On–off system for PI3-kinase–Akt signaling through S -nitrosylation of phosphatase with sequence homology to tensin (PTEN)

Abstract

Nitric oxide (NO) physiologically regulates numerous cellular responses through S-nitrosylation of protein cysteine residues. We performed antibody-array screening in conjunction with biotin-switch assays to look for S-nitrosylated proteins. Using this combination of techniques, we found that phosphatase with sequence homology to tensin (PTEN) is selectively S-nitrosylated by low concentrations of NO at a specific cysteine residue (Cys-83). S-nitrosylation of PTEN (forming SNO-PTEN) inhibits enzymatic activity and consequently stimulates the downstream Akt cascade, indicating that Cys-83 is a critical site for redox regulation of PTEN function. In ischemic mouse brain, we observed SNO-PTEN in the core and penumbra regions but found SNO-Akt, which is known to inhibit Akt activity, only in the ischemic core. These findings suggest that low concentrations of NO, as found in the penumbra, preferentially S-nitrosylate PTEN, whereas higher concentrations of NO, known to exist in the ischemic core, also S-nitrosylate Akt. In the penumbra, inhibition of PTEN (but not Akt) activity by S-nitrosylation would be expected to contribute to cell survival by means of enhanced Akt signaling. In contrast, in the ischemic core, SNO-Akt formation would inhibit this neuroprotective pathway. In vitro model systems support this notion. Thus, we identify unique sites of PTEN and Akt regulation by means of S-nitrosylation, resulting in an "on-off" pattern of control of Akt signaling.apoptosis | ischemia | oxidation N itric oxide (NO) exerts pleiotropic cellular responses on proliferation, apoptosis, neurotransmission, and neurotoxicity in several types of cells by means of protein S-nitrosylation. This modification occurs by means of oxidative reaction between NO and cysteine (Cys) thiol in the presence of an electron acceptor (such as O 2 or a transition metal) or through transnitrosylation from S-nitrosothiol to another Cys thiol (1-3). Several methods have been published to detect S-nitrosylated proteins (SNO-Ps) by using antibodies, photolysis, and mercury affinity (4). In particular, the biotin-switch assay is a modified immunoblot developed by Jaffrey and Snyder that has been commonly used to detect endogenous SNO-Ps; this method has greatly advanced the field (5). Subsequently, other methods have been developed to detect SNO-Ps (6), but some of them involve samples treated with high concentrations of NO donor. In the presence of high concentrations of NO, however, it is possible that some Cys residues are artifactually S-nitrosylated.Antibody arrays have been used to profile protein expression levels with high sensitivity. Each spotted antibody can be validated for its ability to bind proteins in the assay. Samples hybridizing to each antibody on the array can be easily detected. Although a number of proteins have been identified as substrates for S-nitrosylation in the past several years (3-6), we hypothesized that many more candidates modified by physiological levels of NO might still remain to be identified. We therefore teste...

From Nitric Oxide Toward S-Nitrosylation: Expanding Roles in Gametes and Embryos

“…PTEN structure reports disparate sites for S-nitrosylation (Cys 83 [100]), S-sulfhydration, and hydrogen peroxide-induced disulfide bond formation (Cys 71 and Cys 124 [103]). One should keep in mind that in this particular example, S-nitrosylation targets a different site than the Cys 124, mandatory in the phosphatase activity of PTEN.…”

“…Gene suppressor PTEN acts as an inhibitor of the PI-3 kinase/Akt signaling pathway, attenuating cellular growth and survival. S-nitrosylation enabled Akt hyperactivity, and thereby is associated with observed neuroprotective effects [100]. Oxidation of the active site cysteine is acknowledged as a common mechanism for regulating protein tyrosine phosphatases [101].…”

Nitric Oxide Regulates Growth Factor Signaling in Pancreatic Cancer Cells

“…Signaling proteins that are directly modified by S-nitrosylation include Ras, Akt, JNK, PTEN, IκB kinase, and Bcl2. (Azad et al;Lander et al, 1997;Numajiri et al;Park et al, 2000;Reynaert et al, 2004;Yasukawa et al, 2005) …”

Reactive Nitrogen Species and Hydrogen Sulfide as Regulators of Protein Tyrosine Phosphatase Activity

“…Physiologically, the PTEN S-nitrosylation was observed in regions of ischemic mammalian brain with low NO concentrations (core and penumbra regions), while S-nitrosylated Akt was detected only at sites with high NO concentrations (ischemic core). It is hypothesized that PTEN S-nitrosylation (and thus inactivation) prevents Akt S-nitrosylation, improves cell survival by enabling hyperactivity of the Akt pathway, and is associated with neuroprotective effects (58,66).…”