The phosphorylation of choline acetyltransferase

Bruce, Gordon; Hersh, Louis B.

doi:10.1007/bf00964869

Cited by 53 publications

(37 citation statements)

References 26 publications

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“…In the SN, the a-PKC immunoreactivity was localized in dopaminergic neurons projecting to the striatum. These findings are consistent with reports that CAT could be phosphorylated by PKC (Bruce and Hersh, 1989) and that TH is one of substrates of PKC (Albert et al, 1984;Nishizuka, 1986;Onali and Olianas, 1987). In addition, a-PKC may modulate the membrane responsiveness of these neurons, related to the receptors and channels, because a-PKC was present predominantly along the perikaryal membrane within both the CP and the SN.…”

Section: Discussionsupporting

confidence: 93%

Differential localization of four subspecies of protein kinase C in the rat striatum and substantia nigra

et al. 1991

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The distribution of protein kinase C (PKC) subspecies and their colocalization with neurotransmitters were examined in the rat striatum and substantia nigra (SN), using immunocytochemistry. The alpha- and beta I-PKC immunoreactivies were seen predominantly in the perikarya of the neurons in the striatum and SN. In contrast, the beta II- and gamma- PKC immunoreactivities were abundant in both the perikarya and the neuropils in the striatum and only in the neuropils in the SN. From electron microscopic studies, the alpha- and beta I-PKC immunoreactivities were seen adjacent to the plasma membrane, while the beta II-PKC immunoreactivity was observed in the cytoplasm around the Golgi complex. The gamma-PKC immunoreaction was dense throughout the cytoplasm. The double-staining and lesion studies revealed that the alpha-PKC-immunopositive neurons in the striatum were intrinsic cholinergic neurons, and that most of the alpha-PKC-immunoreactive neurons in the SN were dopaminergic neurons. The beta I-PKC- immunoreactive neurons were intrinsic GABAergic neurons in the striatum. Moreover, most of the beta II- and gamma-PKC-immunoreactive neurons were medium-sized neurons projecting to the SN, and over 90% of GABAergic neurons in the caudate-putamen contained beta II-PKC. The beta II-PKC-immunoreactive neurons showed no gamma-PKC immunoreactivity, and the gamma-PKC-immunoreactive neurons were not beta II-PKC immunoreactive. These findings suggest that alpha-PKC is related to the function of the nigral dopaminergic and the striatal cholinergic neurons, and that the beta I-PKC is involved in the function of the striatal intrinsic GABAergic neurons. The beta II- and gamma-PKC may also modulate a specific neuronal function in the striatonigral system.

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Section: Discussionsupporting

confidence: 93%

Differential localization of four subspecies of protein kinase C in the rat striatum and substantia nigra

et al. 1991

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“…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.…”

Section: Discussionmentioning

confidence: 99%

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

Dobránsky

Doherty-Kirby

Kim

et al. 2004

Journal of Biological Chemistry

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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

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“…We demonstrated previously that both 69-and 82-kDa purified recombinant ChAT is comprised of multiple isoforms with the more acidic isoforms being phosphorylated (7), but it has not been determined whether some isoforms bind to membrane more readily than others. Bruce and Hersh (4) reported that phosphorylation of human placental ChAT by multifunctional calcium-calmodulin (CaM) kinase altered its ionic association with synaptic membranes, with phosphorylated ChAT binding less well to membranes than the native protein. However, this finding requires further investigation, because it was only observed over a narrow NaCl concentration range from 5 to 20 mM, with equivalent amounts of unphosphorylated and phosphorylated ChAT bound to membrane fragments in the absence of NaCl or at 30 mM NaCl or above.…”

Section: Figmentioning

confidence: 99%

“…The highest activities induced by phosphorylation were observed following phosphorylation of ChAT by protein kinase C (PKC) (7). In terms of subcellular compartmentalization, it appears that phosphorylation may regulate association of ChAT with plasma membrane or membranes of subcellular organelles (4), and partitioning of enzyme between cytosol and membrane fractions.…”

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confidence: 99%

Functional Characterization of Phosphorylation of 69-kDa Human Choline Acetyltransferase at Serine 440 by Protein Kinase C

Dobránsky

Davis

Rylett

2001

Journal of Biological Chemistry

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Choline acetyltransferase, the enzyme that synthesizes the transmitter acetylcholine in cholinergic neurons, is a substrate for protein kinase C. In the present study, we used mass spectrometry to identify serine 440 in recombinant human 69-kDa choline acetyltransferase as a protein kinase C phosphorylation site, and sitedirected mutagenesis to determine that phosphorylation of this residue is involved in regulation of the enzyme's catalytic activity and binding to subcellular membranes. Incubation of HEK293 cells stably expressing wild-type 69-kDa choline acetyltransferase with the protein kinase C activator phorbol 12-myristate 13-acetate showed time-and dose-related increases in specific activity of the enzyme; in control and phorbol estertreated cells, the enzyme was distributed predominantly in cytoplasm (about 88%) with the remainder (about 12%) bound to cellular membranes. Mutation of serine 440 to alanine resulted in localization of the enzyme entirely in cytoplasm, and this was unchanged by phorbol ester treatment. Furthermore, activation of mutant enzyme in phorbol ester-treated HEK293 cells was about 50% that observed for wild-type enzyme. Incubation of immunoaffinity purified wild-type and mutant choline acetyltransferase with protein kinase C under phosphorylating conditions led to incorporation of [ 32 P]phosphate, with radiolabeling of mutant enzyme being about one-half that of wild-type, indicating that another residue is phosphorylated by protein kinase C. Acetylcholine synthesis in HEK293 cells expressing wild-type choline acetyltransferase, but not mutant enzyme, was increased by about 17% by phorbol ester treatment.Choline acetyltransferase (ChAT, 1 EC 2.3.1.6) catalyzes synthesis of the neurotransmitter acetylcholine (ACh) in cholinergic neurons in peripheral and central nervous systems. These neurons control a wide range of physiological and biochemical processes in most organ systems, including regulation of cardiovascular and motor functions, and cognitive functions such as learning, attention, and memory. Diminished ChAT activity signals degeneration of cholinergic neurons in a number of neurodegenerative disorders. For example, a consistent finding in necropsy brain of subjects with Alzheimer disease is profound loss of ChAT that correlates with diminished cognitive function early in the course of the disease. Decreased ChAT activity can be accounted for, at least in part, by loss of cholinergic neurons, but may also be related to decreased expression of cholinergic phenotypic genes and/or altered regulation of the enzymes catalytic activity leading to decreased function.There is polymorphism in expression of mRNA for ChAT and, in human only, one of these transcripts, denoted the M isoform, has two translation initiation sites yielding proteins with apparent molecular masses of 69 and 82 kDa; all other transcript isoforms encode the 69-kDa form of enzyme only (1, 2). We demonstrated recently that the 82-kDa form of the enzyme is targeted to nucleus of cells, whereas 69-kDa ChAT is local...

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The phosphorylation of choline acetyltransferase

Cited by 53 publications

References 26 publications

Differential localization of four subspecies of protein kinase C in the rat striatum and substantia nigra

Differential localization of four subspecies of protein kinase C in the rat striatum and substantia nigra

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

Functional Characterization of Phosphorylation of 69-kDa Human Choline Acetyltransferase at Serine 440 by Protein Kinase C

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