Noonan syndrome (NS) is one of several autosomal dominant “RASopathies” caused by mutations in components of the RAS-RAF-MEK-ERK MAPK pathway. Germ line mutations in RAF1 (encoding the serine-threonine kinase for MEK1/2) account for ~3-5% of NS, and unlike other NS alleles, RAF1 mutations that confer increased kinase activity are highly associated with hypertrophic cardiomyopathy (HCM). Notably, some NS-associated RAF1 mutations show normal or decreased kinase activity. To explore the pathogenesis of such mutations, I generated “knock-in” mice that express kinase-activating (L613V) or -impaired (D486N) Raf1 mutants, respectively. Knock-in mice expressing the kinase-activated allele Raf1L613V developed typical NS features (short stature, facial dysmorphia, haematological abnormalities), as well as HCM. As expected, agonist-evoked Mek/Erk activation was enhanced in multiple cell types expressing Raf1L613V. Moreover, postnatal Mek inhibition normalized the growth, facial, and cardiac defects in L613V/+ mice, showing that enhanced Mek/Erk activation by Raf1 mutant is critical for evoking NS phenotypes. 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 Raf1D486N-expressing cells compared with controls. In both mouse and human cells, RAF1D486N, as well as other kinase-impaired RAF1 mutants, show increased heterodimerization with BRAF, which is necessary and sufficient to promote increased MEK/ERK activation. Furthermore, kinase-activating RAF1 mutants also require heterodimerization to enhance MEK/ERK activation. Our results suggest that increased heterodimerization ability is the common pathogenic mechanism for NS-associated RAF1 mutations.

Noonan syndrome (NS) is one of several autosomal dominant “RASopathies” caused by mutations in components of the RAS-RAF-MEK-ERK MAPK pathway. Germ line mutations in RAF1 (encoding the serine-threonine kinase for MEK1/2) account for ~3-5% of NS, and unlike other NS alleles, RAF1 mutations that confer increased kinase activity are highly associated with hypertrophic cardiomyopathy (HCM). Notably, some NS-associated RAF1 mutations show normal or decreased kinase activity. To explore the pathogenesis of such mutations, I generated “knock-in” mice that express kinase-activating (L613V) or -impaired (D486N) Raf1 mutants, respectively. Knock-in mice expressing the kinase-activated allele Raf1L613V developed typical NS features (short stature, facial dysmorphia, haematological abnormalities), as well as HCM. As expected, agonist-evoked Mek/Erk activation was enhanced in multiple cell types expressing Raf1L613V. Moreover, postnatal Mek inhibition normalized the growth, facial, and cardiac defects in L613V/+ mice, showing that enhanced Mek/Erk activation by Raf1 mutant is critical for evoking NS phenotypes. 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 Raf1D486N-expressing cells compared with controls. In both mouse and human cells, RAF1D486N, as well as other kinase-impaired RAF1 mutants, show increased heterodimerization with BRAF, which is necessary and sufficient to promote increased MEK/ERK activation. Furthermore, kinase-activating RAF1 mutants also require heterodimerization to enhance MEK/ERK activation. Our results suggest that increased heterodimerization ability is the common pathogenic mechanism for NS-associated RAF1 mutations.

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京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第19145号 / 生博第328号 / 新制||生||44(附属図書館) / 32096 / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 松田 道行, 教授 原田 慶恵, 教授 豊島 文子 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM