Phosphorylation of Rat Brain Choline Acetyltransferase and Its Relationship to Enzyme Activity

Abstract

Choline acetyltransferase catalyzes the formation of acetylcholine from choline and acetyl-CoA in cholinergic neurons. The present study examined conditions for modulation of kinase-mediated phosphorylation of this enzyme. By using a monospecific polyclonal rabbit anti-human choline acetyltransferase antibody to immunoprecipitate cytosolic and membrane-associated subcellular pools of enzyme from rat hippocampal synaptosomes, we determined that only the cytosolic fraction of the enzyme (67,000 +/- 730 daltons) was phosphorylated under basal, unstimulated conditions. The quantity of this endogenous phosphoprotein was dependent, in part, upon the level of intracellular calcium, with 32Pi incorporation into the enzyme in nerve terminals incubated in nominally calcium-free medium only 43 +/- 7% of control. The corresponding enzymatic activity of cytosolic choline acetyltransferase did not appear to be altered by lowered cytosolic calcium, whereas membrane-associated choline acetyltransferase activity was decreased to 58 +/- 11% of control. Depolarization of synaptosomes with 50 microM veratridine neither altered the extent of phosphorylation or specific activity of cytosolic choline acetyltransferase, nor induced detectable phosphorylation of membrane-associated choline acetyltransferase, although the specific activity of the membrane-associated enzyme was increased to 132 +/- 5% of control. In summary, phosphorylation of choline acetyltransferase does not appear to regulate cholinergic neurotransmission by a direct action on catalytic activity of the enzyme.

Arachidonic acid increases choline acetyltransferase activity in spinal cord neurons through a protein kinase C‐mediated mechanism

“…These observations are consistent with the fact that a decrease in phosphorylation rate of cytosolic (i.e. 69-kDa) ChAT due to diminished levels of cytosolic calcium resulted in a marked decrease of ChAT activity (Schmidt and Rylett 1993).…”

Non-neuronal expression of choline acetyltransferase in the rat kidney

“…ChAT immunoreactivity revealed that ChAT was localized to the apical side of principal cells. In human and other animal species, such as rodents and Drosophila, 80-90% of ChAT is in the cytosol, while the remaining 10-20% appears to be bound to the plasma membrane in nerve endings (Benishin and Carroll, 1981;Badamchian et al, 1986;Schmidt and Rylett, 1993;Salem et al, 1994). The significance of this differential subcellular localization of ChAT protein is unknown, but it suggests that there are multiple regulatory systems in cholinergic cells.…”

Protein Kinase C Isoforms Differentially Phosphorylate Human Choline Acetyltransferase Regulating Its Catalytic Activity

“…Based on published reports (9, 10, 26, 39 -41), ChAT is a substrate for multiple kinases in vitro, and in situ ChAT is phosphorylated constitutively and in response to cell perturbations. In hippocampal nerve terminals, ChAT phosphorylation is partially Ca 2ϩ -dependent (39,40), and lowering cytosolic Ca 2ϩ decreases incorporation of [ 32 P]phosphate (39); the one or more protein kinases that phosphorylate ChAT in situ were not identified.…”