In vitro phosphorylation of acetylcholinesterase at non-consensus protein kinase A sites enhances the rate of acetylcholine hydrolysis

Mirta Grifman, Ayelet Arbel, Dalia Ginzberg, David Glick, Sharona Elgavish, Boaz Shaanan, Hermona Soreq*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

35 Scopus citations


Here, we report that the catalytic subunit of cAMP-dependent protein kinase (PKA) but not casein kinase II or protein kinase C phosphorylates recombinant human acetylcholinesterase (AChE) in vitro. This enhances acetylthiocholine hydrolysis up to 10-fold as compared to untreated AChE, while leaving unaffected the enzyme's affinity for this substrate and for various active and peripheral site inhibitors. Alkaline phosphatase treatment enhanced the electrophoretic migration, under denaturing conditions, of part of the AChE proteins isolated from various mammalian sources and raised the isoelectric point of some of the treated AChE molecules, indicating that part of the AChE molecules are also phosphorylated in vivo. Enhancement of acetylthiocholine hydrolysis also occurred with Torpedo AChE, which has no consensus motif for PKA phosphorylation. Further, mutating the single PKA site in human AChE (threonine-249) did not prevent this enhancement, suggesting that in both cases it was due to phosphorylation at non-consensus sites. In vivo suppression of the acetylcholine hydrolyzing activity of AChE and consequent impairment in cholinergic neurotransmission occur under exposure to both natural and pharmacological compounds, including organophosphate and carbamate insecticides and chemical warfare agents. Phosphorylation of AChE may possibly offer a rapid feedback mechanism that can compensate for impairments in cholinergic neurotransmission, modulating the hydrolytic activity of this enzyme and enabling acetylcholine hydrolysis to proceed under such challenges.

Original languageAmerican English
Pages (from-to)179-187
Number of pages9
JournalMolecular Brain Research
Issue number1-2
StatePublished - Nov 1997

Bibliographical note

Funding Information:
We thank Drs. G. Robinson (Urbana, IL) for critically reviewing this manuscript, A. Shafferman and A. Fischer (Ness Ziona, Israel) for the gifts of purified recombinant human AChE, U. Brodbeck (Bern, Switzerland) for purified native AChEs and monoclonal antibodies and Y. Gruenbaum (Jerusalem) for COS-1 cells. This study was supported by the US Army Medical Research and Development Command (Contract DAMD 17-97-1-7007, to H.S.).


  • Acetylcholinesterase
  • Neurotransmission
  • Non-consensus phosphorylation site
  • Protein phosphorylation
  • cAMP-dependent protein kinase A


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