Activating Mutations Affecting the Dbl Homology Domain of SOS2 Cause Noonan Syndrome

Abstract

The RASopathies constitute a family of autosomal-dominant disorders whose major features include facial dysmorphism, cardiac defects, reduced postnatal growth, variable cognitive deficits, ectodermal and skeletal anomalies, and susceptibility to certain malignancies. Noonan syndrome (NS), the commonest RASopathy, is genetically heterogeneous and caused by functional dysregulation of signal transducers and regulatory proteins with roles in the RAS/extracellular signal-regulated kinase (ERK) signal transduction pathway. Mutations in known disease genes account for approximately 80% of affected individuals. Here, we report that missense mutations altering Son of Sevenless, Drosophila, homolog 2 (SOS2), which encodes a RAS guanine nucleotide exchange factor, occur in a small percentage of subjects with NS. Four missense mutations were identified in five unrelated sporadic cases and families transmitting NS. Disease-causing mutations affected three conserved residues located in the Dbl homology (DH) domain, of which two are directly involved in the intramolecular binding network maintaining SOS2 in its autoinhibited conformation. All mutations were found to promote enhanced signaling from RAS to ERK. Similar to NS-causing SOS1 mutations, the phenotype associated with SOS2 defects is characterized by normal development and growth, as well as marked ectodermal involvement. Unlike SOS1 mutations, however, those in SOS2 are restricted to the DH domain.

Keywords: Noonan syndrome; RAS signaling; SOS2; genotype-phenotype correlations.

Figure 1. Mutations affecting residues located in the Dbl homology domain of SOS2 cause Noonan syndrome

(A) Clinical features of subjects carrying a mutated SOS2 allele. (B) Three-dimensional structure (above) and domain organization (below) of SOS proteins and positions of Noonan syndrome- (NS-) causing mutations. Proteins are shown in the inactive conformation of SOS1 (residues 6–1045) (PDB ID: 3KSY) [Gureasko et al., 2010] and the homology model derived for SOS2 (residues 6–1043). Protein domains are color-coded: Histone-like folds (cyan), Dbl homology (DH) (brown), pleckstrin homology (pink), helical linker (gray), RAS exchange motif (REM) (green), and CDC25 (blue). The cyan plane indicates the membrane position. The side chains of mutated residues (this study and [Lepri et al., 2011]) are shown in the SOS structures, while the NS-causing amino acid substitutions identified in SOS2 and the distribution of SOS1 mutations reported in 83 NS subjects [Lepri et al., 2011] are shown above and below the scheme, respectively. (C) The DH/REM interface is shown with the surface of the REM domain colored by its electrostatic potential and the Cα trace of the DH domain as a ribbon with the mutated residues colored in red (upper panel). Interactions at the DH/REM interface occurring in the SOS2 model (lower panel, left) and SOS1 X-ray structure (right). The residues participating in the inter-domain interactions are shown as color-coded atoms (O: red, N: blue, C: gray, S: yellow). The ribbon is colored red at mutated residues. The interface is stabilized by several salt bridges, and is anionic at the DH domain (Asp263, Glu266, Asp269, Glu270, Asp283, Glu366), and cationic at the REM domain (Arg623, Arg686, Arg692, His693, His697, Lys726, Lys733). Met267 interacts hydrophobically with Leu685 and Trp727. Thr264 stabilizes the structure of the DH domain at the interface with REM by forming an H-bond with Cys²⁸⁰ (black dashed line).

Figure 2. NS-associated SOS2 mutants cause enhanced MEK/ERK activation

(A) SOS2 mutants enhance MEK/ERK activation. FLAG-SOS2 and HA-ERK1 expression constructs were co-transfected into 293T cells. Cells were randomly growing (RG), or starved and then stimulated with EGF (20 ng/ml), as indicated, prior to lysis. Left: a representative immunoblot. Right: quantification of MEK and ERK phosphorylation from three biological replicates (mean ± s.d; ** P < 0.01, * P < 0.05, Bonferroni post-test when ANOVA was significant). (B) SOS2 mutants increase RAS activation. Flp-In T-REx 293 cells stably expressing FLAG-SOS2 constructs were starved, stimulated with 20 ng/ml EGF, and RAS loading was assessed. Left: a representative immunoblot. Right: quantification of RAS loading pooled from two biological replicates (mean ± s.d; * P < 0.05, one-tailed Student’s t test).