Src Subfamily Kinases Regulate Nuclear Export and Degradation of Transcription Factor Nrf2 to Switch Off Nrf2-mediated Antioxidant Activation of Cytoprotective Gene Expression

Abstract

Nrf2 (NF-E2-related factor 2) is a nuclear transcription factor that in response to chemical and radiation stress regulates coordinated induction of a battery of cytoprotective gene expressions leading to cellular protection. In this study, we investigated the role of Src kinases in the regulation of Nrf2 and downstream signaling. siRNA-mediated inhibition of Fyn, Src, Yes, and Fgr, but not Lyn, in mouse hepatoma Hepa-1 cells, led to nuclear accumulation of Nrf2 and up-regulation of Nrf2 downstream gene expression. Mouse embryonic fibroblasts with combined deficiency of Fyn/Src/Yes/Fgr supported results from siRNA. In addition, steady-state overexpression of Fyn, Src, and Yes phosphorylated Nrf2Tyr568 that triggered nuclear export and degradation of Nrf2 and down-regulation of Nrf2 downstream gene expression. Exposure of cells to antioxidant, oxidant, or UV radiation increased nuclear import of Fyn, Src, and Yes kinases, which phosphorylated Nrf2Tyr568 resulting in nuclear export and degradation of Nrf2. Further analysis revealed that stress-activated GSK3␤ acted upstream to the Src kinases and phosphorylated the Src kinases, leading to their nuclear localization and Nrf2 phosphorylation. The overexpression of Src kinases in Hepa-1 cells led to decreased Nrf2, increased apoptosis, and decreased cell survival. Mouse embryonic fibroblasts deficient in Src kinases showed nuclear accumulation of Nrf2, induction of Nrf2 and downstream gene expression, reduced apoptosis, and increased cell survival. The studies together demonstrate that Src kinases play a critical role in nuclear export and degradation of Nrf2, thereby providing a negative feedback mechanism to switch off Nrf2 activation and restore normal cellular homeostasis.The INrf2⅐Nrf2 complex serves as a sensor of chemical-and radiation-induced oxidative and electrophilic stress (1, 2). Nrf2 resides predominantly in the cytoplasm where it interacts with actin-associated cytosolic protein, INrf2 (inhibitor of Nrf2) or Keap1 (Kelch-like ECH-associated protein 1). INrf2 functions as a substrate adaptor protein for a Cul3⅐Rbx1-dependent E3 ubiquitin ligase complex that ubiquitinates and degrades Nrf2, thus maintaining steady-state levels of Nrf2 (1, 2). Nrf2 is a nuclear transcription factor that coordinately activates expression of more than 200 cytoprotective genes required for protection of cells against stressors (1, 2). The mechanisms by which Nrf2 is released from INrf2 under stress have been actively investigated. One mechanism is that cysteine thiol groups of INrf2 function as sensors for oxidative stress, which are modified by the chemical inducers, causing formation of disulfide bonds between cysteines of two INrf2 peptides. This leads to a conformational change that renders INrf2 unable to bind with Nrf2 (3, 4). Another mechanism is that antioxidant-induced protein kinase C (PKC) phosphorylates Nrf2Ser-40, leading to dissociation of Nrf2 from INrf2 and nuclear localization of Nrf2 (5, 6). Recently, it has been shown that the two mechanisms work in concer...

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Endoplasmic reticulum stress and Nrf2 signaling in cardiovascular diseases

“…In addition, in the nucleus there are other Nrf2 negative regulators. In particular it has been reported that all the four Src subfamily members, including Fyn, Src, Yes, and Fgr follow Nrf2 in the nucleus, phosphorylate Nrf2Tyr568 that leads to nuclear export and ubiquitination/degradation of Nrf2 [68]. Furthermore, there is evidence that the transcription factor Bach1 is a further negative regulator of Nrf2 since it has been reported to compete with Nrf2 for binding to ARE [69].…”

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“…After discovering Fyn's role in regulating Nrf2, Jaiswal's group further investigated roles of other Src kinase subfamily members (Src, Yes and Fgr) and the Lyn subfamily for controlling Nrf2 gene expression. They elucidated that all four Src subfamily kinases (Src, Yes, Fyn and Fgr) but not Lyn phosphorylate Nrf2 Tyr568 and lead to the nuclear export, ubiquitination and degradation of Nrf2 [20]. While there is controversy as to the direct interaction of GSK-3β with Nrf2, these data still indicate that when Typically, Nrf2 is sequestered to the cytoplasm through binding with Keap1 and continually shuttled to the proteasome for degradation.…”

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