Tau proteins-enhanced Ca/calmodulin (CaM)-dependent phosphorylation by the brain supernatant of diisopropyl phosphorofluoridate (DFP)-treated hen: tau mutants indicate phosphorylation of more amino acids in tau by CaM kinase II

Diisopropyl phosphorofluoridate (DFP) produces organophosphorus ester-induced delayed neurotoxicity (OPIDN) in hen, human, and other sensitive species. A single dose of DFP (1.7 mg/kg, s.c.) produces mild ataxia in 7–14 days in hens, followed by progression to severe ataxia or paralysis. We studied the effect of DFP administration on Ca²⁺/calmodulin-dependent phosphorylation of tau proteins by the brain supernatants of control and DFP-treated hens. Brain supernatants from DFP-treated hens showed enhanced in vitro phosphorylation of htau40 and its various mutants, but no change in the two-dimensional phosphopeptide pattern, when compared to control hen brain supernatants. Analysis of tau mutants phosphorylated by brain supernatant and recombinant CaM kinase II α-subunit showed that (1) brain supernatant CaM kinase II is mainly responsible for the phosphorylation of Ser⁴¹⁶, (2) Ser³⁵⁶, but probably not Ser²⁶², is phosphorylated by CaM kinase II, (3) no amino acid between Lys³⁹⁵–Ala⁴³⁷ except Ser⁴¹⁶ is phosphorylated by CaM kinase II, (4) a number of amino acids in the tau molecule, which are phosphorylated by the brain supernatant in the absence of Ca²⁺/calmodulin are also mildly phosphorylated by CaM kinase II. The enhanced Ca²⁺/calmodulin-dependent phosphorylation of tau proteins by brain supernatant of DFP-treated hens that includes phosphorylation of a number of amino acids is likely to alter the functional properties of tau proteins in OPIDN. The hyperphosphorylated tau may destabilize microtubules, alter axonal transport, and result in degeneration of axons in OPIDN.