Peroxide-dependent sulfenylation of the EGFR catalytic site enhances kinase activity.
ABSTRACT Protein sulfenylation is a post-translational modification of emerging importance in higher eukaryotes. However, investigation of its diverse roles remains challenging, particularly within a native cellular environment. Herein we report the development and application of DYn-2, a new chemoselective probe for detecting sulfenylated proteins in human cells. These studies show that epidermal growth factor receptor-mediated signaling results in H(2)O(2) production and oxidation of downstream proteins. In addition, we demonstrate that DYn-2 has the ability to detect differences in sulfenylation rates within the cell, which are associated with differences in target protein localization. We also show that the direct modification of epidermal growth factor receptor by H(2)O(2) at a critical active site cysteine (Cys797) enhances its tyrosine kinase activity. Collectively, our findings reveal sulfenylation as a global signaling mechanism that is akin to phosphorylation and has regulatory implications for other receptor tyrosine kinases and irreversible inhibitors that target oxidant-sensitive cysteines in proteins.
- SourceAvailable from: Khalilah Reddie[show abstract] [hide abstract]
ABSTRACT: The polarizable sulfur atom in cysteine is subject to numerous post-translational oxidative modifications in the cellular milieu, which regulates a wide variety of biological phenomena such as catalysis, metal binding, protein turnover, and signal transduction. The application of chemical rationale to describe the features of different cysteine oxoforms affords a unique perspective on this rapidly expanding field. Moreover, a chemical framework broadens our understanding of the functional roles that specific cysteine oxidation states can play and facilitates the development of mechanistic proposals, which can be tested in both biochemical and cellular studies.Current opinion in chemical biology 10/2008; 12(6):746-54. · 8.30 Impact Factor
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ABSTRACT: In the past decade tremendous progress has been made toward a new class of therapeutics termed 'targeted covalent drugs', in which structure-based approaches are employed to create small molecules that inactivate their protein target through targeted covalent attachment to a specific cysteine. In the kinase field, this approach is demonstrating promise in overcoming the potency, selectivity, and efficacy challenges currently faced by reversible kinase inhibitors, with several advancing into late stage clinical testing. This design paradigm has been successfully applied to making drug candidates for epidermal growth factor receptor (EGFR), Her2, and Bruton's tyrosine kinase (Btk). Here we review recent pre-clinical and clinical advances with targeted covalent kinase inhibitors, and the potential for broader application of the approach.Current opinion in chemical biology 08/2010; 14(4):475-80. · 8.30 Impact Factor
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ABSTRACT: Stimulation of various cells with growth factors results in a transient increase in the intracellular concentration of H2O2 that is required for growth factor-induced protein tyrosine phosphorylation. The effect of H2O2 produced in response to epidermal growth factor (EGF) on the activity of protein-tyrosine phosphatase 1B (PTP1B) was investigated in A431 human epidermoid carcinoma cells. H2O2 inactivated recombinant PTP1B in vitro by oxidizing its catalytic site cysteine, most likely to sulfenic acid. The oxidized enzyme was reactivated more effectively by thioredoxin than by glutaredoxin or glutathione at their physiological concentrations. Oxidation by H2O2 prevented modification of the catalytic cysteine of PTP1B by iodoacetic acid, suggesting that it should be possible to monitor the oxidation state of PTP1B in cells by measuring the incorporation of radioactivity into the enzyme after lysis of the cells in the presence of radiolabeled iodoacetic acid. The amount of such radioactivity associated with PTP1B immunoprecipitated from A431 cells that had been stimulated with EGF for 10 min was 27% less than that associated with PTP1B from unstimulated cells. The amount of iodoacetic acid-derived radioactivity associated with PTP1B reached a minimum 10 min after stimulation of cells with EGF and returned to base line values by 40 min, suggesting that the oxidation of PTP1B is reversible in cells. These results indicate that the activation of a receptor tyrosine kinase by binding of the corresponding growth factor may not be sufficient to increase the steady state level of protein tyrosine phosphorylation in cells and that concurrent inhibition of protein-tyrosine phosphatases by H2O2 might also be required.Journal of Biological Chemistry 07/1998; 273(25):15366-72. · 4.65 Impact Factor