(Received for publication, August 1, 1996, and in revised form, October 25, 1996)
From the GAD65, the smaller isoform of the
Glutamic acid decarboxylase (GAD)1 (EC
4.1.1.15) catalyzes the GAD65 and GAD67 differ with regard to their steady state saturation
with the co-enzyme pyridoxal 5 The efficacy of neurotransmitter production by GAD65 may also be
regulated by its subcellular location. GAD65 is isolated both in a
cytosolic and a firmly membrane anchored form (15, 16). The enzyme is
synthesized as a cytosolic soluble protein, but undergoes a stepwise
post-translational modification in the NH2-terminal domain
to become hydrophobic and anchored to synaptic vesicle-like
microvesicles (16, 17). The modifications involve palmitoylation of
cysteines 30 and 45, but this modification is not essential for
membrane anchoring of the protein in COS-7 cells (18). Membrane
anchoring is reversible, suggesting that trafficking of GAD65 between
membranes and cytosol may regulate its proximity to synaptic vesicles
and the efficacy by which its product GABA can be accumulated for
secretion (16). Association of GAD65 with vesicles could place the
protein in the proximity of a hypothetical GABA transporter, perhaps
serving the double function of loading the neurotransmitter into the
vesicle that secretes it, as well as removing GABA from the proximity
of the enzyme, to prevent product inhibition of enzyme activity
(19).
GAD purified from rat, pig, and human brain under native conditions
forms dimers (20-22). The native form of both GAD65 and GAD67 is a
non-covalently associated homodimer detected by gel filtration and
native gel electrophoresis.2 Both the GAD67
and GAD65 dimers dissociate into monomers on SDS-PAGE under either
reducing or nonreducing conditions. Reducing SDS-PAGE resolves GAD65
but not GAD67 into two bands of distinct mobility, which have been
designated Two-dimensional gel electrophoretic studies of GAD65 in human and rat
islets (15, 23, 24) and brain (8) in the presence of enzyme inhibitors
suggested to us that the protein might be phosphorylated. The present
study demonstrates that GAD65 is phosphorylated in vivo as
well as in vitro, that phosphorylation distinguishes GAD Oligonucleotide-mediated
mutagenesis of serines in the NH2-terminal region and
deletion of the first 8 and 15 amino acids of rat GAD65 (rGAD65),
respectively, were described previously (18, 25).
Oligonucleotide-mediated mutagenesis of individual serine residues 3, 6, 10, 13, 17, and 20 in human GAD65 (hGAD65) was performed by the
method of Kunkel (26). The S3A/S6A/S10A/S13A mutant was generated in
two steps, by first constructing a S3A/S6A mutant, generating a single
stranded cDNA, and undertaking a second round of mutagenesis to
convert serines 10 and 13 to alanine. The template for these mutants
was the hGAD65 cDNA cloned into a Bluescript S/K vector (18).
For expression in COS-7 cells (Life Technologies, Inc., Gaithersburg,
MD), mutant hGAD65 cDNAs were subcloned into the SV40-based expression vector pSVsport (Life Technologies, Inc.) at the
EcoRI and XbaI sites. For expression in BHK-21
cells (Life Technologies, Inc.), PC12 cells (American Type Tissue
Collection, Rockville, MD), and Baby hamster kidney cells (BHK-77-3) stably expressing
hGAD65 (a generous gift from W. Hagopian, University of Washington) were derived from tk GAD6, a mouse monoclonal antibody specific for
GAD65, and 1701, a rabbit antiserum that recognizes an epitope in the
carboxyl terminus of GAD65 and GAD67, were described previously (30). Rabbit antiserum to glucagon (DAKO, Carpintera, CA) and normal mouse
serum (Sigma) were used as control sera.
For phosphorylation experiments BHK-77-3 cells or
transfected COS-7 cells were grown to 80-90% confluency in a 10-cm
plate, rinsed twice in phosphate-free Dulbecco's modified Eagle's
medium (Life Technologies, Inc.) containing 1% dialyzed fetal calf
serum, and for BHK-77-3 cells 800 nM methotrexate and
incubated in the same medium for 2 h. The cells were then rinsed
three times in the same medium and incubated in 5 ml of fresh medium
containing 4 mCi of [32P]- or
[33P]orthophosphate (ICN, Costa Mesa, CA) for 3 h.
Metabolic labeling of cellular proteins with
[35S]methionine (Amersham) was described previously (31).
Labeled cells were washed twice in phosphate-buffered saline, and lysed in 0.5 ml of HMAP buffer (10 mM HEPES/NaOH, pH 7.4, 1 mM MgCl2, 1 mM
2-aminoethylisothiouronium bromide, 0.2 mM PLP, containing 0.1 mM p-chloromercuriphenyl sulfonic acid, 1 mM phenylmethylsulfonyl fluoride, 10 mM
benzamidine, 0.1 mM Na3VO4, 5 mM ETDA, 5 mM sodium fluoride, and 1% Triton
X-114 (HMAP-PIT buffer) for 30 min. The lysate was centrifuged at
15,000 × g for 30 min. GAD65 was immunoprecipitated from the supernatant by incubation with the GAD6 antibody for 2 h,
and isolation of the immune complexes using protein A-Sepharose (Pharmacia, Uppsala, Sweden). Immunoprecipitates were washed seven times, and eluted from protein A-Sepharose by one of two methods. For
one-dimensional SDS-PAGE on 10% polyacrylamide gels, samples were
boiled for 3 min in SDS sample buffer (23). For two-dimensional gel
electrophoresis using non-equilibrium pH-gradient gel electrophoresis (NEPHGE) in the first dimension and 10% SDS-PAGE in the second dimension, washed immunoprecipitates were incubated at 28 °C for 3 min in an urea/Nonidet P-40 buffer followed by centrifugation to remove
the protein A-Sepharose beads. The NEPHGE/SDS-PAGE two-dimensional analysis was carried out as described elsewhere (15, 23). The
polyacrylamide gels were dried and analyzed by autoradiography (32P, 33P) or fluorography (35S).
Alternatively, proteins were transferred from gels by Western blotting
to Immobilon-P membranes (Millipore, Bedford, MA) and immunostained
with the 1701 antibody as described previously (30). Antibody staining
was visualized with either an alkaline phosphatase-coupled secondary
antibody (Zymed, S. San Francisco, CA), or chemiluminescent detection
using a horseradish peroxidase-coupled secondary antibody (Vector Labs,
Burlingame, CA) and the ECL system (Amersham, Buckinghamshire, United
Kingdom). Western blots were also analyzed by autoradiography.
A 90-100% confluent 10-cm (inner
diameter) plate of BHK-77-3 cells, transfected COS-7 cells, or infected
Sf9 cells was harvested in 0.5 ml of HMAP buffer containing 1 mM phenylmethylsulfonyl fluoride and 0.1 mM
p-chloromercuriphenyl sulfonic acid (HMAP-P buffer). The
cells were lysed on ice using a glass homogenizer, warmed to 37 °C,
and incubated for an additional 5 min at the same temperature in the
presence of 150 µCi of [ In vivo
32P-labeled hGAD65 was isolated by immunoprecipitation
with the GAD6 antibody. The immunoprecipitates were subjected to
SDS-PAGE, transferred to Immobilon-P membranes, and immunostained with
the 1701 antiserum. The GAD65 Tryptic
phosphopeptide analyses were performed essentially as described (32).
Briefly, lysates of in vivo 32P-labeled BHK-77-3
cells (10-cm plate) were immunoprecipitated with GAD6 and the
immunoprecipitates subjected to SDS-PAGE followed by autoradiography of
the unfixed gel. GAD65 was excised from the gel and electroeluted into
0.5 ml of 50 mM NH4HCO3, 0.1% SDS on a C.B.S. Scientific (DelMar, CA) electroelution apparatus (33). The
eluted protein was lyophilized, acetone precipitated, re-dissolved in
50 µl of 50 mM ammonium bicarbonate, and digested with 8 µg of sequencing grade modified trypsin (Promega, Madison, WI)
overnight at 37 °C. The tryptic digest was lyophilized, re-dissolved
in water, and applied to a thin layer cellulose plate (Baker).
Phosphopeptides were separated in the first dimension by
electrophoresis at pH 4.4 (acetone/pyridine/acetic acid/water,
8:1:2:40) or at pH 8.9 (0.1 M ammonium carbonate) (34) on a
Savant TLE apparatus at 900 V, followed by ascending chromatography in
the second dimension in pyridine/1-butanol/acetic acid/water
(50:75:15:60). The phosphopeptides were detected by
autoradiography.
GAD65 was
immunoprecipitated from Triton X-114 lysates of in vivo
labeled BHK-77-3 cells and purified by electroelution as described
above. The lyophilized and acetone precipitated sample was dissolved in
100 µl of 50 mM ammonium bicarbonate. A 25-µl aliquot
was removed for SDS-PAGE analyses. For an extensive degradation by
trypsin, the remaining sample was digested with 4 µg of modified trypsin (Promega), first at 4 °C for 5 min, and then at 37 °C overnight. Aliquots (25 µl) were removed at the two time points and
quenched by boiling in SDS sample buffer. Digested samples were
analyzed in triplicate lanes by Tris/Tricine non-equilibrium gel
electrophoresis (35). The lanes were subjected to drying and
autoradiography or immunoblotting using the 1701 antiserum.
For mild trypsin digestion to generate the 58-kDa tryptic fragment of
GAD65, which contains the COOH-terminal 69/70-585 amino acids (30),
GAD65 in 100 µl of 50 mM ammonium bicarbonate was treated
with 0.6 µg of trypsin at 4 °C and 10-µl aliquots removed at
different time points, quenched as described above, and analyzed by
SDS-PAGE using 10% polyacrylamide gels, followed by immunoblotting with the 1701 antiserum.
Subcellular fractionation
of cells was carried out using a modification of methods described
previously (18). All procedures except the incubation with
[ Analysis of the subcellular distribution of phosphorylated GAD65 was
carried out using in vitro or in vivo
32P-labeled BHK-77-3 cells. In vivo labeled
cells were homogenized in HMAP-P buffer, containing 0.1 mM
Na3VO4, and 10 mM benzamidine/HCl, and 5 mM sodium fluoride (lysis buffer). Cell homogenates
were centrifuged at 800 × g to remove nuclei and
cellular debris. The postnuclear supernatant was centrifuged at
150,000 × g for 1 h to separate cytosol and crude
membrane fractions. The cytosolic fraction was supplemented with 5 mM EDTA and 1% Triton X-114. Sedimented membranes were
resuspended in lysis buffer containing 5 mM EDTA and 0.5 M NaCl, incubated for 1 h, and resedimented by
centrifugation at 150,000 × g for 1 h to separate
washed membranes and a high salt wash fraction. The membrane wash
fraction was supplemented with Triton X-114 to obtain a final
concentration of 1%. The washed membranes were extracted in lysis
buffer containing 1% Triton X-114 for 1 h followed by
centrifugation at 150,000 × g for 1 h. The
cytosol, washed membrane extract, and membrane wash fractions were
subjected to one round of temperature-induced Triton X-114 phase
separations according to Bordier (36), and the detergent and aqueous
phases analyzed by immunoprecipitation, SDS-PAGE, immunoblotting, and
autoradiography. In some experiments the phosphatase inhibitors, sodium
fluoride and EDTA, were both present during homogenization of cells. In
other experiments sodium fluoride and EDTA were absent from the lysis
buffer but added together to the postnuclear spin.
For in vitro labeling experiments, BHK-77-3 cells were lysed
in lysis buffer without sodium fluoride, and labeled with
[ In a different set of experiments, a cytosolic and a crude membrane
fraction were prepared and labeled separately in vitro. BHK-77-3 cells were homogenized in lysis buffer without sodium fluoride, and a cytosolic and a crude membrane fraction prepared as
described above. The membrane fraction was resuspended in the original
volume of hypotonic lysis buffer, and both fractions were labeled
in vitro with [ For comparative analysis of subcellular distribution and amphiphilicity
of wild-type GAD65 and phosphorylation mutants, transiently transfected
COS-7, BHK-21, PC12, or GAD65 was immunoprecipitated
from in vitro labeled BHK-77-3 cell lysates prepared from
two 10-cm tissue culture plates. The washed protein A-Sepharose pellet
was resuspended in 50 mM Tris/HCl, pH 8.5, 0.1 mM EDTA and incubated with 5 units of calf intestinal phosphatase according to the manufacturers instructions (Boehringer Mannheim). Aliquots were removed at different intervals, boiled in SDS
sample buffer, and analyzed by SDS-PAGE, immunoblotting, and
autoradiography.
Activity assays for the
wild-type and mutant GAD65 proteins, generated by transient
transfection in COS-7 cells, were carried out by a modification of the
method described by Blindermann et al. (20). To determine
the Km and Vmax for
glutamate, COS-7 cells were harvested 48 h post-transfection, and
lysed in 50 mM HEPES/NaOH, pH 7.0, 0.1 mM PLP,
1 mM 2-aminoethylisothiouronium bromide, 1 mM
phenylmethylsulfonyl fluoride, 10 mM benzamidine/HCl, 5 mM sodium fluoride, and 0.5% Triton X-114. The lysate was
centrifuged at 15,000 × g for 30 min to remove debris,
and the supernatant was aliquoted and diluted 1:10 in the same buffer
without Triton X-114, but containing 0.2-9 mM
[1-14C]glutamate. Reactions were incubated for 1 h
at 37 °C, and quenched by addition of 6 N HCl. Lysates
from non-transfected COS-7 cells were used as a negative control at
each substrate concentration. GAD65 protein concentrations in the
different lysates were measured as percentage of wild-type protein by
immunoblotting and densitometry analyses (GS-700 Imaging Densitometer,
Bio-Rad) of multiple serial dilutions of each protein. Kinetic
constants were calculated by a nonlinear fit to the Michaelis-Menten
equation using GraFit (37). Km and
Vmax values for PLP were determined in a similar
manner except that cells were lyzed in the absence of PLP, the assay
buffer contained 0.1% bovine serum albumin, and the reactions were
carried out in 20 mM [1-14C]glutamate and at
PLP concentrations between 0.1 and 20 µM.
We
initially analyzed whether hGAD65 expressed in BHK-77-3 and COS-7 cells
could be phosphorylated in cellular lysates. The cells were
homogenized, incubated with [
Under native conditions, GAD65 is a noncovalently associated homodimer
of two 65-kDa subunits. Heating of the protein in SDS in the presence
or absence of reducing agents results in dissociation of the dimer into
monomer subunits.2 Under reducing conditions, high
resolution SDS-PAGE and immunoblotting analyses of GAD65 reveals the
doublet of GAD65 bands designated The first possibility seemed unlikely, because the presence or absence
of phosphatase inhibitors in phosphorylation experiments did not seem
to affect the We next addressed the
question whether GAD65 is phosphorylated in vivo. BHK-77-3
cells in culture were labeled with [32P]orthophosphate,
and cell lysates were prepared and subjected to immunoprecipitation
with GAD6. Immunoblotting of the immunoprecipitates revealed the GAD65
Extensive
two-dimensional gel electrophoretic analyses using NEPHGE or
isoelectric focusing in the first dimension and SDS-PAGE in the second
dimension have been carried out to characterize GAD65 isolated from
human and rat islets of Langerhans (8, 23, 30). GAD65
immunoprecipitated from total islet cell lysates or islet cell
membranes frequently resolved into a series of differently charged
spots in the first dimension, with the least acidic species having a pI
of approximately 6.7 which corresponds to the pI calculated for the
unmodified protein based on its amino acid sequence (6).
We investigated the two-dimensional pattern of phosphorylated GAD65
isolated from in vivo labeled BHK-77-3 cells, and compared it with the [35S]methionine-labeled and/or immunostained
protein on Western blots (Fig. 2). GAD65 was identified
in autoradiograms of total cell lysates by Western blot analysis of
two-dimensional gels using the 1701 antibody, followed by
autoradiography, or by comparison of the mobility of an
immunoprecipitated sample of the protein, which was analyzed in a
parallel run. Western blot analyses of 35S- and
33P-labeled GAD65 revealed a similar pattern of 4 differently charged spots in both GAD65
The acidic charge heterogeneity in GAD65 The two-dimensional gel electrophoretic data strongly suggest that
phosphorylation is an exclusive property of The amino acid residue(s), which
undergo phosphorylation in the GAD65 protein, were characterized by
phosphoamino acid analyses. GAD65 from in vivo
phosphorylated BHK-77-3 cells was purified, subjected to hydrolysis in
6 N HCl, and amino acids were separated by thin layer
electrophoresis. Phosphorylated amino acids were detected by
autoradiography. Those analyses detected only one phosphorylated amino
acid which was identified as phosphoserine (Fig. 3,
panel A). Thus GAD65 seems to undergo phosphorylation exclusively on serine residue(s).
We next assessed the distribution of phosphorylation sites in GAD65 by
two-dimensional phosphopeptide mapping. 32P-Labeled GAD65
was purified from BHK-77-3 cells and subjected to tryptic digestion
followed by two-dimensional separation of peptides by TLE at pH 4.4 or
8.9 in the first dimension, and TLC in the second dimension. Labeled
peptides were detected by autoradiography. TLE at pH 4.4 revealed a
single phosphorylated spot on the two-dimensional plate, suggesting
that the phosphorylation sites may reside in a single peptide (Fig.
3B, top panel). Identical results were obtained for hGAD65
expressed in COS-7 cells (results not shown). TLE at pH 8.9, which
enhances the separation of acidic peptides, resolved the phosphorylated
peptides into two major and one minor phosphorylated spots on the
two-dimensional plate (Fig. 3B, bottom panel). These results
as well as the three differently charged, phosphorylated species
detected in Preliminary observations suggested to us that GAD65 might be
phosphorylated in the NH2-terminal region. Thus
immunoblotting of phosphorylated GAD65 sometimes revealed degraded
fragments of GAD65, which were recognized on Western blots by an
antiserum to a COOH-terminal peptide (1701), yet these bands did not
show up on the autoradiograms of immunoblots. To further assess the location of the phosphorylated peptides, GAD65 isolated from in vivo 32P-labeled lysates of BHK-77-3 cells, was
subjected to a mild trypsin digestion to generate a 58-kDa peptide
previously shown to contain amino acids 69/70-585 (28). The 58-kDa
fragment, which was positive for immunostaining with the 1701 antiserum
(Fig. 4B, panel a), was not phosphorylated
(Fig. 4B, panel b), suggesting that the phosphorylation
sites reside in the first 69/70 amino acids of GAD65. In the SDS-PAGE
analyses of those experiments, most phosphorylated tryptic fragments
migrated with the dye front. Tris/Tricine gel electrophoresis of
purified GAD65 partially digested with trypsin revealed a small
phosphorylated fragment of molecular mass of approximately 8 kDa (Fig.
4B, lanes 5 and 6), which upon exhaustive tryptic
digestion resolved into three smaller bands of molecular mass 2-3 kDa
(Fig. 4B, lanes 6 and 7). Analyses of the
sequence of the first 69/70 amino acids in GAD65 revealed two candidate tryptic fragments in this size range, an NH2-terminal
peptide (aa 1-27, mass 2.7 kDa), and a fragment containing aa 44-69
(mass 2.2 kDa, Arg65 is an unlikely cleavage site because
it is next to a proline).
To
further characterize the identity of the phosphorylated residues in
GAD65, we analyzed the phosphorylation of NH2-terminal deletion mutants of rat GAD65, which have also had the palmitoylated cysteine residues 30 and 45 mutated to alanine (18), as well as
substitution mutants of rat GAD65, in which two or three serines in the
NH2-terminal region have been mutated to alanine (25). Furthermore, site-directed mutagenesis was used to construct hGAD65 mutants in which serine residues in the NH2-terminal region
were individually substituted with alanine.
There are eight serine residues in the first 70 amino acids of hGAD65
at positions 3, 6, 10, 13, 17, 20, 58, and 61. The
NH2-terminal region of rat GAD65 also has serines at
positions 3, 6, 10, 13, 17, 58, and 61 but has a proline at position
20, and additional serines at positions 52 and 64 (Fig.
5A). The collection of mutants enabled
analysis of every serine residue in the first 70 amino acids of GAD65
for phosphorylation.
Wild-type and mutant GAD65 plasmids were transiently expressed in COS-7
cells. The corresponding proteins were immunoprecipitated from in
vivo 32P-labeled cell lysates, using the GAD6
antibody. This antibody recognizes an epitope in the carboxyl-terminal
41 amino acids of GAD65, which is not affected by the mutations (24,
39). Immunoprecipitates were analyzed by SDS-PAGE and autoradiography (Fig. 5B). The rGAD65 S58A/S61A/S64A mutant did not show a
decrease in phosphorylation, strongly suggesting that the
phosphorylated residues do not reside in the aa 44-69 fragment of
GAD65 (Fig. 5, lanes 6 and 7). In contrast,
mutation or deletion of 2-3 serines in the NH2-terminal
peptide of rGAD65, affected the phosphorylation of the protein. Thus, a
significant decrease in 32P-labeling was observed in a
rGAD65 S3A/S6A, rGAD65 S10A/S13A/S17A, and rGAD65 Analysis of single serine substitution mutants in the
NH2-terminal peptide of hGAD65 (Fig. 5B, lanes
8-14) revealed a decrease in phosphorylation signal in the hGAD65
S3A, hGAD65 S6A, hGAD65 S10A, and hGAD65 S13A proteins but not in the
hGAD65 S17A and hGAD65 S20A mutants, consistent with
polyphosphorylation of serines in the NH2-terminal peptide
and suggesting that all 4 serines, 3, 6, 10, 13, can be phosphorylated
relatively independently of each other. Interestingly, the S6A, S10A,
and S13A mutants also showed a slight increase in mobility on SDS-PAGE.
Similar shifts in mobility upon removal of phosphorylation sites have
been reported for other proteins (40). Substitution of all 4 serines,
3, 6, 10, and 13 in hGAD65 abolished The mutational analysis, as well as the two-dimensional phosphopeptide
analysis and protein studies, suggest that all four serines residues 3, 6, 10, and 13 can be phosphorylated in combinations to generate mono-,
di-, and triphosphorylated GAD65 Bao et al. (41, 42) reported
that the enzyme activity of an unspecified soluble isoform of GAD from
porcine brain is regulated by phosphorylation. We assessed whether
phosphorylation of GAD65 affects Km or
Vmax for glutamate and PLP. The analyses for
glutamate were carried out using detergent extracts of wild-type and
phosphorylation mutants of hGAD65 and rGAD65 expressed in COS-7 cells
(Table I). Substitution of the phosphorylated serines in
the NH2-terminal domain for alanine either individually or in combinations, that included all serines 3, 6, 10, and 13 in hGAD65,
did not affect the Km or Vmax
of GAD65 for glutamate (Table I). Deletion of the first 15 amino acids
of the rGAD65 resulted in a decrease of Km for
glutamate from 1.2 to 0.9 mM. This may, however, reflect a
conformational effect of deletion, rather than removal of the
phosphorylated residues, since a decrease in Km for
glutamate was not observed for the hGAD65 tetraserine mutant.
Kinetic constants for glutamate for wild-type and mutant GAD65
proteins
Department of Medicine,
-aminobutyric acid-synthesizing enzyme glutamic acid decarboxylase
is detected as an
/
doublet of distinct mobility on
SDS-polyacrylamide gel electrophoresis. Glutamic acid decarboxylase
(GAD) 65 is reversibly anchored to the membrane of synaptic vesicles in
neurons and synaptic-like microvesicles in pancreatic
-cells. Here
we demonstrate that GAD65
but not
is phosphorylated in
vivo and in vitro in several cell types.
Phosphorylation is not the cause of the
/
heterogeneity but
represents a unique post-translational modification of GAD65
. Two-dimensional protein analyses identified five phosphorylated species
of three different charges, which are likely to represent mono-, di-,
and triphosphorylated GAD65
in different combinations of
phosphorylated serines. Phosphorylation of GAD65
was located at
serine residues 3, 6, 10, and 13, shown to be mediated by a membrane
bound kinase, and distinguish the membrane anchored, and soluble forms
of the enzyme. Phosphorylation status does not affect membrane
anchoring of GAD65, nor its Km or
Vmax for glutamate. The results are consistent
with a model in which GAD65
and -
constitute the two subunits of
the native GAD65 dimer, only one of which,
, undergoes
phosphorylation following membrane anchoring, perhaps to regulate
specific aspects of GAD65 function in the synaptic vesicle
membrane.
-decarboxylation of glutamate generating
-aminobutyric acid (GABA). GABA is a major inhibitory
neurotransmitter in the central nervous system (1), and may also act as
a paracrine signaling molecule in pancreatic islets, where it is
synthesized in
-cells (2, 3). GABA can also function as a metabolic
intermediate and be fueled into the Krebs cycle via the GABA shunt (4,
5). In mammalian species two highly homologous non-allelic forms of the
enzyme have been identified and designated GAD65 and GAD67 in
accordance with their relative molecular masses in kDa (6, 7). GAD65,
but not GAD67, is unusually susceptible to becoming an autoantigen in
two human diseases that affect its major sites of expression,
pancreatic
-cells and GABA-ergic neurons. Thus GAD65 is a major
autoantigen in insulin-dependent diabetes mellitus in man
(8), and the non-obese diabetic mouse (9, 10), and in a rare
neurological disease, stiff-man syndrome in man (11).
-phosphate (PLP). At least 50% of GAD
in brain is present as the PLP free apoenzyme (12, 13). GAD65
constitutes the majority of this enzyme reservoir (14), which can be
activated by influx of co-factor, or perhaps by targeting the protein
to compartments in the cell where PLP is available. In contrast to
GAD65, the majority of GAD67 seems to be perpetually saturated with PLP
(14).
and
(15, 16, 23). The nature of the differences
between
and
is not known. Both
and
are palmitoylated,
their ratio is always identical in different subcellular fractions and
no differences have been detected in their hydrophobicity (15, 16).
and -
, and is a prominent modification of the membrane anchored, but
not the cytosolic form of the enzyme.
Site-directed Mutagenesis
TC3 cells (27), wild type hGAD65,
and the S3A/S6A/S10A/S13A mutant were subcloned into the
cytomegalovirus based vector pcDNA3 (Invitrogen, San Diego, CA) at
the EcoRI, and XBaI sites.
ts13 BHK cells (28), which were
originally isolated as temperature-sensitive BHK-21 cells. BHK-77-3
cells were cultured in Dulbecco's modified Eagle's medium,
supplemented with 10% fetal calf serum, glutamine (2 mM),
penicillin (100 units/ml), streptomycin (100 µg/ml), and 800 nM methotrexate. COS-7 cells were grown in the same medium as BHK-77-3 cells but without methotrexate. Transient expression of
wild-type and mutant hGAD65 or rGAD65 in COS-7 cells was obtained by
LipofectAMINE (Life Technologies, Inc.) assisted transfection using 15 µg of plasmid DNA and 57 µl of LipofectAMINE (per 10-cm plate of
cells) in Opti-MEM I (Life Technologies, Inc.). Cells were refreshed
with Dulbecco's modified Eagle's medium 10-12 h after transfection
and cultured for additional 48 h before harvesting. BHK-21 cells
were cultured in Glasgow minimal essential medium, BHK-21 (Life
Technologies, Inc.), supplemented with 10% tryptose phosphate, 10%
fetal calf serum, penicillin (100 units/ml), and streptomycin (100 µg/ml). Transient expression of protein in BHK-21 cells was achieved
in a similar manner as in COS-7 cells, except using 20 µg of
pCDNA3 constructs. PC12 cells were cultured and transfected as
described by Bonzelius et al. (29).
TC3 cells were
cultured as described previously (27). Transient expression of protein
in
TC3 cells was achieved in a similar manner as in COS-7 cells
except using Cellfectin (Life Technologies, Inc.) mediated transfer of
pcDNA3 constructs. hGAD65 was expressed from a baculovirus
construct in Sf9 cells as described previously (16).
-32P]ATP (30 Ci/mmol,
Amersham). Labeled samples were cooled on ice for 5 min and
supplemented with 100 µl of HMAP-P buffer containing EDTA, sodium
fluoride, Na3VO4, and Triton X-114 to yield
final concentrations similar to those in HMAP-PIT buffer, extracted for
30 min and cleared by centrifugation at 15,000 × g for
30 min. The supernatants were immunoprecipitated and analyzed by SDS-PAGE.
and
bands were excised and
subjected to hydrolysis for 2 h in 6 N HCl at
110 °C using a Waters Picotag hydrolysis station (Waters, Bedford,
MA). Hydrolyzed samples were evaporated to dryness, re-dissolved in
water, and spotted on a cellulose thin layer electrophoresis (TLE)
plate (Baker, Phillipsburg, NJ). The phosphoamino acids were separated by TLE at pH 4.4 (acetone/pyridine/acetic acid/water, 8:1:2:40) on a
Savant TLE apparatus (Savant, Farmingdale, NY) at constant voltage (900 V). The TLE plates were stained with ninhydrin and placed on an x-ray
film (Kodak Biomax, Eastman/Kodak, Rochester, NY). Phosphoamino acids
were detected by autoradiography and identified by co-migration with
ninhydrin stained standards.
-32P]ATP were carried out at 4 °C.
-32P]ATP as described in the section for in
vitro labeling. 5 mM Sodium fluoride was added
immediately after labeling and labeled lysates were subjected to
centrifugation at 800 × g. The rest of the procedure
was carried out as described above for the in vivo labeled
cells.
-32P]ATP. The labeled
cytosolic fraction, and crude membrane suspension were cooled on ice,
supplemented with 5 mM EDTA, 5 mM sodium
fluoride, and 1% Triton X-114, incubated on a shaker for 30 min and
centrifuged at 15,000 × g for 30 min. The supernatants
were analyzed by immunoprecipitation, SDS-PAGE, immunoblotting, and
autoradiography.
TC3 cells were homogenized in lysis buffer,
and subjected to the subcellular fractionation procedure described
above. Furthermore, transiently transfected
TC3 cells were used for
a more detailed Triton X-114 phase distribution study. Briefly
subcellular fractions were subjected to two rounds of
temperature-induced phase transitions using 2% Triton X-114, followed
by a final wash of the aqueous phase as described by Bordier (36). The
rest of the analysis was carried out as described above for the
in vivo labeled material.
GAD65 Is Phosphorylated in Vitro in Whole Cell Lysates
-32P]ATP, and the labeled
protein isolated by immunoprecipitation with a GAD65 specific antibody
(GAD6). The isolated protein was analyzed by SDS-PAGE and
autoradiography. These experiments demonstrated that
32P-labeled GAD65 is specifically immunoprecipitated from
phosphorylated cell lysates of both BHK-77-3 cells and COS-7 cells
(Fig. 1A). Similar phosphorylation
experiments carried out using lysates of Sf9 cells expressing GAD65
showed that GAD65 is also phosphorylated in vitro, using
this material (Fig. 1B, lanes 5 and 6).
Fig. 1.
GAD65 is phosphorylated in vitro
and in vivo and the phosphorylated protein has a mobility
similar to the component on Western blots. A,
autoradiogram of immunoprecipitates of detergent extracts of in
vitro [
-32P]ATP-labeled COS-7 and BHK cells,
using the GAD65 specific monoclonal antibody GAD6 (lanes 1, 3, 5, and 7) and an anti-glucagon antibody (lanes 2, 4, 6, and 8). Lanes 1 and 2 are
COS-7 cells transfected with hGAD65. Lanes 3 and
4 are untransfected COS-7 cells. Lanes 5 and
6 are BHK-77-3 cells stably expressing hGAD65. Lanes
7 and 8 are the parental BHK-21 cells which do not
express GAD. MW, molecular weight markers. Phosphorylated
GAD65 is specifically immunoprecipitated from transfected COS-7 and
BHK-77-3 cells. B, comparison of the autoradiogram signal
with the immunostaining pattern on Western blots for hGAD65
immunoprecipitated from 32P-labeled cells. GAD65 was
immunoprecipitated from cells phosphorylated in vitro using
[
-32P]ATP (lanes 1-6) or from cells
labeled in vivo with [32P]phosphate
(lanes 7 and 8) and the immunoprecipitates
subjected to SDS-PAGE followed by Western blotting, immunostaining of
the Western blots (even lanes), and autoradiography (odd lanes). The mobility of the main phosphorylated GAD65 component (32P)
corresponds with that of the
component on immunoblots
(WB). In addition, a weak phosphorylated component,
, is
seen above
, in some experiments and co-migrates with a similar
signal on the immunoblot (lanes 3 and 4). No
phosphorylated band co-migrated with the GAD65
.
[View Larger Version of this Image (67K GIF file)]
and
(Fig. 1B) as
described earlier (15, 16, 18), and sometimes a more slowly migrating
band designated
(Fig. 1B, lanes 1 and 2).
Analyses of 32P autoradiograms of the immunoblots revealed
one major phosphorylated band which co-migrated with the GAD65
band,
and in some experiments, a weaker band which co-migrated with the
GAD65
band. No 32P-labeled band co-migrated with
GAD65
. The co-migration of phosphorylated GAD65 with
, but not
with
, suggested either that phosphorylation is a cause of the
/
heterogeneity or that phosphorylation is an exclusive property
of
.
/
ratio (data not shown). Furthermore, a treatment
of GAD65 immunoprecipitated from in vitro labeled BHK-77-3
cells with calf intestinal phosphatase, resulted in a significant
decrease in the 32P signal, yet the
/
ratio of the
protein on Western blots was not affected (data not shown). Hence, it
is unlikely that phosphorylation is the principal cause of the observed
/
heterogeneity. This result has been confirmed by
two-dimensional protein analyses (see below).
/
bands (Fig. 1B, lane 8). Autoradiography of the
immunoblots demonstrated that the major in vivo
phosphorylated band co-migrated with
(Fig. 1B, lane 7).
Thus phosphorylation seems to be restricted to GAD65
both in
vivo and in vitro. Phosphorylation of GAD65 was
severalfold more efficient in vivo than in vitro. In
vivo labeled cells were therefore used for all of the following experiments except where indicated.
and
, ranging in pI from
approximately 6.7 to 6.3, and one or two spots in
of a charge
similar to the most acidic spots of
and
. The two-dimensional
pattern observed for GAD65
/
in BHK-77-3 cells is identical to the
pattern described previously for the protein in pancreatic
-cells
(23). Autoradiography of the Western blot of the
33P-labeled GAD65 and alignment of the autoradiographed and
immunostained images demonstrated that all but the most basic spot in
as well as all the spots in
were phosphorylated. The most
basic spot in GAD65
and all the spots in GAD65
were not
33P-labeled. These analyses confirm that phosphorylation is
not the primary cause of the
/
heterogeneity, because they reveal an unmodified
/
doublet at pI 6.7. Phosphorylation, however, seems to give rise to
, because all the
spots are
phosphorylated and there is no unmodified
spot at pI 6.7.
Fig. 2.
Two-dimensional NEPHGE-SDS/PAGE analysis of
hGAD65 from BHK-77-3 cells. Panel a, autoradiogram of a
whole cell lysate of BHK-77-3 cells metabolically labeled with
[35S]methionine. The locations of GAD65 and
were
identified by alignment with GAD65 identified by immunostaining of a
Western blot of an identical gel run in parallel (panel b).
Panel c, immunostaining of a Western blot of GAD65
immunoprecipitated from BHK-77-3 cells, labeled in vivo with
[33P]phosphate, and analyzed on a two-dimensional gel.
Panel d, autoradiogram of the immunostained Western blot in
panel c. There is no phosphorylation of the most basic
spot, but the 33P-signal co-migrates with the other and
more acidic spots in
and all the spots in
. None of the
spots are phosphorylated.
[View Larger Version of this Image (55K GIF file)]
on two-dimensional gels
represents the phosphorylated species. Phosphorylation is, however,
clearly not the cause of the identical charge heterogeneity detected in
GAD65
. Two possibilities can be suggested to explain this result.
The first is that GAD65
undergoes an acidic modification, which does
not affect the mobility on SDS-PAGE, and is distinct from
phosphorylation, yet results in identical charge heterogeneity as
observed for the phosphorylated GAD65
. Although this possibility cannot be formally excluded it seems unlikely, based on extensive two-dimensional analysis of GAD65 isolated from pancreatic
-cells in
different conditions, which preserved the charge heterogeneity or
phosphorylation of GAD65
to a varying degree (15, 23, 30). In every
analysis the charge heterogeneity of GAD65
has been the exact
replica of the GAD65
pattern, inferring that if GAD65
indeed
undergoes a series of distinct acidic modifications they would (i)
result in identical charge heterogeneity as that imposed by
phosphorylation of
in all cases; and (ii) be of identical stability
as the phosphorylation of
in a variety of conditions. The
probability of a distinct acidic modification that so closely resembles
phosphorylation is low. The most likely explanation is that
does
not undergo an acidic modification, but that
and
constitute the
two subunits of the GAD65 dimer. The GAD65 dimer is stable in non-ionic
detergent and urea2 and is therefore unlikely to dissociate
during electrophoresis in the NEPHGE dimension. It will, however, come
apart under the strongly dissociating conditions of the SDS-PAGE
dimension. The charge heterogeneity detected in
in the NEPHGE
dimension on two-dimensional gels would therefore not reflect its own
heterogeneity, but that of the native GAD65 dimer.
. Thus the acidic charge
heterogeneity in
would result from phosphorylation of one, two, and
three amino acid residues, respectively, without significantly
affecting the SDS-PAGE mobility, whereas the phosphorylated spots in
may represent double and/or triple phosphorylation of a different
combination of amino acids in GAD65
resulting in a slight mobility
shift on SDS-PAGE. It is also possible that the modification of GAD65
is restricted to mono- and diphosphorylation of the protein, but that
the triple charge heterogeneity is caused by differences in protonation
of the phosphate groups at the near neutral pH of GAD65 (38).
Fig. 3.
GAD65 is phosphorylated on serine residue(s)
which may reside in the same tryptic fragment. A,
autoradiogram of a TLE analyses of amino acids generated by hydrolysis
of hGAD65 purified from in vivo phosphorylated BHK-77-3
cells. P-S, P-T, and P-Y indicate the location of
phosphoserine, phosphothreonine, and phosphotyrosine standards;
Pi, inorganic phosphate. B, autoradiograms of
two-dimensional TLE/TLC analysis of tryptic fragments of hGAD65 purified from in vivo phosphorylated BHK-77-3 cells using
TLE at pH 4.4 (top panel) and 8.9 (bottom panel),
respectively. TLE at pH 8.9 enhances the separation of differently
charged peptides and resolves the single spot detected at pH 4.4 into
three species indicated by arrows.
[View Larger Version of this Image (38K GIF file)]
/
by two-dimensional gel electrophoresis (Fig. 2)
suggest that GAD65 exists in a mono, di, and perhaps a
triphosphorylated form.
Fig. 4.
Localization of the phosphorylation site(s)
to the amino terminus of GAD65 by one-dimensional tryptic
phosphopeptide mapping. A, tryptic degradation map of the
NH3 terminus of GAD65. B, separation of tryptic
phosphopeptides of GAD65 by SDS-PAGE (panels a and
b) and Tris/Tricine gel electrophoresis (panel
c). Panel a, immunostaining with the COOH-terminal
antibody 1701 of a Western blot of SDS-PAGE of 32P-labeled
GAD65 purified from in vivo labeled BHK-77-3 cells and incubated with low concentrations of trypsin at 37 °C for the indicated times. Panel b, autoradiogram of the immunoblot
shown in panel a. The 58-kDa COOH-terminal fragment of GAD65
detected by immunoblotting of lane 2, is not phosphorylated
(lane 4). Panel c, autoradiogram of
32P-labeled GAD65 purified from BHK-77-3 cells, and
incubated with a high concentration of trypsin at 4 °C for 5 min
(lane 6) or overnight (O/N) at 37 °C
(lane 7). Twice as much sample was loaded in lane
7 to ensure disappearance of the 8-kDa band and detection of any
minor fragments present.
[View Larger Version of this Image (54K GIF file)]
Fig. 5.
GAD65 is phosphorylated on serine residues in
the first 15 amino acids of GAD65. Upper panel,
autoradiogram of wild-type and mutant rat (lanes 1-7) and
human (lanes 8-16). GAD65 immunoprecipitated from
transiently transfected and in vivo phosphorylated COS-7 cells and Western blotted to polyvinylidene difluoride membranes. Lower panel, immunostaining of the same Western blots.
Substitution of serines 3 and 6 (lane 2) results is a
decrease in phosphorylation and additional removal of serines 10 and 13 results in a complete loss of phosphorylation of rat GAD65 (lane
3). Similarly mutation of serines 3 and 6, or serines 10, 13, and
17 to alanine but not mutation of serines 58, 61, and 64 caused a
decrease in phosphorylation compared to the wild-type rat protein
(lanes 4-7). Consistent with the results with rat mutants,
mutation of individual serines 6, 10, and 13 in hGAD65 to alanine
results in a decrease in phosphorylation signal and, furthermore, a
slight mobility shift, whereas mutation of serine 3 results in a
decrease in phosphorylation signal only.
[View Larger Version of this Image (55K GIF file)]
1-8 mutants (Fig.
5B, lanes 2, 4, and 5), suggesting that the
NH2-terminal peptide is phosphorylated on at least 2 serine
residues. Furthermore, deletion of the first 15 amino acids in rGAD65,
containing serines 3, 6, 10, and 13 abolished phosphorylation. The
similar decrease in phosphorylation of the
1-8 mutant, which is
lacking serines 3 and 6, but also the palmitoylated cysteines (aa 30 and 45), and the S3A/S6A mutant, which retains the palmitoylation sites, suggested that palmitoylation does not play a role in
phosphorylation of GAD65, and this result was confirmed by mutational
analysis of hGAD65 (see below).
95% of the phosphorylation
signal (Fig. 5, lanes 15 and 16).
. All these serine residues reside
in the same tryptic fragment (aa 1-27). Hence the three spots detected
by two-dimensional tryptic phosphopeptide analyses at pH 8.9, are
likely to represent the mono-, di-, and triphosphorylated GAD65. The
two-dimensional protein analyses reveal three acidic spots which
co-migrate with nonphosphorylated
on SDS-PAGE, and one or two spots
in
which align(s) with the most acidic spot(s) in
. Since
addition of each phosphate group is likely to result in one charge
shift, the three increasingly acidic 33P-labeled spots that
co-migrate with nonphosphorylated
are likely to represent GAD65
phosphorylated on one, two, and three serines, respectively, in a
combination that does not result in a significant mobility shift in the
SDS-PAGE dimension. Similarly, the spot(s) detected in
, may
represent double, and/or triple phosphorylation of serines in GAD65
in a combination of serines residues that results in a slight mobility
shift in the SDS-PAGE dimension.
Construct
Km (mM) (mean ± S.D.)
Relative Vmax (mean ± S.D.)
hGAD65
2.15
± 0.35
1.0
hGAD65
S3A/S6A/S10A/S13A
2.13 ± 0.16
1.0 ± 0.2
rGAD65
1.02 ± 0.15
1.0
rGAD65
1-8
0.81 ± 0.18
1.0 ± 0.1
rGAD65
1-15
0.71 ± 0.13
0.8 ± 0.1
rGAD65
S3A/S6A
1.21 ± 0.18
1.0 ± 0.5
rGAD65
S10A/S13A/S17A
1.26 ± 0.14
1.0 ± 0.3
rGAD65
C30A/C45A
1.08 ± 0.04
1.3 ± 0.4
The analysis for PLP were carried out using wild-type hGAD65 and the S3A/S6A/S10A/S13A mutant. Both proteins showed identical deviations from Michaelis-Menten kinetics at low PLP concentrations. At PLP concentrations between 0.5 and 20 µM PLP, however, both wild-type and the tetraserine GAD65 mutant obeyed Michaelis-Menten kinetics, were identical with regard to Vmax, and displayed an identical Km value of ~0.9 µM.
In summary, phosphorylation does not seem to regulate kinetic properties of GAD65 with regard to glutamate and PLP. It is of note, however, that a detection of subtle effects of phosphorylation on the kinetics of GAD65 may require the separation of phosphorylated, and nonphosphorylated fractions of the wild-type protein and purification of each to homogeneity.
Phosphorylation of GAD65 Is Mediated by a Membrane-associated Kinase and Phosphorylated Protein Remains Largely Membrane-associatedMembrane anchoring of GAD65 is reversible
(15, 16), but the factors that regulate the trafficking of the protein
between cytosol and membranes are unknown. Washing of isolated
membranes in buffers containing enzyme inhibitors results in a stably
membrane-associated protein that can only be released from membranes by
detergent (15, 16). We analyzed the subcellular localization of
phosphorylated GAD65. 32P in vivo labeled
BHK-77-3 cells were homogenized, and a postnuclear supernatant
separated into cytosolic and crude membrane fractions. The membranes
were subjected to a high salt wash. The cytosol, washed membrane
fraction, and high salt membrane wash were subjected to Triton X-114
phase separation to analyze the effect of phosphorylation on the
hydrophobicity of the protein. GAD65 in the different fractions was
analyzed by immunoprecipitation followed by immunoblotting and
autoradiography of blots (Fig. 6, panels a
and d). As shown previously for the protein in pancreatic
-cells and transiently transfected COS-7 cells (15, 16, 18), GAD65
is found in the cytosol, membrane wash, and washed particulate fraction
of BHK-77-3 cells (Fig. 6, panel d). Similar amounts of
total GAD65 were detected in washed membrane and cytosolic fractions
(Fig. 6, panel d, compare lanes 1 and
2 with lanes 5 and 6). In contrast, phosphorylated GAD65 was detected either exclusively (results not
shown) or predominantly (Fig. 6, panel a) in the washed
membrane fraction. We speculated that cytosolic phosphatases might
rapidly remove the phosphate from the cytosolic form during
homogenization of cells and nuclear spin of lysates. The presence of
the phosphatase inhibitors EDTA and/or sodium fluoride throughout the
subcellular fractionation procedure, rather than only after the
postnuclear spin, did not, however, significantly enhance the detection
of phosphorylated GAD65 in the cytosol, which varied between 0 and 20%. Thus the majority of phosphorylated GAD65 remained firmly membrane anchored following subcellular fractionation and a high salt
wash of membranes (Fig. 6, panels a and d).
Similar data were obtained for the in vitro labeled protein
(Fig. 6, panels b and e). Phosphorylation did not
seem to affect the distribution of GAD65 into Triton X-114 detergent
and aqueous phases indicating that phosphorylation does not
significantly alter the hydrophobicity of GAD65 (Fig. 6, compare
upper and lower panels).
In another set of experiments, membranes and cytosol from unlabeled BHK-77-3 cells were separated before in vitro labeling with [32P]ATP (Fig. 6, panel d). In these experiments equal amounts of GAD65 were present in membranes and cytosolic fractions. 32P labeling was only detected in GAD65 in the membrane fraction demonstrating that GAD65 is phosphorylated by a membrane bound kinase. The subcellular fractionation data show that phosphorylation of GAD65 takes place exclusively in a membrane compartment(s), and that stably phosphorylated protein remains membrane associated.
To address the question whether phosphorylation affects the subcellular
localization of GAD65, we analyzed the subcellular distribution of
wild-type hGAD65 and the hGAD65 S3A/S6A/S10A/S13A mutant, transiently
expressed in COS-7 cells, BHK-21 cells, PC12 cells, and in an
insulinoma cell line TC3 (27). The tetraserine mutant was not
phosphorylated in these cell lines (Fig. 7, lane 4, and results not shown). No difference was detected in the
subcellular distribution, nor the hydrophobicity of the wild-type and
the nonphosphorylated mutant in any of these cell lines (Fig. 7,
lanes 5-16 and results not shown). Thus phosphorylation
does not seem to affect the membrane association of GAD65 in endocrine
or non-endocrine cell lines.
We have demonstrated that GAD65 expressed in a variety of cell lines is phosphorylated both in vitro and in vivo. Phosphorylation is mediated by a membrane bound kinase, and the phosphorylated protein seems to remain largely membrane associated. This study provides the first evidence that the membrane bound form of GAD65 is phosphorylated. Phosphorylation as well as palmitoylation thus distinguish the membrane bound and soluble forms of the enzyme.
The identity of the phosphorylated protein as GAD65 was established by
several criteria. First, the 32P-labeled protein was
immunoprecipitated with a GAD65 specific antibody (GAD6), but not with
control antibodies. Second, the phosphorylated protein was recognized
by a second GAD antibody (1701) on Western blots of immunoprecipitates.
Third, the phosphorylated protein was absent in parent cell lines
negative for GAD65, and finally, two-dimensional gel electrophoretic
analysis of the phosphorylated protein revealed the well characterized
charge and size coordinates of GAD65 from pancreatic -cells and
neurons (8, 15, 23, 24). Phosphorylated GAD65 co-migrated with the
component of the GAD65
/
doublet described earlier (15, 16, 23)
and with a weaker more slowly migrating band on SDS-PAGE,
, but was
not detected in GAD65
. Phosphorylation is the first modification shown to be unique to GAD65
.
GAD65 is polyphosphorylated on the NH2-terminal serine residues 3, 6, 10, and 13. Interestingly, exon 1 (aa 1-25), which encodes the phosphorylated region of GAD65, as well as exons 2 (aa 26-40) and 3 (aa 46-95), share no homology with the analogous regions of the larger isoform of glutamate decarboxylase, GAD67, although the two proteins are 78% identical in the remaining sequences. Thus it seems likely that the phosphorylation of NH2-terminal serines in GAD65 shown here is unique for this isoform.
Two-dimensional NEPHGE/SDS-PAGE analysis of intact GAD65 confirmed the
presence of multiple phosphorylated species, and allowed their further
characterization. The two-dimensional gel electrophoretic analyses of
GAD65 also revealed a heterogeneity in the SDS-PAGE mobility of the two
most acidic phosphorylated species, corresponding to the band
detected by one-dimensional SDS-PAGE. Since alanine substitutions of
individual serine residues in the NH2-terminal region of
GAD65 affected the SDS-PAGE mobility of the protein to a different
degree, we propose that the 33P-labeled spots detected in
and
represent phosphorylation of different combinations of
serine residues 3, 6, 10, and 13.
The nature of differences between the and
forms of GAD65 is
unknown. Both
and
are palmitoylated and analyses of
hydrophobicity and subcellular localization of GAD65 have not revealed
differences in those parameters between
and
(16). SDS-PAGE
analysis of the protein under nonreducing conditions reveals only a
single band with the mobility of GAD65
. Addition of increasing
concentrations of
-mercaptoethanol is accompanied by the appearance
of the
-band, suggesting that the oxidation and/or folding state of
the two forms may differ.2 The two-dimensional gel
electrophoretic analyses show that phosphorylation is not the cause of
the
/
heterogeneity, since both exist in a nonphosphorylated
form. Rather, phosphorylation seems to be an exclusive property of
GAD65
. GAD65
is not phosphorylated and yet consistently displays
an identical acidic charge heterogeneity on two-dimensional gels as
. Based on those results, we propose that
and
constitute the
two subunits of the native non-disulfide linked GAD65 dimer, and that
the protein remains as a dimer in the conditions of the NEPHGE analyses
of the first dimension, but then falls apart in the strongly
dissociating conditions of the second dimensional SDS-PAGE. Thus
studies of native GAD65 would analyze the
/
dimer as one entity
and not discern potential differences in the chemical and physical
parameters of the two subunits. This model is consistent with the
inability of earlier studies to detect differences in Triton X-114
partition patterns of
and
, in spite of their distinct
phosphorylation, and the consequent anticipated increase in the
hydrophilicity of
(15, 16).
The charge shift separation of phosphorylated and non-phosphorylated spots by two-dimensional NEPHGE/SDS-PAGE allow us to roughly estimate the stoichiometry of GAD65 phosphorylation. Based on several two-dimensional experiments, we estimate that 20-40% of total GAD65 in BHK-77-3 cells is phosphorylated, corresponding to 30-60% of the membrane anchored protein.
A common biological function of protein phosphorylation is regulation of enzymatic activity. Bao et al. (41, 42) reported that phosphorylation of a soluble, nonidentified GAD isoform from brain by cAMP-dependent kinase (PKA) caused a decrease in enzyme activity, which could be reversed by treatment of the protein with calcineurin. We determined the Vmax and Km for wild-type GAD65 as well as a series of phosphorylation mutants in cell free lysates generated from transfected COS-7 cells. In our studies, a decrease or loss in phosphorylation observed for the serine substitution mutants did not result in a detectable effect on the Vmax and Km for glutamate or PLP. Furthermore, several lines of evidence suggest that the kinase involved in phosphorylation of GAD65 is not PKA. First, the phosphorylation site we have identified does not contain a PKA consensus sequence; second, the addition of cAMP or staurosporin (a cAMP-dependent kinase inhibitor) to our in vitro labeling reactions did not affect the degree of phosphorylation of GAD65; third, we were unable to detect phosphorylation of purified GAD65 in vitro by PKA. In contrast GAD67 can be phosphorylated by PKA in similar conditions.3 We therefore suggest that the phosphorylation reported by Bao et al. (41, 42) may be a characteristic of the cytosolic isoform, GAD67.
Phosphorylation can play a role in the regulation of membrane association of proteins. Addition of phosphate groups can decrease the membrane avidity of proteins by charge repulsion from membrane phospholipids. Hence phosphorylation of a membrane protein can facilitate its translocation to the cytosol (Ref. 43 and references therein). Conversely, phosphorylation of a cytosolic protein can block its membrane association and retain it in the cytosol (44). GAD65 is an amphiphilic molecule which is isolated in a hydrophilic soluble form, a hydrophobic soluble form, and a hydrophobic firmly membrane anchored form (15, 16). The membrane anchoring of GAD65 is reversible (16). The phosphorylated NH2-terminal peptide is proximal to the region of GAD65 that undergoes a set of hydrophobic modifications and is implicated in the membrane anchoring of the protein (16, 18, 45).
The mechanism of membrane anchoring of GAD65 is not known. Palmitoylation of cysteines 30 and 45 is an exclusive property of membrane anchored GAD65 and was originally proposed to be involved in membrane anchoring (16). However, site-directed mutagenesis of the palmitoylated cysteines does not affect the distribution of the protein between cytosol and membrane compartments in COS-7 cells (18). Palmitoylation is therefore not critical for membrane anchoring, but may assist in targeting GAD65 to the right membrane compartment, and/or play a role in protein-protein interaction in synaptic vesicle membranes. Deletion of amino acids 1-23 in GAD65 and concomitant mutation of the palmitoylated cysteines also does not affect the distribution of the protein between soluble and membrane compartments in COS-7 cells. However, removal of the next 8 amino acids (aa 24-31) abolishes membrane anchoring (18). Studies of GAD65/GAD67 chimeras suggest that a substitution of the first 29 amino acids of GAD67 with the first 27 amino acids of GAD65 is sufficient to target the normally cytosolic GAD67 isoform to the perinuclear membrane compartment in CHO cells (45). Thus amino acids 24-27 seem to be critical for membrane anchoring of GAD65. Because of the proximity of the phosphorylated serine residues to the region implicated in membrane anchoring of GAD65, we speculated that phosphorylation might be a mechanism by which GAD65 is released from membranes. To address this possibility, subcellular fractionation experiments were performed on in vivo or in vitro phosphorylated GAD65. These experiments revealed that phosphorylated GAD65 is predominantly localized in membrane compartments, despite the fact that 40-50% of total GAD65 is recovered in the cytosol. A prominent fraction of membrane associated GAD65 (30-60%) was phosphorylated in our experiments. Yet high salt wash of membranes in the presence of phosphatase inhibitors did not release the phosphorylated protein, strongly suggesting that phosphorylation is either not involved in dissociation of GAD65 from membranes, or if required, it is not sufficient for release.
Phosphorylation could play a role in membrane association by mediating
association of soluble GAD65 with a membrane protein, thus targeting it
to a specific membrane compartment where it can undergo lipid
modification(s) by a membrane associated enzyme and become firmly
membrane anchored. Two lines of evidence argue against this
possibility. First, the hGAD65 S3A/S6A/S10A/S13A in which
phosphorylation is almost completely abolished, is membrane anchored
similar to wild-type GAD65 in transiently transfected COS-7 cells,
TC3 cells, BHK-21 cells, and PC12 cells, and similar results have
been obtained for the rGAD65
1-15 phosphorylation mutant, and the
deletion and substitution mutants, which show a significant decrease in
phosphorylation in COS-7 cells (Refs. 18 and 25, and this study)
demonstrating that phosphorylation of GAD65 is not required for
membrane anchoring in those cells. Second, in vitro
phosphorylation experiments using separated membrane and cytosolic
fractions of BHK-77-3 in the presence of phosphatase inhibitors, showed
that GAD65 was exclusively phosphorylated in the membrane fraction.
Thus phosphorylation is not involved in anchoring cytosolic GAD65 to
membranes, but is an independent modification caused by a membrane
associated kinase, and restricted to the fraction of the enzyme, which
is already membrane anchored.
In summary we have demonstrated that the membrane bound form of GAD65
is phosphorylated on multiple serine residues in the NH2-terminal domain and that this modification is
restricted to GAD65. The biological function of this modification
remains to be established. Phosphorylation of GAD65 does not appear to
alter its kinetic behavior with respect to the substrate glutamate, or
the co-enzyme PLP, nor does it appear to be involved in the membrane
anchoring of the protein in transiently transfected cells.
By necessity, we have studied the effect of phosphorylation of GAD65 on
membrane anchoring and enzyme activity in recombinant cell lines. The
role of phosphorylation of the enzyme in its natural environment,
neurons and primary pancreatic -cells, may be distinct and perhaps
become accessible in the future by genetic manipulations in the
mouse.
We speculate that phosphorylation of membrane anchored GAD65 is involved in regulating or facilitating a specific function of the enzyme in synaptic vesicle membranes. One possibility is that phosphorylation mediates interaction of GAD65 with regulatory proteins in the synaptic vesicle membrane or facilitates transport of the product GABA to the lumen of vesicles by docking the enzyme to a hypothetical GABA transporter.