From the Department of Physiology, University of Western Ontario, and the Neurodegenerative Diseases Group, The John P. Robarts Research Institute, London, Ontario N6A 5C1, Canada
Received for publication, December 26, 2000, and in revised form, February 28, 2001
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ABSTRACT |
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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
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
(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 localized to non-nuclear cellular compartments such as
cytoplasm and plasma membrane (3). 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 (4-6). 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) (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.
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.
Preparation of ChAT Constructs--
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 Expression of Wild-type and S440A Mutant 69-kDa ChAT in HEK293
Cells--
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.
Immunoaffinity Columns and One-step Purification of 69-kDa
ChAT--
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) (7). 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 mM
sodium 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 (0.2 M 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 (7). For
purified ChAT used for in vitro
phosphorylation/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 M
Tris-HCl, pH 8.8, to a final pH of about 8.0.
Phosphorylation of Purified ChAT by PKC--
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 [ In Vivo Treatment of HEK293 Cells with PMA--
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 mM
AEBSF, 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.
Subcellular Fractionation of HEK293 Cells--
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 (3).
ChAT Activity Measurement--
ChAT activity was measured
radioenzymatically by a modified method of Fonnum (8), as published
previously (9). 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 mM
AEBSF, 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 SDS-PAGE--
One-dimensional sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on
7.5 or 10% gels according to the method of Laemmli (10). 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.
Western Blot Analysis--
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).
Sample Preparation and Mass
Spectrometry--
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
(11) 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 to
32P-phosphorylated isoforms identified by autoradiography
were cut from the Coomassie Blue-stained gel, rinsed, incubated with
sequencing grade-modified (TPCK) trypsin (Promega), and reduced, and
S-alkylated by carboxyamidomethylation (12). 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.
ACh Synthesis in HEK293 Cells Expressing Wild-type and
S440A-ChAT--
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 0.4 N 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.
PKC-mediated Phosphorylation of 69-kDa ChAT--
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 (7). 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 Effect of Activation of PKC by PMA on 69-kDa ChAT in Situ--
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. 1A). In terms of effective concentration, an
EC50 value of about 0.3 µM was determined
from the sigmoidal dose-response curve shown in Fig. 1B,
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).
One-step Purification of 69-kDa Human ChAT--
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.
Identification of PKC Phosphorylation Site(s)--
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 [ Phosphorylation of Wild-type and Mutant 69-kDa ChAT--
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. 4B,
[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. 4A).
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 Localization of ChAT--
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. 5A, 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. 5A).
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. 5B, treatment of HEK293
cells expressing either wild-type or S440A-ChAT with 1 µM
PMA for 2 h did not result in changes in enzyme amount in
cytosolic or membrane fractions, as analyzed by densitometry in seven
independent experiments.
ACh Synthesis in PMA-treated HEK293 Cells--
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 (7). 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-induced enhancement of ChAT enzymatic activity.
Numerous reports in the literature indicate that ChAT is predominantly
a cytosolic protein but that some fraction of the total neuronal enzyme
associates both ionically and nonionically with plasma membrane (13).
The proportion of enzyme that is membrane-bound appears to vary between
species and at different stages of development (14, 15). The means by
which ChAT binds nonionically to membranes has not been elucidated; it
does not have obvious hydrophobic domains for association with the
lipid bilayer, and the presence of covalent modifications such as a
glycosylphosphatidylinositol linkage have been controversial (16-18).
Eder-Colli and colleagues (19) reported recently that in
Drosophila neurons amphiphilic ChAT had properties of a
peripheral membrane protein and was removed from plasma membranes by
alkaline carbonate and urea, suggesting that it is anchored to membrane
through association with other, unidentified membrane components.
ChAT also binds ionically to cellular membranes, being liberated
experimentally by alterations in salt concentration and pH. Changes in
phosphorylation state could result in altered charge on the protein and
changes in isoelectric point. 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. In the present studies, we
identified serine 440 as a PKC phosphorylation site that is involved in
membrane association of ChAT. Under the subcellular fractionation
conditions used in the present experiments, about 10-15% of total
wild-type enzyme activity was membrane-bound. Mutation of this
phosphorylation site eliminated membrane binding of ChAT, suggesting
that addition of negative charge to the protein in this position
facilitated interaction with charged groups on cellular membranes, or
resulted in a protein conformation involved in interaction with other
membrane proteins. It is likely that the site(s) on ChAT phosphorylated by CaM kinase is not serine 440 as it is not within a canonical CaM
kinase consensus sequence, with phosphorylation at other sites potentially leading to differences in functional outcome for the phosphorylated enzyme. Testing this hypothesis awaits identification of
CaM kinase phosphorylation site(s) in ChAT and investigation of their
functional roles by mutagenesis studies.
Phosphorylation of ChAT by PKC either in vitro (7) or
following PKC activation in situ resulted in enhanced
catalytic activity. It is likely that the increase in activity of
cytosolic and membrane-associated ChAT observed in PMA-treated cells is
explained by this change in kinetics rather than by translocation of
enzyme protein between subcellular compartments, particularly because
we demonstrated that the amount of ChAT recovered in cytosolic and
membrane fractions of HEK293 cells expressing either wild-type or
S440A-ChAT was not altered by PKC activation. Phosphorylation of
purified recombinant 69-kDa human ChAT by PKC in vitro led
to a 2-fold increase in enzyme activity (7), compared with an increase
in ChAT activity to ~160% of control in cells stably expressing the
enzyme in which PKC was activated by PMA. This suggests that
phosphorylation-related regulation of ChAT activity in the cellular
context is more complex and may not be maximal when compared with that
measured for the enzyme in vitro, with net phosphorylation
in situ being a balance between the function of kinases and
phosphatases. Moreover, it is not know to what level PKC was activated
in cells by PMA, and how this compares to activity of PKC present under
optimized phosphorylation reaction conditions in vitro. This
finding with ChAT is similar to reports for another
neurotransmitter-synthesizing enzyme tyrosine hydroxylase, where
activation of the enzyme in situ in response to experimental
manipulations that would activate protein kinases (20) is generally
less than that observed following phosphorylation of the purified
enzyme in vitro (21).
Mutation of serine 440 to alanine in 69-kDa ChAT did not completely
eliminate phosphorylation of the enzyme by PKC. Incubation of
S440A-ChAT with PKC under phosphorylating conditions resulted in
one-half as much [32P]phosphate incorporation as that
measured for wild-type ChAT. This suggests that there are two (or more)
functional phosphorylation sites for PKC in 69-kDa human ChAT in
vitro with serine 440 being one of these. Only one site was
identified in these studies by MALDI-MS and MALDI-TOF suggesting that
the tryptic peptide bearing the other phosphorylation site(s) was not
recovered in the complex peptide mixture analyzed, or that the ChAT
isoforms phosphorylated on this additional residue(s) were not
analyzed. In the preparation of PKC-phosphorylated ChAT for mass
spectrophotometric analysis, 32P-labeled isoforms of the
enzyme were recovered from a two-dimensional SDS-PAGE gel for tryptic
digestion. It is possible that additional phosphorylated isoforms of
the enzyme that fell below detection on autoradiography were omitted
from analysis. It is also important to note that, in HEK293 cells
expressing S440A-ChAT, PMA treatment resulted in increased activity of
the mutant to about one-half the level of activation measured for the
wild-type enzyme. This suggests that PKC-mediated phosphorylation at
this additional site(s) is also involved in regulation of activity of
ChAT. However, PMA treatment of cells expressing S440A-ChAT did not
lead to mutant ChAT protein or activity becoming associated with the
membrane fraction, as demonstrated in Fig. 5. This indicates that,
although functional PKC phosphorylation site(s) in ChAT other than
serine 440 may be involved in regulation of enzymatic activity, they are not involved in membrane association of the protein.
From the functional standpoint, it has been considered that
membrane-bound ChAT may play a unique role in regulation of ACh biosynthesis being situated to acetylate choline efficiently upon uptake into the nerve terminal by sodium-coupled choline transporters (22). Studies employing a choline analogue that binds irreversibly to
choline binding sites suggested that choline is transferred from the
sodium-coupled choline transporter to membrane-bound ChAT, because this
form of the enzyme was most inhibited in nerve terminals incubated with
the compound (23). It has been difficult, however, to address the role
of membrane versus cytosolic ChAT in ACh synthesis directly,
and studies reported to date have attempted to modulate activity of
selected subcellular pools of the enzyme and then monitor changes in
ACh levels. For example, lowering nerve terminal chloride ion
concentration dramatically reduced membrane-bound ChAT activity without
changing activity of the cytosolic enzyme, but this did not alter
resting ACh synthesis (24). It is important, however, to evaluate
changes in neurotransmitter synthesis under conditions where demand for
its production is increased; this is complicated experimentally as
stimulation of ACh turnover by depolarization, for example, leads to
simultaneous activation of high affinity choline uptake, ACh synthesis
and release, and membrane-bound ChAT activity thereby making it
difficult to dissect out relationships between enzyme activity and
product formation.
Identification of serine 440 as a functional phosphorylation site in
ChAT may be important in the context of human neurological disorders. A
very recent report identified single nucleotide polymorphisms leading
to point mutations in ChAT to be associated with myasthenic syndrome
and apnea, with one of these involving the arginine residue at the +2
position to serine 440 being mutated to a histidine residue (25). This
mutation leads to an enzyme with very high Km for
both of its substrates. Because this arginine forms an important part
of the canonical consensus sequence 440SIR442
for PKC, it is unlikely that this mutant form of ChAT would be recognized and phosphorylated by PKC at serine 440, leading potentially to altered regulation of the enzyme. Furthermore, it has been shown
that there are reduced levels of PKC, or alterations in activity and
distribution of its isozymes, that may play a role in selective
degeneration of some neurons during aging and Alzheimer disease
(26-28). Immunohistochemical studies have shown brain area-specific modifications in selected PKC isoforms in Alzheimer brain when compared
with control subjects (29). After perturbation of cells by a range of
stimuli, individual PKC isoforms translocate from cytoplasm to
different subcellular sites, including nucleus, cytoskeleton, and
plasma membrane, suggesting that they may mediate distinct cellular
functions (30-32). The role of individual PKC isoforms in
phosphorylation-mediated regulation of ChAT and cholinergic neuron
function requires further investigation to establish basic mechanisms,
and to identify modifications that may occur in disease.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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.
-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.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
-I,
,
,
, and
but not by PKC
-II (data not shown).
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Fig. 1.
Activation of 69-kDa human ChAT in
PMA-treated HEK293 cells. HEK293 cells stably expressing
recombinant 69-kDa human ChAT grown at 50% confluency were treated
(A) for varying times with 1 µM PMA, or
(B) for 2 h with varying concentrations of PMA.
Following PMA treatments, ChAT activity was measured in crude lysates
of the cells. Data are expressed as the mean ± S.E. of five
independent experiments with duplicate measurements. Curves were fit to
data using GraphPad Prism. Statistical differences at the level of
p < 0.05 were determined by Student's t
test when compared with control values and denoted by
asterisks.
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Fig. 2.
Immunoaffinity purification of ChAT.
ChAT was purified from crude extract of High-5 cells on a
CTab/Fab-CNBr-Sepharose column as described under "Experimental
Procedures." Crude extract (50 µg of protein) and purified enzyme
(~2 µg of protein) were run on one-dimensional SDS-PAGE gels
followed by Coomassie Blue staining. The purification yield was at
least 90% of enzyme detected by measuring the enzymatic
activity.
-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.
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Fig. 3.
Strategy for identification of PKC
phosphorylation site(s) in ChAT using MALDI-MS. Masses obtained
for 32P-labeled tryptic peptides derived from in-gel
trypsin digestion of PKC-phosphorylated isoforms of ChAT were compared
with theoretical masses for tryptic peptides of the enzyme in the data
base. A combination of this information with predicted consensus
sequences for PKC phosphorylation sites allowed identification of one
PKC phosphorylation site in 69-kDa human ChAT. Further studies used
MALDI-TOF in linear and reflectron mode to analyze a mixture of tryptic
peptides of ChAT; this revealed the presence of one phosphorylated
peptide with a mass shift of about 80 Da (see "Results").
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Fig. 4.
Time course for incorporation of
[32P]phosphate into wild-type and S440A-ChAT by PKC.
Equal amounts (1 µg) of purified wild-type or S440A-ChAT were
phosphorylated in vitro using PKC, as described under
"Experimental Procedures." At varying times up to 1 h, samples
were removed from incubation and prepared for analysis by SDS-PAGE.
Proteins were transferred from gels to nitrocellulose membranes then
subjected to autoradiography followed by immunoblotting. Finally,
ChAT-immunopositive bands were excised, and incorporated
[32P]phosphate was measured by liquid scintillation
counting. A, the stoichiometry of incorporation of
[32P]phosphate into wild-type and mutant ChAT was
determined. B, samples from incubations of both wild-type
and mutant ChAT were analyzed by autoradiography (upper
panels) and immunoblot with anti-ChAT antibody (lower
panels). This experiment was performed two times, with data in
A showing the average of two separate experiments, and data
in B being from a representative experiment.
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Fig. 5.
Comparison of PMA treatment on activity of
cytosolic and membrane-associated ChAT. HEK293 cells expressing
wild-type and S440A-ChAT were treated for 2 h with 1 µM PMA, then cytosolic and membrane fractions were
prepared from cells as described under "Experimental Procedures."
ChAT activity measured in cytosolic and membrane fractions from
wild-type cells was increased to 148 and 165% of control,
respectively. S440A-ChAT was not present in membrane fractions of
control or PMA-treated cells, and cytosolic-associated mutant was
activated 125% compare with control (A). Data are expressed
as the mean ± S.E. of five independent experiments.
p < 0.05, compared with corresponding control value.
The content of ChAT in cytoplasmic and membrane subfractions of control
(1) and PMA-treated (2) cells was compared by
immunoblot (B). This is a representative blot from seven
independent experiments with densitometric analysis. To obtain bands of
intensity suitable for densitometry, lanes were loaded with 20 µg of
protein from cytosolic fractions and films exposed for 5 s during
detection, and 50 µg of protein from membrane fractions and films
exposed for 2 min during detection.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Dr. David Litchfield, Department of Biochemistry, University of Western Ontario for helpful discussion, and Dr. Y. Yang from the Molecular Medicine Research Centre Mass Spectrometry Laboratory, University of Toronto for mass spectrometric analysis.
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FOOTNOTES |
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* This work was supported in part by a grant from the Medical Research Council of Canada (to R. J. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
A Research Associate of the Ontario Neurotrauma Foundation.
§ To whom correspondence should be addressed: Dept. of Physiology, Medical Sciences Bldg., University of Western Ontario, London, Ontario N6A 5C1, Canada. Tel.: 519-661-3464; Fax: 519-661-3827; E-mail: jane.rylett@med.uwo.ca.
Published, JBC Papers in Press, April 12, 2001, DOI 10.1074/jbc.M011702200
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ABBREVIATIONS |
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The abbreviations used are: ChAT, choline acetyltransferase; ACh, acetylcholine; PKC, protein kinase C; MALDI, matrix-assisted laser desorption ionization; TOF, time-of-flight; MS, mass spectrometry; PBS, phosphate-buffered saline; CTab, carboxyl terminus of human ChAT; AEBSF, 4-(2-aminoethyl)benzenesulfonylfluoride hydrochloride; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; PMA, phorbol 12-myristate 13-acetate; HPLC, high performance liquid chromatography; KR, Krebs-Ringer; CaM, calmodulin.
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