title: GSK3beta-mediated Drp1 phosphorylation induced elongated mitochondrial morphology against oxidative stress.
abstract: 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 H(2)O(2)-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 H(2)O(2) insult.
authors.affiliation: Department of Biochemistry, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan, ROC.
authors.forename: Chia-Hua
authors.initials: CH
authors.lastname: Chou
authors.forename: Ching-Chih
authors.initials: CC
authors.lastname: Lin
authors.forename: Ming-Chang
authors.initials: MC
authors.lastname: Yang
authors.forename: Chih-Chang
authors.initials: CC
authors.lastname: Wei
authors.forename: Huei-De
authors.initials: HD
authors.lastname: Liao
authors.forename: Run-Chin
authors.initials: RC
authors.lastname: Lin
authors.forename: Wen-Yu
authors.initials: WY
authors.lastname: Tu
authors.forename: Tsung-Chieh
authors.initials: TC
authors.lastname: Kao
authors.forename: Ching-Mei
authors.initials: CM
authors.lastname: Hsu
authors.forename: Jiin-Tsuey
authors.initials: JT
authors.lastname: Cheng
authors.forename: An-Kuo
authors.initials: AK
authors.lastname: Chou
authors.forename: Chu-I
authors.initials: CI
authors.lastname: Lee
authors.forename: Joon-Khim
authors.initials: JK
authors.lastname: Loh
authors.forename: Shen-Long
authors.initials: SL
authors.lastname: Howng
authors.forename: Yi-Ren
authors.initials: YR
authors.lastname: Hong
mesh_terms: D000595:Amino Acid Sequence; D017209:Apoptosis / Q000187:drug effects; D001343:Autophagy / Q000187:drug effects; D034281:Dynamins / Q000737:chemistry / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006868:Hydrolysis / Q000187:drug effects; D008239:Lysine / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology*; D063154:Mitochondrial Dynamics / Q000187:drug effects; D008954:Models, Biological; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000187:drug effects; D020798:Two-Hybrid System Techniques
chemical_list: D050505:Mutant Proteins; D010768:Phosphoserine; D006861:Hydrogen Peroxide; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3; D020558:GTP Phosphohydrolases; D034281:Dynamins; D008239:Lysine