Phosphorylation of Amyloid Precursor Protein at Threonine-668 is Essential for its Copper-Responsive trafficking in SH-SY5Y neuroblastoma cells. [Signal Transduction]

March 7th, 2014 by Acevedo, K. M., Opazo, C. M., Norrish, D., Challis, L. M., Li, Q.-X., White, A. R., Bush, A. I., Camakaris, J.

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 (T668) in SH-SY5Y neuronal cells. The level of APPT668-p (detected using a phosphosite-specific antibody) exhibited a copper-dependent increase. Using confocal microscopy imaging we demonstrate that the phospho-deficient mutant, T668 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 T668 residue in copper-responsive trafficking by treating SH-SY5Y cells with inhibitors for glycogen synthase kinase 3-β (GSK3-β) and cyclin-dependent kinases (Cdk), the main kinases that phosphorylate APP at T668 in neurons. Our results show that the GSK3-β 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 GSK3-β dependent phosphorylation in SH-SY5Y cells.
  • Posted in Journal of Biological Chemistry, Publications
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