Increased BRAF Heterodimerization Is the Common Pathogenic Mechanism for Noonan Syndrome-Associated RAF1 Mutants

Abstract

Noonan syndrome (NS) is a relatively common autosomal dominant disorder characterized by congenital heart defects, short stature, and facial dysmorphia. NS is caused by germ line mutations in several components of the RAS-RAF-MEK-extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway, including both kinase-activating and kinaseimpaired alleles of RAF1 (ϳ3 to 5%), which encodes a serine-threonine kinase for MEK1/2. To investigate how kinase-impaired RAF1 mutants cause NS, we generated knock-in mice expressing Raf1 D486N . Raf1 D486N/؉ (here D486N/؉) female mice exhibited a mild growth defect. Male and female D486N/D486N mice developed concentric cardiac hypertrophy and incompletely penetrant, but severe, growth defects. Remarkably, Mek/Erk activation was enhanced in Raf1 D486N -expressing cells compared with controls. RAF1 D486N , as well as other kinase-impaired RAF1 mutants, showed increased heterodimerization with BRAF, which was necessary and sufficient to promote increased MEK/ERK activation. Furthermore, kinase-activating RAF1 mutants also required heterodimerization to enhance MEK/ERK activation. Our results suggest that an increased heterodimerization ability is the common pathogenic mechanism for NS-associated RAF1 mutations. N oonan syndrome (NS) is a relatively common (1 in 1,000 to 2,500 live births) autosomal dominant disorder (28,30,45), characterized by short stature, craniofacial dysmorphia, a wide spectrum of congenital cardiac anomalies, and an increased risk of hematopoietic malignancy. Although NS is genetically heterogeneous (1, 4, 49), all known cases are caused by germ line mutations in conserved components of the canonical RAS-RAF-MEK-extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) (here RAS/ERK) cascade, a key regulator of cell proliferation, differentiation, and survival (13, 24). Mutations in PTPN11, which encodes the protein tyrosine phosphatase SHP2, account for approximately one-half of NS cases (46). Other known NS genes include SOS1 (ϳ10%) (38, 47), RAF1 (3 to 5%) (31, 36), KRAS (Ͻ2%) (41, 56), NRAS (9), and SHOC2 (10) (Ͻ1 to 2%). Mutations in some of these genes, as well as in genes encoding other RAS/ERK pathway components, also cause phenotypically related disorders, such as neurofibromatosis type 1 (NF1), Costello syndrome, cardiofacio-cutaneous (CFC) syndrome, and LEOPARD syndrome (named for the major features of this disorder, including multiple lentigines, electrocardiographic conduction abnormalities, ocular hypertelorism, pulmonic stenosis, abnormal genitalia, retardation of growth, and sensorneural deafness); together with NS, these syndromes are now termed "RASopathies" (49). How mutations in the same signaling pathway cause similar yet clearly distinct phenotypes remains unclear. Consequently, a detailed understanding of RASopathy pathogenesis should yield new insights into RAS/ERK pathway regulation.RAF family serine-threonine kinases (22,25,53) function as key RAS effectors, phosphorylating and a...

Ablation of BRaf Impairs Neuronal Differentiation in the Postnatal Hippocampus and Cerebellum

“…Dimer formation in wild-type BRaf and transactivation of CRaf by wild-type BRaf are dependent on the interaction with activated Ras via the RBD domain [13]–[15]. CRaf/BRaf heterodimerization levels have been reported to be significantly reduced in BRaf knockdown cells [56]. Therefore, a BRaf protein lacking exon 3 would be expected to be unable to dimerize.…”

The novel RAF1 mutation p.(Gly361Ala) located outside the kinase domain of the CR3 region in two patients with Noonan syndrome, including one with a rare brain tumor

“…Greater RAF1 kinase activity was determined for amino acid substitutions located in CR2, CR3, and at the end of CR3, while mutations affecting residues 486 and 491 impaired kinase activity (Kobayashi et al, ; Pandit et al, ; Razzaque et al, ). Nevertheless, expression of the NS‐associated RAF1 mutants Ser257Leu, Pro261Ser, Pro261Ala, Val263Ala, and Leu613Val as well as Asp486Asn, Thr491Ile, and Thr491Arg resulted in robust ERK activation indicating increased and prolonged signaling through the RAS‐MEK‐ERK cascade (Pandit et al, ; Razzaque et al, ; Wu et al, ). In a recent study, both kinase‐defective and kinase‐activating RAF1 mutants were found to promote heterodimerization with BRAF providing evidence for a common pathogenic mechanism in this NS subtype (Wu et al, ).…”

The importance of Raf dimerization in cell signaling

“…In work by Wu et al, increased heterodimerization with B-Raf was found to be the common pathogenic mechanism for Rasopathy/Noonan Syndrome (NS) C-Raf mutants. 21 More specifically, Wu and coworkers found that NS C-Raf mutants with impaired (D486N) or intermediate (L613V) kinase activities displayed increased heterodimerization with B-Raf. With the use of the R > H mutation as well as Raf depletion experiments, they went on to show that the upregulation in MEK/ERK signaling induced by these NS C-Raf mutants was dependent upon the interaction with B-Raf.…”