GSK-3β Acts Upstream of Fyn Kinase in Regulation of Nuclear Export and Degradation of NF-E2 Related Factor 2

Abstract

NF-E2-related factor 2 (Nrf2) regulates expression and coordinated induction of a battery of chemoprotective genes in response to oxidative and electrophilic stress. This leads to protection against oxidative stress and neoplastic diseases. Nuclear import and export of Nrf2 play a significant role in control of nuclear levels of Nrf2 and thus the expression of Nrf2 downstream genes. Tyrosine kinase Fyn phosphorylates tyrosine 568 of Nrf2 that leads to the nuclear export of Nrf2. In this study, we investigated the upstream factor(s) in regulation of Fyn and Fynmediated nuclear export of Nrf2. The investigations shed light on a novel mechanism of Nrf2 regulation in response to oxidative stress. We demonstrate that GSK-3␤ acts upstream of Fyn kinase in control of nuclear export of Nrf2. Chemical and short interfering RNA-mediated inhibition of GSK-3␤ led to nuclear accumulation of Nrf2 and transcriptional activation of the Nrf2 downstream gene nqo1. Chemical and short interfering RNA inhibition of GSK-3␤ and Fyn individually and in combination revealed that both kinases follow the same pathway to regulate nuclear export of Nrf2. We further demonstrate that hydrogen peroxide phosphorylates tyrosine 216 of GSK-3␤. This leads to activation of GSK-3␤. The activated GSK-3␤ phosphorylates Fyn at threonine residue(s). Phosphorylated Fyn accumulates in the nucleus and phosphorylates Nrf2 at tyrosine 568. This leads to nuclear export, ubiquitination, and degradation of Nrf2.NF-E2-related factor (Nrf2) 2 is a nuclear transcription factor that binds to antioxidant-response element (ARE) and regulates expression and coordinated induction of a battery of chemoprotective genes in response to antioxidants, oxidants, and radiations (1). This induction involves a mechanism essential for cellular protection against oxidative and electrophilic stress and neoplastic diseases (1). Nrf2-null mice are born normal and viable, indicating that Nrf2 is not required for development and growth of mice (2). Nrf2-null mice express significantly lower levels and no induction of chemoprotective proteins that include NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione S-transferase Ya subunit (GST-Ya), ␥-glutamylcysteinyl synthetase, and heme oxygenase (HO-1) (1). These mice demonstrate slower wound healing and pulmonary emphysema in response to exposure to tobacco smoke (3, 4).A cytosolic inhibitor of Nrf2, INrf2 (inhibitor of Nrf2) or Keap1, retains Nrf2 in the cytoplasm (5, 6). The INrf2-Nrf2 complex serves as cellular sensor of oxidative and electrophilic stress (1). The cellular exposure to oxidants, antioxidants, and radiations antagonizes this interaction and leads to the release of Nrf2 from INrf2 (1). Nrf2 translocates into the nucleus and induces the expression of chemoprotective proteins. Electrophilic adduction of selected cysteines in INrf2 and PKC phosphorylation of Nrf2 are known to contribute to the release of Nrf2 from INrf2 (1). Disruption of INrf2 in mice leads to postnatal death, probably from malnutrition resulting from hyperk...

Nrf2 target genes are induced under marginal selenium-deficiency

“…Fyn kinase is phosphorylated and thereby activated by GSK3b. Phospho-Fyn, in turn, phosphorylates Nrf2 resulting in the nuclear export of Nrf2 [23]. The reduced activity of GSK3b, which may be deduced from the downregulation of Gsk3b under selenium-limiting conditions in the previous study, might lower the amount of active Fyn resulting in nuclear accumulation of Nrf2.…”

“…In the previous study [28], we found the Wnt pathway activated, indicated by the enhanced expression of b-catenin, Dvl2, Lef1 and c-Myc and the down-regulation of Gsk3b. GSK3b, a multifunctional serine/threonine kinase, is not only involved in glycogen metabolism and canonical Wnt signaling but has also been shown to inactivate the Nrf2 pathway [23,49]. Fyn kinase is phosphorylated and thereby activated by GSK3b.…”

Pyk2 cytonuclear localization: mechanisms and regulation by serine dephosphorylation

“…Pyk2 could directly regulate tyrosine phosphorylated resident proteins of the nucleus such as histone variant H2AX [51] or H3/H4 [52]. Pyk2 associates with Src-family kinases, which localize to the nucleus in specific conditions, regulating euchromatin decondensation [53] and protection against oxidative stress through phosphorylation of Nrf2 by Fyn [54,55]. Nuclear Pyk2 may also play a role of a scaffolding protein for p53 [35].…”

Nuclear Factor–Erythroid-2–Related Factor 2 in Aging and Lung Fibrosis

“…76 In addition to the involvement of PKC and phosphatidylinositol 3-kinase in the activation of Nrf2, accumulating evidence suggests a role for mitogenactivated protein kinases 19,77,78 ; however, some reports suggest negative regulation of Nrf2 by these protein kinase pathways. 79 For example, extracellular signal regulated kinase 1/2 signaling cascades have been shown to increase the activation/stabilization of Nrf2, leading to induction of cytoprotective genes. 77 Activation of p38 mitogen-activated protein kinase and Janus kinase has also been implicated in the activation and nuclear translocation of Nrf2.…”

“…19,78 In contrast, phosphorylation of tyrosine residues (Tyr-568) on Nrf2 by glycogen synthetase kinase 3b activated Fyn has been shown to promote nuclear export and degradation of Nrf2. 79 Acetylation and deacetylation of Nrf2 promote nuclearcytoplasmic shuttling and regulation of its transcriptional activity. 69 Acetylation of lysine residues in Nrf2 enhances Nrf2-DNA binding and transcription of target genes.…”