Functional Effects of PTPN11 (SHP2) Mutations Causing LEOPARD Syndrome on Epidermal Growth Factor-Induced Phosphoinositide 3-Kinase/AKT/Glycogen Synthase Kinase 3β Signaling

Abstract

LEOPARD syndrome (LS), a disorder with multiple developmental abnormalities, is mainly due to mutations that impair the activity of the tyrosine phosphatase SHP2 (PTPN11). How these alterations cause the disease remains unknown. We report here that fibroblasts isolated from LS patients displayed stronger epidermal growth factor (EGF)-induced phosphorylation of both AKT and glycogen synthase kinase 3␤ (GSK-3␤) than fibroblasts from control patients. Similar results were obtained in HEK293 cells expressing LS mutants of SHP2. We found that the GAB1/phosphoinositide 3-kinase (PI3K) complex was more abundant in fibroblasts from LS than control subjects and that both AKT and GSK-3␤ hyperphosphorylation were prevented by reducing GAB1 expression or by overexpressing a GAB1 mutant unable to bind to PI3K. Consistently, purified recombinant LS mutants failed to dephosphorylate GAB1 PI3K-binding sites. These mutants induced PI3K-dependent increase in cell size in a model of chicken embryo cardiac explants and in transcriptional activity of the atrial natriuretic factor (ANF) gene in neonate rat cardiomyocytes. In conclusion, SHP2 mutations causing LS facilitate EGF-induced PI3K/AKT/GSK-3␤ stimulation through impaired GAB1 dephosphorylation, resulting in deregulation of a novel signaling pathway that could be involved in LS pathology.Noonan syndrome (NS) (MIM163950) is a relatively frequent (ϳ1/2,000 births) autosomal dominant disease primarily characterized by facial dysmorphism, heart defects, and short stature. LEOPARD syndrome (LS) (MIM151100) is a rarer but related disorder that associates, roughly, NS symptoms with deafness and cutaneous abnormalities. Both NS and LS belong to the family of "neuro-cardio-facial-cutaneous" (NCFC) syndromes, a group of developmental disorders that display different combinations of the above-mentioned symptoms with mental retardation and tumor predisposition (4, 13).At least 80% of LS and 50% of NS patients carry germ line missense mutations in PTPN11, the gene encoding SHP2. SHP2 is a widely expressed protein tyrosine phosphatase (PTP) that contains Src homology 2 (SH2) domains and promotes Rasmitogen-activated protein kinase (MAPK) activation through different molecular mechanisms (14,27,39,47). Other LS or NS forms are caused by mutations in KRAS, SOS1, RAF1, BRAF, and SHOC2 genes, which encode key factors of the Ras-MAPK pathway (6,32,34,37,38,41).Although genetic studies have provided essential advances, how PTPN11 mutations cause the diseases' symptoms remains an open question. Biochemical studies have shown that NS mutations are located at contact points between the catalytic and the SH2 domains and therefore disrupt SHP2 autoinhibitory conformation (11,19), thereby stimulating SHP2 catalytic activity (gain-of-function mutations). Conversely, LS mutations are confined within the catalytic domain and repress SHP2 activity (15,21,40).A key issue in understanding LS/NS pathogenesis is the identification of the signaling pathways that are altered by these mutations. It is now well ac...

New Approaches to Prevent LEOPARD Syndrome-associated Cardiac Hypertrophy by Specifically Targeting Shp2-dependent Signaling

“…Importantly, our novel findings demonstrate that pharmacological inhibition of particular signaling steps upstream of mTOR could become feasible therapeutic approaches in the clinical arena. Our results complement the recent findings of two other groups showing that inhibition of phosphoinositide 3 kinase with LY294002 or wortmannin is also effective against the prohypertrophic effects of LS mutations in Shp2 (8,9). This is not surprising because PI3K is an important activator of Akt and, in turn, is regulated by FAK (28,29).…”

MECHANISMS IN ENDOCRINOLOGY: Novel genetic causes of short stature

“…Mutations in these genes can also cause short stature without obvious clinical features (158). Inhibition of IGF1 release via GH-induced ERK hyperactivation or EGF-induced PI3K/AKT/GSK-3b stimulation may contribute to short stature in patients with PTPN11 mutations (159,160). Genetic aberrations in several other intracellular pathways play a role in short stature syndromes.…”

Developmental SHP2 dysfunction underlies cardiac hypertrophy in Noonan syndrome with multiple lentigines

“…Developmental SHP2 dysfunction underlies cardiac hypertrophy in Noonan syndrome with multiple lentigines Jessica Lauriol, 1 Janel R. Cabrera, 1 Ashbeel Roy, 1 Kimberly Keith, 1 Sara M. Hough, 1 Federico Damilano, 1 Bonnie Wang, SHP2 Y279C and SHP2 T468M mutants promote a hypertrophic phenotype in cardiomyocytes through impaired GAB1 dephosphorylation, leading to increased PI3K/AKT activity (35). Furthermore, we showed previously that hearts from an NSML mouse model using a knockin of the Y279C Ptpn11 allele (Shp2 Y279C/+ mice, also called Ptpn11 Y279C/+ mice) have elevated PI3K/mTOR activity and that rapamycin-mediated mTOR inhibition both prevents and reverses adult-onset HCM (28).…”