Interaction between a Domain of the Negative Regulator of the Ras-ERK Pathway, SPRED1 Protein, and the GTPase-activating Protein-related Domain of Neurofibromin Is Implicated in Legius Syndrome and Neurofibromatosis Type 1

Abstract

Constitutional heterozygous loss-of-function mutations in the SPRED1 gene cause a phenotype known as Legius syndrome, which consists of symptoms of multiple café-au-lait macules, axillary freckling, learning disabilities, and macrocephaly. Legius syndrome resembles a mild neurofibromatosis type 1 (NF1) phenotype. It has been demonstrated that SPRED1 functions as a negative regulator of the Ras-ERK pathway and interacts with neurofibromin, the NF1 gene product. However, the molecular details of this interaction and the effects of the mutations identified in Legius syndrome and NF1 on this interaction have not yet been investigated. In this study, using a yeast two-hybrid system and an immunoprecipitation assay in HEK293 cells, we found that the SPRED1 EVH1 domain interacts with the N-terminal 16 amino acids and the C-terminal 20 amino acids of the GTPase-activating protein (GAP)-related domain (GRD) of neurofibromin, which form two crossing ␣-helix coils outside the GAP domain. These regions have been shown to be dispensable for GAP activity and are not present in p120 GAP . Several mutations in these N-and C-terminal regions of the GRD in NF1 patients and pathogenic missense mutations in the EVH1 domain of SPRED1 in Legius syndrome reduced the binding affinity between the EVH1 domain and the GRD. EVH1 domain mutations with reduced binding to the GRD also disrupted the ERK suppression activity of SPRED1. These data clearly demonstrate that SPRED1 inhibits the Ras-ERK pathway by recruiting neurofibromin to Ras through the EVH1-GRD interaction, and this study also provides molecular basis for the pathogenic mutations of NF1 and Legius syndrome.Spred (Sprouty-related protein with an EVH1 domain) family proteins, initially discovered as c-Kit-and c-Fms-binding proteins, have been shown to suppress the Ras-ERK pathway. Spreds form a subfamily of the Sprouty/Spred family, which is characterized by the Sprouty-related C-terminal cysteine-rich (SPR) 3 domain. In mammals, four Sprouty homologues and three members of the Spred family of proteins, Spred1, Spred2, and Spred3, have been discovered. Mammalian Spreds can negatively regulate the Ras-Raf-ERK pathway (1, 2); however, compared with Spred1 and Spred2, Spred3 has much weaker ERK suppression activity (3). The SPR domain has been shown to be palmitoylated, causing Sprouty and Spred to localize in the membrane fraction (4, 5). Spred1 and Spred2 are composed of an N-terminal enabled/vasodilator-stimulated protein homology 1 (EVH1) domain, a central c-Kit-binding domain, and a cysteine-rich C-terminal SPR domain, whereas Spred3 lacks a functional c-Kit-binding domain.

SPRED1 Interferes with K-ras but Not H-ras Membrane Anchorage and Signaling

“…Another study showed that overexpression of SPRED1 inhibits Ras activation, as evidenced by decreased levels of GTP-bound Ras (5, 8). Consistent with this last model, SPRED1 was reported to reduce GTP-Ras levels and MAPK signaling by corecruiting neurofibromin (NF1), a Ras-inactivating and GTPase-activating protein (GAP), to the plasma membrane (5,(8)(9)(10).…”

“…Our new mechanism does not exclude the possibility that the GTPase-activating protein (GAP) NF1 is in addition recruited to the membrane together with SPRED1, as suggested before (9,10). NF1 recruitment may then in addition deactivate Ras.…”

“…Another study showed that overexpression of SPRED1 inhibits Ras activation, as evidenced by decreased levels of GTP-bound Ras (5, 8). Consistent with this last model, SPRED1 was reported to reduce GTP-Ras levels and MAPK signaling by corecruiting neurofibromin (NF1), a Ras-inactivating and GTPase-activating protein (GAP), to the plasma membrane (5,(8)(9)(10).Despite these mechanistic uncertainties, the important role of SPRED proteins in developmental processes is well appreciated. In vivo studies of SPRED1-or SPRED2-deficient mice have demonstrated that lack of a single SPRED protein causes dysfunctions in bone development, hematopoietic processes, and allergen-induced airway eosinophilia, without affecting viability and fertility of the mice (3,11,12).…”

RASopathy Gene Mutations in Melanoma

“…SPRED1 acts as a tumor suppressor because it enhances NF1 inhibitory activity by recruiting the protein to the plasma membrane and to RAS (Stowe et al, 2012; Hirata et al, 2016) (Figure 1). Mutations in SPRED1 act in an autosomal-dominant manner in Legius and Neurofibromatosis 1-like (NF1-like) syndromes, mild forms of neurofibromatosis type 1 carrying skin features such as multiple café-au-lait macules, but no neurofibromas (Brems et al, 2012).…”

Manifestações Cutâneas das Rasopatias

“…37 A SL poderá associar-se a um risco aumentado de neoplasias, estando descritos casos de leucemia mieloblástica aguda em crianças com SL. 38 Esta associação deve-se ao papel do gene SPRED1 na regulação da hematopoiese, por intermédio da inibição da proteína ERK. A ausência de atividade da proteína Spred-1 provoca uma hiperatividade da via RAS/MAPK.…”