INTRODUCTION

Top Abstract Introduction Materials and Methods Results and Discussion References

Ras protein activation in eukaryotes is mediated through the action of Ras-specific guanine nucleotide exchange factors (GEFs) (1, 17) linking the activation of surface receptors by upstream signals to the accumulation of a Ras-GTP complex able to deliver signals to the nucleus. GEFs are highly conserved in evolution, having been initially identified in lower organisms such as Saccharomyces cerevisiae (CDC25 and SCD25) (4, 5), Schizosaccharomyces pombe (Ste6) (15), and Drosophila (Son of sevenless [Sos]) (2). Three types of Ras-specific GEFs have been described in mammals: the highly homologous GRF1 and GRF2 (6, 10, 11, 21), the closely related Sos1 and Sos2 (3, 7, 14), and GRP (9). sos and GRF2 genes are widely expressed in adult tissues and cell lines, while expression of GRF1 and GRP genes is restricted primarily to the brain. All Ras-specific GEFs share a region of homology with the C-terminal 450 amino acids of CDC25 (CDC25-H domain) constituting the catalytic domain of all these proteins (1).

The GEF protein Sos plays a crucial role in the process of coupling protein tyrosine kinases, via the adapter protein Grb2, to Ras activation, facilitating GDP-GTP exchange. Sos1 and Sos2 proteins are constitutively bound to the SH3 domain of Grb2 through the proline-rich region present in their C. termini. The Grb2-Sos complex binds directly to the activated receptors or to a second adapter protein, such as Shc, through the SH2 domain of Grb2 (8, 12, 20).

Alignment of the murine or human Sos1 and Sos2 proteins uncovers a high overall (65% amino acid identity) degree of similarity. Similarity between Sos1 and Sos2 is highest (up to 75% amino acid identity) at their N-terminal regions. In contrast, the homology between the C-terminal regions of Sos1 and Sos2 is more restricted and scattered (overall similarity of 40%), with conserved regions mostly reduced to the short proline-rich motifs responsible for interaction with the SH3 domain of Grb2. These differences between their C-terminal regions are likely to account for the distinct signaling and functional properties of the two Sos proteins. It has been reported that human Sos2 has a higher affinity for Grb2 than Sos1 (25) and that mouse Sos1 is more stable than Sos2, which appears to be degraded by a ubiquitin-dependent process (18). Other differences between Sos1 and Sos2 are related to their protein tyrosine kinase signaling properties: Sos1 participates in short- and long-term signaling, whereas Sos2-dependent signals are predominantly short term (19).

sos1 is essential for embryonic development, with homozygous null sos1^/ embryos dying at midgestation in utero (19, 24). To evaluate the functional significance of the highly homologous sos2 gene, we created a targeted disruption of this locus in mice. In this report we show that sos2 is completely dispensable for mouse development, since its null mutation resulted in viable mice with no apparent phenotypic effect due to this deficiency.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results and Discussion References

Sos2 targeting vector and chimeric mouse production. Four mouse genomic sos2 clones were identified and isolated from a 129SvJ mouse-derived library (Stratagene, La Jolla, Calif.), using a 690-bp probe derived from the sequence immediately downstream of the CDC25-H domain (bp 3093 to 3783) of murine sos2 cDNA (3) (GenBank accession no. Z11664). Since there is 72.8% homology between sos1 and sos2 at the CDC25-H domain, we used sequences downstream ofinstead of within-this domain to avoid cross-hybridization with sos1.

View larger version (43K): [in this window] [in a new window]   FIG. 1.   Targeted disruption of the murine sos2 gene in ES cells and mice. (A) Schematic representation of the sos2 locus and targeting vector. Boxes in the wild-type allele schematics represent the exons of the sos2 CDC25-H domain. The open boxes in the targeting vector schematics represent the pgk-neo and pgk-tk selectable marker genes. Position of boundaries of individual exons coding for the C-terminal portion of the protein are indicated by vertical marks. The position of the 5' flanking probe used in Southern blotting is indicated. DH, Dbl homology; PH, pleckstrin homology; REM, Ras exchange motif. (B) Homologous recombination of the targeting vector in mice was verified by Southern blotting, digesting genomic DNA with NcoI, and hybridizing with a 5'-flanking probe. The wild-type allele produced a 11.8-kb band, whereas the mutant allele yielded a 7.0-kb band due the introduction of a new NcoI site in the targeting vector. (C) Routine genotyping of mice was performed by PCR using the oligonucleotides indicated (see Materials and Methods for sequences). The LM127 and LM129 primers are specific for the sos2 gene and amplify a fragment of 367 bp. The LM82 primer is specific for the promoter of the neo gene and amplifies a fragment of 410 bp with LM127. Nc, NcoI; Sp, SpeI; Nd, NdeI; Xb, XbaI.

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Genotyping of targeted ES cells, mice, and embryos. Genomic DNA was extracted from cultured ES cells, mouse tail biopsies, and embryo yolk sacs as described by Laird et al. (16). ES cells were incubated at 37°C, and tail biopsies and embryo yolk sacs were incubated at 55°C, in lysis buffer (100 mM Tris-HCl [pH 8.0], 5 mM EDTA, 0.2% sodium dodecyl sulfate [SDS], 200 mM NaCl, 100 µg of proteinase K per ml 4 to 5 h or overnight. DNA was isopropanol precipitated and recovered by lifting the aggregated precipitate from the solution. DNA was washed in 70% ethanol and resuspended in 200 µl of Tris-EDTA (pH 8.0) buffer. For Southern analysis, 20 µl of DNA was digested with SpeI, electrophoresed on 0.6% agarose gels, and blotted to GeneScreen Plus membranes (Dupont, Boston, Mass.). A probe flanking the 5' end of the targeting vector sequence was labeled with a random primer labeling kit (Stratagene) and used in hybridizations. Wild-type and mutated alleles were identified by predicted restriction fragment size differences. Clones displaying homologous recombination were reconfirmed with NcoI digestion and the same 5' probe (Fig. 1B). Digestion of ES cell DNA with enzymes that did not cut within the targeting vector and Southern blotting and hybridization with a neo probe showed only a single band, indicating the there was only a single site of vector insertion in the targeted ES cell clones (not shown).

Histopathological analysis. More than 30 tissues taken from sos2^/ adult mice (three males and one female, 16 weeks old) after necropsy were fixed in formalin and embedded in paraffin. Sections were stained with hematoxylin and eosin. Lesions were not found in any tissues. As controls, we examined the same tissues from two 16-week-old wild-type sos2^+/+ males and three 16-week-old wild-type sos2^+/+ females. Two 16-week-old sos2^+/ males were also examined.

Western blot analysis. Protein extracts were obtained from snap-frozen mouse tissues. Tissues were homogenized in radioimmunoprecipitation buffer (50 mM Tri-HCl [pH 7.5], 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) and centrifuged in a Sorvall S1256 apparatus at 30,000 rpm for 30 min. Supernatant was recovered, and proteins were quantified. Lysates (50 to 70 µg/lane) were loaded onto SDS-7.5% polyacrylamide gels, and the proteins were transferred to polyvinylidene difluoride membranes (Millipore Immobilon-P) by electroblotting. Membranes blocked in TTBS (10 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.05% Tween 20) plus 1% bovine serum albumin were incubated, as appropriate, with 1:100 dilutions of commercial polyclonal antibodies from Santa Cruz Biotechnology, Santa Cruz, Calif.). Antibodies used were anti-C-terminal Sos1 (C-23, sc-256), anti-C-terminal Sos2 (C-19, sc-258), and anti-N-terminal Sos1/Sos2 (D-21, sc-259; recognizing the identical N termini of both Sos1 and Sos2). Western blots were developed using the ProtoBlot Western blotting alkaline phosphatase system (Promega) following procedures recommended by the supplier.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

To investigate the in vivo function of the mammalian Sos2 GEF, we used gene targeting techniques to generate mice deficient in the endogenous sos2 locus. In an approach similar to that used previously for sos1 (19), we inactivated sos2 by targeting its catalytic CDC25-H domain, responsible for the guanine nucleotide exchange activity of the molecule (1).

By means of restriction mapping and partial sequencing, we identified the positions of various exons coding for the CDC25-H domain of sos2 (Fig. 1A). Based on those genomic mapping data, the targeting vector pLM146 (Fig. 1A) was constructed by replacing genomic sequences containing a highly conserved exon (encoding amino acids 855 to 893) of the central region of the Sos2 CDC25-H domain with a pgk promoter-driven neo cassette. The herpes simplex virus tk gene was included downstream of the long arm of homology in pLM146 to provide negative selection for nonhomologous recombination events (Fig. 1A).

The replacement vector pLM146 was electroporated into CJ7 ES cells, which were then selected in the presence of G418-fialuridine. A relatively high frequency of homologous recombination (7 positives out of 80 clones screened) was observed with our construct. Subsequent microinjection of two of the positive ES cell lines was used to generate chimeras that were then mated to C57BL/6 females to give rise to sos2^+/ mice. These sos2 heterozygotes were further inbred and generated sos2-null mice at the expected Mendelian frequency of 1/4. Mutant genotypes were initially confirmed by Southern hybridization (Fig. 1B), and further routine genotyping was carried out by PCR using oligonucleotides hybridizing within both the sos2 gene and the pgk-neo cassette (Fig. 1C).

To confirm that the modification of the sos2 gene resulted in a null mutation, we examined Sos2 expression by Western immunoblot in tissues from wild-type and mutant mice. Using antibodies specific for the C-terminal region of Sos2 protein (Santa Cruz Biotechnology), we were unable to detect in tissues from / mutant mice the presence of the full-length Sos2 protein that is easily observable in their wild-type counterparts (Fig. 2A). In contrast, antibodies specific for Sos1 revealed the presence of similar levels of this protein in both wild-type and mutant sos2 tissues (Fig. 2B), indicating that there is no overexpression of Sos1 to compensate for the absence of Sos2.

View larger version (34K): [in this window] [in a new window]   FIG. 2.   Western blot to detect Sos proteins in extracts from wild-type and sos2/ mouse tissues. Fifty- to 70-µg aliquots of total protein from brains and testes of wild-type and sos2/ mice were electrophoresed on SDS-7.5% polyacrylamide gels. (A) Immunodetection of Sos2 protein. It is present in wild-type and absent in mutant mouse tissues. (B) Immunodetection of Sos1 protein. It is present in all tissues regardless of whether they are deficient for Sos2. There is no apparent increase in the amount of Sos1 protein in tissues that are deficient for Sos2. (C) Immunodetection using antibodies specific for the common N-terminal region of Sos1 and Sos2 proteins. A shortened, truncated Sos2 protein form was not detected in sos2/ mutant mice at the theoretical position indicated (*), confirming the absence of any Sos2 protein form in the mutant sos2 mice. Sizes are indicated in kilodaltons.

It should be noted that in our targeting strategy, the deletion of exon C from the genomic sequences contained in pLM146 (Fig. 1A) creates an out-of frame disruption which might, at least potentially, give rise to a truncated protein (857 residues, expected molecular size of ca. 105 kDa) in the hypothetical case that the resulting mutated locus could be transcribed into a stable or functional RNA, which in turn would need to be able to be translated into a stable protein. To check for the possible occurrence of a shorter Sos2 protein in mutant sos2 animals, we performed Western blot analysis of the same tissues using antibodies specific for the amino-terminal region of Sos2. Using a commercial antibody recognizing specifically the common amino-terminal region of Sos1 and Sos2 in mice and humans, we failed to detect the presence of any low-molecular-weight form of Sos2 protein in tissues of the mutant mice (Fig. 2C). These results confirmed the absence of any form of Sos2 protein in our homozygous / mutant mice.

We have kept mutant sos2 mice in our laboratory for about 1.5 years now. Lesions were not found in any tissues of the sos2^/ mice that could be attributed to the null mutation. Both the heterozygous +/ and homozygous / mutant sos2 mice developed normally, with males and females being fertile. In addition, the mutant mice showed no obvious defects, and their long-term survival rates were indistinguishable from those of their wild-type littermates. Thus, the absence of sos2 in mice did not compromise development or fertility in mice. Finally, histopathological analysis of more than 30 tissues in four adult sos2^/ animals revealed no gross anatomical defects or histopathological changes (data not shown).

Since the absence of the highly homologous Sos1 protein results in embryonic lethality, probably due to impaired placental development (19), we wished to address the effect of the absence of Sos2 on early development. Histopathological analysis of sos2^/ embryos at day 12 of gestation did not show any difference from their wild-type littermates and development of the placenta appeared also to be normal in / animals. This is consistent with the significantly lower levels of sos2 expression relative to sos1 expression observed in wild-type placenta (19).

Our results confirm that in sharp contrast to Sos1, Sos2 is dispensable for embryonal and adult mouse development, as well as for normal growth and fertility of the knockout animals. This very significant functional difference between two otherwise remarkably similar proteins is most likely related to our previously reported observations (19), indicating that Sos1 participates in both short- and long-term signaling through the Ras-mitogen-activated protein kinase pathway, while Sos2-dependent signals are predominantly short term.