Bcr-Abl signaling through the PI-3/S6 kinase pathway inhibits nuclear translocation of the transcription factor Bach2, which represses the antiapoptotic factor heme oxygenase-1

Abstract

The malignant phenotype of chronic myeloid leukemia (CML) is due to the abnormal tyrosine kinase activity of the Bcr-Abl oncoprotein. We have previously reported that expression of the Bach2 transcription factor, which induces apoptosis in response to oxidative stress, is greatly reduced in CML cells. Because these cells are resistant to apoptosis, we tested whether Bach2 could also be regulated through posttranslational mechanisms that promote inhibition of the apoptotic response to mutagenic stimuli in CML. We found that Bach2 is phosphorylated on S521 via the phosphatidylinositol-3/S6 kinase pathway, and substitution of this site to alanine leads to nuclear accumulation of the protein, indicating that this phosphorylation is important for its subcellular localization. Ectopic expression of the S521 mutant imparts greater impairment to CML cell growth than the wildtype factor. Furthermore, we showed that Bach2 transcriptionally represses heme oxygenase-1, an antiapoptotic factor upregulated in CML. Because CML cells are known to produce high levels of intracellular reactive oxygen species, overexpression of heme oxygenase-1 resulting from inhibition of Bach2 activity may contribute to their genomic instability and leukemic phenotype. IntroductionChronic myeloid leukemia (CML) is a myeloproliferative disorder of the hematopoietic stem cell caused by a t(9;22)(q34;q11) translocation that generates the Philadelphia (Ph) chromosome. This genetic defect, also found in 20% to 30% of adult acute lymphoblastic leukemias, results in the expression of Bcr-Abl, a fusion oncoprotein with uncontrolled tyrosine kinase activity. Imatinib mesylate inhibits the Bcr-Abl tyrosine kinase, suppresses the proliferation of CML progenitor cells, and induces apoptosis of Ph-positive cell lines. It is a highly effective drug and has become the first-choice treatment of CML. However, resistance to the inhibitor emerges in some patients, especially in advanced phases of CML (reviewed by Yoshida and Melo 1 and Deininger et al 2 ).Bcr-Abl phosphorylates several substrates that activate multiple signaling pathways, including Ras, signal transducer and activator of transcription-5 (STAT-5), extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK), Janus kinase 2 (Jak-2), phosphatidylinositol-3 kinase (PI-3K), and others. 3 This abnormal signaling leads to the malignant cellular phenotype of CML, including increased proliferation, reduction of adhesion to the bone marrow stroma and extracellular matrix, and inhibition of the apoptotic response to mutagenic stimuli. 4 DNA damage can be caused by oxidative stress arising when reactive oxygen species (ROSs) are not adequately removed from cells. 5 Interestingly, it has been suggested that ROSs are increased in CML cells. [6][7][8] Because a persistent increase of ROSs can lead to accumulation of DNA mutations, it may induce genomic instability as a long-term consequence. 9,10 Thus, BCR-ABL-positive cells can survive under levels of oxidative stress that normally induc...

The Transcription Factor Bach2 Is Phosphorylated at Multiple Sites in Murine B Cells but a Single Site Prevents Its Nuclear Localization

“…Therefore, phosphorylation of these residues may induce a conformational change of the disordered region, thereby regulating its function such as protein interaction. Human BACH2 is phosphorylated at Ser-521, which inhibits BACH2 nuclear accumulation in NIH3T3 cells (27). This site corresponds to Ser-520 of mouse Bach2, which was phosphorylated in our mass spectrometry analysis.…”

“…Although Bach2 has been reported as a phosphoprotein (27), this study is the first systematic analysis of its phosphorylation using normal primary B cells. By using the presence of a slower moving band of Bach2 upon immunoblotting as a proxy of its phosphorylation, we found that Bach2 phosphorylation was reduced by treating cells with the inhibitors of PI3K, Akt, or mTOR but not by those of MEK or p38 MAPK (Figs.…”

“…B cells deficient for Pten show a specific defect in CSR (26), which is very similar to that of Bach2-deficient mice (7). Second, human BACH2 is phosphorylated downstream of the PI3K, resulting in cytoplasmic accumulation in lymphoma cells (27). Interestingly, when mouse mature B cells are activated in vitro, a portion of them show cytoplasmic accumulation of Bach2, which are becoming IgMsecreting plasma cells without undergoing CSR (8).…”

“…We have reported previously that human BACH2 is phosphorylated at Ser-521, which regulates its subcellular localization in NIH3T3 cells (27). Therefore, we mutated the corresponding residue of mouse Bach2, Ser-520, and compared its subcellular localization in the Irf4 Ϫ/Ϫ Irf8 Ϫ/Ϫ pre-B cells.…”

Wearing Red for Signaling: The Heme-Bach Axis in Heme Metabolism, Oxidative Stress Response and Iron Immunology

“…Regarding human Bach2, the phosphorylation of serine 521 is required for the cytoplasmic accumulation of Bach2. This modification is dependent on the PI-3K/S6 kinase pathway, thus raising the possibility that Bach2 is regulated via phosphorylation in response to extracellular cytokines and growth factors (Yoshida et al 2007). In addition to this site, we recently found that additional multiple sites of Bach2 are modified by phosphorylation in response to the PI-3K pathway (Ando R., Shima H., Sato Y., Muto A. and Igarashi K., unpublished observation).…”