Regulation of Nrf2—an update

Nrf2:INrf2 (Keap1) are cellular sensors of oxidative and electrophilic stress. Nrf2 is a nuclear factor that controls the expression and coordinated induction of a battery of genes that encode detoxifying enzymes, drug transporters, antiapoptotic proteins, and proteasomes. In the basal state, Nrf2 is constantly degraded in the cytoplasm by its inhibitor, INrf2. INrf2 functions as an adapter for Cul3/Rbx1 E3 ubiquitin ligase-mediated degradation of Nrf2. Chemicals, including antioxidants, tocopherols including α-tocopherol (vitamin E), and phytochemicals, and radiation antagonize the Nrf2:INrf2 interaction and lead to the stabilization and activation of Nrf2. The signaling events involve preinduction, induction, and postinduction responses that tightly control Nrf2 activation and repression back to the basal state. Oxidative/electrophilic signals activate unknown tyrosine kinases in a preinduction response that phosphorylates specific residues on Nrf2 negative regulators, INrf2, Fyn, and Bach1, leading to their nuclear export, ubiquitination, and degradation. This prepares nuclei for unhindered import of Nrf2. Oxidative/electrophilic modification of INrf2 cysteine 151 followed by PKC phosphorylation of Nrf2 serine 40 in the induction response results in the escape or release of Nrf2 from INrf2. Nrf2 is thus stabilized and translocates to the nucleus, resulting in a coordinated activation of gene expression. This is followed by a postinduction response that controls the “switching off” of Nrf2-activated gene expression. GSK3β, under the control of AKT and PI3K, phosphorylates Fyn, leading to Fyn nuclear localization. Fyn phosphorylates Nrf2 Y568, resulting in nuclear export and degradation of Nrf2. The activation and repression of Nrf2 provide protection against oxidative/electrophilic stress and associated diseases, including cancer. However, deregulation of INrf2 and Nrf2 due to mutations may lead to nuclear accumulation of Nrf2 that reduces apoptosis and promotes oncogenesis and drug resistance.     

Phosphorylation and dephosphorylation of tyrosine 141 regulate stability and degradation of INrf2: a novel mechanism in Nrf2 activation

INrf2-Nrf2 proteins are sensors of chemical/radiation stress. Nrf2, in response to stresses, is released from INrf2. Nrf2 is translocated into the nucleus where it binds to the antioxidant response element and coordinately activates the expression of a battery of genes that protect cells against oxidative and electrophilic stress. An autoregulatory loop between INrf2 and Nrf2 regulates their cellular abundance. Nrf2 activates INrf2 gene expression, and INrf2 serves as an adapter for degradation of Nrf2. In this report, we demonstrate that mutation of tyrosine 141 in bric-a-bric, tramtrack, broad complex domain to alanine rendered INrf2 unstable and nonfunctional. INrf2Y141A mutant degraded rapidly as compared with wild type INrf2, although it could dimerize and bind Nrf2. De novo synthesized INrf2 protein was phosphorylated at tyrosine 141. Tyrosine 141-phosphorylated INrf2 was highly stable. Treatment with hydrogen peroxide, which is an oxidizing agent, led to dephosphorylation of INrf2Y141, resulting in rapid degradation of INrf2. This resulted in stabilization of Nrf2 and activation of ARE-mediated gene expression. These results demonstrate that stress-induced dephosphorylation of tyrosine 141 is a novel mechanism in Nrf2 activation and cellular protection.     

Nuclear import and export signals in control of Nrf2

Nrf2 binds to the antioxidant response element and regulates expression and antioxidant induction of a battery of chemopreventive genes. In this study, we have identified nuclear import and export signals of Nrf2 and show that the nuclear import and export of Nrf2 is regulated by antioxidants. We demonstrate that Nrf2 contains a bipartite nuclear localization signal (NLS) and a leucine-rich nuclear export signal, which regulate Nrf2 shuttling in and out of the nucleus. Immunofluorescence and immunoblot analysis revealed that Nrf2 accumulates in the nucleus within 15 min of antioxidant treatment and is exported out of nucleus by 8 h after treatment. Nrf2 mutant lacking the NLS failed to enter the nucleus and displayed diminished expression and induction of the downstream NAD(P)H:quinone oxidoreductase 1 gene. The Nrf2 NLS sequence, when fused to green fluorescence protein, resulted in the nuclear accumulation of green fluorescence protein, indicating that this signal sequence was sufficient to direct nuclear localization of Nrf2. A nuclear export signal (NES) was characterized in the C terminus of Nrf2, the deletion of which caused Nrf2 to accumulate predominantly in the nucleus. The Nrf2 NES was sensitive to leptomycin B and could function as an independent export signal when fused to a heterologous protein. Further studies demonstrate that NES-mediated nuclear export of Nrf2 is required for degradation of Nrf2 in the cytosol. These results led to the conclusion that Nrf2 localization between cytosol and nucleus is controlled by both nuclear import and export of Nrf2, and the overall distribution of Nrf2 is probably the result from a balance between these two processes. Antioxidants change this balance in favor of nuclear accumulation of Nrf2, leading to activation of chemopreventive proteins. Once this is achieved, Nrf2 exits the nucleus for binding to INrf2 and degradation.     

Hsp90 Interaction with INrf2(Keap1) Mediates Stress-induced Nrf2 Activation

INrf2(Keap1) functions as an adapter for Cul3/Rbx1-mediated degradation of Nrf2. In response to stress, Nrf2 is released from INrf2 and translocates inside the nucleus leading to activation of cytoprotective proteins critical in protection against adverse effects including cancer. We demonstrate here a novel role of heat shock protein 90 (Hsp90) in control of the INrf2 and Nrf2 activation. Hsp90 interacted with INrf2 that leds to stabilization of INrf2 during heat shock stress. Domain mapping showed the requirement of INrf2-NTR and the Hsp90-CLD region for interaction of Hsp90 with INrf2. Heat shock and antioxidants induced Hsp90, and casein kinase 2 (CK2) phosphorylated INrf2Thr55. This led to increased Hsp90-INrf2 interaction, dissociation of the Rbx1/Cul3·INrf2·Nrf2 complex, and activation of Nrf2. Inhibitors of CK2 and Hsp90, and mutation of INrf2Thr55 abolished the Hsp90-INrf2 interaction and downstream signaling. INrf2 is released from Hsp90 once the heat shock or antioxidant stress subsidized, thereby allowing INrf2 to interact with Nrf2 and facilitate Nrf2 ubiquitination and degradation. The results together demonstrate a novel role for the stress-induced Hsp90-INrf2 interaction in regulation of Nrf2 activation and induction of cytoprotective proteins.     

A rev-like NES mediates cytoplasmic localization of HERV-K cORF

The human endogenous retrovirus K (HERV-K)-encoded protein cORF has recently been shown to be a functional homolog of the HIV Rev protein. Rev-mediated RNA export requires interaction between a leucine-rich nuclear export signal (NES) in Rev and the nuclear export receptor Crm1/exportin1. Like Rev, cORF binds to Crm1 and cORF-mediated RNA export depends on Crm1 activity. Here we document that mutation of the putative NES in cORF results in trapping of the protein in the nucleus, suggesting that the cORF NES functions in analogy to the Rev NES.     

Inhibitor of Nrf2 (INrf2 or Keap1) protein degrades Bcl-xL via phosphoglycerate mutase 5 and controls cellular apoptosis

INrf2 (Keap1) is an adaptor protein that facilitates INrf2-Cul3-Rbx1-mediated ubiquitination/degradation of Nrf2, a master regulator of cytoprotective gene expression. Here, we present evidence that members of the phosphoglycerate mutase family 5 (PGAM5) proteins are involved in the INrf2-mediated ubiquitination/degradation of anti-apoptotic factor Bcl-xL. Mass spectrometry and co-immunoprecipitation assays revealed that INrf2, through its DGR domain, interacts with PGAM5, which in turn interacts with anti-apoptotic Bcl-xL protein. INrf2-Cul3-Rbx1 complex facilitates ubiquitination and degradation of both PGAM5 and Bcl-xL. Overexpression of PGAM5 protein increased INrf2-mediated degradation of Bcl-xL, whereas knocking down PGAM5 by siRNA decreased INrf2 degradation of Bcl-xL, resulting in increased stability of Bcl-xL. Mutation of PGMA5-E79A/S80A abolished INrf2/PGAM5/Bcl-xL interaction. Therefore, PGAM5 protein acts as a bridge between INrf2 and Bcl-xL interaction. Further studies showed that overexpression of INrf2 enhanced degradation of PGAM5-Bcl-xL complex, led to etoposide-mediated accumulation of Bax, increased release of cytochrome c from mitochondria, activated caspase-3/7, and enhanced DNA fragmentation and apoptosis. In addition, antioxidant (tert-butylhydroquinone) treatment destabilized the Nrf2-INrf2-PGAM5-Bcl-xL complex, which resulted in release of Nrf2 in cytosol and mitochondria, release of Bcl-xL in mitochondria, increase in Bcl-xL heterodimerization with Bax in mitochondria, and reduced cellular apoptosis. These data provide the first evidence that INrf2 controls Bcl-xL via PGAM5 and controls cellular apoptosis.     

Both ran and importins have the ability to function as nuclear mRNA export factors

The Ran protein regulates nucleocytoplasmic transport mediated by the karyopherin family of nuclear transport factors. Ran is converted to the active, GTP bound form in the nucleus and then binds to a conserved domain found in all karyopherins. This interaction induces cargo binding for exportins and cargo release for importins. In either case, the Ran.GTP is then transported to the cytoplasm by the karyopherin, where it is hydrolyzed to Ran.GDP. To ask whether Ran could function as a nuclear mRNA export factor, we fused Ran to the MS2 coat protein and inserted MS2 RNA-binding sites into an unspliced cat mRNA that is normally sequestered in the nucleus. Coexpression of MS2-Ran induced cat mRNA export and CAT enzyme expression as effectively as, for example, an MS2-Rev fusion protein. MS2-Ran dependent nuclear mRNA export was reduced by inhibitors specific for Crm1, but not blocked as was seen with MS2-Rev. Consistent with the hypothesis that Crm1 is not the only karyopherin cofactor for MS2-Ran mediated mRNA export, we show that not only Crm1 but also CAS, transportin, importin beta and exportin t can all export mRNA from the nucleus when tethered via the MS2 RNA-binding domain. In contrast, two shuttling hnRNPs, hnRNP A1 and hnRNP K, proved unable to function as nuclear RNA export factors when expressed as MS2 fusions. Together, these data argue that karyopherins that normally function to transport proteins into or out of the nucleus are also capable of exporting tethered mRNA molecules.     

Identification of an NTF2-related factor that binds Ran-GTP and regulates nuclear protein export

Active transport of macromolecules between the nucleus and cytoplasm requires signals for import and export and their recognition by shuttling receptors. Each class of macromolecule is thought to have a distinct receptor that mediates the transport reaction. Assembly and disassembly reactions of receptor-substrate complexes are coordinated by Ran, a GTP-binding protein whose nucleotide state is regulated catalytically by effector proteins. Ran function is modulated in a noncatalytic fashion by NTF2, a protein that mediates nuclear import of Ran-GDP. Here we characterize a novel component of the Ran system that is 26% identical to NTF2, which based on its function we refer to as NTF2-related export protein 1 (NXT1). In contrast to NTF2, NXT1 preferentially binds Ran-GTP, and it colocalizes with the nuclear pore complex (NPC) in mammalian cells. These properties, together with the fact that NXT1 shuttles between the nucleus and the cytoplasm, suggest an active role in nuclear transport. Indeed, NXT1 stimulates nuclear protein export of the NES-containing protein PKI in vitro. The export function of NXT1 is blocked by the addition of leptomycin B, a compound that selectively inhibits the NES receptor Crm1. Thus, NXT1 regulates the Crm1-dependent export pathway through its direct interaction with Ran-GTP.     

Yeast poly(A)-binding protein Pab1 shuttles between the nucleus and the cytoplasm and functions in mRNA export

Pab1 is the major poly(A)-binding protein in yeast. It is a multifunctional protein that mediates many cellular functions associated with the 3'-poly(A)-tail of messenger RNAs. Here, we characterize Pab1 as an export cargo of the protein export factor Xpo1/Crm1. Pab1 is a major Xpo1/Crm1-interacting protein in yeast extracts and binds directly to Xpo1/Crm1 in a RanGTP-dependent manner. Pab1 shuttles rapidly between the nucleus and the cytoplasm and partially accumulates in the nucleus when the function of Xpo1/Crm1 is inhibited. However, Pab1 can also be exported by an alternative pathway, which is dependent on the MEX67-mRNA export pathway. Import of Pab1 is mediated by the import receptor Kap108/Sxm1 through a nuclear localization signal in its fourth RNA-binding domain. Interestingly, inhibition of Pab1's nuclear import causes a kinetic delay in the export of mRNA. Furthermore, the inviability of a pab1 deletion strain is suppressed by a mutation in the 5'-3' exoribonuclease RRP6, a component of the nuclear exosome. Therefore, nuclear Pab1 may be required for efficient mRNA export and may function in the quality control of mRNA in the nucleus.     

Ran-binding protein 3 is a cofactor for Crm1-mediated nuclear protein export.

Crm1 is a member of the karyopherin family of nucleocytoplasmic transport receptors and mediates the export of proteins from the nucleus by forming a ternary complex with cargo and Ran:GTP. This complex translocates through the nuclear pores and dissociates in the cytosol. The yeast protein Yrb2p participates in this pathway and binds Crm1, but its mechanism of action has not been established. We show that the human orthologue of Yrb2p, Ran-binding protein 3 (RanBP3), acts as a cofactor for Crm1-mediated export in a permeabilized cell assay. RanBP3 binds directly to Crm1, and the complex possesses an enhanced affinity for both Ran:GTP and cargo. RanBP3 shuttles between the nucleus and the cytoplasm by a Crm1-dependent mechanism, and the Crm1--RanBP3-NES-Ran:GTP quarternary complex can associate with nucleoporins. We infer that this complex translocates through the nuclear pore to the cytoplasm where it is disassembled by RanBP1 and Ran GTPase--activating protein.     

Interaction of the hepatitis B virus X protein with the Crm1-dependent nuclear export pathway

The leucine-rich nuclear export signal (NES) is used to shuttle large cellular proteins from the nucleus to the cytoplasm. The nuclear export receptor Crm1 is essential in this process by recognizing the NES motif. Here, we show that the oncogenic hepatitis B virus (HBV) X protein (HBx) contains a functional NES motif. We found that the predominant cytoplasmic localization of HBx is sensitive to the drug leptomycin B (LMB), which specifically inactivates Crm1. Mutations at the two conserved leucine residues to alanine at the NES motif (L98A,L100A) resulted in a nuclear redistribution of HBx. A recombinant HBx protein binds to Crm1 in vitro. In addition, ectopic expression of HBx sequesters Crm1 in the cytoplasm. Furthermore, HBx activates NFkappaB by inducing its nuclear translocation in a NES-dependent manner. Abnormal cytoplasmic sequestration of Crm1, accompanied by a nuclear localization of NFkappaB, was also observed in hepatocytes from HBV-positive liver samples with chronic active hepatitis. We suggest that Crm1 may play a role in HBx-mediated liver carcinogenesis.