Inactivation of Src Family Tyrosine Kinases by Reactive Oxygen Species in Vivo

University of Texas at Tyler, Tyler, Texas, United States
Journal of Biological Chemistry (Impact Factor: 4.57). 07/2005; 280(25):23918-25. DOI: 10.1074/jbc.M503498200
Source: PubMed


Reactive oxygen species, including H2 O2, and are constantly produced in the human body and are involved in the development of cardiovascular diseases. Emerging evidence
suggests that reactive oxygen species, besides their deleterious effects at high concentrations, may be protective. However,
the mechanism underlying the protective effects of reactive oxygen species is not clear. Here, we reported a novel finding
that H2O2 at low to moderate concentrations (50-250 μm) markedly inactivated Src family tyrosine kinases temporally and spatially in vivo but not in vitro. We further showed that Src family kinases localized to focal adhesions and the plasma membrane were rapidly and permanently
inactivated by H2O2, which resulted from a profound reduction in phosphorylation of the conserved tyrosine residue at the activation loop. Interestingly,
the cytoplasmic Src family kinases were activated gradually by H2O2, which partially compensated for the loss of total activities of Src family kinases but not their functions. Finally, H2O2 rendered endothelial cells resistant to growth factors and cytokines and protected the cells from inflammatory activation.
Because Src family kinases play key roles in cell signaling, the rapid inactivation of Src family kinases by H2 O2 may represent a novel mechanism for the protective effects of reactive oxygen species.

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Available from: Hua Tang, Nov 18, 2015
    • "Previous studies have documented the interaction of SHP2 and FAK, by which inactivation of SHP2 eventually leads to FAK phosphorylation and activation [65]. Furthermore, the participation of SHP2 in the signalosome associated with fluid shear stress-dependent activation of eNOS has been previously reported, as well as the capacity of peroxide to inactivate SHP2 in endothelial cells [66]. LSS-mediated inactivation of SHP2 has been also described to be dependent on its catalytic cysteine [67]. "
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    ABSTRACT: Laminar shear stress (LSS) triggers signals that ultimately result in atheroprotection and vasodilatation. Early responses are related to the activation of specific signaling cascades. We investigated the participation of redox-mediated modifications and in particular the role of hydrogen peroxide (H2O2) in the sulfenylation of redox-sensitive phosphatases. Exposure of vascular endothelial cells to short periods of LSS (12dyn/cm(2)) resulted in the generation of superoxide radical anion as detected by the formation of 2-hydroxyethidium by HPLC and its subsequent conversion to H2O2, which was corroborated by the increase in the fluorescence of the specific peroxide sensor HyPer. By using biotinylated dimedone we detected increased total protein sulfenylation in the bovine proteome, which was dependent on NADPH oxidase 4 (NOX4)-mediated generation of peroxide. Mass spectrometry analysis allowed us to identify the phosphatase SHP2 as a protein susceptible to sulfenylation under LSS. Given the dependence of FAK activity on SHP2 function, we explored the role of FAK under LSS conditions. FAK activation and subsequent endothelial NO synthase (eNOS) phosphorylation were promoted by LSS and both processes were dependent on NOX4, as demonstrated in lung endothelial cells isolated from NOX4-null mice. These results support the idea that LSS elicits redox-sensitive signal transduction responses involving NOX4-dependent generation of hydrogen peroxide, SHP2 sulfenylation, and ulterior FAK-mediated eNOS activation.
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    • "It was suggested that activation or inactivation of SFKs is due to the level of reactive oxygen compounds, or simply H 2 O 2 present, and inactivation of Src and other tyrosine kinases was assumed as the protective mechanism to prevent cells from infl ammatory activation (Kemble and Sun, 2009). Recently this mechanism was explained by the activation of kinases by virtue of oxidation on a conserved cysteine residue, and that is why only three of the SFKs were vulnerable to be activated or deactivated depending on the reduced or oxidized status of the cellular environment (Tang et al., 2005; Chiarugi, 2008; Kemble and Sun, 2009). Several studies indicated that receptor protein tyrosine phosphatase-α (RPTPα) is a positive regulator of SFKs which is critical for ROS signal transduction (Hao et al., 2006b), and overexpression of the RPTPα was reported to result in persistent activation of pp60 c-Src kinase, with concomitant cell transformation and tumourigenesis (Zheng et al., 1992; Hao et al., 2006b). "
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    ABSTRACT: Current evidences demonstrated that the activity of protein kinases can be controlled through oxidative stress induced by reactive oxygen species (ROS) and normalized by antioxidants. Recent studies with ROS, generated by mitochondria, suggested the potential signalling role of these species, where ROS, especially hydrogen peroxide, were proposed as membrane-related signalling components. The protein regulation by cellular redox states has shown that protein tyrosine kinase members, such as Src kinase and some of the members of the Src family kinases (SFKs), are proteins regulated by the cellular oxidation and reduction status. In this context, the oxidant or antioxidant potential of the synthetic Src kinase inhibitors previously synthesized and studied by our research group, such as N-substituted indole-3-imine and -amine derivatives, were investigated employing various acellular in vitro methods including microsomal NADPH-dependent inhibition of lipid peroxidation (LP), interaction of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and scavenging of superoxide anion radicals. Here, we report that some of the synthetic inhibitors designed for Src kinase target have both antioxidant and kinase inhibition properties.
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    ABSTRACT: Signaling functions of superoxide and hydrogen peroxide in enzymatic phosphorylation/dephosphorylation reactions are now well documented, but their mechanisms are still not always clear. Now we propose the novel signaling mechanisms, by which superoxide and hydrogen peroxide mediate the activation and inhibition of phosphorylation/dephosphorylation catalyzed by protein kinases and protein phosphatases. We suggest that as a powerful nucleophile, superoxide is able to mediate phosphorylation of numerous proteins by protein kinases through the deprotonation of protein serine or threonine residues that sharply accelerates the rates of nucleophilic reaction between kinases and phosphorylating proteins. Furthermore the role of superoxide is enhanced due to its "chain" formation in the O(2)(-)--> PI 3-kinase --> protein kinases --> NADPH oxidase --> O(2)(-) cycle. Furthermore we suggest that hydrogen peroxide signaling in the dephosphorylation reactions by protein phosphatases and in the activation of protein kinases is actually mediated by superoxide formed during the conversion of H(2)O(2) into superoxide by the oxidized superoxide dismutase. This proposal is supported by the high rates of superoxide reactions with an anion of the catalytic cysteine residue of protein tyrosine phosphatases and the inability of hydrogen peroxide to react directly with protein serine and threonine residues in the reactions of protein kinases. Understanding of specific role of superoxide in the reactions catalyzed by protein kinases and protein phosphatases can be of importance for the selection of inhibitors of these enzymes playing a big role in numerous physiological and pathological processes.
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