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

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 site-directed 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 ester-treated 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 [32P]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, 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 site-directed 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 ester-treated 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 [32P]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 acetylcholine protein kinase C matrix-assisted laser desorption ionization time-of-flight mass spectrometry phosphate-buffered saline carboxyl terminus of human ChAT 4-(2-aminoethyl)benzenesulfonylfluoride hydrochloride dithiothreitol polyacrylamide gel electrophoresis phorbol 12-myristate 13-acetate high performance liquid chromatography Krebs-Ringer calmodulin 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 (1Oda Y. Nakanishi I. Deguchi T. Mol. Brain Res. 1992; 16: 287-294Crossref PubMed Scopus (72) Google Scholar, 2Misawa H. Matsuura J. Oda Y. Takahashi R. Deguchi T. Mol. Brain Res. 1997; 44: 323-333Crossref PubMed Scopus (55) Google Scholar). We demonstrated recently that the 82-kDa form of the enzyme is targeted to nucleus of cells, whereas 69-kDa ChAT is localized to non-nuclear cellular compartments such as cytoplasm and plasma membrane (3Resendes M.C. Dobransky T. Ferguson S.S.G. Rylett R.J. J. Biol. Chem. 1999; 274: 19417-19421Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Whereas cytosolic/membrane-associated ChAT is clearly involved in catalyzing ACh biosynthesis, the functional role of the nuclear form of the enzyme remains to be elucidated. A critical issue in production of the neurotransmitter ACh is subcellular distribution and regulation of catalytic activity of its biosynthetic enzyme ChAT. Factors controlling ChAT enzyme activity, and the role that post-translational modifications play in this in healthy neurons and during pathological processes such as Alzheimer disease is poorly understood. It has been demonstrated previously that ChAT undergoes phosphorylation both in vitro and in nerve terminals by calcium-dependent protein kinases (4Bruce G. Hersh L.B. Neurochem. Res. 1989; 14: 613-620Crossref PubMed Scopus (47) Google Scholar, 5Schmidt B.M. Rylett R.J. J. Neurochem. 1993; 61: 1774-1781Crossref PubMed Scopus (42) Google Scholar, 6Habert E. Birman S. Mallet J. J. Neurochem. 1992; 58: 1447-1453Crossref PubMed Scopus (25) Google Scholar). Results obtained recently in our laboratory showed that ChAT serves as a substrate for a number of protein kinases, but that it's enzymatic activity is regulated by phosphorylation by only some of the kinases. The highest activities induced by phosphorylation were observed following phosphorylation of ChAT by protein kinase C (PKC) (7Dobransky T. Davis W.L. Xiao G.H. Rylett R.J. Biochem. J. 2000; 349: 141-151Crossref PubMed Scopus (37) Google Scholar). In terms of subcellular compartmentalization, it appears that phosphorylation may regulate association of ChAT with plasma membrane or membranes of subcellular organelles (4Bruce G. Hersh L.B. Neurochem. Res. 1989; 14: 613-620Crossref PubMed Scopus (47) Google Scholar), and partitioning of enzyme between cytosol and membrane fractions. The current studies are aimed at identification of phosphorylation sites of 69-kDa human ChAT by PKC, and characterization of their functional role in regulation of enzymatic activity and/or subcellular compartmentalization of the enzyme within the cell. Using matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) analysis and MALDI-TOF (time-of-flight) in linear and reflectron mode, we identified serine 440 as a PKC phosphorylation site, and determined that phosphorylation of this amino acid plays a role in membrane-association of the enzyme and participates in regulation of it's catalytic activity. The cDNA for human 69-kDa ChAT (N1-ChAT) in pcDNA3 was kindly provided by Dr. H. Misawa (Tokyo Metropolitan Institute for Neuroscience, Tokyo). The mutant S440A-ChAT was prepared by site-directed mutagenesis of Ser440 → Ala in wild-type 69-kDa human ChAT by polymerase chain reaction using the forward primer 5′-GAGAGCGCGGCCATCCGCCGA-3′ and the reverse primer 5′-TCGGCGGATGGCCGCGCTCTC-3′ coupled with forward primer 5′-AAAAGGTACCGCCACCATGGCAGCAAAAACTCCCAGCAGTGA-3′ and reverse primer 5′-TTTTGGATCCAGTCAAGGTTGGTGTCCC-3′ to give the full-length mutant cDNA with KpnI and BamHI restriction sites at the 5′- and 3′-ends, respectively. Following restriction endonuclease digestion of the ends, the fragment was ligated into pcDNA3.1. Integrity of the mutation and the full-length cDNA was confirmed by sequencing. Monolayers of HEK293 cells were transfected with plasmid DNA containing inserts encoding wild-type and mutant 69-kDa human ChAT using the LipofectAMINE 2000 method (Life Technologies, Inc.). G418-resistant stable transformants were selected and tested for ChAT enzyme activity by radioenzymatic assay and ChAT protein by immunoblot. Cells were maintained in modified Eagle's medium containing 10% fetal calf serum, 50 units/ml penicillin/streptomycin, and 0.5 mg/ml G418 in humidified 5% CO2 at 37 °C. Two different immunoaffinity columns were used for preparation of purified native ChAT in an one-step purification protocol. The antibody used was a rabbit polyclonal antibody prepared to a peptide encoding the last 13 amino acids at the carboxyl terminus of human ChAT (called CTab) (7Dobransky T. Davis W.L. Xiao G.H. Rylett R.J. Biochem. J. 2000; 349: 141-151Crossref PubMed Scopus (37) Google Scholar). The first column was prepared by attachment of Fab (antigen-binding fragments) of CTab to CNBr-Sepharose. Fab fragments were prepared by proteolytic cleavage of whole affinity-purified CTab antibody with immobilized papain (>5000 units/g of Sepharose CL-6B) using PBS, pH 7.0, supplemented with 50 mm cysteine-HCl and 5 mm Na4EDTA. Proteolytic treatment was performed with 1000 units papain per mg of antibody for 5 h at 37 °C with shaking, then 10 ml of 20 mm Tris-HCl, pH 8.0, was added to the suspension, mixed, and centrifuged at 2000 × g for 5 min. The was to a column to the fragments and Fab present in the column the were at °C of mm pH 8.0, then a The the column the purified of Fab fragments to be used for preparation of the immunoaffinity column with this was using the the binding of mg of Fab of The of this column was at least mg of purified ChAT of The was of whole purified CTab antibody to be coupled mg/ml was in 5 ml of antibody binding pH and added to 5 ml of of the gel was with 5 gel of and one of pH this mg of in 5 ml of was and the antibody was to the h at with binding sites to with The purification of this immunoaffinity column is at least 5 mg of purified ChAT The was used for purification of recombinant ChAT with both of immunoaffinity For purification of ChAT used for of phosphorylation cellular were prepared cells for enzyme purification as previously (7Dobransky T. Davis W.L. Xiao G.H. Rylett R.J. Biochem. J. 2000; 349: 141-151Crossref PubMed Scopus (37) Google Scholar). For purified ChAT used for in cellular of HEK293 cells stably expressing ChAT were prepared by × in mm Tris-HCl, pH mm mm at and centrifuged at × g for min. The both and HEK293 were with mm Tris-HCl, pH mm mm then an immunoaffinity column at were then with 10 column of containing mm and by 5 column of ChAT protein was with mm pH and with a of pH to a pH of about of wild-type and mutant ChAT with PKC was performed at °C. The kinase reaction of 20 mm pH mm mm mm mm mm 10 mg/ml mg/ml was added to the purified enzyme preparation and for then the phosphorylation reaction was by of electrophoresis For the the of purified ChAT was using as a of purified ChAT was phosphorylated by PKC for in the of of as were then proteins were to only was in the as by membranes were to and following membranes were for to were the and was by Cells were at h medium was added to the cells, then the phorbol ester phorbol 12-myristate 13-acetate was a mm in in the medium and added to cells for and at Following were prepared for of ChAT activity by × in mm Tris-HCl, pH mm at 10 mm and For ChAT subcellular localization and activation cells were for h with and mutant 69-kDa HEK293 cells were with then into and by at × g for 5 min. Cells were in mm Tris-HCl, pH mm 10 mm mm mm at and centrifuged at × g for 5 min. The containing was with and in mm pH mm mm mm mm The was with the then centrifuged at for h with the yielding the and the containing the membranes. were in then in containing by × were not used in the present study, this subcellular 69-kDa ChAT (3Resendes M.C. Dobransky T. Ferguson S.S.G. Rylett R.J. J. Biol. Chem. 1999; 274: 19417-19421Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). ChAT activity was by a modified method of Biochem. J. PubMed Scopus Google Scholar), as previously R.J. S. J. Neurochem. 1993; 61: PubMed Scopus Google Scholar). of HEK293 cells were for of ChAT activity and membrane with 50 mm Tris-HCl, pH containing 50 mm mm mm at 10 mm and mm gel electrophoresis was performed or 10% to the method of PubMed Scopus Google Scholar). proteins were with or to for For were in in 10% acid and 50% for 20 in the of to be prepared for mass were in for an h to of For proteins were membranes in a using mm mm containing membranes were protein with of in acid to proteins and membranes were with in PBS, then with the CTab antibody for h at were then with containing and bound antibody was with and by ChAT was phosphorylated by PKC as then phosphorylated isoforms were by using the method of J. Biol. Chem. Full Text PDF Google with containing pH and the gel isoforms identified by were the with and by Chem. PubMed Scopus Google Scholar). The were by reverse and containing were identified by of all fractions. of present in these were obtained by then with peptide masses for ChAT using the The protein and were performed at the Institute at MALDI-TOF analysis was performed at the at the of In this protein in gel were by and and purification of peptide fragments were in acid in our masses were determined by MALDI-TOF in reflectron and linear HEK293 cells and cells stably expressing wild-type and mutant in were in the or of for h at 37 °C to of ACh synthesis Cells were then with Krebs-Ringer and with in for at 37 °C. Following cells were with then in acid for min. were to then centrifuged at × g for 10 to cellular protein for were with of to acid then with 10 mg/ml in to choline and choline in the and and other choline in the Choline into then to were in choline kinase reaction kinase in pH then for at 37 °C to of In a study, we demonstrated that 69-kDa human ChAT is a substrate for a number of protein kinases, including PKC, with phosphorylation of purified recombinant ChAT in vitro by PKC leading to a in catalytic activity of the enzyme (7Dobransky T. Davis W.L. Xiao G.H. Rylett R.J. Biochem. J. 2000; 349: 141-151Crossref PubMed Scopus (37) Google Scholar). In the present study, we this to determine isoforms of PKC ChAT. Using incorporation of and to by PKC we observed that 69-kDa human ChAT was phosphorylated by PKC and but not by PKC not the of activation of PKC ChAT activity in of HEK293 cells stably expressing 69-kDa human ChAT were with the phorbol ester in this resulted in a time- and activation of the recombinant enzyme. ChAT activity was increased by 10 with the at 10% of control and a by h In terms of an of about was determined the in with in ChAT activity obtained at about The of to be with of in increases in ChAT activity not columns prepared by binding of Fab fragment of the antibody CTab to or whole purified antibody to of purified enzyme in a purification preparation of purified native protein is of the enzyme isoforms as in cells, as demonstrated by of not The of ChAT obtained the one-step immunoaffinity purification using is as demonstrated with a of at least of ChAT activity with activity in the The 69-kDa form of human ChAT 10 for phosphorylation by PKC, including and The for identification of functional PKC phosphorylation by mass spectrometry is in recombinant ChAT was under phosphorylating conditions with PKC and then by to identification of phosphorylated isoforms by Following digestion of the ChAT the were by and two were identified to These were and by a peptide with mass of whereas the other a peptide with mass of with the peptide masses for ChAT two with the mass of and peptide with a mass of the These all the PKC the phosphorylation serine analysis using MALDI-TOF in linear and reflectron an in the mass of this peptide mass of to a mass of of the of a this of serine 440 as a phosphorylation site, we functional analysis mutagenesis of this the role of phosphorylation of 69-kDa human ChAT at serine we prepared a site-directed mutant in serine 440 was to alanine (called this we the course of phosphorylation of purified wild-type and S440A-ChAT by in in was into both of the enzyme with phosphorylation by min. analysis that at wild-type ChAT about of per of enzyme whereas S440A-ChAT about one-half this These a phosphorylation of two or sites of 69-kDa human ChAT by PKC, with Ser440 as one of these phosphorylation compartmentalization of wild-type and S440A-ChAT and the of activation of PKC in by this was determined in HEK293 cells stably expressing the two of the enzyme. of cells into and membrane in distribution of wild-type and ChAT. The wild-type enzyme was present in both with of enzyme activity in cytoplasm and the being following with mm only enzyme activity was in the membrane that ChAT protein was with the membranes. In all activity of the S440A-ChAT was in the with enzyme activity in the membrane in 5 treatment of cells with for h resulted in activation of wild-type ChAT in both membrane of and of treatment also led to activation of the S440A-ChAT in cytosol of but not in of ChAT activity in the membrane these cells 5 for ChAT were performed and membrane control and cells to determine the in ChAT activity was related to a in the of enzyme protein in of the or of enzyme between cytoplasm and in 5 treatment of HEK293 cells expressing wild-type or S440A-ChAT with for h not in in enzyme in or membrane as by in The for cells expressing wild-type S440A-ChAT to in the or of was ACh was not in wild-type HEK293 cells, with all into the cells to or other choline not by In expression of wild-type or S440A-ChAT in HEK293 cells choline that only about of into the cells was to these with the remainder being to or as of cells expressing wild-type and S440A-ChAT with for h to with resulted in ACh synthesis to of control for wild-type ChAT and at of control for We recently that purified recombinant 69-kDa human ChAT is a substrate for PKC, and that phosphorylation of the enzyme in vitro led to a in activity (7Dobransky T. Davis W.L. Xiao G.H. Rylett R.J. Biochem. J. 2000; 349: 141-151Crossref PubMed Scopus (37) Google Scholar). In the present study, we demonstrated for the first that 69-kDa human ChAT undergoes regulation of its catalytic activity in to activation of cellular PKC by phorbol ester the enzyme is phosphorylated by PKC at residue serine 440 within a functional for PKC, and mutation of serine 440 to alanine resulted in loss of binding of ChAT to membranes and of of ChAT enzymatic activity. in the that ChAT is predominantly a protein but that some of the enzyme both and with plasma membrane S. PubMed Scopus (42) Google Scholar). The of enzyme that is appears to between and at different of J. Brain Res. 1993; PubMed Scopus Google Scholar, J. Neurochem. PubMed Scopus Google Scholar). The by ChAT to membranes has not been it not for association with the and the of modifications such as a been J. Neurochem. PubMed Scopus Google Scholar, Brain Res. 1993; PubMed Scopus Google Scholar, Y. Brain Res. 1992; PubMed Scopus Google Scholar). and G. J. J. J. PubMed Scopus Google recently that neurons ChAT of a peripheral membrane protein and was plasma membranes by and that it is to membrane association with membrane ChAT also to cellular being by in and in phosphorylation in altered the protein and in We demonstrated previously that both and 82-kDa purified recombinant ChAT is of isoforms with the isoforms being phosphorylated (7Dobransky T. Davis W.L. Xiao G.H. Rylett R.J. Biochem. J. 2000; 349: 141-151Crossref PubMed Scopus (37) Google Scholar), but it has not been determined some isoforms to membrane and Hersh (4Bruce G. Hersh L.B. Neurochem. Res. 1989; 14: 613-620Crossref PubMed Scopus (47) Google that phosphorylation of human ChAT by kinase altered its association with with phosphorylated ChAT binding to membranes the native this finding it was only observed a range 5 to 20 with of and phosphorylated ChAT bound to membrane fragments in the of or at mm or In the present we identified serine 440 as a PKC phosphorylation that is involved in membrane association of ChAT. the subcellular conditions used in the present about of wild-type enzyme activity was Mutation of this phosphorylation membrane binding of that of to the protein in this with cellular or resulted in a protein involved in with other membrane It is that the ChAT phosphorylated by kinase is not serine 440 as it is not within a kinase with phosphorylation at other sites leading to in functional for the phosphorylated enzyme. this identification of kinase phosphorylation in ChAT and of their functional by mutagenesis of ChAT by PKC in vitro (7Dobransky T. Davis W.L. Xiao G.H. Rylett R.J. Biochem. J. 2000; 349: 141-151Crossref PubMed Scopus (37) Google or following PKC activation in resulted in catalytic activity. It is that the in activity of and ChAT observed in cells is by this in by of enzyme protein between subcellular we demonstrated that the of ChAT in and membrane of HEK293 cells expressing wild-type or S440A-ChAT was not altered by PKC of purified recombinant 69-kDa human ChAT by PKC in vitro led to a in enzyme activity (7Dobransky T. Davis W.L. Xiao G.H. Rylett R.J. Biochem. J. 2000; 349: 141-151Crossref PubMed Scopus (37) Google Scholar), with an in ChAT activity to of control in cells stably expressing the enzyme in PKC was by that regulation of ChAT activity in the cellular is and may not be with that for the enzyme in with being a between the function of kinases and it is not to PKC was in cells by and this to activity of PKC present under phosphorylation reaction conditions in finding with ChAT is to for another enzyme activation of the enzyme in in to that protein kinases E. J. Neurochem. PubMed Scopus Google is that observed following phosphorylation of the purified enzyme in vitro C. T. T. T. S. H. T. T. 1999; PubMed Scopus Google Scholar). Mutation of serine 440 to alanine in 69-kDa ChAT not phosphorylation of the enzyme by Incubation of S440A-ChAT with PKC under phosphorylating conditions resulted in one-half as incorporation as that for wild-type ChAT. that are two functional phosphorylation sites for PKC in 69-kDa human ChAT in vitro with serine 440 being one of one was identified in these studies by and MALDI-TOF that the peptide the other phosphorylation was not in the peptide or that the ChAT isoforms phosphorylated this were not In the preparation of ChAT for mass isoforms of the enzyme were a gel for It is that phosphorylated isoforms of the enzyme that were It is also to in HEK293 cells expressing treatment resulted in increased activity of the mutant to about one-half the of activation for the wild-type enzyme. that phosphorylation at this is also involved in regulation of activity of ChAT. treatment of cells expressing S440A-ChAT not to mutant ChAT protein or activity with the membrane as demonstrated in functional PKC phosphorylation in ChAT other serine 440 may be involved in regulation of enzymatic activity, are not involved in membrane association of the the functional it has been that ChAT may play a role in regulation of ACh being to choline into the nerve by choline J. Neurochem. PubMed Scopus (47) Google Scholar). a choline that to choline binding sites that choline is the choline to this form of the enzyme was most in nerve terminals with the R.J. J. Neurochem. 1989; PubMed Scopus Google Scholar). It has been to the role of membrane ChAT in ACh synthesis and studies to to activity of selected subcellular of the enzyme and then in ACh For example, nerve ChAT activity activity of the enzyme, but this not ACh synthesis B.M. Rylett R.J. 1993; PubMed Scopus Google Scholar). It is to in neurotransmitter synthesis under conditions for its production is this is as of ACh by for example, to activation of high choline ACh synthesis and and ChAT activity it to between enzyme activity and of serine 440 as a functional phosphorylation in ChAT may be in the of human disorders. A identified leading to in ChAT to be with and with one of these the residue at the to serine 440 being to a residue R. S. S. PubMed Scopus Google Scholar). mutation to an enzyme with high for both of its this an of the PKC, it is that this mutant form of ChAT be and phosphorylated by PKC at serine leading to altered regulation of the enzyme. Furthermore, it has been that are of PKC, or in activity and distribution of its that may play a role in degeneration of some neurons during and Alzheimer disease E. Scholar, G. T. Brain Res. PubMed Scopus Google Scholar, Google Scholar). studies brain modifications in selected PKC isoforms in Alzheimer brain with control subjects E. G. S. R. T. J. PubMed Google Scholar). of cells by a range of PKC isoforms cytoplasm to different subcellular including and plasma that may cellular functions C. J. PubMed Scopus Google Scholar, C. Y. 1997; PubMed Scopus Google Scholar, J. H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The role of PKC isoforms in regulation of ChAT and cholinergic function to and to identify modifications that may in disease. We Dr. of of for and Dr. Y. the of for mass

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