Focal Adhesion Kinase Overexpression Enhances Ras-dependent Integrin Signaling to ERK2/Mitogen-activated Protein Kinase through Interactions with and Activation of c-Src

David D Schlaepfer,Tony Hunter

doi:10.1074/jbc.272.20.13189

David D Schlaepfer, Tony Hunter

Open Access

https://doi.org/10.1074/jbc.272.20.13189

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Abstract

Cell adhesion to extracellular matrix proteins such as fibronectin (FN) triggers a number of intracellular signaling events including the increased tyrosine phosphorylation of the cytoplasmic focal adhesion protein-tyrosine kinase (PTK) and also the stimulation of the mitogen-activated protein kinase ERK2. Focal adhesion kinase (FAK) associates with integrin receptors, and FN-stimulated phosphorylation of FAK at Tyr-397 and Tyr-925 promotes the binding of Src family PTKs and Grb2, respectively. To investigate the mechanisms by which FAK, c-Src, and Grb2 function in FN-stimulated signaling events to ERK2, we expressed wild type and mutant forms of FAK in human 293 epithelial cells by transient transfection. FAK overexpression enhanced FN-stimulated activation of ERK2 approximately 4-fold. This was blocked by co-expression of the dominant negative Asn-17 mutant Ras, indicating that FN stimulation of ERK2 was Ras-dependent. FN-stimulated c-Src PTK activity was enhanced by wild type FAK expression, whereas FN-stimulated activation of ERK2 was blocked by expression of the c-Src binding site Phe-397 mutant of FAK. Expression of the Grb2 binding site Phe-925 mutant of FAK enhanced activation of ERK2, whereas a kinase-inactive Arg-454 mutant FAK did not. Expression of wild type and Phe-925 FAK, but not Phe-397 FAK, enhanced p130(Cas) association with FAK, Shc tyrosine phosphorylation, and Grb2 binding to Shc after FN stimulation. FN-induced Grb2-Shc association is another pathway leading to activation of ERK2 via Ras. The inhibitory effects of Tyr-397 FAK expression show that FAK-mediated association and activation of c-Src is essential for maximal signaling to ERK2. Moreover, multiple signaling pathways are activated upon the formation of an FAK.c-Src complex, and several of these can lead to Ras-dependent ERK2 mitogen-activated protein kinase activation.

Highlights

The family of transmembrane integrin receptors mediate cell adhesion to the extracellular matrix and trigger intracellular signaling events such as the stimulation of the mitogenactivated protein kinase ERK2 [1,2,3,4,5]
There may be more than one signaling pathway upstream of Ras, since antibody-mediated clustering of integrins in suspended cells can generate signals to ERK2 in the absence of Focal adhesion kinase (FAK) tyrosine phosphorylation [37], whereas presentation of fibroblasts to an insoluble FN matrix stimulates FAK tyrosine phosphorylation, transient c-Src association, and Grb2 binding in a time course that parallels ERK2 activation
FAK Overexpression Enhances but Dominant Negative Ras (Asn-17) Inhibits ERK2 Activation by FN Stimulation—To test the role of FAK in adhesion-mediated signal transduction events to ERK2, site-directed mutagenesis was used to create single-site phenylalanine replacements of FAK tyrosine residues (397, 407, 861, and 925) that have been shown to be phosphorylated in vivo [23, 24, 27]

Summary

Introduction

The family of transmembrane integrin receptors mediate cell adhesion to the extracellular matrix and trigger intracellular signaling events such as the stimulation of the mitogenactivated protein kinase ERK2 [1,2,3,4,5]. In human 293 epithelial cells, FAK overexpression enhanced c-Src kinase activity and FN-stimulated signaling to ERK2, whereas dominant negative Ras expression blocked ERK2 activation without affecting FAK tyrosine phosphorylation or c-Src activity.

Results

Conclusion