The Sos1 and Sos2 Ras-specific exchange factors: differences in placental expression and signaling properties
2000; Springer Nature; Volume: 19; Issue: 4 Linguagem: Inglês
10.1093/emboj/19.4.642
ISSN1460-2075
AutoresXiaolan Qian, Luis Miguel Pedrero Esteban, William C. Vass, Cheerag D. Upadhyaya, Alex G. Papageorge, Kate Yienger, Jerrold M. Ward, Douglas R. Lowy, Eugenio Santos,
Tópico(s)Pregnancy and preeclampsia studies
ResumoArticle15 February 2000free access The Sos1 and Sos2 Ras-specific exchange factors: differences in placental expression and signaling properties Xiaolan Qian Xiaolan Qian Laboratory of Cellular Oncology Bethesda, National Cancer Institute, Bethesda, MD, 20892 USA Search for more papers by this author Luis Esteban Luis Esteban Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, MD, 20892 USA Search for more papers by this author William C. Vass William C. Vass Laboratory of Cellular Oncology Bethesda, National Cancer Institute, Bethesda, MD, 20892 USA Search for more papers by this author Cheerag Upadhyaya Cheerag Upadhyaya Laboratory of Cellular Oncology Bethesda, National Cancer Institute, Bethesda, MD, 20892 USA Search for more papers by this author Alex G. Papageorge Alex G. Papageorge Laboratory of Cellular Oncology Bethesda, National Cancer Institute, Bethesda, MD, 20892 USA Search for more papers by this author Kate Yienger Kate Yienger Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, MD, 20892 USA Search for more papers by this author Jerrold M. Ward Jerrold M. Ward Veterinary and Tumor Pathology Section, National Cancer Institute, Frederick, MD, 21702 USA Search for more papers by this author Douglas R. Lowy Corresponding Author Douglas R. Lowy Laboratory of Cellular Oncology Bethesda, National Cancer Institute, Bethesda, MD, 20892 USA Search for more papers by this author Eugenio Santos Eugenio Santos Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, MD, 20892 USA Present address: Centro de Investigatión del cáncer, CSIC-USAL, Campus Unamuno, Universidad Salamanca, 37007 Salamanca, Spain Search for more papers by this author Xiaolan Qian Xiaolan Qian Laboratory of Cellular Oncology Bethesda, National Cancer Institute, Bethesda, MD, 20892 USA Search for more papers by this author Luis Esteban Luis Esteban Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, MD, 20892 USA Search for more papers by this author William C. Vass William C. Vass Laboratory of Cellular Oncology Bethesda, National Cancer Institute, Bethesda, MD, 20892 USA Search for more papers by this author Cheerag Upadhyaya Cheerag Upadhyaya Laboratory of Cellular Oncology Bethesda, National Cancer Institute, Bethesda, MD, 20892 USA Search for more papers by this author Alex G. Papageorge Alex G. Papageorge Laboratory of Cellular Oncology Bethesda, National Cancer Institute, Bethesda, MD, 20892 USA Search for more papers by this author Kate Yienger Kate Yienger Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, MD, 20892 USA Search for more papers by this author Jerrold M. Ward Jerrold M. Ward Veterinary and Tumor Pathology Section, National Cancer Institute, Frederick, MD, 21702 USA Search for more papers by this author Douglas R. Lowy Corresponding Author Douglas R. Lowy Laboratory of Cellular Oncology Bethesda, National Cancer Institute, Bethesda, MD, 20892 USA Search for more papers by this author Eugenio Santos Eugenio Santos Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, MD, 20892 USA Present address: Centro de Investigatión del cáncer, CSIC-USAL, Campus Unamuno, Universidad Salamanca, 37007 Salamanca, Spain Search for more papers by this author Author Information Xiaolan Qian1, Luis Esteban2, William C. Vass1, Cheerag Upadhyaya1, Alex G. Papageorge1, Kate Yienger2, Jerrold M. Ward3, Douglas R. Lowy 1 and Eugenio Santos2,4 1Laboratory of Cellular Oncology Bethesda, National Cancer Institute, Bethesda, MD, 20892 USA 2Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, MD, 20892 USA 3Veterinary and Tumor Pathology Section, National Cancer Institute, Frederick, MD, 21702 USA 4Present address: Centro de Investigatión del cáncer, CSIC-USAL, Campus Unamuno, Universidad Salamanca, 37007 Salamanca, Spain *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:642-654https://doi.org/10.1093/emboj/19.4.642 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Targeted disruption of both alleles of mouse sos1, which encodes a Ras-specific exchange factor, conferred mid-gestational embryonic lethality that was secondary to impaired placental development and was associated with very low placental ERK activity. The trophoblastic layers of sos1−/− embryos were poorly developed, correlating with high sos1 expression in wild-type trophoblasts. A sos1−/− cell line, which expressed readily detectable levels of the closely related Sos2 protein, formed complexes between Sos2, epidermal growth factor receptor (EGFR) and Shc efficiently, gave normal Ras·GTP and ERK responses when treated with EGF for ≤10 min and was transformed readily by activated Ras. However, the sos1−/− cells were resistant to transformation by v-Src or by overexpressed EGFR and continuous EGF treatment, unlike sos1+/− or wild-type cells. This correlated with Sos2 binding less efficiently than Sos1 to EGFR and Shc in cells treated with EGF for ≥90 min or to v-Src and Shc in v-Src-expressing cells, and with less ERK activity. We conclude that Sos1 participates in both short- and long-term signaling, while Sos2-dependent signals are predominantly short-term. Introduction The ras genes encode widely expressed membrane-associated GTPases that function as molecular switches that transduce a wide variety of growth and differentiation signals induced by extracellular ligands (Lowy and Willumsen, 1993; Macara et al., 1996; Campbell et al., 1998). Receptors activated by such ligands stimulate Ras by activating Ras-specific guanine nucleotide exchange factors (GNEFs), which convert Ras from inactive Ras·GDP to active Ras·GTP. In mammals, three types of Ras-specific GNEFs have been described: the closely related Sos1 and Sos2 proteins, the highly homologous GRF1 and GRF2, and GRP (Bowtell et al., 1992; Cen et al., 1992; Shou et al., 1992; Guerrero et al., 1996; Fam et al., 1997; Tung et al., 1997; Ebinu et al., 1998; Pierret et al., 1998; Tognon et al., 1998). As is true of ras, the sos genes are widely expressed, while the full-length grf1 and grp gene products have a more restricted tissue distribution. Each type of Ras-specific GNEF seems to respond to distinct upstream signals. GRP is activated by a diacylglycerol-dependent mechanism (Ebinu et al., 1998; Tognon et al., 1998), while GRF1 and GRF2 appear to respond to calcium-dependent signals (Farnsworth et al., 1995; Tung et al., 1997) and heterotrimeric G proteins (Mattingly and Macara, 1996; Kiyono et al., 1999). The Sos proteins have been implicated mainly in signals induced by activated protein tyrosine kinases, which are coupled to Sos via adaptor proteins. Sos1 and Sos2 proteins are constitutively bound to the Grb2 adaptor, through an interaction between the SH3 domains in Grb2 and proline-rich regions in the C-termini of both Sos proteins. Upon protein tyrosine kinase activation, Sos is induced to form a complex with the kinase either by binding to the SH2 domain of Grb2, or less directly by binding a second adaptor protein such as Shc, which, following its activation, simultaneously binds to the SH2 domain of Grb2 and to the activated protein tyrosine kinase (Basu et al., 1994; Quilliam et al., 1995; Downward, 1996; Cherfils and Chardin, 1999). sos1 is essential for intrauterine development, with homozygous null animals dying in mid-gestation in association with reported yolk sac and embryonic heart defects (Wang et al., 1997). Since Sos2 is also widely expressed, the lethality of sos1−/− embryos implies that the role of sos1 in development is distinct from that of sos2. However, it is not clear whether this arises primarily because sos2 may not be expressed sufficiently in some cells whose normal function depends on Sos, or may be secondary to putative differences between Sos1 and Sos2 signaling. In this report, we present experimental support for both possibilities. A placental defect that seems to account for the lethality of sos1−/− embryos occurs in an area that expresses higher levels of Sos1 than Sos2. On the other hand, we present in vitro results with two protein tyrosine kinases, Src and the epidermal growth factor receptor (EGFR), which suggest that Sos1 can participate in both short-term and long-term signaling, while Sos2-dependent signals are predominantly short-term. This mechanism may also contribute to the extra-embryonic defects seen in sos1−/− placentas. Results Targeting of the Sos1 locus To inactivate the mouse sos1 locus, the genomic sequences containing an exon coding for a highly conserved N-terminal portion (40 amino acids) of its catalytic CDC25-H domain in J1 mouse embryonic stem (ES) cells (Pichel et al., 1996) were replaced with a phosphoglycerate kinase promoter-driven neomycin cassette, in the targeting vector pLM82 (Figure 1). Chimeric animals carrying the targeted sos1 gene were then used to generate heterozygous mice carrying one normal and one mutant sos1 allele, which were phenotypically normal. Southern analysis of the offspring from multiple crosses between sos1+/− heterozygotes did not show any sos1−/− newborn animals, indicating that such homozygous null animals were not viable (Figure 1B). In contrast, a PCR-based genotyping assay (Figure 1C) allowed detection of sos1−/− embryos from such crosses throughout early gestation. In agreement with a previous report (Wang et al., 1997), sos1−/− embryos were never found past day 11 of embryonic development, with various stages of necrosis and death of sos1−/− embryos observed between day post-conception 9 (DPC9) and DPC11 (not shown). Figure 1.Targeted disruption of the murine mSos1 gene in ES cells and mice. (A) Schematic representation of the mSos1 locus and targeting vector. Boxes in the wild-type allele schematics represent the exons of the mSos1 CDC25-H domain. The open boxes in the targeting vector schematics represent the pgk-neo and pgk-tk selectable markers. The position of the 3′-flanking probe used in Southern blotting is indicated. (B) Homologous recombination of the targeting vector in ES cells was verified by Southern blotting, digesting genomic DNA with EcoRV and hybridizing with the 3′-flanking probe. The wild-type allele produced a 14 kb band, whereas the mutant allele yielded a 6.9 kb band due to the introduction of a new EcoRV site in the targeting vector. (C) Genotyping of embryos arising from heterozygous crosses was performed by RT–PCR using the oligonucleotides indicated, whose sequences are given in Materials and methods. The LM87 and LM107 primers are specific for the sos1 gene and amplify a fragment of 612 bp. The LM82 primer is specific for the Neo-PGK promoter and amplifies a fragment of 410 bp with LM87. Download figure Download PowerPoint Whereas no gross primary developmental abnormalities were seen in early stage sos1−/− embryos, placental defects were observed consistently with this genotype (Figure 2). We noted that by DPC10 the sos1−/− placental labyrinth layer (Figure 2D–F) was invariably extremely thin compared with wild type (Figure 2A–C). The spongiotrophoblast and labyrinth trophoblast layers in the sos1−/− placentas were highly disorganized, and the embryonic vasculature in the labryrinth was incomplete (compare Figure 2A and B with D and E). Labyrinth trophoblasts in sos1−/− placentas were dysplastic in appearance; they typically had pleomorphic nuclei and less cytoplasm than normal trophoblasts (Figure 2F). Abnormal, multinucleated giant trophoblasts containing abundant cytoplasm were also seen frequently, with advanced cases having hemorrhage in the yolk sac and the placental labyrinth (Figure 2D). We infer that the collapse of the normal labyrinth structure led to interference with the nutrition of the embryo and its subsequent death (Rinkenberger et al., 1997). In contrast to Wang et al. (1997), we did not observe cardiac- or other organ-specific abnormalities in the embryos before they died secondary to the placental defects. Figure 2.Placental labyrinth defects in sos1−/− animals. The left column (A–C) displays control preparations of mouse wild-type sos1 (+/+) placental/embryo structures at DPC12 (H&E-stained) at low, medium and high magnification, respectively. The right column (D–F) shows the corresponding sos1−/− preparations at DPC12. The wild type (A) contains embryo and developed placenta, while −/− (D) shows hemorrhage (dark red areas) into the labyrinth caused by the collapse of the labyrinth structure, and a dead embryo (not visible). The wild type (B) shows the chorionic plate (pale zone at top of figure), labyrinth, prominent spongiotrophoblast layer, giant cells and decidual layers, while −/− (E) has a thinner labyrinth, and it is difficult to discern the spongiotrophoblast layer. The wild type (C) shows a normal labyrinth, with maternal erythrocytes (M), labyrinth trophoblasts (LTB) and strings of embryonic nucleated erythrocytes within embryonic blood vessels. The −/− (F) has fewer nucleated embryonic erythrocytes (arrows) within fewer embryonic blood vessels, dysplastic labyrinth trophoblasts and a multinucleated giant cell (indicated by the letter G). Download figure Download PowerPoint The apparent relevance of sos1 to the normal development of the labyrinth trophoblast and spongiotrophoblasts is underscored further by our observation that Sos1 is highly expressed in this layer of normal placentas. By in situ hybridization of sos1+/+ embryos and placentas at DPC12, we found that the spongiotrophoblasts gave the strongest Sos1 RNA signal, followed by the labyrinth trophoblasts (Figure 3A, left side), whose levels were similar to those in the central nervous system and other embryonic tissues (not shown). Immunohistochemistry with specific antibodies confirmed that Sos1 protein was expressed most abundantly in spongiotrophoblasts (Figure 3B, panels a and b). The level of Sos2 in placental tissues appeared to be lower than that of Sos1, with positive Sos2 immunostaining in a few cells of the decidua and in giant cell trophoblasts (Figure 3A and B). Figure 3.Sos1 and Sos2 expression in normal murine placental tissues. In situ hybridization (A) and histochemistry (B) of wild-type DPC12 placental tissues. (A) Left column: in situ hybridization with sos1 antisense (upper panel) and sense (negative control, lower panel) probes, ×50. Right column: in situ hybridization with sos2 antisense (upper panel) and sense (negative control, lower panel) probes. The results show a high sos1 mRNA signal in spongiotrophoblasts, less in labyrinth trophoblasts and none in the decidua. There was less sos2 expression. (B) Immunohistochemistry of normal placenta with Sos1- and Sos2-specific antibodies at low (a and c) and high (b and d) magnification, hematoxylin-stained. (a and b) The results show high expression of Sos1 protein within spongiotrophoblasts and less in the labyrinth. (c and d) Sos2 signal is seen in giant cell trophoblasts but not in spongiotrophoblasts and labyrinth trophoblasts. (e) Negative control of immunohistochemical staining using the same reagents except for the primary Sos1 and Sos2 antibodies (rabbit IgG). Download figure Download PowerPoint Sos1- and Sos2-dependent ERK activation and complex formation in placental lysates of wild-type and mutant Sos1 embryos The results obtained above indicated that homozygous disruption of sos1 was associated with primary placental abnormalities. Since the ERK MAP kinases are located downstream from Ras, ERK activity is often used as a marker of Sos-dependent activity. To determine if there might be a correlation between the sos1 genotype and placental ERK activity, extracts from the fetal portions of placentas were examined (Figure 4A). When the ERK activity of individual DPC10 placentas was analyzed, as determined by the ability of ERK2 immunoprecipitates to phosphorylate myelin basic protein (MBP), it was highest in sos1+/+ placentas (PF1, PF2 and PF4), lowest in an sos1−/− placenta (PF3) and intermediate in an sos1−/− placenta (PF5). The ERK activity in the maternal portion of the sos1+/− placenta (PM4) was much lower than that from the fetal portion (PF4), indicating that possible contamination with the maternal portion of the placenta would not contribute to the ERK activity identified in the fetal portion of the placentas. Figure 4.Analysis of signaling events in placenta. For (A) and (B), individual E10 placentas from Sos1+/− matings were dissected and separated into fetal portion (PF) and maternal portion (PM). (A) Placental ERK activity. Anti-ERK2 immunocomplexes were assayed for kinase activity using MBP as substrate. The middle and lower panels, with the control immunoblots, indicate that the extracts contained comparable amounts of ERK2 and EGFR protein, respectively. (B) Complexes between Sos1 and phosphorylated RTKs and other tyrosine-phosphorylated proteins. Placental extracts were immunoprecipitated with Sos1-specific antibody followed by immunoblotting with anti-phosphotyrosine antibody (PY). (C) Sos1 versus Sos2 complexes with phosphorylated proteins. Extracts from the pooled fetal portions of several wild-type (+/+) E10 placentas were immunoprecipitated with anti-Sos-1 or anti-Sos2 antibodies followed by anti-PY blot. The lower part, with the reblots, shows strong Sos1 and Sos2 signals precipitated by the homologous antibody. Download figure Download PowerPoint The results argue that most of the ERK activity in the DPC10 fetal placenta is Sos1 dependent. Consistent with this interpretation, the relative amount of Sos1 bound to phosphotyrosine-containing proteins of ∼170 kDa, which we interpret to represent primarily activated receptor tyrosine kinases (RTKs), correlated with the relative ERK activity in the individual placental extracts (Figure 4B). The sos1+/+ placenta (PF4) had the strongest signal, while the sos1−/− placenta (PF5) had the weakest. An intermediate signal was seen in the sos1+/− placenta (PF3), with the signal from the maternal portion of the sos1+/− placenta being lower (PM4). In another experiment, wild-type DPC10 extracts pooled from several placentas were used to compare the relative proportion of Sos1 and Sos2 that bound to tyrosine-phosphorylated proteins, including RTKs (Figure 4C). The Sos1 immunoprecipitates gave a much stronger signal than the Sos2 immunoprecipitates (Figure 4C, upper panels), although the control immunoblots displayed similar intensities of precipitated Sos1 and Sos2 protein (lower panels). These results suggest preferential binding of Sos1 to activated RTKs, and other tyrosine-phosphorylated proteins in the placenta, compared with Sos2. Transformation of rodent fibroblasts by v-Src and EGFR is dependent on Sos1 To study sos function in greater detail, DPC9 embryos from matings of sos1+/− animals were used to generate stable, permanent cell lines that were sos1−/−(−/− cells), sos1+/− (+/− cells) and wild type (+/+ cells). Three different −/− cell lines were derived. When preliminary analysis indicated that their growth characteristics were similar, one −/− line was selected for more detailed characterization. As expected, the −/− line contained Sos2 but not Sos1, the +/+ and +/− lines contained both Sos1 and Sos2, with the +/− line having less Sos1 than the +/+ line, and each line contained similar levels of Sos2 (data not shown). The loss of endogenous Sos1 was associated with a slower rate of growth. In 10% fetal calf serum, the generation time of the −/− cells was ∼40 h, while that of the +/+ and +/− lines was ∼25–30 h. Cell transformation by oncogenes represents an alternative approach to examine the integrity of growth-dependent pathways. Since Sos1 lies genetically and biochemically upstream of Ras, the −/− cells should be sensitive to transformation by Ras. To verify that this was the case, −/− cells were infected with various dilutions of Harvey murine sarcoma virus, which contains a mutationally activated rasH gene. The −/− cells were transformed readily by this virus, although they were not quite as sensitive as the +/− or +/+ cells (Table I). Table 1. Transforming activities induced by oncogenes Retrovirus sos1 genotype +/+ +/− −/− v-RasH 6.3 6.5 5.3 v-Src 6.5 5.1 0a EGFR (+EGF) 5.1 3.4 0a Mo-MuLV pseudotyped transforming viruses were prepared by superinfecting NIH 3T3 cells transfected with rescuable v-rasH-, v-Src- or EGFR-expressing retroviral vectors as described (Velu et al., 1987, 1989; DeClue et al., 1991a). Mouse embryo cells with the sos1 +/+, +/− or −/− genotype were infected with 10-fold serial dilutions of virus, and cultured in 2% FBS–DMEM with or without EGF (10 ng/ml) for 7–10 days, when foci of transformed cells were counted. As expected, foci were seen in cells infected with the EGFR virus only if the cells were treated with EGF. The numbers in the table indicate the log10 of the focus-forming titer. Data shown are representative of two experiments. The log10 titers in NIH 3T3 cells, as determined in the same experiment, were 7.3, 6.1 and 5.8, for v-RasH, v-Src and EGFR, respectively. a No foci were seen in the Sos1 −/− cells infected with the v-Src or EGFR virus. In contrast to Ras, many RTK and non-receptor (cytoplasmic) tyrosine kinases (CTKs) have been placed upstream from Sos and/or Ras. Several approaches have led to the conclusion that in many mammalian cell systems transformation by protein tyrosine kinases requires the activation of endogenous Ras (Smith et al., 1986; DeClue et al., 1991b; Nori et al., 1991; Stacey et al., 1991), although in some mammalian systems transformation by the Src protein tyrosine kinase appears to be Ras independent (Aftab et al., 1997; Oldham et al., 1998). To determine whether the presence of Sos2 was sufficient for transformation by CTK and RTK, the −/− cells were infected with a retrovirus encoding a mutationally activated Src protein, which is a CTK, or one encoding the wild-type human EGFR, an RTK. The EGFR virus has been shown previously to induce conditional cell transformation when the cells expressing the EGFR were treated with exogenous ligand such as EGF (Velu et al., 1987). Unlike the Ras-expressing virus, neither the EGFR-encoding virus, in the presence of EGF, nor the Src-encoding virus induced morphological transformation of the −/− cells (Table I), although the cells had been infected successfully by the viruses, as verified by the ability of cell-free filtrates of culture fluid from the infected cells to induce morphological transformation of NIH 3T3 cells, which contain Sos1 (data not shown). Furthermore, both viruses were able to induce the expected transformed phenotypes in +/+ and +/− cells (Table I). The +/− cells were less sensitive than the +/+ cells to transformation, perhaps because the +/− cells have less Sos1 protein than the +/+ cells. The above results suggested that Sos1 is required for cell transformation by Src and EGFR, and that the lack of Sos1 in the −/− cells accounts for their inability to be transformed by these tyrosine kinases. To verify this possibility, a plasmid encoding wild-type Sos1 was transfected into the −/− cells that were infected with the Src retrovirus. This led to the development of numerous areas of constitutive focal transformation for the Src-containing cells (Figure 5). Figure 5.Reconstitution of Sos1 in −/− cells restores the transforming activity of v-Src. −/− cells were transiently transfected with empty vector or a plasmid expressing mouse Sos1 followed by infection with the v-Src or v-Ras virus. The photomicrographs are from the resulting lines. (A) −/− cells transfected with Sos1, not infected with virus (negative control); (B) −/− cells transfected with empty vector and infected with v-Src virus; (C) −/− cells transfected with empty vector and infected with v-Ras virus; and (D) −/− cells transfected with Sos1 and infected with v-Src virus. Download figure Download PowerPoint Short-term treatment with EGF induces similar signaling in +/+ or +/− cell lines The biological observations described above indicated that Sos2 was not able to support transformation by Src and EGFR under the conditions tested, and conversely that the presence of Sos1 was required for transformation by Src and EGFR. These results made it likely that there might be differences between signaling by Sos1 and Sos2, a possibility also raised by the data obtained with placenta extracts (Figure 4C). We therefore compared EGF-dependent Sos1 and Sos2 signaling. We first tested the ability of the cells to respond to short-term EGF treatment. Since the other Ras-specific exchange factors, GRF and GRP, were not expressed in the cell lines (data not shown), the change in Ras·GTP should be a determinant of Sos function. When the change in endogenous Ras·GTP was determined after 5 min EGF treatment (10 ng/ml), a similar increase was seen in the +/+, +/− and −/− cell lines (Figure 6A). As with the placental extracts, we also used the ability of EGF to stimulate ERK2 activity as an index of Sos activity. Consistent with the Ras·GTP results, the EGF-dependent activation of endogenous ERK2 was similar in the +/+ and −/− cells (Figure 6B). Figure 6.Ligand-induced acute signaling in wild-type and Sos1 −/− cells. (A) EGF-dependent changes in Ras·GTP. Subconfluent Sos1 +/+, +/− and −/− cells were deprived of serum for 16 h, then metabolically labeled with [32P]orthophosphate for 10 h and stimulated with 10 ng/ml EGF for 5 min. Cells were then lysed and analyzed for Ras·GTP and Ras·GDP (Zhang et al., 1992). The results represent the average of three experiments. (B) Ligand-induced ERK activity. Serum-deprived cells were treated with or without ligand for 5 min as indicated. Anti-ERK2 immunocomplexes were assayed for kinase activity using MBP as substrate. Data shown are representative of two experiments. Download figure Download PowerPoint Long-term ERK activity differs between +/+ and −/− cell lines Since no obvious differences were obtained between the +/+ and −/− cells subjected to short-term growth factor treatment, we speculated that longer term signaling might be impaired in the −/− cells. To examine this possibility, we used ERK2 activity to compare EGF-dependent signaling in +/+ and −/− cells overexpressing EGFR. As with the analysis of parental cells, there was a strong activation of ERK2 activity when the +/+ and −/− lines overexpressing EGFR were treated with EGF for 10 min (Figure 7A). When the +/+ cells were incubated with EGF for 90 min or longer, the activity of ERK2 remained higher than prior to EGF treatment, although there was as expected a progressive decrease in ERK2 activity, since EGFR activation is followed by its down-regulation and degradation (Figure 7A and B). It is noteworthy, however, that starting with the 90 min time point, the ERK2 activity in the −/− line was much lower than in the +/+ line at each time point (Figure 7A). Figure 7.Comparison of EGF-induced long-term ERK activity in Sos1 +/+ or +/− cells with Sos1-knockout cells. Sos1 +/+, +/− or −/− embryo cells were infected with virus encoding human EGFR. Cells were treated with EGF (10 ng/ml) for the time periods indicated. (A) A 100 μg aliquot of protein from Sos1 +/+ and −/− cell lysates was immunoprecipitated with anti-ERK2 antibody followed by immune complex kinase assay using MBP as a substrate. (B) Equal amounts of protein from EGF-treated Sos1 (−/−) or (+/+) embryo cells were analyzed by immunoblotting with the indicated antibody. (C) A 100 μg aliquot of protein from EGF-treated Sos1 +/− and −/− cell lysates was immunoprecipitated with anti-ERK2 antibody followed by immune complex kinase assay as in (A). The amount of radioactivity present in the phosphorylated MBP was quantitated by a phosphoimager, and the fold increase is shown underneath the figure. (D) The upper part of the gel in (C) was immunoblotted with anti-ERK2 antibody, as a loading control. Download figure Download PowerPoint In contrast to mouse Sos1, mouse Sos2 has been shown to be subject to constitutive ubiquitin-dependent degradation (Nielsen et al., 1997), raising the possibility that the more rapid decrease in ERK activity in the −/− cells might have resulted from a decrease in the steady-state level of Sos2. However, the more rapid decrease in ERK activity was not secondary to a decrease in Sos2 levels upon EGF treatment, as the levels of Sos1 and Sos2 remained stable throughout the 24 h observation period (Figure 7B). Consistent with the interpretation that the absence of Sos1 accounted for the lower EGF-dependent ERK activity seen at longer time points in the −/− cells, the ERK activity was also higher in the +/− cell line at these times (Figure 7C and D). Sos1 forms long-term stable complexes with activated EGFR and Shc; Sos2 complex formation is of shorter duration To explore the mechanism responsible for the reduced longer term ERK signaling in the −/− cells, complex formation was analyzed between Sos and other signaling partners. As noted in the Introduction, EGF treatment induces complex formation between Sos and the activated EGFR. The binding is indirect, being mediated by the Grb2 that is bound to Sos. Some binding occurs via a tripartite complex composed of a Grb2 molecule bound directly to activated EGFR and to Sos, while other binding is mediated by a tetrapartite compl
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