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

Abstract

Accumulating evidence suggests that protein tyrosine phosphorylation-based signaling pathways are under the regulation of reactive oxygen species. Although protein tyrosine phosphatases are directly regulated by reversible oxidation, it is not clear whether protein tyrosine kinases (PTKs) are also directly regulated by reduction/oxidation (redox). In this study we report a mechanism of direct oxidative inactivation specific for the PTKs in the Src and fibroblast growth factor receptor (FGFR) families, key enzymes in mammalian signal transduction. Src is fully active when reduced and retains 8 -25% of the full activity toward various substrates when oxidized. This inactivation is caused by oxidation of a specific cysteine residue (Cys-277), which results in homodimerization of Src linked by a disulfide bridge. Cys-277 is located in the Gly loop in the catalytic domain. This cysteine residue is conserved only in 8 of the >90 PTKs in the human kinome, including 3 of the 10 Src family kinases and all 4 kinases of the FGFR family. FGFR1 is also reversibly regulated by redox because of this cysteine residue, whereas Csk, a PTK that lacks a cysteine residue at the corresponding position, is not similarly regulated. These results demonstrate a mechanism of direct redox regulation conserved in certain specific PTKs. R eactive oxygen species (ROS), such as hydrogen peroxide and superoxide, can alter the function of proteins by oxidizing free sulfhydryl groups to sulfenic, sulfinic, or sulfonic acids (1, 2). Cellular responses to ROS are historically considered a damage-control mechanism to certain pathological situations that lead to oxidative stress. However, recent studies indicate that certain growth factors and cell adhesion also stimulate the production of ROS, which serve as secondary messengers to regulate downstream signaling pathways (3, 4). Numerous protein phosphorylation pathways respond to ROS (5-9), and identifying the proteins and the residues sensitive to oxidation will help elucidate the mechanism of cross-talk between redox and protein tyrosine phosphorylation.The level of protein tyrosine phosphorylation is the function of opposing actions of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). All PTPs contain a catalytic Cys residue in the active site, and oxidation of this residue leads to the inactivation of the PTP activity (10). This response is recognized as a major mechanism by which ROS regulate the level of protein tyrosine phosphorylation. However, whether PTKs also directly respond to ROS is not established. PTK Src is a key regulator of cell survival, cytoskeleton reorganization, DNA synthesis, and cell division (11,12). A number of studies suggest that Src also plays an important role in cellular response to ROS, because Src specific inhibitors and dominantnegative Src mutants strongly attenuate cellular response to ROS (13-16). However, how ROS regulate Src activity has been controversial, likely reflecting the complexity of Src regulation. Src contains regulatory str...

FLT3-driven redox-modulation of Ezrin regulates leukaemic cell migration

“…Subsequent studies by Senga et al (2000) identified a number of cysteine residues in Src necessary for the enzymes stability and transformative ability. Giannoni et al (2005) focused on Cys245 and Cys487 which were demonstrated to undergo intramolecular Although these studies support a model whereby Src is activated in response to ROS, data consistent with an inhibitory role for ROS in Src activity has also been proffered by Kemble et al (2009). In contrast to the majority of published studies, yet echoing the earlier results of Cunnick et al (1998), Kemble proposes that, upon oxidation, intermolecular disulfide bridges form between Cys277 residues on two Src molecules, resulting in inactive Src dimers.…”

“…This is in contrast to the cell-free assays used by Kemble et al (2009), in which a redox-inactivation of Src was also observed. However Tang Irrespective of its consequence for Src kinase activity, the susceptibility of Src to redox regulation is a characteristic shared by other members of the Src family kinases (SFK).…”

“…In addition to this and as documented by Kemble et al (2009) there is evidence to suggest that FGFR1 is negatively redox regulated while report that VEGFR undergoes oxidative activation.…”

S-Nitrosylation at Cysteine 498 of c-Src Tyrosine Kinase Regulates Nitric Oxide-mediated Cell Invasion

“…Reactive oxygen species, such as superoxide and hydrogen peroxide, serve as secondary messengers to regulate signaling pathways of certain growth factors and cell adhesion. A number of studies have suggested that cysteine modification of c-Src plays an important role for the reactive oxygen speciesmediated signal transductions; however, whether c-Src is activated or inactivated by oxidation remains unclear (31). Upon cell adhesion, c-Src is activated by the formation of an intramolecular S-S bond between Cys 245 and Cys 487 , which are highly conserved residues among the Src family kinases and are localized in the SH2 domain and kinase domain, respectively (17).…”

Oxidation-induced intramolecular disulfide bond inactivates mitogen-activated protein kinase kinase 6 by inhibiting ATP binding

“…In the case of mitogen-activated protein kinase kinase kinase 1, a member of the MAP3K family, oxidation selectively induces glutathionylation of Cys 1238 near the conserved GXGXXG(or S/A) motif (i.e., P loop) in the kinase subdomain I (i.e., GLGAFSSC 1238 ) that is known to directly bind ATP (15). As for Src, a recent paper showed that oxidation induced the formation of a homodimer via an intermolecular disulfide bond between two Cys 277 in the middle of the conserved P loop (i.e., GQGC 277 FG in rat Src) (33). In both cases above, oxidation of a specific cysteine in the kinase domain inactivates the kinases, which is fully reversible upon treatment with a reducing agent.…”