Tau Becomes a More Favorable Substrate for GSK-3 When It Is Prephosphorylated by PKA in Rat Brain

Abstract

Microtubule-associated protein tau is abnormally hyperphosphorylated in Alzheimer's disease (AD) and other tauopathies and is believed to lead to neurodegeneration in this family of diseases. Here we show that infusion of forskolin, a specific cAMP-dependent protein kinase A (PKA) activator, into the lateral ventricle of brain in adult rats induced activation of PKA by severalfold and concurrently enhanced the phosphorylation of tau at Ser-214, Ser-198, Ser-199, and or Ser-202 (Tau-1 site) and Ser-396 and or Ser-404 (PHF-1 site), which are among the major abnormally hyperphosphorylated sites seen in AD. PKA activation positively correlated to the extent of tau phosphorylation at these sites. Infusion of forskolin together with PKA inhibitor or glycogen synthase kinase-3 (GSK-3) inhibitor revealed that the phosphorylation of tau at Ser-214 was catalyzed by PKA and that the phosphorylation at both the Tau-1 and the PHF-1 sites is induced by basal level of GSK-3, because forskolin activated PKA and not GSK-3 and inhibition of the latter inhibited the phosphorylation at Tau-1 and PHF-1 sites. Inhibition of cdc2, cdk5, or MAPK had no significant effect on the forskolin-induced hyperphosphorylation of tau. Forskolin inhibited spatial memory in a dose-dependent manner in the absence but not in the presence of R p -adenosine 3 ,5 -cyclic monophosphorothioate triethyl ammonium salt, a PKA inhibitor. These results demonstrate for the first time that phosphorylation of tau by PKA primes it for phosphorylation by GSK-3 at the Tau-1 and the PHF-1 sites and that an associated loss in spatial memory is inhibited by inhibition of the hyperphosphorylation of tau. These data provide a novel mechanism of the hyperphosphorylation of tau and identify both PKA and GSK-3 as promising therapeutic targets for AD and other tauopathies.

Activation of Glycogen Synthase Kinase-3 Inhibits Long-Term Potentiation with Synapse-Associated Impairments

“…The hippocampus was homogenized as described above, and the homogenate was centrifuged at 12,000 ϫ g for 20 min at 4°C. The resulting supernatant was assayed for GSK-3 activity using phospho-GS peptide 2 (Millipore) as described previously (Liu et al, 2004). Briefly, 7.5 g of protein was incubated for 30 min at 30°C with 20 M peptide substrate and 200 M ␥-32 P ATP (1500 cpm/pmol ATP) in 30 mM Tris, pH 7.4, 10 mM MgCl 2 , 10 mM NaF, 1 mM Na 3 VO 4 , 2 mM EGTA, and 10 mM ␤-mercaptoethanol in a total volume of 25 l. The reaction was stopped with 300 mM o-phosphoric acid (25 l).…”

Tau-Based Treatment Strategies in Neurodegenerative Diseases

“…cAMP-dependent protein kinase A (PKA) has been shown to phosphorylate tau at S262/S356 and S214 in vitro and in vivo. 102,103 Phosphorylation by PKA results in reduced microtubule-binding affinity and promotes the subsequent phosphorylation of different diagnostic phospho-epitopes. PKA phosphorylation at S214 is required to prime tau for its sequential phosphorylation by GSK-3␤, thereby giving rise to the AD-specific AT100 phospho-epitope.…”

“…102 Activation of PKA by forskolin resulted in spatial memory deficits in rats. 103 Isoquinoline sulfonamide derivatives and staurosporines are known to inhibit PKA with low selectivity and also affect PKG, PKC, and the myosin light chain kinase (MLCK). PKA inhibitors with higher selectivity are KT-5720 and PKI.…”

Physiological regulation of tau phosphorylation during hibernation

“…priming effect) (Liu et al 2004), such that even basal levels of GSK-3b activity may result in tau hyperphosphorylation. Additionally, PKA can also phosphorylate tau at several sites (i.e.…”