okadaic-acid and 4-aminophenylphosphate

We isolated and characterized several phosphoseryl/phosphothreonyl phosphatase activities (P1-P11) from frontal lobe of six autopsied human brains. Of these, PP1 (P3) was a major tau phosphatase. The enzyme required metal ions and was maximally activated by Mn2+. Western blots with antibodies to known protein phosphatases showed PP1 and PP2B immunoreactivity. However, the removal of PP2B by immunoabsorption or its inhibition with EGTA did not result in appreciable loss of P3 activity. These observations suggest that P3 was an enriched PPI. Dephosphorylation of Alzheimer disease hyperphosphorylated tau (AD P-tau) by PP1 was site-specific. PPI preferentially dephosphorylated pT212 (40%), pT217 (26%), pS262 (33%), pS396 (42%) and pS422 (31%) of AD P-tau. Dephosphorylation of tau at pT181, pS199, pS202, pT205, pS214, and pS404, was undetectable. Of the sites dephosphorylated, pT212 was only a substrate for PP1, as purified/enriched PP2A and PP2B from the same brains did not dephosphorylate this site.

Topics: Adenosine Diphosphate; Alzheimer Disease; Aniline Compounds; Blotting, Western; Brain Chemistry; Calcineurin; Calmodulin; Chromatography, Agarose; Chromatography, DEAE-Cellulose; Chromatography, Gel; Enzyme Inhibitors; Humans; Okadaic Acid; Organophosphorus Compounds; Phosphoprotein Phosphatases; Phosphorylation; Phosphothreonine; Polylysine; Protein Phosphatase 1; Recombinant Proteins; Substrate Specificity; tau Proteins; Triazines

Topics: Aniline Compounds; Animals; Blastocyst; Egg Proteins; Embryo, Nonmammalian; Enzyme Activation; Enzyme Inhibitors; Marine Toxins; Morula; Okadaic Acid; Oocytes; Organophosphorus Compounds; Oxazoles; Phosphoprotein Phosphatases; Sea Urchins; Zygote

The catalytic subunit of Ca2+/calmodulin-dependent protein phosphatase (calcineurin A) was overexpressed about 50-fold in Dictyostelium discoideum cells transformed with a vector containing the cDNA for D. discoideum calcineurin A under control of the actin-6 promoter. In crude lysates from the overexpressing cell line, high Ca2+/calmodulin-stimulated phosphatase activity was detected. Calcineurin A was purified by anion exchange chromatography and calmodulin-Sepharose affinity chromatography, and the enzymatic activity of the isolated protein was characterized. Its phosphatase activity was strictly dependent on the addition of divalent metal ions such as Mg2+ or Mn2+. Disulphide-reducing agents increased the activity more than 10-fold. Ca2+/calmodulin stimulated the activity by a factor of 2.5-5. Despite the high extra Ca2+/calmodulin-dependent phosphatase activity, the overexpressing cell line showed no phenotypic aberrations.

Topics: Aniline Compounds; Animals; Calcineurin; Calcineurin Inhibitors; Calmodulin; Caseins; Cations, Divalent; Cattle; Chlorides; Chromatography, Affinity; Chromatography, DEAE-Cellulose; Dictyostelium; Dithiothreitol; DNA, Complementary; Enzyme Inhibitors; Gene Expression; Magnesium Chloride; Manganese Compounds; Okadaic Acid; Organophosphorus Compounds; Phosphopeptides; Protozoan Proteins; Recombinant Fusion Proteins; Sulfhydryl Reagents; Transformation, Genetic

A specific inhibitor of protein tyrosine phosphatase (PTPase), RK-682 (3-hexadecanoyl-5-hydroxymethyl-tetronic acid) was isolated from microbial metabolites. In vitro, RK-682 inhibited dephosphorylation activity of CD45 and VHR with IC50 54 and 2.0 microM, respectively. In situ, sodium orthovanadate and RK-682 enhanced the phosphotyrosine level of Ball-1 cells, a human B cell leukemia, but not the phosphoserine/threonine level. The PTPase inhibitors, however, had the different arrest point on the cell cycle progression. Sodium orthovanadate inhibited the cell cycle progression at G2/M boundary phase, on the other hand, RK-682 inhibited the G1/S transition.

Topics: Aniline Compounds; Antifungal Agents; Blotting, Western; cdc25 Phosphatases; Cell Cycle; Cell Cycle Proteins; Dual Specificity Phosphatase 3; Enzyme Inhibitors; Ethers, Cyclic; G1 Phase; Humans; Kinetics; Leukemia, B-Cell; Okadaic Acid; Organophosphorus Compounds; Phosphoprotein Phosphatases; Phosphoproteins; Phosphotyrosine; Protein Tyrosine Phosphatases; Pyrans; Spiro Compounds; Streptomyces; Tumor Cells, Cultured; Vanadates