Functional Characterization of Phosphorylation of 69-kDa Human Choline Acetyltransferase at Serine 440 by Protein Kinase C
2001; Elsevier BV; Volume: 276; Issue: 25 Linguagem: Inglês
10.1074/jbc.m011702200
ISSN1083-351X
AutoresTomáš Dobránsky, Wanda L. Davis, R. Jane Rylett,
Tópico(s)Microbial Natural Products and Biosynthesis
ResumoCholine 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 supernatant was subsequently applied to a Protein G-Sepharose column to separate the Fc fragments and undigested IgG. Fab fragments, present in the column flow-through from the Protein G-Sepharose column, were dialyzed overnight at 4 °C against three changes of 40 mm sodium phosphate, pH 8.0, then loaded onto a DEAE-Sepharose column. The flow-through from the DEAE-Sepharose column contained the purified fraction of Fab fragments to be used for preparation of the immunoaffinity column with CNBr-Sepharose; this was accomplished using the standard protocol from Amersham Pharmacia Biotech allowing the binding of ∼7 mg of Fab fragments/ml of gel. The capacity of this column was at least 3 mg of purified ChAT protein/ml of gel. The second approach was based on immobilization of whole purified CTab antibody on Protein G-Sepharose. Antibody to be coupled (15 mg/ml gel) was dissolved in 5 ml of antibody binding buffer (50 mmsodium borate, pH 8.2) and added to 5 ml of Protein G-Sepharose. After 30 min of gentle rocking, the gel was washed with 5 gel volumes of antibody-binding buffer and one volume of cross-linking buffer (0.2m triethanolamine, pH 8.2). Immediately after this wash, 33 mg of dimethyl pimelimidate dissolved in 5 ml of cross-linking buffer was added, and the antibody was covalently attached to the Protein G-Sepharose (by 1 h gently rocking at room temperature) with antigen binding sites facing outward to interact with antigen. The purification capacity of this immunoaffinity column is at least 5 mg of purified ChAT protein/ml resin. The same protocol was used for purification of recombinant ChAT with both types of immunoaffinity columns. For purification of ChAT used for MALDI-MS determination of phosphorylation sites, crude cellular extracts were prepared from baculovirus-infected, ChAT-expressing High-5 cells for enzyme purification as described 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 vitrophosphorylation/stoichiometry studies, total cellular extracts of HEK293 cells stably expressing ChAT were prepared by sonication (3 × 15 s) in lysis buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.1% Triton X-100, 1 mm AEBSF, leupeptin/aprotinin/pepstatin at 10/25/10 μg/ml), and centrifuged at 15,000 × g for 30 min. The supernatants from both High-5 and HEK293 cell lysates were diluted 1:1 with loading buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm AEBSF), then loaded onto an immunoaffinity column at 0.25 ml/min. Columns were then washed with 10 column volumes of loading buffer containing 500 mm NaCl and 0.2% Nonidet P-40, followed by 5 column volumes of loading buffer. Purified ChAT protein was eluted with 100 mm glycine-HCl buffer, pH 2.7, and immediately neutralized with a small volume of 2 mTris-HCl, pH 8.8, to a final pH of about 8.0. Phosphorylation of wild-type and mutant ChAT with PKC (Amersham Pharmacia Biotech) was performed at 30 °C. The kinase reaction buffer (30 μl of 20 mm HEPES, pH 7.2, 1 mm sodium orthovanadate, 25 mm glycerol 3-phosphate, 1 mm DTT, 1 mm CaCl2, 15 mm MgCl2, 10 mg/ml 1,2-dioleyl-sn-glycerol, 100 mg/ml phosphatidylserine) was added to the purified enzyme preparation (1–3 mg/ml) and incubated for varying times, then the phosphorylation reaction was stopped by addition of electrophoresis sample buffer. For the stoichiometry experiments, the concentration of purified ChAT was estimated from Coomassie Blue-stained gels using bovine serum albumin as a standard. One microgram (14.5 pmol) of purified ChAT was phosphorylated by PKC (0.4 milliunit) for varying times in the presence of 0.6 nmol of [γ-32P]ATP (15 μCi), as described above. Samples were run on one-dimensional SDS-PAGE gels, then proteins were transferred to nitrocellulose membrane; only residual radioactivity was retained in the gels after transfer as monitored by Cerenkov counting. Nitrocellulose membranes were apposed to film, and following brief (5 min) autoradiography, membranes were processed for immunoblotting. Subsequently, areas corresponding to radioactive bands on autoradiography were cut from the membranes, and incorporated [32P]phosphate was quantified by Cerenkov counting. Cells were treated at ∼50% confluence. 2 h before treatment, fresh medium was added to the cells, then the phorbol ester phorbol 12-myristate 13-acetate (PMA) was diluted from a 1 mm stock in Me2SO in the same medium and added to cells for varying times and at varying concentrations. Following treatment, cell lysates were prepared for measurement of total ChAT activity by sonication (3 × 15 s) in lysis buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.1% Triton X-100, 1 mmAEBSF, leupeptin/aprotinin/pepstatin at 10/25/10 μg/ml, 500 μm sodium orthovanadate, 10 mm sodium fluoride, and 250 μm eserine sulfate). For ChAT subcellular localization and activation studies, cells were treated for 2 h with 1 μm PMA. Wild-type and mutant 69-kDa ChAT-expressing HEK293 cells were washed twice with ice-cold PBS then scraped into PBS and pelleted by centrifugation at 700 × g for 5 min. Cells were gently resuspended in lysis buffer (10 mm Tris-HCl, pH 7.5, 0.05% Nonidet P-40, 3 mm MgCl2, 10 mm NaCl, 1 mm AEBSF, 1 mm sodium orthovanadate; leupeptin/aprotinin/pepstatin at 10/25/10 μg/ml) and centrifuged at 500 × g for 5 min. The pellet containing crude nuclei was washed once with lysis buffer and three times in wash buffer (10 mm HEPES, pH 6.8, 300 mm sucrose, 3 mm MgCl2, 25 mm NaCl, 1 mm AEBSF, 500 μm sodium orthovanadate). The original post-nuclear supernatant was combined with the washes then centrifuged at 35,000 rpm for 1 h with the supernatant yielding the cytosolic fraction and the pellet containing the membranes. Membrane pellets were washed three times in lysis buffer then solubilized in lysis buffer containing 1% Nonidet P-40 by sonication (3 × 15 s). Purified nuclei were not used in the present study, because this subcellular fraction contains little 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 measured radioenzymatically by a modified method of Fonnum (8Fonnum F. Biochem. J. 1969; 115: 465-479Crossref PubMed Scopus (965) Google Scholar), as published previously (9Rylett R.J. Goddard S. Lambros A. J. Neurochem. 1993; 61: 1388-1397Crossref PubMed Scopus (20) Google Scholar). Lysates of ChAT-expressing HEK293 cells were diluted for measurement of ChAT activity (cytosolic fraction 1:100 and membrane fractions 1:10) with 50 mm Tris-HCl, pH 7.5, containing 50 mm NaCl, 1 mm MgCl2, 2 mm EDTA, 0.5% bovine serum albumin, 1 mmAEBSF, leupeptin/aprotinin/pepstatin at 10/25/10 μg/ml, 500 μm sodium orthovanadate, 10 mm NaF, 250 μm eserine sulfate, and 1 mm DTT. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on 7.5 or 10% gels according to the method of Laemmli (10Laemmli E.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207192) Google Scholar). After electrophoresis, proteins were either stained with Coomassie Brilliant Blue or transferred to nitrocellulose for immunoblotting. For staining, gels were incubated in 0.05% Coomassie Blue R-250 in 10% acetic acid and 50% methanol for 20 min; in the case of samples to be prepared for mass spectrometry, gels were washed in acetic acid/methanol for an additional 3 h to ensure adequate removal of SDS. For immunoblotting, proteins from SDS-PAGE gels were transferred onto nitrocellulose membranes in a semidry electroblotting apparatus using transfer buffer (48 mm Tris, 39 mm glycine) containing 20% methanol. Nitrocellulose membranes were stained after protein transfer with 0.02% solution of Ponceau-S in 1% acetic acid solution to visualize transferred proteins and standards. Subsequently, membranes were saturated with 8% non-fat milk powder in PBS, then probed with the anti-ChAT CTab antibody (1:2000) for 1 h at room temperature. Membranes were then washed with PBS containing 0.5% Triton X-100, and bound primary antibody was reacted with peroxidase-coupled secondary antibodies and detected by chemiluminescence (ECL kit, Amersham Pharmacia Biotech). Immunoaffinity-purified ChAT (10 μg) was phosphorylated by PKC as described above, then phosphorylated isoforms were separated by two-dimensional SDS-PAGE using the method of Garrels (11Garrels J.I. J. Biol. Chem. 1979; 245: 7961-7977Abstract Full Text PDF Google Scholar) with isoelectric focusing gels containing 2% Ampholines, pH 3.5–10, and the second dimension run on 7.5% separating gel (thickness 1 mm). Protein spots corresponding to32P-phosphorylated isoforms identified by autoradiography were cut from the Coomassie Blue-stained gel, rinsed, incubated with sequencing grade-modified (TPCK) trypsin (Promega), and reduced, andS-alkylated by carboxyamidomethylation (12Shevchenko A. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7818) Google Scholar). The resulting tryptic peptides were separated by C18 reverse phase HPLC, and fractions containing [32P]phosphopeptides were identified by Cerenkov counting of all fractions. Masses of peptides present in these fractions were obtained by MALDI-MS (TofSpec SE, Micromass, Inc., Beverly, MA), then compared with theoretical tryptic peptide masses for ChAT using the program GPMAW (Lighthouse Data, Denmark). The in-gel protein trypsinization, HPLC, and MALDI-MS analyses were performed at the Howard Hughes Medical Institute Biopolymer Facility/W. M. Keck Biotechnology Resource Laboratory at Yale University. Additional MALDI-TOF analysis was performed at the Molecular Medicine Research Centre Mass Spectrometry Laboratory at the University of Toronto. In this case, protein samples in SDS-polyacrylamide gel were processed by in-gel trypsin digestion, and recovery and purification of peptide fragments were carried out on C18-ZipTips (Whatman) eluted in 65% (v/v) acetonitrile:1% acetic acid (v/v):water in our laboratory. Peptide masses were determined by MALDI-TOF in reflectron and linear mode. Wild-type HEK293 cells and cells stably expressing wild-type and mutant ChAT, plated in 12-well plates, were preincubated in the presence or absence of 1 μm PMA for 2 h at 37 °C prior to determination of ACh synthesis capacity. Cells were then washed with Krebs-Ringer (KR) solution, and incubated with [3H]choline (0.5 μm; 1 μCi/well) in KR solution for 15 min at 37 °C. Following incubation, cells were washed with KR, then extracted in 2% trichloroacetic acid for 30 min. Cell extracts were transferred to microcentrifuge tubes, then centrifuged at 12,000 × g for 10 min to recover cellular protein for measurement. Supernatants were shaken with 4 volumes of diethyl ether twice to remove trichloroacetic acid then extracted with 10 mg/ml sodium tetraphenylboron in 3-heptanone to recover choline and choline esters in the organic phase and separate them from phosphorylcholine and other choline metabolites remaining in the aqueous phase. Choline esters where back-extracted into 0.4n HCl, then reduced to dryness. Dried samples were solubilized in choline kinase reaction buffer (choline kinase 0.02 unit, DTT, ATP, MgCl2 in glycylglycine buffer pH 8.0), then incubated for 30 min at 37 °C to allow separation of [3H]ACh from unmetabolized [3H]choline. In a previous 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 2-fold increase 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 extended this observation to determine which isoforms of PKC phosphorylate ChAT. Using incorporation of [32P]phosphate and autoradiography to monitor covalent modification by PKC isoforms, we observed that 69-kDa human ChAT was phosphorylated by PKC α, β-I, γ, δ, ε, and ξ but not by PKC β-II (data not shown). To test the effect of activation of PKC on ChAT activity in situ, monolayers of HEK293 cells stably expressing 69-kDa human ChAT were treated with the phorbol ester PMA. As shown in Fig.1, this resulted in a time- and dose-dependent activation of the recombinant enzyme. ChAT activity was significantly increased by 10 min with the effect becoming maximal at 157 ± 10% of control and reaching a plateau by 2 h (Fig. 1 A). In terms of effective concentration, an EC50 value of about 0.3 μm was determined from the sigmoidal dose-response curve shown in Fig. 1 B, with maximal increase in ChAT activity obtained at about 1 μm (158% ± 8%). The effect of PMA appeared to be biphasic with concentrations of PMA above 2 μm resulting in smaller increases in ChAT activity (data not shown). Immunoaffinity columns prepared by covalent binding of Fab fragment of the anti-ChAT antibody CTab to CNBr-Sepharose or whole purified antibody to Protein G-Sepharose allowed isolation of highly purified enzyme in a single purification step. This preparation of purified native protein is comprised of the same enzyme isoforms as produced in cells, as demonstrated by immunoblots of two-dimensional SDS-PAGE gels (data not shown). The purity of ChAT obtained from the one-step immunoaffinity purification protocol using High-5 cell lysate is greater than 95% as demonstrated on Coomassie Blue-stained gels (Fig.2), with a yield of at least 90% of total ChAT activity when compared with total activity in the crude extract. The 69-kDa form of human ChAT contains 10 putative canonical consensus sequences for phosphorylation by PKC, including SYK (position 126–128), SYR (161), TNR (255), THR (283), SSR (346), SRK (347), SIR (440), SEK (476), SNR (532), and SSK (586). The strategy adopted for identification of functional PKC phosphorylation site(s) by mass spectrometry is outlined in Fig.3. Purified recombinant ChAT was incubated under phosphorylating conditions with PKC and [γ-32P]ATP then separated by two-dimensional SDS-PAGE to allow identification of phosphorylated isoforms by autoradiography. Following tryptic digestion of the 32P-labeled ChAT isoforms, the resulting peptides were separated by HPLC, and two fractions were identified to contain 32P-labeled phosphopeptide. These were recovered and analyzed by MALDI-MS. One fraction contained a peptide with mass of 1390.43 (±0.2%), whereas the other contained a peptide with mass of 1237.30 (±0.2%). Comparison with the theoretical tryptic peptide masses for ChAT revealed two peptides with the calculated mass of 1391.764 having sequences LVPTYESASIRR (residues 432–443) and RLVPTYESASIR (residues 431–442). Another peptide with a calculated mass of 1235.663 had the sequence LVPTYESASIR (residues 432–442). These three peptides all contained the PKC consensus sequence SIR (residues 440–442) therefore identifying the candidate phosphorylation site serine 440. Further analysis using MALDI-TOF in linear and reflectron mode revealed an 80-Da shift in the mass of this peptide from predicted mass of 1391.764 to a measured mass of 1472.03, indicative of the presence of a phosphate group. Based on this confirmation of serine 440 as a putative phosphorylation site, we pursued functional analysis through mutagenesis of this residue. To investigate the biological role of phosphorylation of 69-kDa human ChAT at serine 440, we prepared a site-directed mutant in which serine 440 was changed to alanine (called S440A-ChAT). To characterize this mutant, we monitored the time course of phosphorylation of purified wild-type and S440A-ChAT by PKC. As illustrated in autoradiographs in Fig. 4 B, [32P]phosphate was rapidly incorporated into both forms of the enzyme with maximal phosphorylation occurring by 30 min. Stoichiometric analysis revealed that at steady state wild-type ChAT contained about 2.8 mol of phosphate per mol of enzyme protein, whereas S440A-ChAT contained about one-half this amount (Fig. 4 A). These results predict a phosphorylation of two or more sites of 69-kDa human ChAT by PKC, with Ser440 serving as one of these phosphorylation sites. Subcellular compartmentalization of wild-type and S440A-ChAT and the effect of activation of PKC in situ by PMA on this measure was determined in HEK293 cells stably expressing the two forms of the enzyme. Interestingly, subfractionation of cells into cytosolic and membrane components revealed striking differences in distribution of wild-type and S440A-mutant ChAT. The wild-type enzyme was present in both fractions, with 88% of total enzyme activity found in cytoplasm and the remaining 12% being membrane-associated; following washing with 350 mm NaCl, only residual enzyme activity (∼0.5%) was recovered in the membrane fraction, suggesting that ChAT protein was ionically associated with the membranes. In contrast, all activity of the S440A-ChAT was recovered in the cytosolic fraction, with no measurable enzyme activity found in the membrane fraction. As shown in Fig. 5 A, treatment of cells with 1 μm PMA for 2 h resulted in activation of wild-type ChAT in both membrane (165% of control) and cytosolic (148% of control) compartments. PMA treatment also led to activation of the S440A-ChAT in cytosol (125% of control) but did not result in appearance of detectable ChAT activity in the membrane fraction from these cells (Fig. 5 A). Immunoblots for ChAT were performed on cytosolic and membrane samples from control and PMA-treated cells to determine whether the increase in ChAT activity was related to a change in the amount of enzyme protein in any of the fractions, or translocation of enzyme between cytoplasm and membrane. As indicated in Fig. 5 B, treatment of HEK293 cells expressing either wild-type or S440A-ChAT with 1 μmPMA for 2 h did not result in changes in enzyme amount in cytosolic or membrane fractions, as analyzed by densitometry in seven independent experiments. The capacity for cells expressing wild-type versus S440A-ChAT to synthesize [3H]ACh from [3H]choline in the absence or presence of PMA stimulation was tested. ACh was not synthesized in wild-type HEK293 cells, with almost all [3H]choline taken up into the cells converted to phosphorylcholine or other choline metabolites not extracted from aqueous solution by sodium tetraphenylboron. In contrast, expression of wild-type or S440A-ChAT in HEK293 cells shifted choline metabolism so that only about 10–20% of [3H]choline transported into the cells was converted to these metabolites, with the remainder being metabolized to [3H]ACh or remaining as unmetabolized [3H]choline. Treatment of cells expressing wild-type and S440A-ChAT with 1 μm PMA for 2 h prior to incubation with [3H]choline resulted in ACh synthesis increasing to 117% of control for wild-type ChAT and remaining at 97% of control for S440A-ChAT. We found recently that purified recombinant 69-kDa human ChAT is a substrate for PKC, and that phosphorylation of the enzyme in vitro led to a 2-fold increase 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 time that 1) 69-kDa human ChAT undergoes rapid regulation of its catalytic activity in response to activation of cellular PKC by phorbol ester PMA, 2) the enzyme is phosphorylated by PKC at residue serine 440 within a functional consensus sequence for PKC, and 3) mutation of serine 440 to alanine resulted in loss of binding of ChAT to membranes and attenuation of PMA-ind
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