Reversible oxidation and inactivation of the tumor suppressor PTEN in cells stimulated with peptide growth factors

Abstract

hydrogen peroxide ͉ peptide growth factor receptor ͉ peroxiredoxin

Akt activation by arachidonic acid metabolism occurs via oxidation and inactivation of PTEN tumor suppressor

“…Oxidation of PTEN by ROS/H 2 O 2 has been reported in macrophages stimulated with lipopolysaccharide and phorbol ester (Leslie et al, 2003), in HEK293 cells stimulated with insulin (Kwon et al, 2004), in HeLa cells stimulated with epidermal growth factor (EGF) (Kwon et al, 2004), and in fibroblasts stimulated with platelet-derived growth factor (PDGF) (Seo et al, 2005). Despite the limited degree of PTEN oxidation in these studies, there are ample functional consequences.…”

“…Detection of oxidized PTEN by alkylation with biotinconjugated maleimide Serum-starved cells were treated for 10 min with 0-60 mM AA and oxidized PTEN was alkylated with biotin-maleimide as described (Kwon et al, 2004). Briefly, treated cells were washed, frozen, transferred to vacuum and incubated for 1 h at 251 with 1 ml of oxygen-free extraction buffer (50 mM sodium phosphate, pH 7.0/1 mM ethylenediaminetetraacetic acid (EDTA)/10 mM NEM/10 mM iodoacetic acid/1% Triton X-100/5 mM NaF/50 mg/ml leupeptin/50 mg/ml aprotinin).…”

“…The active-site cysteine of PTPs can be oxidized to several species by various ROS (Tonks, 2005). To address whether PTEN was oxidized to a species other than Cys124-Cys71 disulfide during oxidative metabolism of AA, we followed a protocol that allows oxidized cysteine residues to be selectively tagged with N-ethylmaleimide (NEM)-biotin and the oxidized proteins can be isolated by immunoprecipitation with neutravidin beads (Kwon et al, 2004). We found increased oxidized PTEN in the neutravidin immunoprecipitation from cells treated with AA compared to control (Figure 3e, lane 1 versus 2).…”

Redox-based regulation of signal transduction: Principles, pitfalls, and promises

“…The subsequent addition of reductant coupled to the use of a thiol-specific labeling agent such as biotinylated IAA or biotinylated NEM will then identify the oxidized Cys (Figure 3, top panel) [47]. This methodology, also referred to as "the thiol trapping technique" [220] has been successfully applied to cells in culture and demonstrated reversible oxidation and inactivation of tyrosine phosphatases in response to stimulation with growth factor receptors [29,116,117,219]. A similar strategy has been developed to detect Snitrosylated proteins, widely known as the "biotin switch method" (Figure 3, middle panel) [56,221].…”

“…Inactivation of tyrosine phosphatases by H 2 O 2 has been demonstrated in a number of settings, including physiological scenarios, such as stimulation of cells with the growth factor, epidermal growth factor, (EGF), or PDGF [17]. Reversible inactivation of the tyrosine phosphatases, PTP-1B [116], PTEN [117], and SHP-2 [29] also have been reported, and a formation of a sulfenic acid intermediate was reported as the oxidative event, although the formation of sulfenyl amide species (Cys-S-N-R) and glutathionylated species also have been shown [118,119]. Elegant biochemical analyses were done to demonstrate the sulfenic and sulfenyl amide forms of PTP1B in vitro, and the glutathionylated form of PTP1B has been shown in intact cells.…”