EGF‐induced ERK‐activation downstream of FAK requires rac1‐NADPH oxidase

Abstract

Reactive oxygen species (ROS) function as signaling molecules mainly by reversible oxidation of redox-sensitive target proteins. ROS can be produced in response to integrin ligation and growth factor stimulation through Rac1 and its effector protein NADPH oxidase. One of the central roles of Rac1-NADPH oxidase is actin cytoskeletal rearrangement, which is essential for cell spreading and migration. Another important regulator of cell spread is focal adhesion kinase (FAK), a coordinator of integrin and growth factor signaling. Here, we propose a novel role for NADPH oxidase as a modulator of the FAK autophosphorylation site. We found that Rac1-NADPH oxidase enhanced the phosphorylation of FAK at Y397. This site regulates FAK's ability to act as a scaffold for EGF-mediated signaling, including activation of ERK. Accordingly, we found that EGF-induced activation of FAK at Y925, the following activation of ERK, and phosphorylation of FAK at the ERK-regulated S910-site depended upon NADPH oxidase. Furthermore, the inhibition of NADPH oxidase caused excessive focal adhesions, which is in accordance with ERK and FAK being modulators of focal adhesion dissociation. Our data suggest that Rac1 through NADPH oxidase is part of the signaling pathway constituted by FAK, Rac1, and ERK that regulates focal adhesion disassembly during cell spreading.

The role of small GTPase Rac1 in stress signaling

“…PM association of Rac1 initiates downstream signaling pathways such as integrin signaling for membrane ruffling and ERK signaling for focal adhesion (Zheng et al 2010;Flinder et al 2011). Consistent with previous studies documented for human hepatocyte cell line HepG2 (Ozaki et al 2000) our present study confirmed that as a response of growth factor stimulation, endogenous Rac1 translocates to the PM in melanoma cells ( Figure 17) and presumably induces its downstream signaling pathway which can thus stimulates heat shock factor activation and HSP70 expression.…”

“…Rac1 has numerous roles in normal physiology and disease state of cell functionality such as cell cycle regulation (Michaelson et al 2008), lamellipodia formation, membrane ruffling (Steffen et al 2013), regulation of NADPH oxidase activity in NADPH complex (Flinder et al 2011), cellular adhesion (Lawson & Burridge 2014), proliferation (Woodcock et al 2010), survival, differentiation and malignant transformation (Wertheimer et al 2012), cell migration, actin polymerization, spreading (Kovac et al 2013). Rac1 is also important in various oncogenesis pathways including initiation, progression, invasion and metastasis (Davis et al 2013) and stress sensing (Han et al 2001).…”

Rosmarinic Acid Inhibits Cell Growth and Migration in Head and Neck Squamous Cell Carcinoma Cell Lines by Attenuating Epidermal Growth Factor Receptor Signaling

“…Elevated ROS is a main trigger of cell proliferation and migration [59,60]. Here, we show that cells pretreated with RA reduced EGF-induced total ROS production and H 2 O 2 , specifically, in HNSCC cell lines (Figure 7).…”

Selective Inhibition by Apocynin of the Proliferation and Adhesion to Fibronectin of v-H-<i>ras</i>-Transformed 3Y1 Cells

“…It is intriguing that recent studies have revealed that integrin engagement with an extracellular matrix resulted in the recruitment of focal adhesion kinase to the cytoplasmic membrane and activated Nox. 20) This event may be an alternate route for activating Nox independently of H-ras activation. Our results imply that combined 1-integrin expression and Nox-derived ROS production deteriorate normal cell proliferation and adhesion to extracellular matrices.…”