MODULATION OF THE PHOSPHORYLATION STATE OF TAU IN-SITU - THE ROLES OFCALCIUM AND CYCLIC-AMP

Alterations in situ in the phosphorylation state of the microtubule-as sociated protein tau were examined in response to increasing intracell ular levels of Ca2+ through N-methyl-D-aspartate (NMDA)-receptor activ ation, or activating cyclic AMP (cAMP)-dependent protein kinase (cAMP- PK), in rat cerebral-cortical slices. Increasing intracellular concent rations of Ca2+ by treatment of the brain slices with the glutamate an alogue NMDA in depolarizing conditions (55 mM KCl) resulted in dephosp horylation of tau. Addition of KCl + NMDA to the slices resulted in a 40 % decrease in P-32 incorporation into tan, whereas addition of KCl or NMDA. alone had no effect on tau phosphorylation. The KCl + NMDA-in duced dephosphorylation of tau was blocked by the non-competitive NMDA -receptor antagonist MK801. To determine the involvement of the Ca2+/c almodulin-dependent. phosphatase,calcineurin, in the KCl + NMDA-induce d dephosphorylation of tau, slices were pretreated with the calcineuri n inhibitor Cyclosporin A. Pretreatment of the rat brain slices with C yclosporin A completely abolished the dephosphorylation of tau induced by the addition of KCl + NMDA. The dephosphorylation of tau in situ w as site-selective, as indicated by the loss of P-32 label from only a few select peptides. Activation of cAMP-PK by stimulating adenylate cy clase in rat cerebral-cortical slices with forskolin resulted in a 73% increase over control levels in P-32 incorporation into immunoprecipi tated tau. Two-dimensional phosphopeptide mapping revealed that most o f the sites on tau phosphorylated in brain slices in response to incre ased cAMP levels were the same as those phosphorylated on isolated tau by purified cAMP-PK. Although the state of tau phosphorylation is cer tainly regulated by many protein phosphatases and kinases in vivo, to our knowledge this study provides the first direct evidence of a speci fic protein phosphatase and kinase that modulate the phosphorylation s tate of tau in situ.