title: A Glaucoma-Associated Variant of Optineurin, M98K, Activates Tbk1 to Enhance Autophagosome Formation and Retinal Cell Death Dependent on Ser177 Phosphorylation of Optineurin.
abstract: Certain missense mutations in optineurin/OPTN and amplification of TBK1 are associated with normal tension glaucoma. A glaucoma-associated variant of OPTN, M98K, induces autophagic degradation of transferrin receptor (TFRC) and death in retinal cells. Here, we have explored the role of Tbk1 in M98K-OPTN-induced autophagy and cell death, and the effect of Tbk1 overexpression in retinal cells. Cell death induced by M98K-OPTN was dependent on Tbk1 as seen by the effect of Tbk1 knockdown and blocking of Tbk1 activity by a chemical inhibitor. Inhibition of Tbk1 also restores M98K-OPTN-induced transferrin receptor degradation. M98K-OPTN-induced autophagosome formation, autophagy and cell death were dependent on its phosphorylation at S177 by Tbk1. Knockdown of OPTN reduced starvation-induced autophagosome formation. M98K-OPTN expressing cells showed higher levels of Tbk1 activation and enhanced phosphorylation at Ser177 compared to WT-OPTN expressing cells. M98K-OPTN-induced activation of Tbk1 and its ability to be phosphorylated better by Tbk1 was dependent on ubiquitin binding. Phosphorylated M98K-OPTN localized specifically to autophagosomes and endogenous Tbk1 showed increased localization to autophagosomes in M98K-OPTN expressing cells. Overexpression of Tbk1 induced cell death and caspase-3 activation that were dependent on its catalytic activity. Tbk1-induced cell death possibly involves autophagy, as shown by the effect of Atg5 knockdown, and requirement of autophagic function of OPTN. Our results show that phosphorylation of Ser177 plays a crucial role in M98K-OPTN-induced autophagosome formation, autophagy flux and retinal cell death. In addition, we provide evidence for cross talk between two glaucoma associated proteins and their inter-dependence to mediate autophagy-dependent cell death.
authors.affiliation: Centre for Cellular and Molecular Biology, Council of Scientific and Industrial Research, Hyderabad, 500 007, India.
authors.forename: Kapil
authors.initials: K
authors.lastname: Sirohi
authors.affiliation: Centre for Cellular and Molecular Biology, Council of Scientific and Industrial Research, Hyderabad, 500 007, India.
authors.forename: Asha
authors.initials: A
authors.lastname: Kumari
authors.affiliation: Centre for Cellular and Molecular Biology, Council of Scientific and Industrial Research, Hyderabad, 500 007, India.
authors.forename: Vegesna
authors.initials: V
authors.lastname: Radha
authors.affiliation: Centre for Cellular and Molecular Biology, Council of Scientific and Industrial Research, Hyderabad, 500 007, India.
authors.forename: Ghanshyam
authors.initials: G
authors.lastname: Swarup
mesh_terms: D000818:Animals; D001343:Autophagy; D015153:Blotting, Western; D018797:Cell Cycle Proteins; D002478:Cells, Cultured; D019084:Fluorescent Antibody Technique, Indirect; D005901:Glaucoma / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D026901:Membrane Transport Proteins; D051379:Mice; D018613:Microscopy, Confocal; D010588:Phagosomes / Q000502:physiology*; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000235:genetics; D011990:Receptors, Transferrin / Q000378:metabolism*; D012165:Retinal Ganglion Cells / Q000378:metabolism / Q000473:pathology*; D015398:Signal Transduction; D035582:Transcription Factor TFIIIA / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: D018797:Cell Cycle Proteins; D026901:Membrane Transport Proteins; C409460:OPTN protein, human; D034741:RNA, Small Interfering; D011990:Receptors, Transferrin; D035582:Transcription Factor TFIIIA; C498588:Tbk1 protein, mouse; D017346:Protein Serine-Threonine Kinases