©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Differential Interactions of Human Sos1 and Sos2 with Grb2 (*)

(Received for publication, May 3, 1995; and in revised form, June 13, 1995)

Shao-song Yang Linda Van Aelst (1) Dafna Bar-Sagi (§)

From the Department of Molecular Genetics and Microbiology, School of Medicine, State University of New York, Stony Brook, New York 11794 and Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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)(^1)(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.

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.


MATERIALS AND METHODS

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 beta-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 10^6) 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^3HANCE (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.


RESULTS AND DISCUSSION

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 beta-galactosidase activity. As shown in Fig. 1A, beta-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.


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 beta-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.



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 beta-galactosidase activity. Induction of beta-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 beta-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.

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 beta-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 beta-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.


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 beta-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.



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 [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 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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants CA55360 and CA28146. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by 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 Molecular Genetics and Microbiology, State University of New York, Stony Brook, NY 11794-8621. Tel.: 516-632-9737; Fax: 516-632-8891.

^1
The abbreviations used are: GNEF, guanine nucleotide exchange factor; HA, hemagglutinin; DMEM, Dulbecco's modified Eagle's medium; GST, glutathione S-transferase; GAD, GAL4 transcriptional activation domain; GBD, GAL4 DNA binding domain; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Dr. P. Chardin for hSos2 cDNA and Susan Kaplan for technical assistance.


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