Signal Strength Dictates Phosphoinositide 3-Kinase Contribution to Ras/Extracellular Signal-Regulated Kinase 1 and 2 Activation via Differential Gab1/Shp2 Recruitment: Consequences for Resistance to Epidermal Growth Factor Receptor Inhibition

Abstract

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.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://mcb.asm.org/ .

ACKNOWLEDGMENTS

We thank U. Schaeper for providing the Gab1-ΔGrb2 construct, S. Roche for the plasmid encoding GST-Grb2, and C. Delmas, C. Toulas, and M. O. Jauberteau for providing the glioblastoma cell lines. We are grateful to the vector core of the University Hospital of Nantes, supported by the Association Française contre les Myopathies, for providing the adenovirus vectors.

This work was supported by grants from Association pour la Recherche sur le Cancer and Ligue Nationale Contre le Cancer (comités de Aude, Haute-Garonne, Tarn et Tarn-et-Garonne).