THE INTERRELATIONSHIP BETWEEN SELECTIVE TAU-PHOSPHORYLATION AND MICROTUBULE ASSOCIATION

The purpose of this study was to examine the modulation of tau phospho rylation mediated by protein kinase A, a kinase with low intrinsic act ivity, and by the constitutively active glycogen synthase kinase, as w ell as to examine the subsequent effects on tau-microtubule associatio n in differentiated human SH-SY5Y neuroblastoma cells. Activation of p rotein kinase A with forskolin and rolipram significantly increased ta u phosphorylation at Ser262/356 only in the presence of okadaic acid, indicating that phosphates at these sites are normally turned over rap idly. In contrast, glycogen synthase kinase appears to maintain tau ph osphorylation at Thr181 and Ser396/404 since inhibition of glycogen sy nthase kinase with lithium reduced phosphorylation at these sites. Lit hium treatment also significantly decreased tau and tyrosinated alpha- tubulin levels. Perturbation of microtubules with nocodazole or taxol induced tau dephosphorylation at Tau-1 sites, Thr181 and Ser396/404, i ndicating that both constitutive kinase activity and microtubule state modulate tan phosphorylation at these sites. Nocodazole- or taxol-ind uced tau dephosphorylation was blocked by the protein phosphatase 2A/1 inhibitor okadaic acid, but not by the protein phosphatase 2B inhibit or cyclosporin A. In addition, osmotic stress, such as treatment with 20 mM NaCl, selectively increased tau phosphorylation at the Tau-1 epi tope. To investigate the effect of phosphorylation on tau association with microtubules and microtubule stability in situ, a Triton X-100 ex traction assay was utilized to separate the detergent-soluble cytosoli c components from the detergent-insoluble cytoskeletal components. In control cells or cells treated with lithium very little tau was detect ed in the cytosolic fraction. Activation of protein kinase A in the pr esence of okadaic acid elevated tau levels in the detergent-soluble fr action, which contained all the tau phosphorylated at Ser262/356, and also decreased microtubule stability, as indicated by decreased acetyl ated alpha-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 microtub ule-binding, which was mediated in part by protein kinase A, prevented the association of tau with microtubules in situ. (C) 1998 Elsevier S cience B.V. All rights reserved.