Protein kinase C isoforms differentially phosphorylate human choline acetyltransferase regulating its catalytic activity.
Choline acetyltransferase (ChAT) synthesizes acetylcholine in cholinergic neurons; regulation of its activity or response to physiological stimuli is poorly understood. We show that ChAT is differentially phosphorylated by protein kinase C (PKC) isoforms on four serines (Ser-440, Ser-346, Ser-347, and Ser-476) and one threonine (Thr-255). This phosphorylation is hierarchical, with phosphorylation at Ser-476 required for phosphorylation at other serines. Phosphorylation at some, but not all, sites regulates basal catalysis and activation. Ser-476 with Ser-440 and Ser-346/347 maintains basal ChAT activity. Ser-440 is targeted by Arg-442 for phosphorylation by PKC. Arg-442 is mutated spontaneously (R442H) in congenital myasthenic syndrome, rendering ChAT inactive and causing neuromuscular failure. This mutation eliminates phosphorylation of Ser-440, and Arg-442, not phosphorylation of Ser-440, appears primarily responsible for ChAT activity, with Ser-440 phosphorylation modulating catalysis. Finally, basal ChAT phosphorylation in neurons is mediated predominantly by PKC at Ser-476, with PKC activation increasing phosphorylation at Ser-440 and enhancing ChAT activity.
Dobransky
Tomas
T
Department of Physiology, University of Western Ontario and Cell Biology Research Group, and Robarts Research Institute, London, Ontario N6A 5C1, Canada.
Doherty-Kirby
Amanda
A
Kim
Ae-Ri
AR
Brewer
Dyanne
D
Lajoie
Gilles
G
Rylett
Rebecca J
RJ
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D002460:Cell Line; D002795:Choline O-Acetyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D020294:Myasthenic Syndromes, Congenital / Q000201:enzymology / Q000235:genetics; D009474:Neurons / Q000201:enzymology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D013912:Threonine / Q000737:chemistry
D007527:Isoenzymes; D011994:Recombinant Proteins; D013912:Threonine; D012694:Serine; D002795:Choline O-Acetyltransferase; D011493:Protein Kinase C