Noonan syndrome-associated Raf1 mutants with increased or decreased kinase activity differentially activate Erk and cause distinct syndromic phenotypes

Benjamin G. Neel1,2, Xue Wu1,2, Jeremy Simpson3,4, Jenny H. Hong1,2, Kyoung-Han Kim3, Nirusha K. Thavarajah 2, Peter H. Backx3, and Toshiyuki Araki2
1Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada
2Campbell Family Cancer Research Insitute, Ontario Cancer Institute and Princess Margaret Hospital,  University Health Network, Toronto, ON, Canada
3Department of Physiology and Medicine, Heart and Stroke/Richard Lewar Centre, University of Toronto, Toronto, ON, Canada
4Department of Human Health & Nutritional Sciences, University of Guelph, Guelph, ON, Canada

Noonan syndrome (NS) is one of several  “RASopathies” caused by mutations in different components of the RAS-RAF-MEK-ERK MAPK pathway. Germ line mutations in RAF1 (encoding the serine-threonine kinase RAF1) account for ~3-5% of NS. Unlike other NS alleles, RAF1 mutations that confer increased kinase activity are highly associated with HCM.  Surprisingly, other NS-associated RAF1 mutations show normal or decreased kinase activity. To explore the pathogenesis of NS-associated RAF1 mutations, we generated  “knock-in” mice that express kinase-activating (L613V) and impaired (D486N) RAF1 mutants, respectively. Like NS patients, L613V/+ mice had short stature, craniofacial dysmorphia and hematologic abnormalities. Valvuloseptal development was normal, but L613V/+ mice exhibited eccentric cardiac hypertrophy and aberrant cardiac fetal gene expression, and decompensated following pressure overload. D486N heterozygotes and, surprisingly, homozygotes, were obtained at the expected Mendelian ratio. Male D486N/+ mice did not show NS phenotypes, whereas D486N/+ females had mildly decreased body size. D486N/D486N mice showed cardiac hypertrophy, but not dilatation, and one third had severe growth defects. Agonist-evoked MEK/ERK activation was enhanced in multiple cell types from either Raf1L613V or Raf1D486N mutants, although L613V had a much stronger effect. Moreover,  in response to agonist stimulation, both types of  Raf1 mutant formed more heterodimers with Braf than did wild-type Raf1, and shRNA and mutational analysis indicate that heterodimers are required for mutant action. Hence, NS-associated kinase-active and impaired  RAF1 alleles act as gain-of-function mutants on the RAS-RAF-MEK-ERK pathway by promoting increased heterodimerization with BRAF. Finally, post-natal MEK inhibition normalized all NS defects in L613V/+ mice. We conclude that different NS genes have intrinsically distinct pathological effects, that enhanced MEK-ERK activity is critical for HCM and other RAF1-mutant NS phenotypes, and suggest a mutation-specific approach to  RASopathy therapy.