RIT1 belongs to the RAS family of small GTPases. Germline and somatic RIT1 mutations have been identified in Noonan syndrome (NS) and cancer, respectively. By using heterologous expression systems and purified recombinant proteins, we identified the p21-activated kinase 1 (PAK1) as novel direct effector of RIT1. We found RIT1 also to directly interact with the RHO GTPases CDC42 and RAC1, both of which are crucial regulators of actin dynamics upstream of PAK1. These interactions are independent of the guanine nucleotide bound to RIT1. Disease-causing RIT1 mutations enhance protein-protein interaction between RIT1 and PAK1, CDC42 or RAC1 and uncouple complex formation from serum and growth factors. We show that the RIT1-PAK1 complex regulates cytoskeletal rearrangements as expression of wild-type RIT1 and its mutant forms resulted in dissolution of stress fibers and reduction of mature paxillin-containing focal adhesions in COS7 cells. This effect was prevented by co-expression of RIT1 with dominant-negative CDC42 or RAC1 and kinase-dead PAK1. By using a transwell migration assay, we show that RIT1 wildtype and the disease-associated variants enhance cell motility. Our work demonstrates a new function for RIT1 in controlling actin dynamics via acting in a signaling module containing PAK1 and RAC1/CDC42, and highlights defects in cell adhesion and migration as possible disease mechanism underlying NS. Noonan syndrome (NS) belongs to the RASopathies, a group of developmental diseases caused by mutations in genes encoding RAS-MAPK pathway components. Germline mutations in RIT1 have been identified in NS. RIT1 belongs to the RAS superfamily, however, the cellular function of RIT1 remains elusive. We show that RIT1 binds p21-activated kinase 1 (PAK1), an effector of the RHO GTPases RAC1 and CDC42, which are important regulators of cytoskeletal dynamics. NS-associated RIT1 mutants enhance complex formation between RIT1, RAC1/CDC42 and PAK1. Expression of wild-type or mutant forms of RIT1 caused loss of stress fibers and mature focal adhesions and enhanced cell motility. Our data suggest that dysfunction in actin dynamics is a novel aspect in the pathophysiology of RASopathies.

Costello syndrome (CS) is a developmental disorder characterized by postnatal reduced growth, facial dysmorphism, cardiac defects, mental retardation and skin and musculo-skeletal defects. CS is caused by HRAS germline mutations. In the majority of cases, mutations affect Gly¹² and Gly¹³ and are associated with a relatively homogeneous phenotype. The same amino acid substitutions are well known as somatic mutations in human tumors and promote constitutive HRAS activation by impairing its GTPase activity. In a small number of cases with mild phenotype, a second class of substitutions involving codons 117 and 146 and affecting GTP/GDP binding has been described. Here, we report on the identification and functional characterization of two different three-nucleotide duplications resulting in a duplication of glutamate 37 (p.E37dup) associated with a homogeneous phenotype reminiscent of CS. Ectopic expression of HRAS^E37dup in COS-7 cells resulted in enhanced growth factor-dependent stimulation of the MEK-ERK and phosphoinositide 3-kinase (PI3K)-AKT signaling pathways. Recombinant HRAS^E37dup was characterized by slightly increased GTP/GDP dissociation, lower intrinsic GTPase activity and complete resistance to neurofibromin 1 GTPase-activating protein (GAP) stimulation due to dramatically reduced binding. Co-precipitation of GTP-bound HRAS^E37dup by various effector proteins, however, was inefficient because of drastically diminished binding affinities. Thus, although HRAS^E37dup is predominantly present in the active, GTP-bound state, it promotes only a weak hyperactivation of downstream signaling pathways. These findings provide evidence that the mildly enhanced signal flux through the MAPK and PI3K-AKT cascades promoted by these disease-causing germline HRAS alleles results from a balancing effect between a profound GAP insensitivity and inefficient binding to effector proteins.

Costello syndrome is a congenital disorder comprising a characteristic face, severe feeding difficulties, skeletal, cardiac and skin abnormalities, intellectual disability and predisposition to malignancies. It is caused by heterozygous germline HRAS mutations mostly affecting Gly¹² or Gly¹³, which impair HRAS-GTPase activity and result in increased downstream signal flow independent of incoming signals. Functional analyses of rarer HRAS mutations identified in individuals with attenuated Costello syndrome phenotypes revealed altered GDP/GTP nucleotide affinities (p.K117R) and inefficient effector binding (p.E37dup). Thus, both phenotypic and functional variability associated with HRAS mutations are evident. Here, we report on a novel heterozygous HRAS germline mutation (c.187_207dup, p.E63_D69dup) in a girl presenting with a phenotype at the milder end of the Costello syndrome spectrum. The p.E63_D69dup mutation impaired co-precipitation of recombinant HRAS with NF1 GTPase-activating protein (GAP) suggesting constitutive HRAS^E63_D69dup activation due to GAP insensitivity. Indeed, we identified strongly augmented active HRAS^E63_D69dup that co-precipitated with effectors RAF1, RAL guanine nucleotide dissociation stimulator and phospholipase C1. However, we could not pull down active HRAS^E63_D69dup using the target protein PIK3CA, indicating a compromised association between active HRAS^E63_D69dup and PIK3CA. Accordingly, overexpression of HRAS^E63_D69dup increased steady-state phosphorylation of MEK1/2 and ERK1/2 downstream of RAF, whereas AKT phosphorylation downstream of phosphoinositide 3-kinase (PI3K) was not enhanced. By analyzing signaling dynamics, we found that HRAS^E63_D69dup has impaired reagibility to stimuli resulting in reduced and disrupted capacity to transduce incoming signals to the RAF-MAPK and PI3K-AKT cascade, respectively. We suggest that disrupted HRAS reagibility, as we demonstrate for the p.E63_D69dup mutation, is a previously unappreciated molecular pathomechanism underlying Costello syndrome.