PHOSPHORYLATION OF TAU IN-SITU - INHIBITION OF CALCIUM-DEPENDENT PROTEOLYSIS

In this study, the in situ phosphorylation and subsequent calcium-acti vated proteolysis of tau protein were examined in human neuroblastoma (LA-N-5) cells, which were differentiated into a neuronal phenotype. T he phosphorylation of tau was increased by treating the cells with for skolin and rolipram, which elevate cyclic AMP levels els, by treating with the phosphatase inhibitor okadaic acid, or by treating with a com bination of both treatments. Phosphorylated tau migrated slightly slow er on sodium dodecyl sulfate-polyacrylamide gels than tau from untreat ed cells. Immunostaining with the phosphate-sensitive monoclonal antib ody Tau-1 was also decreased in cells treated with okadaic acid, indic ating an increase in the phosphorylation of specific Ser-Pro motifs wi thin the molecule. Calcium-dependent, in situ proteolysis of tau prote in was induced by treating the cells with the calcium ionophore A23187 , tau protein was proteolyzed to a significantly lesser extent in cell s treated with forskolin and rolipram, okadaic acid, or both than in c ells in which phosphorylation was not increased, Partially purified ta u protein from cells treated with a combination of forskolin, rolipram , and okadaic acid was also more resistant to proteolysis by calpain i n vitro compared with tau isolated from control cells. These data sugg est a possible role for phosphorylation in the regulation of tau metab olism and in pathological conditions in which the balance between prot ein kinases and phosphatases is disrupted.