A Novel Dual Kinase Function of the RET Proto-oncogene Negatively Regulates Activating Transcription Factor 4-mediated Apoptosis
A Novel Dual Kinase Function of the RET Proto-oncogene Negatively Regulates Activating Transcription Factor 4-mediated Apoptosis
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DOI：
10.1074/jbc.m114.619833
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发表时间：
2015-05-01
影响因子：
4.8
通讯作者：
Gagel, Robert F.
中科院分区：
文献类型：
--
作者：
Bagheri-Yarmand, Rozita;Sinha, Krishna M.;Gagel, Robert F.
RET原癌基因是一种酪氨酸激酶受体，因其在细胞存活中的重要作用而广为人知。生殖系错义突变，引起组成性活性致癌RET，被发现可引起多发性内分泌瘤2型，一种影响神经内分泌器官的显性遗传性癌症综合征。然而，RET促进细胞存活和防止细胞死亡的机制仍然难以捉摸。我们证明，除了细胞质定位，RET是位于细胞核和功能作为酪氨酸-苏氨酸双特异性激酶。在甲状腺髓样癌衍生细胞中通过shRNA敲低RET刺激转录激活因子4（ATF 4）的表达，ATF 4是一种应激诱导凋亡的主要转录因子，通过激活其靶向促凋亡基因NOXA和NOXA A。RET敲低也增加了对顺铂诱导的细胞凋亡的敏感性。我们观察到RET与ATF 4在酪氨酸和苏氨酸残基处发生物理相互作用并磷酸化。事实上，RET激酶活性是抑制NOXA基因的ATF 4依赖性激活所必需的，因为阻断苏氨酸磷酸化的位点特异性取代突变增加了ATF 4稳定性并激活了其靶点NOXA和NOXA A。此外，染色质免疫沉淀试验显示，ATF 4占用增加的NOXA启动子在TT细胞与酪氨酸激酶抑制剂或ATF 4诱导剂eeyarestatin以及在RET-耗尽TT细胞。这些发现揭示了RET作为一种新型的双重激酶，具有核定位，并提供了RET通过与ATF 4直接相互作用和磷酸化依赖性失活在甲状腺髓样癌发病过程中抑制促凋亡基因的机制。
The RET proto-oncogene, a tyrosine kinase receptor, is widely known for its essential role in cell survival. Germ line missense mutations, which give rise to constitutively active oncogenic RET, were found to cause multiple endocrine neoplasia type 2, a dominant inherited cancer syndrome that affects neuroendocrine organs. However, the mechanisms by which RET promotes cell survival and prevents cell death remain elusive. We demonstrate that in addition to cytoplasmic localization, RET is localized in the nucleus and functions as a tyrosine-threonine dual specificity kinase. Knockdown of RET by shRNA in medullary thyroid cancer-derived cells stimulated expression of activating transcription factor 4 (ATF4), a master transcription factor for stress-induced apoptosis, through activation of its target proapoptotic genes NOXA and PUMA. RET knockdown also increased sensitivity to cisplatin-induced apoptosis. We observed that RET physically interacted with and phosphorylated ATF4 at tyrosine and threonine residues. Indeed, RET kinase activity was required to inhibit the ATF4-dependent activation of the NOXA gene because the site-specific substitution mutations that block threonine phosphorylation increased ATF4 stability and activated its targets NOXA and PUMA. Moreover, chromatin immunoprecipitation assays revealed that ATF4 occupancy increased at the NOXA promoter in TT cells treated with tyrosine kinase inhibitors or the ATF4 inducer eeyarestatin as well as in RET-depleted TT cells. Together these findings reveal RET as a novel dual kinase with nuclear localization and provide mechanisms by which RET represses the proapoptotic genes through direct interaction with and phosphorylation-dependent inactivation of ATF4 during the pathogenesis of medullary thyroid cancer.