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 3β (GSK-3β) 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-3β 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-3β stimulation through impaired GAB1 dephosphorylation, resulting in deregulation of a novel signaling pathway that could be involved in LS pathology.

This work was supported by grants from the Agence Nationale de la Recherche (Programme Maladies Rares), Association pour la Recherche sur le Cancer, Ligue Nationale contre le Cancer (sections Aude, Haute-Garonne, Tarn, and Tarn-et-Garonne), E-Rare (NS-EuroNet), and Fondation pour la Recherche Médicale.

We are grateful to T. Levade for cell line immortalization. We thank E. Olson, R. Hipskind, S. Roche, C. Racaud-Sultan, and B. Mariamé for reagents; E. Agius for valuable assistance in organotypic culture; M. Mus, A. Bros, and N. Malet for valuable technical help; A. Montagner and M. Dance for helpful discussions; and F. L'Faqihi and V. Duplan-Eche for FACS analysis.