title: Phosphorylation of amyloid precursor protein at threonine 668 is essential for its copper-responsive trafficking in SH-SY5Y neuroblastoma cells.
abstract: Amyloid precursor protein (APP) undergoes post-translational modification, including O- and N-glycosylation, ubiquitination, and phosphorylation as it traffics through the secretory pathway. We have previously reported that copper promotes a change in the cellular localization of APP. We now report that copper increases the phosphorylation of endogenous APP at threonine 668 (Thr-668) in SH-SY5Y neuronal cells. The level of APPT668-p (detected using a phospho-site-specific antibody) exhibited a copper-dependent increase. Using confocal microscopy imaging we demonstrate that the phospho-deficient mutant, Thr-668 to alanine (T668A), does not exhibit detectable copper-responsive APP trafficking. In contrast, mutating a serine to an alanine at residue 655 does not affect copper-responsive trafficking. We further investigated the importance of the Thr-668 residue in copper-responsive trafficking by treating SH-SY5Y cells with inhibitors for glycogen synthase kinase 3-β (GSK3β) and cyclin-dependent kinases (Cdk), the main kinases that phosphorylate APP at Thr-668 in neurons. Our results show that the GSK3β kinase inhibitors LiCl, SB 216763, and SB 415286 prevent copper-responsive APP trafficking. In contrast, the Cdk inhibitors Purvalanol A and B had no significant effect on copper-responsive trafficking in SH-SY5Y cells. In cultured primary hippocampal neurons, copper promoted APP re-localization to the axon, and this effect was inhibited by the addition of LiCl, indicating that a lithium-sensitive kinase(s) is involved in copper-responsive trafficking in hippocampal neurons. This is consistent with APP axonal transport to the synapse, where APP is involved in a number of functions. We conclude that copper promotes APP trafficking by promoting a GSK3β-dependent phosphorylation in SH-SY5Y cells.
authors.affiliation: Department of Genetics, The University of Melbourne, Victoria 3010, Australia.
authors.forename: Karla M
authors.initials: KM
authors.lastname: Acevedo
authors.affiliation: Florey Institute of Neuroscience and Mental Health, Victoria 3052, Australia, and.
authors.forename: Carlos M
authors.initials: CM
authors.lastname: Opazo
authors.affiliation: Department of Genetics, The University of Melbourne, Victoria 3010, Australia.
authors.forename: David
authors.initials: D
authors.lastname: Norrish
authors.affiliation: Department of Genetics, The University of Melbourne, Victoria 3010, Australia.
authors.forename: Leesa M
authors.initials: LM
authors.lastname: Challis
authors.affiliation: Department of Pathology, The University of Melbourne, Victoria 3010, Australia.
authors.forename: Qiao-Xin
authors.initials: QX
authors.lastname: Li
authors.affiliation: Department of Pathology, The University of Melbourne, Victoria 3010, Australia.
authors.forename: Anthony R
authors.initials: AR
authors.lastname: White
authors.affiliation: Florey Institute of Neuroscience and Mental Health, Victoria 3052, Australia, and.
authors.forename: Ashley I
authors.initials: AI
authors.lastname: Bush
authors.affiliation: Department of Genetics, The University of Melbourne, Victoria 3010, Australia,. Electronic address: j.camakaris@unimelb.edu.au.
authors.forename: James
authors.initials: J
authors.lastname: Camakaris
mesh_terms: D000276:Adjuvants, Immunologic / Q000494:pharmacology; D000627:Aminophenols / Q000494:pharmacology; D016564:Amyloid beta-Protein Precursor / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001369:Axons / Q000378:metabolism*; D045744:Cell Line, Tumor; D003300:Copper / Q000378:metabolism*; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D018021:Lithium Chloride / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D051379:Mice; D020125:Mutation, Missense; D009447:Neuroblastoma / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects / Q000235:genetics; D021381:Protein Transport / Q000187:drug effects; D013569:Synapses / Q000378:metabolism*
chemical_list: C417520:3-(3-chloro-4-hydroxyphenylamino)-4-(4-nitrophenyl)-1H-pyrrole-2,5-dione; C545487:APP protein, human; D000276:Adjuvants, Immunologic; D000627:Aminophenols; D016564:Amyloid beta-Protein Precursor; D007211:Indoles; D008301:Maleimides; C417521:SB 216763; D003300:Copper; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; D038362:Glycogen Synthase Kinase 3; D018021:Lithium Chloride
keywords: Amyloid Precursor Protein; Confocal Microscopy; Copper; Glycogen Synthase Kinase 3; Phosphorylation; Trafficking