From the Departments of Physiology and
Biochemistry, University of Western Ontario, and
§ Robarts Research Institute,
London, Ontario N6A 5C1, Canada
Received for publication, November 27, 2002, and in revised form, December 16, 2002
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ABSTRACT |
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Choline acetyltransferase synthesizes
acetylcholine in cholinergic neurons. In the brain, these neurons are
especially vulnerable to effects of Cholinergic neurons in brain are especially vulnerable to effects
of both soluble/oligomeric and deposited/fibrillar forms of Choline acetyltransferase (ChAT; EC 2.3.1.6) produces the
neurotransmitter ACh in cholinergic neurons. ChAT undergoes covalent modification post-translationally by protein kinase-mediated
phosphorylation (14-18), and we showed previously that it is a
substrate for a number of protein kinases (17). Catalytic activity of
this enzyme, its subcellular distribution, and potentially its
interaction with other cellular proteins can be regulated in a
phosphorylation-dependent manner. For example,
phosphorylation of ChAT by protein kinase C (PKC) on Serine 440 led to a significant increase in its activity and ionic binding
to plasma membrane in cells (18).
Phosphorylation of ChAT could be altered by changes in activity or
subcellular redistribution of protein kinases brought about by neuronal
perturbations or pathology such as Alzheimer's disease and traumatic
brain injury. This could alter ACh biosynthesis and cholinergic
neurotransmission and cause dysfunction of cholinergic neurons. A In the present study, we tested the hypothesis that short term exposure
of IMR32 neuroblastoma cells stably expressing human 69-kDa ChAT to
A 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). The mutant
ChAT-T456A was prepared by site-directed mutagenesis of
Thr456 Culture and Treatment of IMR32 Cells--
Human neuroblastoma
IMR32 cells were transfected with plasmids containing wild-type 69-kDa
human ChAT or mutants ChAT-T456A or ChAT-S440A in pcDNA3.1 using
LipofectAMINE 2000 (Invitrogen). 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 µg/ml gentamycin, and 0.5% G418 in humidified 5%
CO2 at 37 °C. For experiments, monolayers of cells were
treated at ~70% confluence. At 4 h before treatment, fresh
medium was added to cells, and then A Analysis of Immunoprecipitation of ChAT or VCP--
Cleared cell lysates
were mixed with CTab anti-ChAT antibody (17) (5 µl of 2 mg/ml stock)
or anti-VCP antibody (2 µl of crude rabbit antiserum) for 1 h on
ice; VCP antibody was obtained from Dr. L. Samelson (NCI-Frederick,
National Institutes of Health, Frederick, MD) (29). Immune complexes
were captured onto Protein-G Sepharose beads for 1 h at 4 °C
and washed three times with radioimmune precipitation buffer (10 mM Tris-HCl, pH 7.5, 140 mM NaCl, 1% Nonidet
P-40, 1% sodium deoxycholate, 0.1% SDS). Electrophoresis sample
buffer (3.3% SDS, 5% Western Blot Analysis--
Proteins were transferred from
SDS-PAGE gels to nitrocellulose membranes in a semidry electroblotting
apparatus using transfer buffer (48 mM Tris, 39 mM glycine) containing 20% methanol. For detection of ChAT
or VCP, blotting membranes were saturated with 8% nonfat milk powder
in phosphate-buffered saline and probed with anti-ChAT CTab antibody
(1:2,000) or anti-VCP antibody (1:2,000) for 1 h at room
temperature. Membranes were washed with phosphate-buffered saline
containing 0.5% Triton X-100, and bound antibodies were detected by
incubation for 1 h with peroxidase-coupled secondary antibodies
(1:5,000; Amersham Biosciences) and the ECL kit (Amersham Biosciences).
Phosphorylation of Purified ChAT by CaM Kinase II and
PKC--
Bacterially expressed recombinant 69-kDa human ChAT was
immunoaffinity-purified as described previously (18). To identify amino
acid residues phosphorylated by CaM kinase II, 5 µg (0.5 µl) of
purified ChAT protein was mixed with 20 µl of kinase buffer (50 mM Tris-HCl, pH 7.2, 0.4 mM dithiothreitol, 0.5 mM CaCl2, 5 mM MgCl2, 1 µM calmodulin, 100 µM ATP) and 2 milliunits
of purified CaM kinase II (a gift from Dr. H. Schulman, Stanford
University) for 30 min at 30 °C. Phosphorylation reactions were
stopped by the addition of electrophoresis sample buffer. To determine
effects of hierarchical phosphorylation of ChAT by PKC and CaM kinase II on ChAT enzymatic activity, a PKC phosphorylation reaction was
carried out for 15 min at 30 °C as described previously (17), followed by phosphorylation by CaM kinase II for an additional 15 min.
ChAT activity was measured immediately at 37 °C.
CaM Kinase II Assay in Permeabilized Cells--
IMR32 cells
expressing 69-kDa human ChAT were used to assess whether CaM kinase II
is activated by treatment with A In-gel Tryptic Digestion of Proteins and Sample
Preparation--
Following SDS-PAGE, proteins were stained briefly
with Coomassie Blue, and then gels were destained and washed for 3 h with at least five solvent changes (50% methanol, 10% acetic acid) to ensure adequate removal of SDS. Gels were subsequently washed with
three changes of H2O, and then bands corresponding to ChAT or co-immunoprecipitated proteins were excised from gels and washed with two changes of acetonitrile. Gel slices were reduced with 10 mM dithiothreitol at 50 °C for 30 min and alkylated by
55 mM iodoacetamide at room temperature for 20 min,
followed by washing three times with 100 mM ammonium
bicarbonate. After two changes of acetonitrile, gel pieces were dried
by vacuum centrifuge and rehydrated in trypsin digestion buffer (50 mM ammonium bicarbonate, 5 mM
CaCl2) containing 12.5 ng/µl trypsin (Roche Molecular
Biochemicals) (34). After a 45-min incubation on ice, excess trypsin
solution was removed, 15 µl of digestion buffer without trypsin was
added, and samples were incubated for 18 h at 37 °C. Tryptic
peptides were extracted from the gel pieces with two changes of 100 µl of ammonium carbonate buffer by shaking in an orbital shaker for 45 min. After a brief centrifugation, the supernatants with eluted peptides were pooled and concentrated by vacuum centrifugation to a
final volume of 15 µl. Pooled extracts were acidified with glacial
acetic acid at a final concentration of 1%.
Two-dimensional Tryptic Phosphopeptide Mapping and Identification
of Phosphorylated Residues--
Two-dimensional thin layer
phosphopeptide maps of ChAT were prepared as described previously (35).
Following in-gel tryptic digestion of proteins, samples were applied to
cellulose TLC plates by sequential spotting of 0.5-µl droplets and
electrophoresed in the first dimension in water/acetic acid/formic acid
(89.7:7.8:2.5, v/v/v, pH 1.9) at 1000 V for 45 min. Plates were
air-dried and developed in the second dimension in
water/n-butyl alcohol/pyridine/acetic acid (30:37.5:25:7.5,
v/v/v/v). Phosphopeptides were visualized by autoradiography using
Eastman Kodak Co. XAR-5 film at
Phosphoamino acid analysis was also performed on phosphopeptides eluted
from cellulose plates or directly on mixtures of phosphopeptides recovered after in-gel tryptic digestion. Tryptic peptides were lyophilized, resuspended in 70 µl of 6 M HCl, and boiled
at 110 °C for 1 h. One- or two-dimensional phosphoamino acid
mapping were performed as described by Boyle et al.
(37).
In Vitro Parent Ion Scanning--
Experiments were performed on
a Q-TOF2 mass spectrometer (Micromass), equipped with a nanoflow
source. The instrument was calibrated with
[Glu1]Fibrinopeptide B (Sigma), and following desalting
on a ZipTipC18TM (4 µl), the concentrated
protein digest sample was loaded into a borosilicate capillary (type F;
Micromass). A voltage between 600 and 1000 V was applied to the
capillary in order to produce nanomolar flow. Parent ion scanning was
performed over the m/z range of 300-1500
monitoring neutral loss of 97.9769 and 49.9885 with a collision energy
of 32 V. Masses that resulted in such neutral losses were subsequently
further fragmented to obtain sequence information. Resultant spectra
were background-subtracted and deconvoluted using MaxEnt3 software
provided in the Micromass MassLynx version 3.5 software package. The
sequence of the peptides was determined using PepSeq version 3.3 software also provided in the MassLynx 3.5 software.
Peptide Sequencing by Mass Spectrometry--
Amino acid
sequences of tryptic phosphopeptides of ChAT isolated from control and
A MALDI-TOF MS--
Proteins co-immunoprecipitating with ChAT were
identified initially by MALDI-TOF mass spectrometry. Samples were mixed
1:1 (v/v) with matrix solution containing 1:1 ethanol/acetonitrile saturated with Characterization of A Short Term Exposure to A Identification of a New Phosphorylation Site in ChAT in Cells
Treated with A
To identify the threonine residue phosphorylated in ChAT with
A
A doubly charged peak at 658.3 m/z (M = 1314.6) was also found in samples. This corresponds to the
phosphorylated form of the tryptic peptide 432-442 of 69-kDa human
ChAT (LVPTYESASIR). This peptide contains a serine residue
(Ser440) that was found previously to be phosphorylated by
PKC (18).
ESI-MS/MS Identification of Threonine 456 as a Putative CaM Kinase
II Phosphorylation Site in ChAT in Vitro--
Immunoaffinity-purified
69-kDa ChAT was incubated under phosphorylating conditions with CaM
kinase II and then resolved by SDS-PAGE and digested with trypsin for
analysis by ESI-MS to identify phosphorylated peptide(s) and amino acid
residue(s). Using parent ion scanning, a doubly charged peak at 621.6 m/z was found to produce a neutral loss of 98 that is indicative of loss of a phosphate group under moderate
fragmentation conditions (41). This m/z value was
subjected to full fragmentation in order to obtain sequence information. This peptide sequenced to SApTPEALAFVR (where pT represents phosphothreonine), as can be seen in Fig.
5, indicating the presence of a
phosphorylated threonine residue. This sequence corresponds to tryptic
fragment 454-465 of 69-kDa ChAT that contains a putative consensus
sequence for CaM kinase II; this is the same threonine residue
(Thr456) found to be phosphorylated in IMR32 cells treated
with A Assay of CaM Kinase II Activity in IMR-32 Cells with A Hierarchical Activation of ChAT with Phosphorylation by PKC and CaM
Kinase II--
We investigated the relationship between
phosphorylation of ChAT by PKC and CaM kinase II and activation of the
enzyme using both purified recombinant ChAT and IMR32 cells treated
with A
As shown in Fig. 7B, H7 also inhibited activation of ChAT in
IMR32 cells treated with A
To address this further, we investigated the temporal relationship of
serine and threonine phosphorylation of ChAT in IMR32 cells treated
with A A
A combined mass spectrometry and immunoblot approach was taken to begin
to identify proteins co-immunoprecipitating with ChAT. For mass
spectrometric analysis, bands were excised from SDS-PAGE gels and
digested with trypsin. Based on MALDI-TOF peptide mass fingerprint data
of the tryptically digested protein, we identified one
co-immunoprecipitating protein with an apparent molecular mass of about
90 kDa to be human VCP. Sequence coverage of 40% was obtained, and 38 peptides were matched to VCP in two independent analysis. Partial
sequence data of tryptic peptides obtained by ESI-MS/MS also matched to
VCP sequences. Fig. 9B confirms the identity of VCP by
immunoblot with anti-VCP antibody in ChAT-immunoprecipitates only from
cells expressing wild-type 69-kDa ChAT that were treated with
A Increased concentrations of A Although mechanisms by which A Using phosphopeptide mapping, phosphoamino acid analysis, and mass
spectrometry, we identified Thr456 as a novel
phosphorylation site in 69-kDa ChAT following short term treatment of
IMR32 cells with A To date, there have been no reports identifying proteins that ChAT
interacts with in the cell. Previously, it was suggested that neurons
contain endogenous modulator(s) of ChAT that may be proteins (55, 56).
Other compounds that may regulate ChAT appear to be small molecules or
lipid products, such as phosphomonoesters (57) and dihydrolipoic acid
(58). In the present study, a number of proteins immunoprecipitated
with ChAT following treatment of cells expressing ChAT with
A The findings reported in the present study identify a new action of
A-amyloid (A
) peptides.
Choline acetyltransferase is a substrate for several protein kinases.
In the present study, we demonstrate that short term exposure of IMR32
neuroblastoma cells expressing human choline acetyltransferase to
A
-(1-42) changes phosphorylation of the enzyme, resulting in
increased activity and alterations in its interaction with other
cellular proteins. Using mass spectrometry, we identified threonine 456 as a new phosphorylation site in choline acetyltransferase from A
-(1-42)-treated cells and in purified recombinant ChAT
phosphorylated in vitro by
calcium/calmodulin-dependent protein kinase II (CaM kinase
II). Whereas phosphorylation of choline acetyltransferase by protein
kinase C alone caused a 2-fold increase in enzyme activity, phosphorylation by CaM kinase II alone did not alter enzyme activity. A
3-fold increase in choline acetyltransferase activity was found with
coordinate phosphorylation of threonine 456 by CaM kinase II and
phosphorylation of serine 440 by protein kinase C. This phosphorylation
combination was observed in choline acetyltransferase from
A
-(1-42)-treated cells. Treatment of cells with A
-(1-42) resulted in two phases of activation of choline acetyltransferase, the
first within 30 min and associated with phosphorylation by protein
kinase C and the second by 10 h and associated with
phosphorylation by both CaM kinase II and protein kinase C. We also
show that choline acetyltransferase from A
-(1-42)-treated cells
co-immunoprecipitates with valosin-containing protein, and mutation of
threonine 456 to alanine abolished the A
-(1-42)-induced effects.
These studies demonstrate that A
-(1-42) can acutely regulate the
function of choline acetyltransferase, thus potentially altering
cholinergic neurotransmission.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amyloid
(A
)1 peptides released
from amyloid precursor protein (APP). Shifts in production of soluble
APP
by
-secretase to production of A
-(1-40) and A
-(1-42)
with activation of
- and
-secretase in Alzheimer's disease and
following traumatic head injury are associated with decreased function
and communication by cholinergic neurons (1-3). A complex relationship
exists between cholinergic neuron function and APP processing and A
peptide production (4-6). Short term exposure to low (picomolar or
nanomolar) concentrations of soluble/oligomeric A
peptide leads to
presynaptic cholinergic dysfunction with a reduction in the
availability of acetylcholine (ACh) precursors choline (7) and
acetyl-coenzyme A (8, 9) coupled to decreased ACh synthesis and release
from hippocampal slices or neuronal cultures (9-13). These acute
effects of A
peptides on neurotransmission and synaptic efficacy
probably differ from the neurotoxicity produced by long term exposure
and high (micromolar) concentrations of the peptides that cause death
of cholinergic neurons. Mechanisms underlying acute and long term effects of A
peptides on cholinergic function have not been resolved.
peptides modulate a range of cellular signal transduction pathways and
protein kinases (19-21). Whereas a number of potential cell surface
receptors for A
peptides have been identified (20-23), it is
unclear how these peptides mediate their cellular actions either
acutely or in the longer term. It is known, however, that A
peptides
can alter cell calcium homeostasis, leading to increased cytosolic free
calcium levels (24, 25). Within a certain concentration range, this
could activate a number of calcium-dependent processes, including calcium-dependent protein kinases such as PKC and
-calcium/calmodulin-dependent protein kinase II (CaM
kinase II). Since ChAT is known to be a substrate for both of these
protein kinases (17), it is likely that A
peptides could affect
cholinergic neurotransmission through regulation of function of this enzyme.
peptides would lead to altered function of the enzyme.
Interestingly, we observed that treatment of cells with A
-(1-42),
but not A
-(1-40), changed the state of phosphorylation of ChAT,
revealing a new putative CaM kinase II phosphorylation site.
Furthermore, phosphorylation at this site, when coordinated with
phosphorylation of Ser440 by PKC, leads to a hierarchical
activation of ChAT and phosphorylation-dependent association of the enzyme with other cellular proteins, including valosin-containing protein (VCP; p97, Cdc48).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Ala in wild-type 69-kDa ChAT using the
QuikChange kit (Stratagene), with the forward primer
5'-CAGATCGGCCGCTCCAGAGGC-3' and the reverse primer
5'-GCCTCTGGAGCGGCCGATCTG-3'. The presence of the mutation was verified
at the nucleotide level by automated DNA sequencing and at the protein
level by ESI-MS/MS sequencing. Mutant ChAT-S440A was prepared
previously (18).
peptides (1-40 or 1-42) or
reverse peptides (40-1 or 42-1) used as negative controls were
diluted in culture medium to final concentrations of 100 nM
from 100 µM stocks and added to cells for varying times
up to 18 h. For protein kinase inhibition studies, cell-permeable inhibitors (Calbiochem) of PKC (H7; 50 µM), CaM kinase II
(KN-93; 5 µM), p38-mitogen-activated protein kinase
(SB202190; 10 µM), or MEK-1/MEK-2 (U0126; 50 µM) were added to the media 2 h before the
addition of A
peptides. A
peptides (Bachem) were dissolved in
double-distilled H2O (1-40 and 40-1) or 0.1% ammonium
hydroxide (1-42 and 42-1) at 100 µM and incubated at
37 °C for 4 days (26); aliquots were stored at
80 °C until use.
For protein phosphorylation studies in cells, culture medium was
changed to phosphate-free modified Eagle's medium (Sigma) containing
[32P]orthophosphate (200 µCi/ml) at 3 h before the
end of the treatment with A
peptides; in the case of treatment times
shorter than 3 h, phosphate-free modified Eagle's medium and
[32P]orthophosphate were added with A
peptides at the
beginning of the incubation interval. Following treatment, lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5%
Triton X-100, 1 mM 4-(2-aminoethyl)-benzenesulfonyl
fluoride, leupeptin/aprotinin/pepstatin at 10/25/10 µg/ml, 500 µM sodium orthovanadate, 10 mM sodium
fluoride, and 700 units/ml DNase I) was added to cells and incubated
for 30 min on ice. Lysates were centrifuged (15,000 × g for 10 min), and supernatants were used for analysis of
activity or phosphorylation of ChAT or for analysis of proteins that
co-immunoprecipitate with ChAT. ChAT activity was measured
radioenzymatically using a modification of the method of Fonnum (27),
as published previously (28).
-Amyloid Peptides--
CD spectra of
A
-(1-40), A
-(1-42), and the corresponding reverse peptides were
recorded on a Jasco spectopolarimeter, model J-810, at 25 °C in a
0.1-cm path length cell at 0.2-nm intervals over the wavelength range
190-260 nm. Peptides were analyzed at a concentration of 50 µM in double-distilled H2O (1-40 and 40-1) or 0.1% ammonium hydroxide (1-42 and 42-1). To assess structural composition of A
-(1-40) and A
-(1-42) peptide solutions by
electron microscopy, carbon-formvar-coated grids were floated on a drop of each sample to allow peptides to adhere. After 3 min, grids were
blotted lightly and then floated on either 1% phosphotungstic acid or
2% uranyl acetate. Following staining, grids were blotted and
air-dried, and then representative images were acquired by examining
grids in a Philips EM300 electron microscope operated at 60 kV.
-mercaptoethanol) was added to the beads, and
immune complexes were dissociated by heating samples at 60 °C for 5 min. After centrifugation (10,000 × g for 2 min), supernatant proteins were separated by one-dimensional SDS-PAGE on
7.5% gels (30).
-(1-40), A
-(1-42), and reverse
control peptides A
-(40-1) and A
-(42-1) using the method of
Heasley and Johnson (31). Briefly, synthetic CaM kinase II-specific
peptide KKALRRQETVDAL was used as a substrate to monitor CaM kinase II
activity in digitonin-permeabilized cells. Cells grown in 24-well
plates were treated in the presence or absence of A
peptides and
then rinsed twice with Dulbecco's modified Eagle's medium buffered
with 20 mM HEPES, pH 7.2. Permeabilization solution (100 µl composed of 137 mM NaCl, 5.4 mM KCl, 1 mg/ml glucose, 20 mM HEPES, pH 7.2, 50 µg/ml digitonin,
10 mM MgCl2, 5 mM EDTA, 2.5 mM CaCl2, 25 mM
-glycerophosphate, 100 µM cold ATP, 0.4 nM
[
-32P]ATP) (10 µCi/well) supplemented with 1 mM CaM kinase substrate peptide was added to cells for 10 min at 30 °C. A parallel set of control cells were treated in the
same manner but without substrate peptide added to obtain a measure of
background [32P]phosphate incorporation. KN-93, a
cell-permeable inhibitor of CaM kinase II, or its inactive analog KN-92
(5 µM) was added as an additional control. Incubation was
terminated by the addition of 10 µl of ice-cold 25% trichloroacetic
acid, and then 40 µl of acidified cellular lysate was spotted onto
25-mm phosphocellulose discs (Whatman P-81) (32). Filter discs were
washed three times with 75 mM phosphoric acid and once with
75 mM Tris-HCl buffer, pH 7.5, and then dried and placed in
scintillation mixture for determination of radioactivity. Proteins were
digested in 0.2 M NaOH and assayed as described by Bradford
(33) to determine specific activity of peptide phosphorylation
expressed as pmol/min/mg protein.
70 °C. Phosphopeptides required
for further analysis were eluted from TLC plates with
water/acetonitrile (4:1, v/v) (36) and then reduced to dryness in a
vacuum centrifuge and reconstituted in 2% acetonitrile and 1% acetic
acid. This solution of peptides was used directly for MALDI-TOF mass
spectrometric analysis. For ESI-MS/MS sequencing, peptides were
purified on ZipTipC18TM according to the
manufacturer's instructions (Millipore Corp.) and eluted from the tip
resin with 65% acetonitrile and 1% acetic acid.
-treated cells or for identification of unknown proteins that
co-immunoprecipitate with ChAT were obtained by mass spectrometry
performed on a Micromass Q-TOF2 mass spectrometer equipped with a
nanospray source and an online Waters CapLC (Waters). In all cases, 1 µl of sample was injected from the autosampler. The instrument was
calibrated with [Glu1]Fibrinopeptide B (Sigma). A
gradient consisting of 5-65% B in 15.5 min (A = 0.1% formic
acid, B = acetonitrile with 0.1% formic acid) flowing at 1 µl/min was used to elute peptides from a 300-µm inner diameter
reversed-phase precolumn (LC-packings, San Francisco, CA) to the mass
spectrometer. Survey spectra were acquired in the
m/z range of 400-2000. Doubly or triply charged
precursor ions were automatically selected for fragmentation by the
quadrupole mass filter. In some cases, specific masses were identified
for fragmentation. Fragmentation was achieved by collision with argon gas in the collision cell. The collision energy was automatically varied depending on the charge state of the parent peptide. Resultant spectra were background-subtracted and deconvoluted using MaxEnt3 software provided in the Micromass MassLynx 3.5 software package. The
sequence of the peptides was determined using PepSeq version 3.3 software also provided in the MassLynx 3.5 software.
-cyano-4-hydroxycinnamic acid. Each sample (1 µl)
was spotted onto the MALDI target plate in triplicate. MALDI-TOF MS was
performed on a MALDI-R mass spectrometer (Micromass). Calibration was
performed externally using angiotensin I (Sigma), renin substrate (Sigma), adenocorticotrophic hormone clip 18-39 (Sigma) for a three-point calibration. In addition, for each sample, the lockmass method was used as additional calibration with the standard
adenocorticotrophic hormone clip 18-39. The peptide mass fingerprint
spectra were matched to the NCBI nonredundant data base entries by
using the following programs, available on the World Wide Web:
Profound (www.proteometrics.com) and Mascot (www.matrixscience.com).
The mass tolerance was set to 60 ppm, and two missed cleavage sites were tolerated with the search restricted to human proteins.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Peptides--
Stock solutions of A
peptide used to treat cells in the present studies were analyzed by CD
and electron microscopy to obtain information that would allow relative
comparisons to be made about peptide conformations. As shown in Fig.
1A, CD analysis revealed that
the secondary structure of A
-(1-40) and A
-(1-42) were
qualitatively identical in that they were both composed predominantly
of
-sheet conformation indicated by minimum molar ellipticity at 215 nm. Based on the shape of the CD spectra, negligible random coil or
-helical content was present in these two peptides. To obtain quantitatively similar signal strength on CD spectra, the concentration of A
-(1-40) and A
-(1-42) used were 50 and 30 µM,
respectively. By comparison, the reverse peptides A
-(40-1) and
A
-(42-1) had a predominantly random coil conformation. In support
of the CD data, as illustrated in Fig. 1B, electron
microscopic analysis confirmed that solutions of both A
-(1-40) and
A
-(1-42) contained characteristic A
-fibrils.
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Fig. 1.
Characterization of A
peptides by CD and electron microscopy. A, CD
spectra are expressed as the mean residue ellipticity in units of
degrees·cm2·dmol
1 after subtraction of
the appropriate buffer base-line spectra. Peptides were analyzed at a
concentration of 50 µM (A
-(1-40), A
-(40-1), and
A
-(42-1)) or 30 µM (A
-(1-42)). Preparations of
A
-(1-40) and A
-(1-42) had comparable
-sheet structure with
typical spectrum minimum at 215 nm and maximum at 195 nm. Related to
the concentration differences in the peptides analyzed to yield
quantitatively similar CD spectra, A
-(1-42) solutions may contain a
somewhat higher concentration of
-sheet conformation. CD spectra of
the reverse peptides revealed a random coil conformation with no
-helical or
-sheet structural features. B, electron
microscopic analysis of A
-(1-40) and A
-(1-42) revealed the
presence of fibrillar amyloid in both solutions.
-(1-42) Enhances Activity and
Phosphorylation of ChAT--
IMR32 cells expressing 69-kDa human ChAT
were incubated with A
-(1-42) for varying times, and then
incorporation of [32P]phosphate and enzyme activity were
monitored. As illustrated in Fig.
2A, catalytic activity of ChAT
was increased within 30 min of the addition of A
-(1-42) to
cultures, with this effect being maximal at 10 h (2-fold increase
in ChAT-specific activity). The effect of A
-(1-42) on ChAT activity
followed a biphasic time course, with the response diminishing beyond
10 h. Immunoblots shown in Fig. 2C (upper
panel) demonstrate that cellular ChAT concentration was
unchanged over the treatment interval. Phosphorylation of ChAT was also
increased up to 3-fold by treatment of cells with A
-(1-42) in a
manner that paralleled the time course for change in activity of the
enzyme, as shown in Fig. 2B; the corresponding autoradiography data are provided in Fig. 2C
(lower panel). Control cells were treated with
inactive peptide A
-(42-1); ChAT activity measured in cells with the
addition of A
-(42-1) did not differ from cells with no A
peptide
added. ChAT activity was not altered in cells treated for up to 18 h with A
-(1-40) or its reverse peptide A
-(40-1) (data not
shown). However, a 2-fold increase in [32P]phosphate
incorporation into ChAT was found in IMR32 cells treated with
A
-(1-40) between 2 and 6 h (data not shown).
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Fig. 2.
A -(1-42) treatment
of IMR32 cells enhances activity and phosphorylation of ChAT.
IMR32 cells were treated with 100 nM A
-(1-42) for
varying times. A, ChAT-specific activity was increased
significantly within 30 min of the addition of A
-(1-42). Enzyme
activity returned to control levels at longer times and then increased
transiently about 2-fold between 10 and 14 h (mean ± S.E.,
n = 5). B, incorporation of
[32P]orthophosphate into ChAT essentially paralleled the
changes in ChAT activity induced by A
-(1-42) treatment. Intensities
of bands on ChAT immunoblots were quantified by densitometry for
normalization of [32P]phosphate incorporation data. For
this latter measure, pieces of nitrocellulose membrane corresponding to
the location of ChAT were excised, and radioactivity was determined by
Cerenkov counting, or alternatively membranes was exposed to film for
autoradiography. Data are expressed as the mean ± S.E. of five
independent experiments. Statistical differences at the level of
p < 0.05 were determined by one-way analysis of
variance with post hoc Tukey's multiple
comparison test. *, differences relative to untreated controls; #,
differences between treatment of cells for 30 min and 10 h.
C, representative immunoblots for ChAT (upper
panel) and autoradiography for [32P]phosphate
incorporation into ChAT (lower panel) are shown,
with similar data obtained in at least four separate experiments.
-(1-42)--
IMR32 cells expressing ChAT were
treated with A
peptides for 10 h, and then ChAT was recovered
by immunoprecipitation and subjected to in-gel tryptic cleavage. In
control cells and cells treated with A
-(1-40) and the reverse
sequence control peptides A
-(40-1) and A
-(42-1), a single
phosphopeptide was observed, as illustrated in Fig.
3A. In contrast, treatment of
cells with A
-(1-42) resulted in the appearance of a second
phosphorylated ChAT peptide. Further analysis of these phosphopeptides
following acid hydrolysis showed that all samples contained
phosphorylated serine residue(s). Interestingly, phosphorylated ChAT
obtained from cells treated with A
-(1-42) contained
phosphothreonine as well as phosphoserine residues (Fig.
3B).
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Fig. 3.
Treatment of IMR32 cells with
A -(1-42) results in phosphorylation of ChAT
at a new site. A, ChAT was immunoprecipitated from
lysates of untreated cells (C) and cells treated for 10 h with A
-(1-40), A
-(40-1), A
-(42-1), and A
-(1-42) and
separated by SDS-PAGE. Following in-gel tryptic digestion, tryptic
peptides of ChAT were analyzed by two-dimensional phosphopeptide
mapping. Under all conditions tested, a phosphopeptide(s) with
phosphorylation on serine residue(s) was observed. By comparison, an
additional phosphopeptide corresponding to phosphorylation on threonine
residue(s) was observed only in cells treated with A
-(1-42).
B, tryptic peptides were also subjected to acid hydrolysis,
and the phosphoamino acids produced were separated by two-dimensional
electrophoresis. Circles indicate the migration of
ninhydrin-stained phosphoamino acid standards, with closed
circles corresponding to phosphoserine and broken circles
corresponding to phosphothreonine. ×, sample origins;
Pi, free [32P]phosphate liberated
during acid hydrolysis of phosphopeptides. Positive spots remaining
near sample origins represent incompletely hydrolyzed phosphopeptides.
Data illustrated are representative of between three and five
independent experiments.
-(1-42) treatment, ChAT was immunoprecipitated from IMR32 cells
after 10 h of treatment with A
peptides. Following isolation by
SDS-PAGE, ChAT-containing bands were subjected to in-gel tryptic digestion, and samples were prepared for mass spectrometric analysis by
ESI-MS/MS. Mass spectra revealed a doubly charged peak at 621.6 m/z present in ChAT recovered from cells treated
with A
-(1-42) (Fig. 4B)
but not in cells treated with A
-(1-40) (Fig. 4A) or A
-(42-1) or in ChAT from untreated control cells (data not shown). Although other peaks were detected in tryptic digests of samples treated with A
-(1-42), none of these corresponded to ChAT peptides with potential phosphorylation sites. The amplitude of the signal detected for this tryptic phosphopeptide from ChAT from
A
-(1-42)-treated cells was very low when compared with purified
ChAT phosphorylated by incubation with protein kinases in
vitro (see below). This suggests that a relatively low proportion
of the enzyme is phosphorylated in situ. Consequently,
sequence information was obtained by fragmenting at this
m/z for extended periods. Subsequent analysis of
the fragmentation pattern revealed the C-terminal sequence VR as well
as the partial sequence EAL. This allowed identification of this
peptide as amino acid residues 454-464 of 69-kDa human ChAT with the
sequence SATPEALAFVR and a mass of 1160.626. The immonium ion region also revealed the presence of the correct amino
acids for the peptide encoding residues 454-464 of 69-kDa human ChAT
(Pro, Val, Thr, Leu, Glu, Phe, and Arg). This sequence contains a
putative consensus sequence for CaM kinase II involving phosphorylation
of Thr456; the canonical consensus sequence for CaM kinase
II is RXX(*S/*T) (38-40), with the corresponding
ChAT sequence of 453RSA*T456. To confirm this
assignment, Thr456 was changed to an alanine residue
(ChAT-T456A) by site-directed mutagenesis to provide a plasmid for use
as an investigative tool; the presence of the mutation was verified by
DNA sequencing of the plasmid and ESI-MS/MS sequencing of the tryptic
peptide encoding amino acid residues 454-464 in T456A-ChAT and absence
of the tryptic peptide encoding the wild-type sequence. As illustrated
in Fig. 4, C and D, analysis of tryptic peptides
of ChAT-T456A obtained from IMR32 cells expressing the mutant enzyme
and treated with A
-(1-42) by phosphopeptide and phosphoamino acid
analysis revealed a single phosphopeptide, with serine being the
residue phosphorylated. This finding is in sharp contrast to the
results obtained with cells expressing wild-type ChAT shown in Fig. 3,
A and B.
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Fig. 4.
ESI-MS analysis of phosphorylated ChAT in
IMR32 cells treated with A -(1-42).
ESI-MS spectra in the range of 620-625 m/z for
tryptically digested ChAT from control and A
peptide-treated IMR32
cells are shown. A and B, spectra obtained for
ChAT from cells treated with A
-(1-40) and A
-(1-42),
respectively. A doubly charged peak at 621.6 that corresponds to the
phosphorylation of peptide 454-464 of human 69-kDa ChAT is seen in the
A
-(1-42) sample but not in the sample from cells treated with
A
-(1-40). Samples from untreated control samples and cells treated
with reverse peptide A
-(42-1) yielded spectra similar to
A, indicating lack of phosphorylation of this ChAT peptide.
All samples were prepared in duplicate, and multiple injections were
examined with identical results. C and D,
two-dimensional patterns for phosphoamino acids and phosphopeptides of
ChAT-T456A, respectively, from cells treated with A
-(1-42). This
demonstrates the lack of threonine phosphorylation as compared with
that observed in wild-type ChAT (shown in Fig. 3).
-(1-42).
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Fig. 5.
ESI-MS parent ion scanning of CaM
kinase II-phosphorylated purified recombinant ChAT. Parent ion
scanning in the m/z range of 300-1000 revealed a
doubly charged peptide at m/z = 621.6 that
fragmented to produce an ion that resulted from a neutral loss of
97.9769 ± 20 mDa. The sequence of the doubly charged
phosphopeptide at 621.6 m/z was found to be
SApTPEALAFVR, which contains a phosphorylated threonine residue. Parent
ion scanning was performed in duplicate. Sequencing was performed on
the processed MS/MS spectrum from the fragmentation of 621.6 m/z over 30 min.
Peptide
Treatment--
We tested whether CaM kinase II was activated in IMR32
cells treated with A
peptides. Cells expressing wild-type ChAT were grown with A
-(1-42), A
-(1-40), or reverse peptides A
-(42-1) or A
-(40-1) and then digitonin-permeabilized and incubated with substrate peptide encoding a CaM kinase II phosphorylation consensus sequence. As illustrated in Fig.
6A, 10-h treatment with
A
-(1-42) selectively increased phosphorylation of the CaM kinase II
substrate peptide by more than 2-fold, indicating activation of this
protein kinase. Phosphorylation of the substrate peptide in cells
treated with the other three A
peptides did not differ from
untreated control cells. This corresponds to the time point when
A
-(1-42)-mediated increases in activity and phosphorylation of ChAT
are maximal. Time course experiments revealed that phosphorylation of
CaM kinase II substrate peptide was not different from control at 30 min or 2 h, significantly increased (160% compared with control)
by 6 h, and returned to control levels at 14 and 18 h after
the addition of A
-(1-42) (data not shown). To further confirm that
phosphorylation of the substrate peptide in A
-(1-42)-treated cells
was related to activation of CaM kinase II, we tested the effects of
the CaM kinase II inhibitor KN-93 and its inactive analogue KN-92. The addition of 5 µM KN-93, but not KN-92, to cells during
treatment with A
-(1-42) markedly reduced subsequent phosphorylation
of the CaM kinase II substrate peptide (Fig. 6B).
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Fig. 6.
Activation of CaM kinase II in IMR32 cells
treated with A -(1-42). A,
cells were treated for 10 h with A
peptides and then
permeabilized with digitonin and incubated with CaM kinase II-specific
substrate peptide in the presence of [32P]ATP.
A
-(1-42) treatment selectively increases phosphorylation of the CaM
kinase II peptide by 2-fold compared with that measured in control
cells or cells treated with other forms of A
peptides. B,
treatment of cells with A
-(1-42) for 30 min did not result in
enhanced phosphorylation of CaM II-specific peptide compared with
control. The CaM kinase II inhibitor KN-93 fully inhibited
phosphorylation of the substrate peptide at 10 h of A
-(1-42)
treatment, whereas its inactive analog KN-92 was without effect.
[32P]Phosphate incorporation in cells incubated in the
absence of added CaM kinase II substrate peptide was used as a measure
of background, with this value subtracted from values obtained in the
presence of substrate peptide. Results are expressed as mean ± S.E. of four independent experiments with triplicate determinations.
Statistical significance at the level of p < 0.05, denoted by asterisks, was determined by one-way analysis of
variance and Dunnet's post hoc test.
-(1-42). As demonstrated in Fig.
7A, purified ChAT activity was
increased about 3-fold by CaM kinase II-mediated phosphorylation only
when the enzyme was also phosphorylated by PKC. Phosphorylation of ChAT
by PKC alone led to a 2-fold increase in enzyme activity, whereas
phosphorylation by CaM kinase II alone did not alter ChAT activity.
Moreover, inhibition of PKC by H7 (10 µM) blocked
activation of ChAT observed when the enzyme was sequentially incubated
with PKC and CaM kinase II under phosphorylating conditions. Incubation of purified ChAT with PKC and CaM kinase II in the presence of KN-93
resulted in a 2-fold increase in ChAT activity similar to that observed
for phosphorylation of ChAT by PKC alone.
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Fig. 7.
Hierarchical activation of purified ChAT and
ChAT in A -(1-42)-treated IMR32 cells related
to phosphorylation by PKC and CaM kinase II. A,
purified recombinant ChAT (1 µg/sample) was phosphorylated by either
PKC or CaM kinase II or by both kinases. Phosphorylation by CaM kinase
II alone did not alter ChAT activity compared with control, whereas
phosphorylation by PKC alone led to a 2-fold increase in ChAT activity
that was blocked by H7. When ChAT was phosphorylated by both PKC and
CaM kinase II, activity of the enzyme increased 3-fold. Importantly,
inhibition of CaM kinase II by KN-93 attenuated enhancement of ChAT
activity to the level obtained with phosphorylation by PKC alone.
Moreover, inhibition of PKC by 10 µM H7 blocked
activation of ChAT when it was incubated under phosphorylating
conditions with PKC and CaM kinase II. B, IMR32 cells were
incubated with A
-(1-42) for 30 min or 10 h in the absence or
presence of protein kinase inhibitors to test involvement of these
kinases in the two phases of activation of ChAT observed in these
studies. None of the inhibitors altered basal ChAT activity. At 30 min
of treatment with A
-(1-42), ChAT activity was increased about
1.6-fold, with this effect blocked by H7. At 10 h of treatment
with A
-(1-42), ChAT activity was increased about 2-fold. This
effect was attenuated to a 1.6-fold increase by KN-93 and completely
blocked by H7. The MEK-1/MEK-2 and p38-kinase inhibitors U0126 and
SB202190 did not alter A
-(1-42)-mediated changes in ChAT activity.
C, phosphoamino acid analysis revealed basal phosphorylation
of ChAT on serine but not threonine residue(s) under control
conditions. This was not altered by any of the kinase inhibitors.
Treatment of cells with A
-(1-42) for 30 min increased phosphoserine
levels, but phosphothreonine was not observed. H7 decreased
phosphoserine levels. At 10 h of A
-(1-42) treatment, there was
an increase in phosphoserine levels and phosphorylation on threonine
residue(s). KN-93 blocked threonine phosphorylation, whereas H7 blocked
serine phosphorylation. For A and B, results are
expressed as mean ± S.E. of four or five independent experiments
with duplicate or triplicate determinations. Statistical differences at
the level of p < 0.05 were determined by one-way
analysis of variance with post hoc Tukey's
multiple comparison test. *, differences relative to controls; #,
differences relative to PKC alone (A) or between treatment
of cells for 30 min and 10 h (B). Data shown in
C are representative of at least three separate
experiments.
-(1-42) for either 30 min or 10 h. On the other hand, the CaM kinase II inhibitor KN-93 partially attenuated activation of ChAT by 10 h of A
treatment and had no
effect on ChAT activation at 30 min of treatment. Whereas
Thr456 is situated in a consensus sequence that could be
recognized by CaM kinase II, it is also positioned at
1 from a
proline residue, creating the possibility that this proline-directed
threonine residue could be phosphorylated by other protein kinases such as mitogen-activated protein kinase. To test this, we used inhibitors of MEK-1/MEK-2 (U0126) and p38-mitogen-activated protein kinase (SB202190) to probe their involvement in phosphorylation of
Thr456 in A
-(1-42)-treated IMR32 cells. The addition of
U0126 or SB202190 to IMR32 cells during A
treatment also had no
effect on A
-mediated activation of ChAT at either time point.
-(1-42) and the effect of kinase inhibitors on
phosphorylation of ChAT in A
-treated cells. As shown in Fig. 7C, H7 reduced serine phosphorylation to control levels at
both 30 min and 10 h, whereas KN-93 eliminated threonine
phosphorylation in cells treated with A
-(1-42) for 10 h. U0126
or SB202190 had no effect on phosphorylation at either 30-min or 10-h
time points. We also tested the effect of A
-(1-42) treatment on
activity of ChAT-S440A and ChAT-T456A mutants in comparison with that
of wild-type enzyme. As shown in Fig. 8,
ChAT-specific activity does not differ in the mutant ChAT-expressing
cell lines compared with that of wild-type enzyme in the absence of
A
-(1-42) treatment. For cells expressing wild-type ChAT, enzyme
activity was increased by about 1.6-fold at 30 min and by about 2-fold
at 10 h after the addition of 100 ng/ml A
-(1-42). By
comparison, A
-(1-42) treatment of IMR32 cells expressing mutant
forms of ChAT did not result in a change in activity of the S440A
mutant of ChAT at either time point tested but did increase activity of
the T456A mutant of ChAT by about 1.6-fold at both 30 min and
10 h.
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Fig. 8.
Phosphorylation of 69-kDa ChAT on
Ser440 in association with Thr456 regulates
hierarchical activation of enzyme. ChAT-specific activities were
compared in IMR32 cells expressing the wild-type enzyme or mutants
ChAT-S440A or ChAT-T456A treated in the absence or presence of
A -(1-42) treatment for 30 min or 10 h. Activity of wild-type
ChAT was increased by about 1.6- and 2-fold in cells treated with
A
-(1-42) for 30 min and 10 h, respectively. This effect was
completely blocked in A
-(1-42)-treated cells expressing ChAT-S440A.
ChAT activity was increased to about 1.6-fold by 30 min of A
-(1-42)
treatment of cells expressing ChAT-T456A, similar to that observed with
the wild-type enzyme. However, the further A
-(1-42)-induced
increase in ChAT activity observed for the wild-type enzyme at 10 h was blocked in cells expressing ChAT-T456A. Results are expressed as
mean ± S.E. of four independent experiments with triplicate
determinations. Statistical differences at the level of
p < 0.05 were determined by one-way analysis of
variance with post hoc Tukey's multiple
comparison test. *, differences relative to controls; #, differences
between treatment of cells for 30 min and 10 h in wild-type enzyme
and ChAT-T456A mutant.
Peptide Treatment Alters Interaction of ChAT with Other
Cellular Proteins--
ChAT was immunoprecipitated from IMR32 cells
treated with A
-(1-42), and co-immunoprecipitating proteins were
separated and analyzed by one-dimensional SDS-PAGE. It is apparent from
the Coomassie-stained gel of ChAT immunoprecipitates shown in Fig. 9A that a number of other
proteins co-immunoprecipitated with both wild-type ChAT and mutant
ChAT-S440A from cells treated with A
-(1-42). These were not
observed in lanes corresponding to treatment of cells with A
-(1-40)
or A
-(42-1) or from cells expressing mutant ChAT-T456A treated with
A
-(1-42).
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Fig. 9.
Phosphorylation of ChAT on Thr456
in A -(1-42)-treated IMR32 cells promotes
novel interactions between ChAT and other cellular proteins.
Lysates were prepared from cells expressing wild-type or mutant
ChAT-S440A or ChAT-T456A treated for 10 h with A
peptides and
then incubated with CTab anti-ChAT antibody to immunoprecipitate ChAT.
A, proteins in samples obtained by immunoprecipitating ChAT
from cell lysates were separated by SDS-PAGE, and then gels were
stained with Coomassie Blue to observe patterns of
co-immunoprecipitating proteins. Selected bands were excised from gels
and subjected to in-gel tryptic digestion, with the resultant peptides
analyzed by mass spectrometry. MALDI-TOF mass fingerprint analysis and
partial ESI-MS/MS sequence analysis of samples from two independent
samples identified a band with apparent molecular mass of ~90 kDa to
be VCP. B, the identity of VCP as a protein that
co-immunoprecipitates with ChAT in A
-(1-42)-treated IMR32 cells
expressing wild-type ChAT was confirmed by immunoblot using an anti-VCP
antibody. C, in separate samples, VCP was immunoprecipitated
from cell lysates using the anti-VCP antibody, and the presence of ChAT
was identified as a protein that co-immunoprecipitates with VCP by
immunoblot using the CTab anti-ChAT antibody.
-(1-42) but not other A
peptides; this finding was obtained in
three independent experiments. To further verify this interaction, VCP
was immunoprecipitated from lysates of IMR32 cells expressing wild-type
ChAT treated with A
peptides using an antibody to VCP. As
illustrated in Fig. 9C, a ChAT immunopositive band was
observed in the anti-VCP immunoprecipitate from cells treated with
A
-(1-42).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
peptides are released into the
brain by cleavage of APP in neurodegenerative diseases such as
Alzheimer's disease and following traumatic head injury. In the
present study, we tested the hypothesis that exposure of IMR32 neuroblastoma cells stably expressing ChAT to A
peptides would alter
function of the enzyme. We demonstrate for the first time that 1)
phosphorylation patterns and activity of ChAT are changed by short-term
exposure of cells to A
-(1-42), 2) exposure of cells to A
-(1-42)
leads to activation of CaM kinase II and phosphorylation on a threonine
residue in ChAT, 3) Thr456 in ChAT is phosphorylated by CaM
kinase II in vitro and in cells following treatment with
A
-(1-42), 4) the increase in ChAT activity observed with A
-
(1-42)-treatment is hierarchically organized with phosphorylation of
Ser440 by PKC being required, and this effect is amplified
by CaM kinase II-mediated phosphorylation of Thr456 at
10 h; 5) treatment of cells with A
-(1-42) leads to
Thr456 phosphorylation-dependent
protein-protein interactions between ChAT and other cellular proteins,
with one of these identified as VCP.
peptides mediate either acute or long
term actions on neurons are not resolved, it is known that they
interact with several cell surface receptors including
-7 nicotinic
ACh receptor (21, 42), p75 nerve growth factor receptor (43),
G-protein-linked formyl-peptide receptor (44), and advanced
glycosylation end product receptor (45). Signal transduction pathways
and cellular responses recruited by binding of A
peptides to these
receptors have not been worked out, but some responses are mediated by
pertussis toxin-sensitive G-protein-coupled mechanisms (22).
Interaction of A
peptides with some receptors alters cellular
calcium homeostasis with enhanced influx of extracellular calcium or
release from intracellular stores (24, 48). Also, A
peptides can
form calcium ionophores in plasma membrane, leading to calcium
conductances across plasma membrane (48, 49). In addition,
intracellular accumulation A
-(1-42) but not other A
peptides,
possibly mediated by interaction with the
-7 nicotinic ACh receptor
(50), led to neuronal toxicity through a p53-Bax cell death pathway
(51). In regard to the present studies, it is known that IMR32 cells
express
-7 nicotinic ACh receptors (50) and may express low levels
of p75 nerve growth factor receptors (51); it is not known whether
other putative binding sites for A
peptides are expressed by these
cells. Increased cytosolic free calcium levels caused by A
peptide
treatment could activate Ca2+-dependent protein
kinases such as PKC and CaM kinase II. ChAT is a substrate for both of
these kinases (17). The present study extends these findings by
demonstrating a link between activation of CaM kinase II and PKC and
regulation of ChAT activity and phosphorylation of ChAT and its
interaction with other proteins. We also showed that A
-(1-42)
increased CaM kinase II activity in permeabilized IMR32 cells, and,
although we did not measure PKC activation in permeabilized cells, we
found increased PKC activity in cell lysates (data not shown).
Activation of PKC isoforms by A
peptides was reported previously
(52-54). A key observation in the present study is that only
A
-(1-42) caused changes in ChAT phosphorylation, activity, and
protein interactions. Mechanisms underlying this selectivity are
unclear, but other studies have demonstrated actions mediated
specifically by A
-(1-42) and not other A
peptides. To confirm
that this did not relate to differences in conformation between
A
-(1-42) and A
-(1-40), we analyzed samples of peptides by CD
and electron microscopy and found both preparations to be comprised of
A
-fibrils and to have similar
-sheet structure. It is likely that
the differences in cellular responses to A
-(1-42) and A
-(1-40)
observed in the present study relate to differences in ability of the
peptides to stimulate receptors that initiate the cellular events.
-(1-42). Residue Thr456 in ChAT is
phosphorylated in A
-(1-42)-treated cells over a time course
coinciding with increased activity of CaM kinase II. Since other
protein kinases including members of the mitogen-activated protein
kinase family could be activated by A
-treatment (21), we used
specific inhibitors to determine that these are probably not
mediating phosphorylation of Thr456 in this situation.
Also, kinase inhibitors and site-directed mutagenesis of critical
residues in ChAT were used to establish a relationship between
phosphorylation of Ser440 by PKC and phosphorylation of
Thr456 by CaM kinase II in enhanced catalytic activity of
ChAT. Phosphorylation of ChAT on Ser440 by PKC alone
increased ChAT activity by 2-fold, with this increased to about 3-fold
when Thr456 is also phosphorylated by CaM kinase II (Fig.
2B). Importantly, phosphorylation by CaM kinase II alone did
not alter ChAT activity. This suggests that phosphorylation of ChAT on
Ser440 leads to the initial activation of ChAT by 30 min.
This increased ChAT activity is not maintained over the next few hours,
but a second phase of activation occurs by 10-14 h after the addition of A
-(1-42) paralleling increased CaM kinase II activity,
phosphorylation of Thr456, and increased serine
phosphorylation. Mechanisms underlying this delayed increase in ChAT
activity and activation of CaM kinase II are unclear. One possibility,
however, is that cellular events initiated with acute addition of
A
-(1-42) result in production/release of cellular mediators
producing effects several hours later. These delayed effects include
activation of CaM kinase II, enhanced activity of ChAT, and interaction
of ChAT with VCP and other cellular proteins.
-(1-42) and associated with phosphorylation of Thr456.
We identified one of these by mass spectrometry, with confirmation by
immunoblotting, to be VCP. VCP, a member of the AAA-ATPase family of
proteins, is a multifunctional protein in yeast and mammalian cells
(59-61) with roles in diverse cellular functions including cell cycle
regulation, clathrin-mediated receptor endocytosis, protein
ubiquitination, and proteasome function. Other cellular proteins with
which VCP is known to interact include BRCA1 (62) and histone
deacetylase-6 (46). Interestingly, it appears that association of VCP
with ChAT is phosphorylation-dependent, occurring under
conditions where both Thr456 and Ser440 are
phosphorylated. VCP does not co-immunoprecipitate with ChAT in
A
-(1-42)-treated cells expressing mutant ChAT-S440A. The functional significance of interaction of ChAT with VCP is unclear. A predominant cellular function of VCP is its role in the link between
acetylation/deacetylation/ubiquitination and proteosomal degradation of
proteins (46, 61). One function for an acetyltransferase is to add
acetyl-residues to proteins to protect them from ubiquitination and
targeting for degradation (46, 47). Although a function for the
interaction between ChAT and VCP remains to be determined, two
hypothesis could be tested. In the first, ubiquitinated-ChAT binds to
VCP, which then serves as a chaperone to target the enzyme to the
proteosome. In the second, ChAT may serve as an acetylase, transferring
acetate to other proteins to alter their function or delay their
degradation. As an acetylase, ChAT catalyzes the transfer of acetyl
groups to hydroxyl moieties of choline and potentially other small
molecules through an O-acetylation reaction. It has never
been demonstrated that ChAT can catalyze the N-acetylation
reaction that would normally be found with acetylation of proteins on
lysine residues.
-(1-42) on cholinergic neuron function. Although loss of ChAT
activity as a consequence of the toxic effects of A
peptides on
cholinergic neurons has been demonstrated, acute effects of A
peptides on function of the enzyme ChAT have not been examined. Exposure of primary cultures of rat septal neurons to A
-(1-42), but
not A
-(1-40), at concentrations used in the present study for
12-24 h suppressed ACh synthesis but did not alter ChAT activity (9).
Also, exposure of SN56 cells to 100 nM A
-(1-42) for
48 h significantly reduced ChAT activity (10). As demonstrated in
the present study, activation of cell signaling pathways and alteration
of the intracellular milieu in response to exposure to A
-(1-42)
leads to short term changes in the phosphorylation state of ChAT that
could result in acute changes in cholinergic neurotransmission.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. L. Samelson (NCI-Fredrick) for providing the VCP antibody, Dr. H. Schulman (Stanford University) for the gift of CaM kinase II, Dr. P. Ferguson (University of Western Ontario) for circular dichroism spectra of amyloid peptides, and Dr. Susan Koval and Judy Sholdice (University of Western Ontario) for electron microscopy images of amyloid peptides.
![]() |
FOOTNOTES |
---|
* This research was supported by operating grants from the Ontario Neurotrauma Foundation (to R. J. R.) and the Ontario Research Development Challenge Fund, Genome Canada, and NSERC (to G. L.).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.
¶ Funded by a Research Associate salary award from 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.: 519-663-5777 (ext. 34078); Fax: 519-663-3789; E-mail: jane.rylett@fmd.uwo.ca.
Published, JBC Papers in Press, December 16, 2002, DOI 10.1074/jbc.M212080200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
A,
-amyloid;
ACh, acetylcholine;
APP, amyloid precursor protein;
ChAT, choline
acetyltransferase;
CTab, anti-ChAT carboxyl-terminal peptide antibody;
CaM kinase II, calcium/calmodulin-dependent protein kinase
II;
ESI, electrospray ionization;
MS, mass spectrometry;
MALDI, matrix-assisted laser desorption/ionization;
TOF, time-of-flight;
PKC, protein kinase C;
VCP, valosin-containing protein.
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REFERENCES |
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