Biochemical and Biophysical Research Communications
Role for tyrosine phosphorylation of SUV39H1 histone methyltransferase in enhanced trimethylation of histone H3K9 via neuregulin-1/ErbB4 nuclear signaling
Introduction
Protein-tyrosine phosphorylation plays key roles in a variety of intracellular signaling pathways involved in cell proliferation, differentiation, gene expression, cell adhesion, and metabolic changes [1]. While receptor-type tyrosine kinases localize to the plasma membrane, most of non-receptor-type tyrosine kinases are present in the cytoplasm but some are at various intracellular locations. We have shown that tyrosine kinases residing in the nucleus are capable of phosphorylating their nuclear substrates to regulate chromatin structural changes and gene expression [[2], [3], [4], [5], [6], [7], [8], [9]].
The receptor-type tyrosine kinase ErbB4 is a member of the epidermal growth factor receptor subfamily and is essential for development or maintenance of the heart, the mammary glands, and the nervous system [[10], [11], [12]]. Notably, ErbB4 has a unique characteristic that ligand stimulation generates the soluble ErbB4 intracellular kinase domain. Stimulation with neuregulin-1 (NRG-1) sequentially cleaves the ErbB4 extracellular domain (4ECD) by the tumor necrosis factor-α-converting enzyme (TACE) and the ErbB4 intracellular domain (4ICD) by γ-secretase, resulting in the release of 4ICD into the cytoplasm [13,14]. 4ICD acts as a non-receptor-type tyrosine kinase having one functional nuclear localization signal (NLS) and three putative nuclear export signals and can shuttle between the cytoplasm and the nucleus, suggesting the possibility of ErbB4's nuclear functions [14,15]. In fact, 4ICD was shown to act as a transcriptional regulator in the nucleus through protein-protein interactions [16,17]. Importantly, we showed that nuclear 4ICD enhances the levels of trimethylation of histone H3 at lysine 9 (H3K9me3), which is crucial for heterochromatin formation and epigenetic gene silencing [17]. These functions of nuclear ErbB4 depend on its kinase activity, suggesting the involvement of tyrosine phosphorylation of its unidentified substrate(s).
In this study, we showed the histone methyltransferase SUV39H1 as a nuclear substrate of 4ICD and the importance of tyrosine phosphorylation of SUV39H1 for NRG-1/ErbB4-induced H3K9me3 in chromatin dynamics.
Section snippets
Cells and transfection
COS-1 and HeLa S3 cells (JCRB) were cultured in Iscove's modified Dulbecco's medium (IMDM) supplemented with 1% fetal bovine serum (FBS) and 4% bovine serum. T47D cells (provided by M. Tagawa, Chiba Cancer Center Research Institute) were cultured in IMDM supplemented with 5% FBS. Cells were transfected with plasmid DNA using polyethylenimine [18]. Stimulation with NRG-1 was performed as described previously [17]. The ErbB-family inhibitor AG1478 (20 μM) was added 30 min before NGR-1
Requirement of SUV39H1 for nuclear ErbB4 kinase-dependent H3K9me3
We showed that nuclear 4ICD enhances the levels of H3K9me3, which is critical for heterochromatin formation and epigenetic gene silencing [17]. To explore how nuclear ErbB4 enhances H3K9me3 levels, we examined the role of the histone methyltransferase SUV39H1 [21] that selectively trimethylates H3K9 in nuclear ErbB4-dependent H3K9me3. We established a stable SUV39H1 knockdown HeLa S3 cell line using shRNA against SUV39H1 (HeLa S3/shSUV39H1) (Fig. 1B). Consistent with previous studies [22,23],
Discussion
Our previous study demonstrated for the first time that NRG-1/ErbB4 nuclear signaling enhances the levels of H3K9me3 in a manner dependent on 4ICD's tyrosine kinase activity [17]. Now, this study shows that NRG-1/ErbB4 nuclear signaling enhances H3K9me3 levels through SUV39H1 phosphorylation at Tyr-297, -303, and -308. Our previous study also showed that the enhanced levels of H3K9me3 by NRG-1/ErbB4 nuclear signaling further lead to transcriptional repression of the human telomerase reverse
Conflicts of interest
The authors declare that there is no conflict of interest.
Acknowledgements
We thank Dr. H. Miyoshi and Dr. M. Tagawa for the invaluable plasmid and cells. This work was supported in part by grants-in-aid for scientific research from the MEXT, Japan (15K07922) and the scholarship donation from the Daiichi Sankyo Co., Ltd.
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