Effect Of Tau Mutations At PKA Sites On Its Phosphorylation

Alzheimer disease (AD) is one of main causes of dementia in the elderly population. The major neuropathological hallmarks of Alzheimer disease (AD) are senile plaque and the intracellular neurofibrillary tangle (NFT) , which is mainly composed of hyperphosphorylated microtubule associated protein tau. The amount of NFTs correlates positively with the severity of cognitive impairment in AD. The main functions of tau are to promote microtubule assembly, maintain the stability of the microtubules,maintain axonal transport. Hyperphosphorylated tau impairs the function of tau binding with microtubule then results in the destabilisation of microtubules, ultimately leads to the degeneration of the affected neurons. Phosphorylation of tau protein is regulated by several kinases, especially cAMP-dependent protein kinase (PKA) and glycogen synthase kinase 3β(GSK-3β) . The activity of protein kinases is critical for the regulation of tau phosphorylation. Recent study demonstrated that tau becomes a more favorable substrate for GSK-3βwhen it is prephosphorylated by PKA in rat brain, but the site-specific modulation of tau prephosphorylation has not been well investigated.To determine how the phosphorylation of PKA specific sites affect tau's biochemical function and further investigate the combined role of PKA and GSK-3βin Alzheimer disease. We reconstructed and expressed tau plasmid. After HEK293/wt cells or N2a/wt cells were transfected with these tau plasmids, N2a/wt cells were treated by forskolin which is a specific PKA activator and HEK293/wt cells were treated by forskolin, or forskolin combining SB, a specific GSK-3βinhibitor. The expression, phosphorylation and assembly of the tau plasmid were examined by western blotting or immunofluorescence.The results showed that forskolin enhanced the phosphorylation of tau at Ser262. Ser404 site of tau can't be phosphorylated when transfected both the tauS409A and the tauS409E . The phosphorylation level of Ser262 was decreased, when transfected the tau^S409A, and the phosphorylation level did not change as compared to control, when transfected the tau^S409E. The phosphorylation level of Thr231 was also decreased, when HEK293/wt cell were transfected constructs of tau^S214A, tau^S262A and tau^S409A. After Forskolin treatment, tau phosphoryaltion at Ser^396/404 was obviousely enhanced in HEK293/wt cell transfected the tauWT but at Ser396/404 was significant decreased in HEK293/wt cell transfected the tau^S214A, tau^S262A and tau^S409A .Conclusions All of data demonstrated: 1) after tau at Ser409 site was phosphorylated,. tau at Ser262 site was more easy to be phosphorlsted by forskolin and mutant tau at Ser409 caused tau at Ser404 site not to be phosphorylated.. 2) Phosphorylated of tau at. Ser214 site can promote tau phosphorylation at Thr231.3) Prephosphorylation of Ser214, Ser262 and Ser409 by PKA can upregulate tau phosphorylation at Ser396/404 site.