Insulin-stimulated Hydrogen Peroxide Reversibly Inhibits Protein-tyrosine Phosphatase 1B in Vivo and Enhances the Early Insulin Action Cascade

Abstract

The insulin signaling pathway is activated by tyrosine phosphorylation of the insulin receptor and key postreceptor substrate proteins and balanced by the action of specific protein-tyrosine phosphatases (PTPases). PTPase activity, in turn, is highly regulated in vivo by oxidation/reduction reactions involving the cysteine thiol moiety required for catalysis. Here we show that insulin stimulation generates a burst of intracellular H 2 O 2 in insulin-sensitive hepatoma and adipose cells that is associated with reversible oxidative inhibition of up to 62% of overall cellular PTPase activity, as measured by a novel method using strictly anaerobic conditions. The specific activity of immunoprecipitated PTP1B, a PTPase homolog implicated in the regulation of insulin signaling, was also strongly inhibited by up to 88% following insulin stimulation. Catalase pretreatment abolished the insulin-stimulated production of H 2 O 2 as well as the inhibition of cellular PTPases, including PTP1B, and was associated with reduced insulin-stimulated tyrosine phosphorylation of its receptor and high M r insulin receptor substrate (IRS) proteins. These data provide compelling new evidence for a redox signal that enhances the early insulin-stimulated cascade of tyrosine phosphorylation by oxidative inactivation of PTP1B and possibly other tyrosine phosphatases. Protein-tyrosine phosphatases (PTPases)1 play a key role in the regulation of reversible tyrosine phosphorylation in the insulin action pathway. Insulin signaling is initiated by the phosphorylation of specific tyrosyl residues of the cell surface insulin receptor, which activates its exogenous kinase activity and promotes the phosphorylation of IRS proteins on specific tyrosine residues (1). These activation steps are balanced, in turn, by specific cellular PTPases that dephosphorylate and inactivate the receptor kinase and reverse the adapter function of the receptor substrate proteins (2). The cellular role of PTPases is apparent from the observation that highly purified insulin receptors and IRS proteins retain their tyrosine phosphorylation and activation state in vitro (3, 4), while in intact or permeabilized cells, receptor activation and substrate tyrosine phosphorylation are rapidly reversed (5-7).Since PTPases are high turnover number enzymes, physiological suppression of PTPase catalytic activity has been postulated to be a key feature of their regulation within the cellular environment to allow tyrosine phosphorylation to proceed in a balanced manner (8). PTPases have in common a conserved ϳ230-amino acid domain that contains the cysteine residue that catalyzes the hydrolysis of protein phosphotyrosine residues by the formation of a cysteinyl-phosphate intermediate (9, 10). Several laboratories have recently provided evidence that reactive oxygen species, including H 2 O 2 , can oxidize and inactivate PTPases in vivo (11,12). Since only the reduced form of the catalytic site is enzymatically active, stepwise and progressively irreversible oxidative inhibition is em...

Regulation of Insulin Signaling through Reversible Oxidation of the Protein-tyrosine Phosphatases TC45 and PTP1B

“…5A). Furthermore, and of particular importance to our study, it has been shown that treatment with insulin triggers H 2 O 2 production in HepG2 cells (21). As shown in Fig.…”

“…The low pK a promotes the function of this Cys residue as a nucleophile in catalysis but renders it highly susceptible to oxidation with concomitant inhibition of PTP activity (15)(16)(17). It is now known that multiple PTPs are transiently oxidized by H 2 O 2 (18 -20) and also in response to certain cellular stimuli (17,(21)(22)(23)(24), illustrating that this mode of regulation may apply broadly across the enzyme family (25).…”

The Major Target of the Endogenously Generated Reactive Oxygen Species in Response to Insulin Stimulation Is Phosphatase and Tensin Homolog and Not Phosphoinositide-3 Kinase (PI-3 Kinase) in the PI-3 Kinase/Akt Pathway

“…Recent reports (Mahadev et al, 2001a;Mahadev et al, 2001b) have demonstrated that insulin-stimulated ROS generation modulated the steady-state tyrosine phosphorylation of the insulin receptor and its cellular substrates by reversible inhibition of PTPases. In addition, the transient increase of ROS in response to insulin regulates the activity of PI-3 kinase/Akt and cellular glucose transport.…”

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

“…Protein tyrosine phosphatases (PTPs) have been suggested to be direct targets of H 2 O 2 in the propagation of its signal in cells stimulated with various growth factors (7)(8)(9). Growth factor signaling is initiated by activation of the corresponding receptor tyrosine kinase and consequent phosphorylation both of the receptor itself and of various other substrates.…”