Abstract
The purpose of this study was to examine the modulation of tau phosphorylation mediated by protein kinase A, a kinase with low intrinsic activity, and by the constitutively active glycogen synthase kinase, as well as to examine the subsequent effects on tau-microtubule association in differentiated human SH-SY5Y neuroblastoma cells. Activation of protein kinase A with forskolin and rolipram significantly increased tau phosphorylation at Ser262/356 only in the presence of okadaic acid, indicating that phosphates at these sites are normally turned over rapidly. In contrast, glycogen synthase kinase appears to maintain tau phosphorylation at Thr181 and Ser396/404 since inhibition of glycogen synthase kinase with lithium reduced phosphorylation at these sites. Lithium treatment also significantly decreased tau and tyrosinated
α-tubulin levels. Perturbation of microtubules with nocodazole or taxol induced tau dephosphorylation at Tau-1 sites, Thr181 and Ser396/404, indicating that both constitutive kinase activity and microtubule state modulate tau phosphorylation at these sites. Nocodazole- or taxol-induced tau dephosphorylation was blocked by the protein phosphatase 2A/1 inhibitor okadaic acid, but not by the protein phosphatase 2B inhibitor cyclosporin A. In addition, osmotic stress, such as treatment with 20 mM NaCl, selectively increased tau phosphorylation at the Tau-1 epitope. To investigate the effect of phosphorylation on tau association with microtubules and microtubule stability in situ, a Triton X-100 extraction assay was utilized to separate the detergent-soluble cytosolic components from the detergent-insoluble cytoskeletal components. In control cells or cells treated with lithium very little tau was detected in the cytosolic fraction. Activation of protein kinase A in the presence of okadaic acid elevated tau levels in the detergent-soluble fraction, which contained all the tau phosphorylated at Ser262/356, and also decreased microtubule stability, as indicated by decreased acetylated
α-tubulin levels. In conclusion, the phosphorylation state of tau in differentiated SH-SY5Y cells is regulated by glycogen synthase kinase, microtubule dynamics and osmotic stress at overlapping sites which apparently have little influence on tau-microtubule association. In contrast, phosphorylation of tau at Ser262/356 within the microtubule-binding, which was mediated in part by protein kinase A, prevented the association of tau with microtubules in situ.
