Phosphorylation of tau in situ: inhibition of calcium-dependent proteolysis.
In this study, the in situ phosphorylation and subsequent calcium-activated proteolysis of tau protein were examined in human neuroblastoma (LA-N-5) cells, which were differentiated into a neuronal phenotype. The phosphorylation of tau was increased by treating the cells with forskolin and rolipram, which elevate cyclic AMP levels, by treating with the phosphatase inhibitor okadaic acid, or by treating with a combination of both treatments. Phosphorylated tau migrated slightly slower on sodium dodecyl sulfate-polyacrylamide gels than tau from untreated cells. Immunostaining with the phosphate-sensitive monoclonal antibody Tau-1 was also decreased in cells treated with okadaic acid, indicating an increase in the phosphorylation of specific Ser-Pro motifs within the molecule. Calcium-dependent, in situ proteolysis of tau protein was induced by treating the cells with the calcium ionophore A23187. Tau protein was proteolyzed to a significantly lesser extent in cells treated with forskolin and rolipram, okadaic acid, or both than in cells in which phosphorylation was not increased. Partially purified tau protein from cells treated with a combination of forskolin, rolipram, and okadaic acid was also more resistant to proteolysis by calpain in vitro compared with tau isolated from control cells. These data suggest a possible role for phosphorylation in the regulation of tau metabolism and in pathological conditions in which the balance between protein kinases and phosphatases is disrupted.
Litersky
J M
JM
Department of Psychiatry and Behavioral Neurobiology, University of Alabama at Birmingham 35294-0017, USA.
Johnson
G V
GV
D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000502:physiology*; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000494:pharmacology; D006801:Humans; D009474:Neurons / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism*; D010766:Phosphorylation; D011480:Protease Inhibitors / Q000494:pharmacology*; D011760:Pyrrolidinones / Q000494:pharmacology; D020889:Rolipram; D014407:Tumor Cells, Cultured; D016875:tau Proteins / Q000378:metabolism*
D011480:Protease Inhibitors; D011760:Pyrrolidinones; D016875:tau Proteins; D005576:Colforsin; D000001:Calcimycin; D000242:Cyclic AMP; D010447:Peptide Hydrolases; D020889:Rolipram; D002118:Calcium