Intracellular Reactive Oxygen Species Activate Src Tyrosine Kinase during Cell Adhesion and Anchorage-Dependent Cell Growth

Abstract

Src tyrosine kinases are central components of adhesive responses and are required for cell spreading onto the extracellular matrix. Among other intracellular messengers elicited by integrin ligation are reactive oxygen species, which act as synergistic mediators of cytoskeleton rearrangement and cell spreading. We report that after integrin ligation, the tyrosine kinase Src is oxidized and activated. Src displays an early activation phase, concurrent with focal adhesion formation and driven mainly by Tyr527 dephosphorylation, and a late phase, concomitant with reactive oxygen species production, cell spreading, and integrin-elicited kinase oxidation. In addition, our results suggest that reactive oxygen species are key mediators of in vitro and in vivo v-Src tumorigenic properties, as both antioxidant treatments and the oxidant-insensitive C245A and C487A Src mutants greatly decrease invasivity, serum-independent and anchorage-independent growth, and tumor onset. Therefore we propose that, in addition to the known phosphorylation/dephosphorylation circuitry, redox regulation of Src activity is required during both cell attachment to the extracellular matrix and tumorigenesis.The activation of integrins by the binding of ECM ligands (i.e., fibronectin, vitronectin, and laminin) induces many cellular responses, including attachment, spreading, migration, proliferation, and survival (27). Recently, we demonstrated that integrin engagement induces a transient increase in intracellular reactive oxygen species that peaks at 45 min of cell adhesion. This oxidative burst is at least threefold greater than that arising after growth factor stimulation (10). Intracellular ROS generated following integrin engagement are necessary for integrin signaling during fibroblast adhesion and spreading. 5Ј-LOX is mainly responsible for integrin-mediated ROS generation, with NADPH oxidase playing a marginal role. ROS are also central to the integration of signals from integrin and GFs, acting as synergistic mediators for each signaling pathway through both p125FAK (10) and Rho (24) redox regulation.Important observations on the role of ROS as physiological regulators of tyrosine kinase receptor signaling cascades have shed new light on the possible mechanisms underlying the growth-regulating and tumor-promoting activities of ROS and on the antiproliferative and antitumoral action of some antioxidant agents (1). A growing body of evidence indicates that GF-induced oxygen species are necessary for optimal propagation of mitogenic and antiapoptotic signals through mechanisms that are incompletely understood.The effect of ROS production is the reversible oxidation of proteins (13). Thiols, by virtue of their ability to be reversibly oxidized, are recognized as key targets of oxidative stress. Redox-sensitive proteins include protein tyrosine phosphatases, as their active-site cysteines are the targets of specific oxidation by various oxidants, including H 2 O 2 . This modification can be reversed by intracellular reducing agents (35). Th...

Direct and specific inactivation of protein tyrosine kinases in the Src and FGFR families by reversible cysteine oxidation

“…Our study defines a mechanism by which a group of specific kinases are directly regulated by ROS. A recent study demonstrates that Src is directly oxidized in cells under oxidative stress (22), but the Cys residue responsible for oxidation has not been identified.…”

“…Thus, in cell-based studies it is often difficult to differentiate the direct effect of redox regulation on Src activity from the indirect effects through Src regulators. For example, both activation (22) and inactivation (23,24) of Src in response to oxidative stress have been reported. This inconsistency is likely because the activation or inactivation is accompanied by an increase in Src phosphorylation level on Tyr-416 or Tyr-527.…”

Peroxiredoxin 1 – an antioxidant enzyme in cancer

“…The low‐to‐moderate ROS level is indispensable to normal cellular proliferation, differentiation and survival 3. The net emission of ROS depends upon the balance between free radical production (pro‐oxidative process) and its elimination by antioxidants (antioxidant defense process) 4.…”

Cancer Cell Metabolism

“…ROS arise as a natural by-product of mitochondrial oxidative phosphorylation, oxygen metabolism, and NADPH/NADPH oxidase functions. ROS-dependent posttranslational modifications of the kinase and phosphatase systems that regulate cell growth are important for proliferation and intracellular signaling (Lee et al 2002;Giannoni et al 2005;Cao et al 2009). Similarly, ROS-driven activation of HIF1-responsive genes is an essential mechanism by which normal cells manage their intracellular redox balance.…”