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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.

SHP2 was recently found to down-regulate PI3K activation by dephosphorylating Gab1 but the mechanisms explaining the positive role of the Gab1/SHP2 pathway in EGF-induced Ras activation remain ill defined. Substrate trapping experiments now suggest that SHP2 dephosphorylates other Gab1 phosphotyrosines located within a central region displaying four YXXP motifs. Because these sites are potential docking motifs for Ras-GAP, we tested whether SHP2 dephosphorylates them to facilitate Ras activation. We observed that a Gab1 construct preventing SHP2 recruitment promoted membrane relocation of RasGAP. Moreover, a RasGAP-inactive mutant restored the activation of Ras in cells transfected with SHP2-inactivating Gab1 mutant or in SHP2-deficient fibroblasts, supporting the hypothesis that RasGAP is a downstream target of SHP2. To determine whether Gab1 is a RasGAP-binding partner, a Gab1 mutant deleted of four YXXP motifs was produced. The deletion suppressed RasGAP redistribution and restored the defective Ras activation caused by SHP2-inactivating mutations. Moreover, Gab1 was found to interact with RasGAP SH2 domains, only under conditions where SHP2 is not activated. To identify Ras-GAP-binding sites, Tyr to Phe mutants of Gab1 YXXP motifs were produced. Gab1 constructs mutated on Tyr(317) were severely affected in RasGAP binding and were the most active in compensating for Ras-defective activation and blocking RasGAP redistribution induced by SHP2 inactivation. We have thus localized on Gab1 a Ras-negative regulatory tyrosine phosphorylation site involved in RasGAP binding and showed that an important SHP2 function is to down-regulate its phosphorylation to disengage RasGAP and sustain Ras activation.

Methyl-β-cyclodextrin (MβCD) was used to explore a role for cholesterol-enriched plasma membrane microdomains in coupling lysophosphatidic acid (LPA) stimulation to phosphoinositide 3-kinase (PI3K) activation. Cholesterol depletion strongly inhibited the production of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate in Vero cells stimulated with LPA. In agreement, the phosphorylation of Akt/protein kinase B, but not of Erk kinases, was suppressed by MβCD. MβCD did not interfere with the overall phospholipid metabolism, and its effects were reversed in cholesterol add-back experiments. Finally, PI3K was detected in lipid rafts prepared from control but not MβCD-treated cells, suggesting that these microdomains contribute to LPA signalling by compartmentalising component(s) of the PI3K pathway. [Copyright &y& Elsevier]

Phosphoinositide 3-kinase (PI3K) participates in extracellular signal-regulated kinase 1 and 2 (ERK1-2) activation according to signal strength, through unknown mechanisms. We report herein that Gab1/Shp2 constitutes a PI3K-dependent checkpoint of ERK1-2 activation regulated according to signal intensity. Indeed, by up- and down-regulation of signal strength in different cell lines and through different methods, we observed that Gab1/Shp2 and Ras/ERK1-2 in concert become independent of PI3K upon strong epidermal growth factor receptor (EGFR) stimulation and dependent on PI3K upon limited EGFR activation. Using Gab1 mutants, we observed that this conditional role of PI3K is dictated by the EGFR capability of recruiting Gab1 through Grb2 or through the PI3K lipid product PIP3, according to a high or weak level of receptor stimulation, respectively. In agreement, Grb2 siRNA generates, in cells with maximal EGFR stimulation, a strong dependence on PI3K for both Gab1/Shp2 and ERK1-2 activation. Therefore, Ras/ERK1-2 depends on PI3K only when PIP3 is required to recruit Gab1/Shp2, which occurs only under weak EGFR mobilization. Finally, we show that, in glioblastoma cells displaying residual EGFR activation, this compensatory mechanism becomes necessary to efficiently activate ERK1-2, which could probably contribute to tumor resistance to EGFR inhibitors. [ABSTRACT FROM AUTHOR]