A novel dual kinase function of the RET proto-oncogene negatively regulates activating transcription factor 4-mediated apoptosis.

Abstract

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.

• Authors: Rozita Bagheri-Yarmand, Krishna M Sinha, Anupama E Gururaj, Zamal Ahmed, Yasmeen Q Rizvi, Su-Chen Huang, John E Ladbury, Oliver Bogler, Michelle D Williams, Gilbert J Cote, Robert F Gagel
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 290 Issue 18 Pg. 11749-61 (May 01 2015) ISSN: 1083-351X [Electronic] United States
• PMID: 25795775 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)
• Copyright: © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Chemical References

• Apoptosis Regulatory Proteins
• BBC3 protein, human
• MAS1 protein, human
• PMAIP1 protein, human
• Protein Kinase Inhibitors
• Proto-Oncogene Mas
• Proto-Oncogene Proteins
• Proto-Oncogene Proteins c-bcl-2
• Activating Transcription Factor 4
• Threonine
• Proto-Oncogene Proteins c-ret
• Cisplatin

Topics

• Activating Transcription Factor 4 (chemistry, metabolism)
• Active Transport, Cell Nucleus (drug effects)
• Apoptosis (drug effects)
• Apoptosis Regulatory Proteins (genetics)
• Cell Line, Tumor
• Cell Nucleus (drug effects, metabolism)
• Cisplatin (pharmacology)
• Gene Expression Regulation (drug effects)
• Humans
• Phosphorylation (drug effects)
• Promoter Regions, Genetic (genetics)
• Protein Kinase Inhibitors (pharmacology)
• Proteolysis (drug effects)
• Proto-Oncogene Mas
• Proto-Oncogene Proteins (genetics)
• Proto-Oncogene Proteins c-bcl-2 (genetics)
• Proto-Oncogene Proteins c-ret (metabolism)
• Threonine (metabolism)
• Transcription, Genetic (drug effects)