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

Thomas Edouard,Frank Lezoualc'H,Armelle Yart,Maithé Tauber,Béatrice Parfait,Francoise Conte-Auriol,Jean-Philippe Combier,Sophie Bel-Vialar,Jean-Pierre Salles,Stanislas Lyonnet,Audrey Nédélec,Mélanie Métrich,Patrick Raynal

doi:10.1128/mcb.00646-09

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

Highlights

LEOPARD syndrome (LS), a disorder with multiple developmental abnormalities, is mainly due to mutations that impair the activity of the tyrosine phosphatase SHP2 (PTPN11)
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
We analyzed in detail the mechanism of PI3K/AKT upregulation by LS mutants and obtained the following data: (i) reducing GAB1 expression using small interfering RNA (siRNA) prevents PI3K/AKT upregulation in LS patient cells, (ii) adenovirus-driven expression of GAB1-YF3 suppresses AKT upregulation in LS cells, (iii) the amount of GAB1/PI3K complex is higher in LS cells than in cells from healthy subjects or Noonan syndrome (NS) cells, and (iv) recombinant LS SHP2 mutants are inactive in dephosphorylating a peptide carrying a GAB1 PI3K-binding site, whereas WT SHP2 or NS mutants dephosphorylate this peptide more efficiently than a standard protein tyrosine phosphatase (PTP) substrate

Summary

Introduction

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. 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. 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. NS mutations could alter other signaling pathways (Ca2ϩ/NFAT, RhoA, and Src) [17, 42, 44]

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