 min at 4 °C while gently rotating. The
immunoprecipitation of HA-hSos proteins was accomplished using an
anti-HA monoclonal mouse antibody 12CA5 (20) precoupled to
protein A-Sepharose as described(21) . The immunoprecipitations
were done for 3 h at 4 °C. The immune complexes were collected by
centrifugation (4 min at 3,000
g), washed five times
with co-IP buffer, and eluted with SDS sample buffer. The precipitated
proteins were resolved on a 12.5% SDS-polyacrylamide gel using the
buffer system of Blattler et al.(22) . Gels were
enhanced with EN
HANCE (DuPont), dried, and exposed to XAR
film (Kodak). Immunoblotting was carried out as described(23) .
Bound antibodies were detected using the ECL detection system (Amersham
Corp.).
GST-Grb2 and hSos Binding Assay
Grb2-glutathione S-transferase (GST-Grb2) fusion construct was a gift from Dr.
Joseph Schlessinger (New York University). The GST-Grb2 fusion protein
was overexpressed in Escherichia coli strain HB101 and
purified by binding to glutathione-agarose beads as
described(24) . Cell lysates were prepared as described above.
Following preincubating with GST for 30 min at 4 °C, the lysates
were incubated with 1 µg of immobilized GST-Grb2 proteins for 90
min at 4 °C. The bound proteins were resolved on a 7.5%
SDS-polyacrylamide gel, and the amount of Sos protein bound was
quantified by both PhosphorImager (Fuji) and densitometry (Howtek)
scanning.
-galactosidase activity. As shown in Fig. 1A,
-galactosidase induction was detected
only when the Grb2-GBD fusion was coexpressed with full-length
hSos2-GAD fusion or with a GAD fusion corresponding to a proline-rich
COOH-terminal domain of hSos2, indicating that the COOH-terminal domain
of Sos2 is both necessary and sufficient for association with Grb2.
-galactosidase activity as described under ``Materials and
Methods.'' Each bar corresponds to average of three
independent transformants. Errorbars indicate the
standard error of the mean.
-galactosidase
activity. Induction of
-galactosidase activity was significantly
reduced by single point mutations in either of the SH3 domains of Grb2 (Fig. 1B). Furthermore, a Grb2 double mutant containing
point mutations in both SH3 domains was completely impaired in its
ability to induce
-galactosidase activity. These results indicate
that the interaction between hSos2 and Grb2 is primarily mediated by
the SH3 domains and that both SH3 domains are involved in the stable
association between the two proteins. Since similar characteristics
have been ascribed to the interactions of hSos1 with Grb2(5) ,
we conclude that mechanisms utilized by hSos1 and hSos2 to bind Grb2
are, in principle, similar.
-galactosidase activity in
the two-hybrid system correlates well with the affinity between the two
interacting proteins observed in vitro(30) . Therefore
we initially employed this system to compare the interactions of hSos1
and hSos2 with Grb2. Fig. 2A shows that the induction
of
-galactosidase activity was approximately 5-fold higher in host
cells expressing hSos2IV and Grb2 fusion proteins than in host cells
expressing hSos1IV and Grb2 fusion proteins, suggesting that hSos2 may
have a higher binding affinity for Grb2 in comparison with hSos1.
-galactosidase activity as described
under ``Materials and Methods.'' Values are the averages of
three separate experiments, and errorbars indicate
the standard error of the mean. SNF4 was used as a negative control. B, COS-1 cells were transfected with either HA-hSos1 or
HA-Sos1 expression vectors and labeled with
[
S]methionine. Equal amounts of the cell lysates
(
3 µg/µl) prepared form the radiolabeled cells were
incubated with 1 µg of GST-Grb2. Bound proteins were eluted and
resolved by SDS-PAGE. The relative intensities of the hSos bands was
determined using a Fuji PhosphorImager. The levels of expression of
hSos1 and hSos2 were identical as determined by immunoprecipitation
with anti-HA antibody (inset). Results shown are from a single
experiment. Identical results were obtained in two independent
experiments. C, COS-1 or 293 cells were transfected with
either HA-hSos1 or HA-Sos2 expression vectors. Anti-HA
immunoprecipitates (IP) were resolved on 12.5% SDS-PAGE. After
transferring the proteins onto nitrocellulose membrane, the membrane
was cut into half along the 87-kDa molecular mass marker. The tophalf was blotted with anti-HA antibody, and the bottomhalf was probed with anti-Grb2
antiserum.
S]methionine-labeled transfected cells were
incubated with fixed amounts of GST-Grb2 immobilized on glutathione
beads. Bound proteins were resolved by SDS-PAGE, and the amount of hSos
polypeptides bound to Grb2 was quantified by using a PhosphorImager.
The identity of the hSos1 and hSos2 polypeptides was confirmed by
immunoblotting using anti-HA antibodies. Comparison between the amounts
of hSos1 and hSos2 bound to GST-Grb2 (after normalization for
expression levels and differences in methionine content) indicates that
the apparent Grb2 binding affinity of hSos2 is higher relative to that
of hSos1 (Fig. 2B). Similar results were obtained from
the quantification of Coomassie Blue-stained hSos1 and hSos2 bands by
densitometry scanning (not shown).
We thank Dr. P. Chardin for hSos2 cDNA and
Susan Kaplan for technical assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.