GSK3beta-Mediated Drp1 Phosphorylation Induced Elongated Mitochondrial Morphology against Oxidative Stress

et al. (2012) GSK3beta-Mediated Drp1 Phosphorylation Induced Elongated Mitochondrial Morphology against Oxidative Stress. PLoS ONE 7(11): e49112. doi:10.1371/journal.pone.0049112 GSK3beta-Mediated Drp1 Phosphorylation Induced Elongated Mitochondrial Morphology against Oxidative Stress Chia-Hua Chou 0 Ching-Chih Lin 0 Ming-Chang Yang 0 Chih-Chang Wei 0 Huei-De Liao 0 Run- Chin Lin 0 Wen-Yu Tu 0 Tsung-Chieh Kao 0 Ching-Mei Hsu 0 Jiin-Tsuey Cheng 0 An-Kuo Chou 0 Chu- 0 I Lee 0 Joon-Khim Loh 0 Shen-Long Howng 0 Yi-Ren Hong 0 Dhyan Chandra, Roswell Park Cancer Institute, United States of America 0 1 Department of Biochemistry, Faculty of Medicine, College of Medicine, Kaohsiung Medical University , Kaohsiung, Taiwan , R.O.C. , 2 Department of Biological Sciences, National Sun Yat-Sen University , Kaohsiung, Taiwan , R.O.C. , 3 Laboratory of Genetic Research, Kaohsiung Armed Forces General Hospital , Kaohsiung, Taiwan , R.O.C. , 4 Graduate Institute of Medicine, Kaohsiung Medical University , Kaohsiung, Taiwan , R.O.C. , 5 Department of Anesthesiology, Kaohsiung Chang Gung Memorial Hospital and Chang-Gung University College of Medicine , Kaohsiung, Taiwan , R.O.C. , 6 Department of Medical Laboratory Science and Biotechnology, Fooyin University , Kaohsiung, Taiwan , R.O.C. , 7 Department of Neurosurgery, Kaohsiung Medical University Hospital , Kaohsiung , Taiwan, R.O.C Multiple phosphorylation sites of Drp1 have been characterized for their functional importance. However, the functional consequence of GSK3beta-mediated phosphorylation of Drp1 remains unclear. In this report, we pinpointed 11 Serine/ Threonine sites spanning from residue 634,736 of the GED domain and robustly confirmed Drp1 Ser693 as a novel GSK3beta phosphorylation site. Our results suggest that GSK3beta-mediated phosphorylation at Ser693 does cause a dramatic decrease of GTPase activity; in contrast, GSK3beta-mediated phosphorylation at Ser693 appears not to affect Drp1 inter-/intra-molecular interactions. After identifying Ser693 as a GSK3beta phosphorylation site, we also determined that K679 is crucial for GSK3beta-binding, which strongly suggests that Drp1 is a novel substrate for GSK3beta. Thereafter, we found that overexpressed S693D, but not S693A mutant, caused an elongated mitochondrial morphology which is similar to that of K38A, S637D and K679A mutants. Interestedly, using H89 and LiCl to inhibit PKA and GSK3beta signaling, respectively, it appears that a portion of the elongated mitochondria switched to a fragmented phenotype. In investigating the biofunctionality of phosphorylation sites within the GED domain, cells overexpressing Drp1 S693D and S637D, but not S693A, showed an acquired resistance to H2O2-induced mitochondrial fragmentation and ensuing apoptosis, which affected cytochrome c, capase-3, -7, and PARP, but not LC3B, Atg-5, Beclin-1 and Bcl2 expressions. These results also showed that the S693D group is more effective in protecting both non-neuronal and neuronal cells from apoptotic death than the S637D group. Altogether, our data suggest that GSK3beta-mediated phosphorylation at Ser693 of Drp1 may be associated with mitochondrial elongation via down-regulating apoptosis, but not autophagy upon H2O2 insult. - Funding: This study was sponsored by National Science Council-98-2320-B-037-024-MY3; National Science Council -98-2321-B-037-061; National Science Council99-2321-B-037-001; National Health Research Intitute-EX100-9809SI (Taiwan); National Sun Yet-San University-Kaohsiung Medical University Joint Research Project-101-020 to Yi-Ren Hong and National Science Council-100-2314-B-037-010 (Taiwan) to Shen-Long Howng. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase originally found to inactivate the enzyme glycogen synthase by phosphorylation [1]. It is well documented that GSK-3 is crucial for cell development, metabolic homeostasis, neuronal growth and differentiation, cell polarity, cell fate and apoptosis [2]. Two mammalian GSK3 isoforms, GSK3a and GSK3beta, that share 97% amino acid identity in their catalytic domain were cloned [3]. GSK3beta has attracted significant attention, in part due to its multifaceted roles in multiple key pathophysiological pathways involved in Alzheimers disease (AD) and several neurodegenerative diseases [4]. We previously explored GSK3beta and its interacting proteins, HdynIV (since renamed dynamin related protein 1, Drp1) [5]. Functional characterization showed that this Drp1 variant lacks a proline-rich domain on its carboxyl-terminus, which was identified as a critical region for interacting with GSK3 [6]. Drp1 is one of the dynamin related proteins, which is a large protein reported to be comprised of an amino (...truncated)