Fig 2: Details of the SPRED1-NF1 Interaction Interface in the Ternary Complex and the Impact of Pathogenic Mutations in SPRED1 and NF1 on the SPRED1-NF1 Interaction(A and B) Enlarged view of the SPRED1(EVH1)-NF1(GRD) interface formed by residues present in the (A) GAPex and (B) GAPc regions of NF1 in the ternary complex. SPRED1(EVH1) and NF1(GRD) are colored light magenta and green, respectively. The residues that participate in the protein-protein interaction are indicated in stick representation. Intermolecular hydrogen bonds and salt bridges are indicated by dashed black lines.(C) Schematic representation of the SPRED1-NF1 interaction interface as identified by PDBSum (http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=index.html/). The interactions are indicated in colors using the notation described for Figure 2C.(D) The missense mutations reported in the SPRED1(EVH1) domain in Legius syndrome. The text color represents the mutation type: red, confirmed pathogenic; yellow, suspected pathogenic; black, unclassified or uncertain. The mutations tested in this study are indicated in bold.(E) Binding affinities (measured using ITC) for Legius syndrome pathogenic mutations (stick representation) in SPRED1. NF1 is indicated in an electrostatic surface representation.(F) Binding affinities (measured using ITC) for neurofibromatosis type 1 pathogenic mutations (stick representation) in NF1. SPRED1 is shown in an electrostatic surface representation.See also Figure S3.

Fig 3: Domain Organization and Overall Structure of the Ternary Complex Formed by Neurofibromin (GRD), SPRED1(EVH1), and KRAS Proteins(A) Domain organization of KRAS, neurofibromin (NF1), and SPRED1 showing the presence of various domains in the full-length protein. The red line drawn above each protein indicates the domains involved in complex formation and used for our structural studies. In NF1, the GAPc and GAPex regions are highlighted in light and dark green, respectively.(B) The overall structure of the complex formed by GMPPNP-bound KRAS (light brown), NF1(GRD) (light green), and SPRED1(EVH1) (light pink) proteins. In the left panel, the ternary complex is indicated in ribbon representation, whereas in the right panel, proteins are indicated in surface representation. In both panels, GMPPNP and Mg2+ bound to KRAS are indicated as sticks and spheres, respectively.See also Figure S1 and Table S1.

Fig 4: Identification of SPRED1 Phosphorylation that Disrupts SPRED1-NF1 Interaction(A) Proteins precipitated from extracts of HEK293T cells transiently transfected with FLAG-NF1 and EGFR(L858R) constructs were assessed by western blot for endogenous SPRED1 binding. WCL, whole-cell lysate; IP, immunoprecipitation.(B) Identification of SPRED1 phosphorylation sites downstream of EGFR(L858R) in HEK293T cells by SPRED1-FLAG and EGFR(L858R) transient transfection, anti-FLAG IP, and LC-MS/MS.(C) Amino acid sequence alignment of SPRED1 flanking serine 105 across indicated species shows evolutionary conservation.(D) Proteins precipitated from extracts of HEK293T cells transiently transfected with phosphomimetic and phosphodeficient SPRED1(S105) mutant constructs were assessed by western blot for endogenous NF1 binding.(E) Enlarged view of the NF1 electrostatic surface that interacts with SPRED1(S105). The S105E mutation results in a significant loss of binding affinity between SPRED1 and NF1.See also Figure S5.

Fig 5: Phosphomimetic and Phosphodeficient SPRED1(S105) Alter RAS-GTP Signaling following EGF Stimulation and K562 Proliferation, along with a Model of SPRED1(S105) Phosphorylation(A) HEK293T cells were transiently transfected with indicated SPRED1-FLAG constructs, serum starved for 16 h, and stimulated with 10 ng/mL EGF for the indicted time points. Downstream signaling was then assessed by western blot and RAS-GTP pull-down assay.(B) K562 cells were infected with SPRED1-IRES-GFP, SPRED1-IRES-GFP mutants, and empty vector expressing retrovirus. Three days after infection, baseline GFP-positive cells were measured by flow cytometry and normalized to 1. During this competition assay, GFP-positive cells were monitored over time to measure the effect of SPRED1 expression on proliferation. The statistical significance of the difference between indicated samples was determined using a two-way ANOVA: ***p < 0.001.(C) Cancer cell lines were infected with SPRED1-FLAG-expressing retrovirus, selected with 1 μg/mL puromycin, anti-FLAG IP, and LC-MS/MS as described above. The y axis: MS1 chromatographic peak areas, arbitrary units relative to control.(D) Oncogenic EGFR-mediated SPRED1(S105) phosphorylation model. Phosphorylated SPRED1(S105) is unable to bind NF1 and inhibit RAS-GTP signaling.See also Figure S6.