Article Figures & Data
Figures
- Fig. 1.
The Sos N terminus is required for transformation by myristoylated Sos1. The structures of wild-type Sos1, myristoylated Sos1 (MyrSos1), and the derived deletion mutants are shown. These Sos1 constructs were transfected into NIH 3T3 cells and assayed for focal transformation as described in Materials and Methods. Data represent mean number of foci per 0.5 μg of DNA from five separate experiments.
- Fig. 2.
Subcellular fractionation of cells expressing of myristoylated Sos mutant proteins. Stable clones of NIH 3T3 cells expressing control wild-type (wt) Sos1, full-length MyrSos1, or deleted versions of MyrSos1 proteins (see Fig. 1 for structures) were metabolically labeled with [35S]methionine (A) or unlabeled (B) and fractionated as described in Materials and Methods. The cytosolic (lanes C) and membrane (lanes M) fractions were immunoprecipitated with anti-Sos antibodies. The labeled samples (A) were analyzed by autoradiography after SDS-PAGE. Alternatively, to avoid the interference of background signals near the region of the gel in which the deleted MyrSos1 proteins migrated, the nonlabeled samples (B) were subjected to SDS-PAGE and analyzed by immunoblotting using anti-Sos antibodies. Wild-type Sos1 served as a control in each panel. Similar results were seen in two separate experiments.
- Fig. 3.
Reduced transforming activity of MyrSos1 and MyrSos1-ΔC with substitution mutations in the Dbl and PH domains. (A) Structures of substitution mutations in Dbl and PH domains. A seven-residue cluster mutation was introduced into the Sos Dbl homology domain (indicated as Dbl∼ in this and other figures). A single-point mutation was introduced into the N-terminal or C-terminal region of the Sos PH domain, (PH∗ or PH#, respectively). PH∗/# represents the presence of both point mutations in the PH domain, and Dbl∼PH∗/# stands for the combination of all mutations. (B and C) The full-length MyrSos1 and C-terminally truncated MyrSos1-ΔC carrying the designated substitution mutations were transfected into NIH 3T3 cells. Panel B shows the focal transforming efficiency of MyrSos1 and its derivatives; panel C shows that of MyrSos1-ΔC and its derivatives. Data shown are summarized from four separate experiments (four readings per experiment). For each mutant, the bar shows the relative transforming activity and standard error compared with that of MyrSos1 (B) or MyrSos1-ΔC (C).
- Fig. 4.
Reduced MAPK activity of MyrSos1 Dbl and PH mutants in COS-7 cells. Cells were transiently cotransfected with HA-MAPK and either empty vector, MyrSos1, or the indicated MyrSos1 substitution mutants. The basal HA-MAPK activity was determined following anti-HA immunoprecipitation using MBP as the substrate (top panel). Compared with MyrSos1, the amounts of MBP were 39% for the PH∗/# mutant, 42% for the Dbl∼ mutant, 26% for the Dbl-PH∗/# mutant, and 5% for the empty vector. The expression of exogenous HA-MAPK was examined by anti-HA immunoblotting (bottom panel). Data shown are representative for three separate experiments.
- Fig. 5.
The Sos1 N terminus interferes with MyrSos1-induced focal transformation. (A) MyrSos1 was cotransfected either with vector or interfering DNA constructs as indicated. The resultant focus-forming activity from MyrSos1-vector (mean number of foci per 0.5 μg of DNA, as in Fig. 1) was designated 100%. The relative (bar, standard error) focus-forming activities from cotransfection of MyrSos1 with the interfering DNAs relative to vector control represent the average from five separate experiments. (B) MyrSos1 or plasmid pPA90, which contains v-ras H, was cotransfected with either vector or MyrSos1-NT. The resultant focus-forming activities from MyrSos1-vector and pPA90-vector were each designated 100%. The relative (bar, standard error) focus-forming activities from cotransfection of pPA90 or MyrSos1 with MyrSos1-NT compared with their activities with the vector control represent the average from three separate experiments. The mean numbers of foci induced by the pPA90-vector and MyrSos1-vector were 1,860/0.5 μg of DNA and 920/0.5 μg of DNA, respectively.
- Fig. 6.
Inhibition of mitogen-dependent growth by a myristoylated Sos N-terminal fragment: importance of Dbl and PH domains. NIH 3T3 cells transfected with either vector or interfering DNA constructs were grown in low serum containing G418 and various mitogens as described in Materials and Methods. After 2 weeks, the dishes were stained with hematoxylin, and the colonies were counted. (A) Stained dishes obtained after growth in EGF. (B) Quantitation of colony formation. For each mitogen (EGF, PDGF, LPA, and 10% serum), the mean number of colonies (a colony contained >30 cells) obtained from vector-transfected cells was designated 100%. The relative colony-forming efficiencies of cells expressing MyrSos1-NT and MyrSos1-NT(Dbl∼PH∗/#) are shown. The data represent the average of three independent experiments.
- Fig. 7.
The myristoylated Sos N terminus inhibits EGF-induced MAPK activity in NIH 3T3 cells; importance of Dbl and PH domains. NIH 3T3 cells transiently cotransfected with HA-MAPK and empty vector, MyrSos1-NT, or MyrSos1-NT(Dbl∼PH∗/#) were serum starved and then treated with EGF (50 ng/ml) for 5 min at 37°C. The immunocomplexes were formed and analyzed for in vitro MAPK assays as described in Materials and Methods and in the legend to Fig. 4. Compared with the vector alone (100%), the amounts of MBP were 48% for MyrSos1-NT and 71% for MyrSos1-NT(Dbl∼PH∗/#). Expression of the exogenous HA-MAPK in these transiently transfected cells was confirmed by anti-HA blotting. Similar results were obtained in two other experiments.
- Fig. 8.
The myristoylated Sos N terminus inhibits EGF-induced Sos-dependent Ras activation; importance of Dbl and PH domains. The proportion of Ras in the GTP-bound form in cell cultures stably transfected with empty vector (A), MyrSos1-NT(Dbl∼PH∗/#) (B), and MyrSos1-NT (C) was determined following a 5-min treatment with or without EGF as indicated. In vivo levels of Ras-GTP were quantitated as described previously (43). Sos1 immunoblot (insert) of corresponding cellular lysates of each cell line demonstrates endogenous level of Sos1 and ectopic of MyrSos1 polypeptides.
- Fig. 9.
EGF-induced association of Sos1 with the activated EGFR requires the Sos N terminus and C terminus. (A and B) Complex formation between exogenous (exo.) Sos proteins and the EGFR. Extracts from NIH 3T3 cells stably transfected with the designated epitope-tagged Sos fragment were serum deprived and treated with EGF for 5 min (+) or left untreated (−). Cell extracts were prepared and immunoprecipitated with antitag antibodies (anti-c-Myc [9E10] [A] and anti-HA [B, left panel]). The endogenous (endo.) Sos1 protein was also immunoprecipitated with anti-Sos antibodies to serve as an internal control (B, right panel). The immunoprecipitates were subjected to SDS-PAGE and transferred to membranes for immunoblotting. The membranes were first probed with antiphosphotyrosine antibody (top panels) and then reprobed with anti-Sos antibodies after stripping (bottom panels). (C) Association between endogenous Sos1 proteins with EGFR. Cell extracts from EGF-treated or untreated control cells and stable NIH 3T3 transfected clones expressing MyrSos1-NT or MyrSos1-NT(Dbl∼PH∗/#) (Fig. 8) were immunoprecipitated with anti-Sos1 antibody follows by antiphosphotyrosine immunoblotting (top). The stripped membranes were then reprobed with anti-Sos antibody (bottom). All data shown are representative of three independent experiments.
