Germ Line Gain of Function with SOS1 Mutation in Hereditary Gingival Fibromatosis

Mutation of human SOS1 is responsible for hereditary gingival fibromatosis type 1, a benign overgrowth condition of the gingiva. Here, we investigated molecular mechanisms responsible for the increased rate of cell proliferation in gingival fibroblasts caused by mutant SOS1 in vitro. Using ectopic expression of wild-type and mutant SOS1 constructs, we found that truncated SOS1 could localize to the plasma membrane, without growth factor stimuli, leading to sustained activation of Ras/MAPK signaling. Additionally, we observed an increase in the magnitude and duration of ERK signaling in hereditary gingival fibromatosis gingival fibroblasts that was associated with phosphorylation of retinoblastoma tumor suppressor protein and the up-regulation of cell cycle regulators, including cyclins C, D, and E and the E2F/DP transcription factors. These factors promote cell cycle progression from G1 to S phase, and their up-regulation may underlie the increased gingival fibroblast proliferation observed. Selective depletion of wild-type and mutant SOS1 through small interfering RNA demonstrates the link between mutation of SOS1, ERK signaling, cell proliferation rate, and the expression levels of Egr-1 and proliferating cell nuclear antigen. These findings elucidate the mechanisms for gingival overgrowth mediated by SOS1 gene mutation in humans. Mutation of human SOS1 is responsible for hereditary gingival fibromatosis type 1, a benign overgrowth condition of the gingiva. Here, we investigated molecular mechanisms responsible for the increased rate of cell proliferation in gingival fibroblasts caused by mutant SOS1 in vitro. Using ectopic expression of wild-type and mutant SOS1 constructs, we found that truncated SOS1 could localize to the plasma membrane, without growth factor stimuli, leading to sustained activation of Ras/MAPK signaling. Additionally, we observed an increase in the magnitude and duration of ERK signaling in hereditary gingival fibromatosis gingival fibroblasts that was associated with phosphorylation of retinoblastoma tumor suppressor protein and the up-regulation of cell cycle regulators, including cyclins C, D, and E and the E2F/DP transcription factors. These factors promote cell cycle progression from G1 to S phase, and their up-regulation may underlie the increased gingival fibroblast proliferation observed. Selective depletion of wild-type and mutant SOS1 through small interfering RNA demonstrates the link between mutation of SOS1, ERK signaling, cell proliferation rate, and the expression levels of Egr-1 and proliferating cell nuclear antigen. These findings elucidate the mechanisms for gingival overgrowth mediated by SOS1 gene mutation in humans. Hereditary gingival fibromatosis (HGF) 2The abbreviations used are: HGF, hereditary gingival fibromatosis; CCNE1, cyclin E1; CCNE2, cyclin E2; EGF, epidermal growth factor; EGFR, EGF receptor; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; PCNA, proliferation cell nuclear antigen; Rb, retinoblastoma tumor suppressor protein; pRB, phospho-Rb; SOS1, Son of Sevenless-1; GEF, guanine nucleotide exchange factor; MEK, MAPK/ERK kinase; HA, hemagglutinin; TRITC, tetramethylrhodamine isothiocyanate; TFDP1, -2, transcription factors DP1 and -2; TBP, TATA-box-binding protein; siRNA, small interference RNA. 2The abbreviations used are: HGF, hereditary gingival fibromatosis; CCNE1, cyclin E1; CCNE2, cyclin E2; EGF, epidermal growth factor; EGFR, EGF receptor; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; PCNA, proliferation cell nuclear antigen; Rb, retinoblastoma tumor suppressor protein; pRB, phospho-Rb; SOS1, Son of Sevenless-1; GEF, guanine nucleotide exchange factor; MEK, MAPK/ERK kinase; HA, hemagglutinin; TRITC, tetramethylrhodamine isothiocyanate; TFDP1, -2, transcription factors DP1 and -2; TBP, TATA-box-binding protein; siRNA, small interference RNA. is a genetic condition characterized by a slowly progressive, benign fibrous enlargement of keratinized gingiva (1Gorlin R.J. 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This truncation abolishes four C-terminal proline-rich motifs, which are required for Grb2 binding. The form of HGF due to SOS1 mutation is designated HGF1. SOS1 functions as a guanine nucleotide exchange factor (GEF) that couples receptor tyrosine kinases to the Ras signaling pathway and controls cell proliferation, differentiation, vesicle trafficking, and regulation of the actin cytoskeleton (5Marshall M.S. Trends Biochem. Sci. 1993; 18: 250-254Abstract Full Text PDF PubMed Scopus (194) Google Scholar). Under the control of two classes of regulatory proteins, GEF and GTPase-activating proteins, Ras functions as a molecular switch between GDP/GTP cycling (6Bourne H.R. Sanders D.A. McCormick F. Nature. 1991; 348: 125-132Crossref Scopus (1836) Google Scholar). Three Ras-GEFs; SOS, guanine nucleotide-releasing factor, and guanyl nucleotide-releasing protein, have been characterized in controlling Ras activation by catalyzing GDP release and association with GTP (7Bar-Sagi D. Hall A. 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Cell. 2003; 112: 685-695Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar). Upon activation of receptor tyrosine kinase, Grb2-SOS1 complexes are recruited to the plasma membrane leading to the exchange of GDP for GTP and Ras activation (14Chardin P. Camonis J.H. Gale N.W. Van Aelst L. Schlessinger J. Wigler M.H. Bar-Sagi D. Science. 1993; 260: 1338-1343Crossref PubMed Scopus (657) Google Scholar). Although the Grb2-SOS1 complex functions exclusively as an Ras activator, SOS1 can also function as a GEF that is specific to the GTPase Rac1. These two distinct catalytic functions of SOS1 are mutually exclusive and reciprocally related (7Bar-Sagi D. Hall A. Cell. 2000; 103: 227-238Abstract Full Text Full Text PDF PubMed Scopus (702) Google Scholar). The activation of Ras signal stimulates downstream signaling pathways, including the mitogen-activated protein kinase (MAPK) family, a ubiquitous signal transduction pathway (15Schlessinger J. Bar-Sagi D. 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Translocation of activated ERK from the cytoplasm to the nucleus is necessary for activation and stabilization of Elk1, c-Jun, c-Myc, and c-Fos. These transcription factors then regulate the expression of genes, such as cyclin D1 and p21WAF1/CIP1, which are critical for the progression from G1 to S phase (26Brunet A. Roux D. Lenormand P. Dowd S. Keyse S. Pouyssegur J. EMBO J. 1999; 18: 664-674Crossref PubMed Scopus (517) Google Scholar, 27Lenormand P. Brondello J.M. Brunet A. Pouyssegur J. J. Cell Biol. 1998; 142: 625-633Crossref PubMed Scopus (193) Google Scholar). Increased cell proliferation has been reported for HGF1 gingival fibroblasts in both monolayer and three-dimensional matrix cultures (28Lee E.J. Jang S.I. Pallos D. Kather J. Hart T.C. J. Dent. Res. 2006; 85: 1050-1055Crossref PubMed Scopus (20) Google Scholar). Here we report that mutant SOS1 contributes an increased and sustained activation of ERK signaling in HGF1 fibroblasts under serum-starved conditions. Sustained ERK signaling leads to increased expression of cell cycle regulators and transcription factors. RNA interference-mediated SOS1 depletion was used to confirm the association between SOS1 mutation, ERK activation, and gingival fibroblast proliferation. These findings document a gain-of-function SOS1 mutation and provide a molecular mechanism for gingival overgrowth in HGF1. Isolation of Fibroblasts and Cell Cultures—Human gingival tissues were obtained with informed consent from three normal subjects (control) and three patients (HGF1). All patients were heterozygous carriers of the SOS1 g.126,142–126,143insC mutation. Three sets of gingival fibroblasts from HGF1 patients and age-matched normal controls (ages 20–24 years) were isolated and grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 1× antibiotic-antimycotic solution (growth medium) at 37 °C in a 5% CO2-humidified incubator and maintained up to 10 passages (28Lee E.J. Jang S.I. Pallos D. Kather J. Hart T.C. J. Dent. Res. 2006; 85: 1050-1055Crossref PubMed Scopus (20) Google Scholar). HeLa cells were obtained from ATCC (Manassas, VA). Plasmid Construction, Transfection, and Subcellular Fractionation—The full-length human SOS1 expression plasmid (pCGN-HASOS1) was a gift from D. Bar-Sagi (State University of New York, Stony Brook, NY). The vector control plasmid was generated by HindIII digestion to release the HA-SOS1 insert and self-ligated. The QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) was used with oligonucleotide (5′AGCATCTGCACCAAATTCTTCCcAAGAACACCGTTAACACCTCC-3′, GenBank™ accession number L13857) to generate the mutated pCGN-HASOS1 construct that carried the HGF1 mutation (small case and cytosine insertion). The mutation was confirmed by DNA sequencing. For transient transfection, 15 × 104 cells/well were seeded in 6-well plates a day before experiments. Expression constructs (2 μg) were transfected into primary gingival fibroblasts using JetPEI reagent (ISC Bioexperss, Kaysville, UT) or into HeLa cells by Lipofectamine 2000 (Invitrogen). Transfection was terminated 48–72 h post-transfection, and total cellular lysates were obtained (29Jang S.I. Steinert P.M. J. Biol. Chem. 2002; 277: 42268-42279Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). To study the subcellular distribution of SOS1, 100 × 104 HeLa cells were plated in 10-cm dishes 24 h before transfection. Eight micrograms of indicated expression constructs were used, and transfected cells were harvested 48 h post-transfection. Cellular fractions were isolated using the ProteoExtract Subcellular Proteome Extraction kit (EMD Biosciences, La Jolla, CA). Cell Proliferation and Ras Activation Assays—For proliferation assays, 1 × 104 cells/well of primary gingival fibroblasts were seeded in 48-well plates. After 24 h the growth medium was replaced with Dulbecco's modified Eagle's medium containing 20 mm HEPES (starving medium) for overnight. The medium was then replaced with growth medium and maintained for 9 days. Cells from triplicate wells were trypsinized at each time point, and the total cell number was determined by using a Coulter Counter (Beckman Z series). To monitor Ras activation, cells were seeded in 150-mm culture dishes, grown to 85% confluence, and serum-starved for 16 h. After treatment with EGF (25 ng/ml) for 0.5–6.0 h at 37 °C, cellular extracts were collected in lysis buffer (25 mm Tris-HCl, pH 7.5, 150 mm NaCl, 10 mm MgCl2, 1 mm EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, 10% glycerol, 2.0 mm phenylmethanesulfonyl fluoride, 20 μm Na3VO4, 10 μm NaF, 1 μg/ml pepstatin A, and 10 μg/ml aprotinin plus protease inhibitor tablet (Complete tablet, Roche Applied Science). Ras activity was measured using a Ras Activation kit (Upstate, Charlottesville, VA). For each reaction, 600 μg of whole cell lysate was incubated with Raf-1 Ras binding domain-agarose (15 μg) for 1 h at 4 °C. The complexes were collected by centrifugation and washed five times with buffer (25 mm Tris-HCl, pH 7.5, 150 mm NaCl, 10 mm MgCl2, 1 mm EDTA, 10% glycerol, 20 μm NaF, and 1% Nonidet P-40). Protein complexes were released with SDS sample buffer, separated by 4–12% NuPAGE, and transferred to a polyvinylidene difluoride membrane. Proteins were detected by mouse anti-Ras antibody (0.05 μg/ml) and goat anti-mouse horseradish peroxidase-conjugated secondary antibody (BioRad, 1:5000). Culture Treatment, Western Blot Analyses, and Indirect Immunofluorescence Staining—To monitor the level of phosphorylated ERK1/2, the cultures were either maintained in growth medium or switched to starving medium for 16 h before addition of EGF (Upstate) with indicated concentrations and incubation times. In some experiments, cultures were treated with AG1478 (EGFR inhibitor, 10 μm, EMD Biosciences) or PD98059 (MEK inhibitor, 10 μm, EMD Biosciences) 30 min prior to the addition of EGF as indicated (Fig. 2, legend). At each time point, cultures were washed with cold phosphate-buffered saline, and whole cellular extracts were prepared by adding lysis buffer directly into monolayer cultures. Isolation of nuclear extracts and Western blotting were conducted as previously described (29Jang S.I. Steinert P.M. J. Biol. Chem. 2002; 277: 42268-42279Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). After washing with phosphate-buffered saline with 0.1% Tween 20, the primary antibodies were detected with a polyclonal goat anti-rabbit or anti-mouse IgG coupled with horseradish peroxidase. Primary antibodies included mouse anti-HA (16B12, 1:1500, Covance, Berkeley, CA), mouse anti-β-actin (1:800, Sigma), mouse anti-SOS1(N) (1:250, epitope at N-terminal, BD Bioscience Pharmingen), rabbit anti-SOS1(C) (1:500, epitope at C-terminal), rabbit anti-Egr-1 (1:300), rabbit anti-EGFR (1:300), rabbit anti-phospho-retinoblastoma (pRb, Ser-780), rabbit anti-retinoblastoma (Rb), and mouse anti-PCNA (1:500) from Santa Cruz Biotechnology, rabbit anti-extracellular signal-regulated kinase (ERK1/2, 1:2000), and rabbit anti-phospho-ERK1/2 (1:2000) from Upstate. Signals were detected by using ECL Western blotting Detection Reagents (Amersham Biosciences) and exposed to x-ray film (XAR, Kodak). All experiments were conducted at least three times and quantitated using a FluorChem digital imaging system (Alpha Innotech, San Leandro, CA) and National Institutes of Health Image 1.63 software. Results were adjusted for loading controls. Immunofluorescent staining was conducted as previously described (30Jang S.I. Kalinin A. Takahashi K. Marekov L.N. Steinert P.M. J. Cell Sci. 2005; 118: 781-793Crossref PubMed Scopus (42) Google Scholar). After transfection, gingival fibroblasts were maintained in either growth or starving medium for 16 h and fixed with paraformaldehyde (3.7%) for 15 min at room temperature. Cells were incubated with anti-HA (1:1000) for 1 h at room temperature. After washing, the cells were incubated with goat anti-mouse conjugated with TRITC IgG antibody for 30 min. After washing, the cell nuclei were stained with 4′,6-diamidino-2-phenylindole, dihydrochloride (Sigma) for 5 min at room temperature and washed three additional times. Slides were examined with fluorescence microcopy (Olympus, IX71), and images were processed using Adobe Photoshop CS. Real-time PCR—Total RNA from equivalent cell densities of control and HGF1 fibroblast cultures maintained either in starving medium for 16 h or switched to growth medium for 1 day were prepared by TRIzol solution (Invitrogen) and the “RNeasy Mini kit” (Qiagen). RNA quality and quantity were determined using a Bioanalyzer (Agilent 2100, Wilmington, DE). Reverse transcription-PCR experiments were conducted as described previously (32Liu W. Akhand A.A. Kato M. Yokoyama I. Miyata T. Kurokawa K. Uchida K. Nakashima I. J. Cell Sci. 1999; 112: 2409-2417Crossref PubMed Google Scholar) with modification. TaqMan probes and PCR primers were purchased from ABI Biosystems (Foster City, CA). These included SOS1 (Hs00362308_m1), E2F1 (Hs00153451_m1), E2F2 (Hs00 231667_m1), transcription factor DP1 (TFDP1, Hs00955488_m1), transcription factor DP2 (TFDP2, Hs00232366_m1), cyclin E1 (CCNE1, Hs00233356_m1), and cyclin E2 (CCNE2, Hs00180319_m1). The RNase P or TATA-box-binding protein (TBP) was used as endogenous control for normalization. QPCR Human Reference Total RNA (Stratagene) was used as a calibrator in all quantitative reverse transcription-PCR experiments. Relative levels of the indicated transcripts in each sample were calculated as 2–ΔΔCt, where ΔΔCt = [target gene's Ct – TBP Ct]sample – [target gene's Ct – TBP Ct]calibrator. Experiments were performed at least three times with triplicate samples. RNA Interference Knock-down and Focus Oligoarray Analysis—Duplex RNA of SOS1 target sequences flanking the cytosine insertion were synthesized with the Silencer siRNA Construction Kit (Ambion). Transient transfection experiments were conducted as previously described (30Jang S.I. Kalinin A. Takahashi K. Marekov L.N. Steinert P.M. J. Cell Sci. 2005; 118: 781-793Crossref PubMed Scopus (42) Google Scholar). Briefly, 18 × 104 cells/well of gingival fibroblasts were plated in 6-well plates 24 h before transfection. Double-stranded siRNA alone or together with SOS1 expression constructs (1 μg/35-mm) was introduced into HeLa cells by Lipofectamine 2000 (Invitrogen). The siRNA for Luciferase (5′-CTTACGCTGAGTACTTCGA) (31Lewis D.L. Hagstrom J Loomis A. G.E. Wolff J.A. Herweijer H. Nat. Gene. 2002; 32: 107-108Crossref PubMed Scopus (525) Google Scholar) was used as a control. Total cellular lysates were collected after 48-h transfection and subjected to Western blot analyses. To monitor the effects of knock-down on proliferation, fibroblasts were seeded at 8000/well into 48-well plates. Cultures were transfected with indicated siRNA (50 nm) overnight and replaced with fresh growth medium (day 0). At each time point, the total number of cells was determined as described above. Experiments were performed at least three times with triplicate samples. To monitor gene expression in response to EGF treatment, normal (control), and HGF1 fibroblast cultures were serum-starved for 16 h and treated with EGF (50 ng/ml) for 24 h. Total RNA from EGF-treated and untreated cultures was prepared as described above. The complementary RNA probe was synthesized (TrueLabeling-Am kit, SuperArray Bioscience, Frederick, MD) together with biotin-16-UTP labeling. The complementary RNA probes were incubated with cell cycle microarray membranes, and the expression levels of each gene were detected with chemiluminescence using a copalyl diphosphate-star substrate (SuperArray Bioscience). Membranes were exposed to x-ray film (Kodak, XAR film) and signal analyzed by the GEArray Expression Analysis Suite (SuperArray Bioscience) with subtraction of background and normalization with housekeeping genes of glyceraldehyde-3-phosphate dehydrogenase and β-actin. Statistical Analyses—For comparisons of proliferation assays, real-time PCR experiments and quantification of phospho-ERK levels, the Student's t test was used to compare the mean values between HGF1 and controls. Values of p < 0.05 were considered significant. Expression of Mutant SOS1 in Gingival Fibroblasts—The insertion mutation of SOS1 in HGF1 results in an early termination as illustrated in Fig. 1A. To verify the presence of truncated SOS1 in HGF1 fibroblasts, antibody with the epitope against the N-terminal region of SOS1 (SOS1(N)), detected one band (∼170 kDa) in controls and two bands (170 and 130 kDa) in HGF1 patients (Fig. 1B, upper panel). The 170-kDa band corresponds to the full-length SOS1 and the smaller ∼130-kDa band approximates the calculated molecular weight of the truncated mutant SOS1. To confirm this, expression constructs of wild-type and mutant SOS1 were transfected into primary gingival fibroblasts. As shown in Fig. 1B (lower panel), the expressed wild type (arrow a, lanes 2 and 5) and mutant (arrow b, lanes 3 and 6) bands were detected at the same positions as the endogenous full-length (lanes 1 and 3) and truncated SOS1 (lanes 4 and 5). Western blotting results revealed the intensity of the mutant (lower) band was 40% less than the full-length (upper) band in HGF1 samples (Fig. 1B). The lower amount of mutant SOS1 could result from the instability of either its transcript or its protein product. Real-time PCR experiments were conducted to monitor the total levels, of both wild-type and mutant SOS1 transcript in HGF1 fibroblasts. Overall levels of SOS1 transcript were 40% lower in HGF1 fibroblasts than in control fibroblasts (Fig. 1C). The TBP transcript served as an internal control. The lower levels of mutant SOS1 protein in HGF1 fibroblasts likely reflect a less stable mutant SOS1 transcript. Because all three sets of controls and HGF1 patients showed similar results, only results from one set of control and HGF1 fibroblasts are presented. Sustained Activation of MAPK in HGF1 Gingival Fibroblasts—Because SOS1 plays a critical role in the Ras/MAPK/ERK signaling pathway (22Nguyen T.T. Scimeca J.C. Filloux C. Peraldi P. Carpentier J.L. Van Obberghen E. J. Biol. Chem. 1993; 268: 9803-9810Abstract Full Text PDF PubMed Google Scholar), the effect of the SOS1 mutation on Ras signaling was evaluated. Although Ras activity was low in serum-starved control fibroblasts, it increased rapidly after EGF treatment and subsequently deceased, approaching basal levels by 30 min (Fig. 2A). In contrast, Ras activity was 5-fold higher in HGF1. Upon EGF stimulation, Ras activity in HGF1 fibroblasts showed higher levels than control in all respective time intervals suggesting mutant SOS1 remained active even under serum-starved conditions, leading to higher Ras activity in response to growth factor stimuli. We next studied how the SOS1 mutation altered signal transduction through the MAPK/ERK pathway. As shown in Fig. 2B, panel a (lanes 2–5), transient activation of ERK1/2 with increasing concentrations of EGF resulted in a dose-related increase in both control and HGF1 fibroblasts. The magnitude of ERK1/2 activation was at least 30% greater in HGF1 than in control fibroblasts at each respective dosage of EGF treatment (Fig. 2B, panel b). In untreated cells, whereas the ERK1 signal was weak but detectable only in HGF1 samples, the ERK2 signal alone showed ∼3-fold greater intensity in HGF1 than in control fibroblasts (Fig. 2B, panel a, lane 1). These data suggest Ras remains active and leads to a sustained activation of ERK1/2 signal in serum-starved HGF1 fibroblasts. Activation of ERK1/2 by EGF was evaluated in the presence of the selective pharmacological inhibitors AG1478 and PD98059 (32Liu W. Akhand A.A. Kato M. Yokoyama I. Miyata T. Kurokawa K. Uchida K. Nakashima I. J. Cell Sci. 1999; 112: 2409-2417Crossref PubMed Google Scholar, 33Pang L. Sawada T. Decker S.J. Saltiel A.R. J. Biol. Chem. 1995; 270: 13585-13588Abstract Full Text Full Text PDF PubMed Scopus (895) Google Scholar). The presence of PD98059 or AG1478 resulted in a similar degree of reduction on phospho-ERK signal in both cell types (Fig. 2C, panel a, lanes 1–4). The overall phospho-ERK1/2 level remained higher in HGF1 than in control fibroblasts (Fig. 2C, panel b). The duration of ERK activation was studied through the continued induction of EGF. In the presence of EGF, the phospho-ERK signal increased and peaked at the 1-h time point in both cell types (Fig. 2D). The signal gradually returned to basal levels after 6 h (Fig. 2D, panel a). Although the pattern of phospho-ERK response to EGF induction was similar in both cell types, the level was 50% higher in HGF1 than in control fibroblasts (Fig. 2D, panel b). The effect of PD98059 on the duration of EGF-induced ERK signaling is shown in Fig. 2E. Activation of ERK signaling was reduced in the presence of MEK inhibitor in both cell types (Fig. 2E, panel a). However, the pattern of reduction was different in HGF1 compared with control cells (Fig. 2E, panel b). After 20-min incubation with inhibitor, 30% of phospho-ERK remained in control fibroblasts, whereas >80% of phospho-ERK remained in HGF1 fibroblasts. The phospho-ERK signal was sustained up to 2 h in HGF1 but not in control fibroblasts. At 4 h, phospho-ERK decreased to basal levels in controls, however, significant levels of active ERK signal remained in HGF1 fibroblasts. These results demonstrate that, in the absence of growth factors, Ras together with its downstream ERK1/2 signaling remained active and sustained in HGF1 fibroblasts. Transient induction by EGF elicited a stronger response, both in magnitude and duration, indicating the functional consequence of the SOS1 mutation in HGF1 fibroblasts. Subcellular Localization of Mutant SOS1 in Gingival Fibroblasts—Because binding of growth factors to their receptors triggers the recruitment of the SOS1-Grb2 complex from the cytoplasm to plasma membrane (34Daub H. Wallasch C. Lankenau A. Herrlich A. Ullrich A. EMBO J. 1997; 16: 7032-7044Crossref PubMed Scopus (588) Google Scholar), we examined the distribution of SOS1 in subcellular fractions. In HGF1 fibroblasts, the endogenous full-length SOS1 was found in both cytosol and membrane fractions, with only trace amounts of mutant SOS1 detected in the membrane fraction (data not shown). This is due to the low expression level of the endogenous mutant SOS1 in HGF1 fibroblasts (Fig. 1B). Therefore, the HA-tagged full-length and truncated SOS1 constructs were expressed in HeLa cells, which have higher transfection efficiency than primary gingival fibroblasts (data not shown). Under starving conditions, <40% of full-length SOS1 was distributed in the membrane/organelle fraction (Fig. 3A, lane 4), whereas >60% of truncated SOS1 was found in membrane/organelle fraction (lane 6). The SOS1(C) antibody, which only detects full-length SOS1, detected <30% of endogenous full-length SOS1 was distributed in the membrane/organelle fraction (compare lanes 1 and 2 with lanes 5 and 6). The identity of the additional (upper) band found in the membrane/organelle fraction (lanes 2 and 6) remains unknown. These results demonstrate the relative ratio of distribution is higher for truncated SOS1 in the membrane/organelle fraction. Immunofluorescence staining revealed that, under growth conditions, both wild-type and mutant SOS1 localized in the cytoplasm and cellular membrane (Fig. 3B, panels a and b, arrows). However, in serum-starved fibroblasts, whereas wild-type SOS1 restricted in the cytoplasm (Fig. 3B, panel c), mutant SOS1 localized in both the cytoplasm and plasma membrane (Fig. 3B, panel d, arrows). These observations were supported by the immunostaining on the distribution of endogenous SOS1 in serum-starved fibroblasts (Fig. 3C). These results suggest that, in the absence of growth factors, truncated SOS1 localized in the plasma membrane even though it lacks the carboxyl-proline-rich domains. Mutant SOS1 Leads to Stronger ERK Signaling in HGF1 Fibroblasts—To test if the presence of mutant SOS1 leads to increa