(Received for publication, November 17, 1994; and in revised form, January 19, 1995 )
From the
Previously we showed that the activity of the -aminobutyric
acid-synthesizing enzyme L-glutamate decarboxylase (GAD) in
crude brain extract is inhibited by ATP and protein phosphatase
inhibitors. We suggested that GAD activity is regulated by protein
phosphorylation. In this paper we further present evidence to support
our hypothesis that protein kinase A and calcineurin may be involved in
regulation of GAD activity through phosphorylation and
dephosphorylation of GAD, respectively. In addition, the effect of
neuronal stimulation on GAD activity in cultured neurons is also
included. A model to link neuronal excitation and activation of GAD by
Ca
-dependent phosphatase is proposed.
-Aminobutyric acid (GABA) (
)has been established
as the major inhibitory neurotransmitter in the vertebrate central
nervous system(1, 2) . The rate-limiting step in GABA
biosynthesis is the decarboxylation of L-glutamate by L-glutamate decarboxylase (EC 4.1.1.15; GAD)(3) . The
level of GABA in the brain is regulated by GAD(4) . Many
neurological disorders, such as seizures, Huntington's chorea,
Parkinson's disease, stiff-man syndrome, and schizophrenia, have
been shown to be related to alterations of the GAD level in the
brain(5, 6, 7, 8, 9, 10) .
Despite its importance, the precise mechanism underlying the regulation
of GAD activity is still not clear.
Reversible phosphorylation is a common mechanism by which the functions of many proteins are regulated in the central nervous system (for review, see (11) ). Regulation of neurotransmitter synthesis by phosphorylation of their synthesizing enzymes has been shown in other neurotransmitter systems(12, 13, 14, 15, 16) . Previously we showed that GAD activity is inhibited by conditions favoring protein phosphorylation, and the inhibition is reversed by phosphatase treatment(17) . This observation suggests that GAD activity is regulated by reversible phosphorylation.
To study
further the mechanism of the regulation of GAD activity by protein
phosphorylation, we conducted experiments to determine the extent of
GAD phosphorylation and GAD activity in crude as well as purified
preparations using various protein kinases, phosphatase, and their
specific inhibitors. In addition, the effect of neuronal stimulation on
GAD activity in cultured neurons is also included. Based on the results
presented in this paper and those reported in the literature, a model
is proposed to link neuronal excitation to activation of GAD through
Ca-dependent dephosphorylation.
For GAD assay, the synaptosomes were
prepared, lysed, and incubated under various conditions as described
above except that no [P]ATP was included. After
centrifugation at 100,000
g, pyridoxal 5`-phosphate
was added to the supernatants to give a final concentration of 0.2
mM. Aliquots of the supernatants were used for GAD assays and
protein determinations as described previously(17) . No ATP and
cAMP were included in the control condition.
For the GAD assay, the procedure was the same as described above
except that no [P]ATP was included, and GAD was
not eluted from the resin. Instead, the resin was suspended in 50
mM potassium phosphate, pH 7.2, containing a mixture of
phosphatase inhibitors, 1 mM AET, and 0.2 mM pyridoxal 5`-phosphate. The suspension was used for the GAD assay
as described previously(17) . Neither kinases nor phosphatases
were added in the control condition.
Figure 1:
Effect of protein
kinase inhibitors, phosphatase inhibitors, and Ca on
P incorporation into GAD in the synaptosomes. Lanes
A-F are autoradiograms of GAD from synaptosomal fractions
under various conditions. Phosphorylation was performed in the lysed
synaptosomal preparations under the following conditions: lane
A, buffer only; lane B, +PKA inhibitor, PKI; lane C, +PKC inhibitor; lane D,
+Ca
; lane E, +Ca
plus 30 nM okadaic acid or lane F,
Ca
plus 10 µM okadaic acid. At the end
of incubation, GAD was isolated from synaptosomal preparations,
purified by an anti-GAD immunoaffinity column, and identified by
SDS-PAGE autoradiography as detailed under ``Experimental
Procedures.'' Arrows indicate both subunits of GAD, 59
kDa and 64 kDa, are phosphorylated. The molecular mass is expressed in
kDa.
It was found that the
purified GAD was phosphorylated by PKA ( Fig. 2lane A)
but not PKC (Fig. 2, lane D). Phosphorylated GAD could
be dephosphorylated by treatment with calcineurin (Fig. 2, lane B). Similarly, phosphorylated GAD could be
dephosphorylated by calf intestinal alkaline phosphatase, but to a
lesser degree (Fig. 2, lane C). It was estimated that
there is 0.47 P/subunit of GAD protein from PKA-mediated
phosphorylation.
Figure 2:
Demonstration of P
incorporation in purified GAD preparation by PKA but not PKC and
subsequent dephosphorylation by calcineurin. Purified GAD in the form
of a GAD
anti-GAD resin complex was treated under various
conditions. Elution of GAD from the anti-GAD immunoaffinity column and
detection of phosphorylated GAD by SDS-PAGE were detailed under
``Experimental Procedures.'' In addition, about 0.5 µg of
GAD purified by an anti-GAD immunoaffinity column was also analyzed on
the same SDS-PAGE and visualized by silver staining as described
previously (17) . Lane A, immunoaffinity-purified GAD
treated with the catalytic subunit of PKA in the presence of
[
-
P]ATP, cAMP and Mg
-ATP. Lane B,
P-labeled GAD in lane A treated
with calcineurin in the presence of Ca
/calmodulin. Lane C,
P-labeled GAD in lane A treated
with alkaline phosphatase. Lane D, immunoaffinity-purified GAD
treated with PKC in the presence of
[
-
P]ATP, Mg
-ATP, and PKC
activators. Lane E, purified GAD showing both 59- and 64-kDa
subunits.
In our previous report we showed that GAD activity in crude
brain preparations is inhibited by ATP or phosphatase
inhibitors(17) . Furthermore, direct phosphorylation of GAD has
also been demonstrated in lysed synaptosomal preparations in the
presence of [-
P]ATP but not
[
-
P]ATP, suggesting that inhibition of GAD
activity by ATP is through phosphorylation of the GAD molecule and not
by competing with pyridoxal 5`-phosphate for the binding to GAD
apoprotein as proposed(21, 22) . In this paper, we
have presented evidence to suggest that PKA and calcineurin might be
the protein kinase and protein phosphatase responsible for
phosphorylation and dephosphorylation of GAD, respectively. Moreover,
the activity of GAD appears to be inversely correlated with the extent
of GAD phosphorylation; namely, GAD is inhibited when it is
phosphorylated and is activated when it is dephosphorylated. Several
lines of evidence support our hypothesis that PKA is involved in GAD
phosphorylation and regulation of GAD activity. First, phosphorylation
of GAD could be demonstrated in both crude as well as purified GAD
preparations by catalytic subunit of PKA. Second, GAD phosphorylation
by PKA is completely blocked by PKI, a specific inhibitor of PKA.
Third, GAD activity is reduced by PKA-mediated phosphorylation, and
this inhibition can be blocked by PKI.
It seemed unlikely that
Ca-dependent kinases, such as PKC and calcium
calmodulin-dependent kinase, might play a significant role in GAD
phosphorylation and regulation of GAD activity. This notion is based on
the following observations. First, the conditions used for
phosphorylation contained 4 mM EGTA, and hence it is unlikely
that Ca
-dependent enzymes, e.g. PKC or
calmodulin-dependent kinase, are still active under such conditions.
However, it must be pointed out that in the presence of activator, e.g. phorbol ester, PKC activity can be increased greatly even
in the presence of a very low concentration of Ca
(23) . Also, it is known that calmodulin-dependent kinase
II can undergo pseudo-irreversible activation by autophosphorylation,
which renders calmodulin-dependent kinase II independent of
Ca
and calmodulin(24) . Hence it is
conceivable that even in the presence of EGTA, PKC and
calmodulin-dependent kinase II could be still active and participate in
GAD phosphorylation. Second, PKC fails to catalyze incorporation of
P into GAD protein either in a crude synaptosomal
preparation or in a highly purified GAD preparation. Furthermore, PKC
or PKC inhibitory peptide has little effect on GAD activity. Third, it
has been shown from immunocytochemical and in situ hybridization studies that calmodulin-dependent kinase II and GAD
protein or GAD mRNA have different regional and cellular distributions
in the brain(25, 26) , suggesting that
calmodulin-dependent kinase II is unlikely to be localized in
GABA-ergic neurons and thus involved with phosphorylation and
regulation of GAD activity.
There are four major types of
serine/threonine protein phosphatases: type 1 (protein phosphatase-1)
and type 2, which includes three subtypes A, B, and C (protein
phosphatases 2A, 2B, and 2C)(27) . These protein phosphatases
can be distinguished by their sensitivity to a protein phosphatase
inhibitor, okadaic acid(20, 28) . Okadaic acid is a
potent inhibitor for protein phosphatases 1 (IC =
20 nM) and 2A (IC
= 0.2 nM).
Protein phosphatase 2B is inhibited by okadaic acid at a higher
concentration (IC
= 5 µM), whereas 2C
is unaffected by okadaic acid at a concentration as high as 10
µM(20, 28) . Since dephosphorylation of
GAD is inhibited by okadaic acid at 10 µM but not at 30
nM (Fig. 1, lanes E and F), it is
likely that protein phosphatase 2B (calcineurin) is the phosphatase
responsible for GAD dephosphorylation. This is consistent with our
previous observation that EGTA or a calcineurin-specific inhibitor,
deltamethrin, prevented GAD activation in the crude
extract(17) . In addition, it has also been shown that
activation of GAD is Ca
-facilitated(22) . The
fact that calcineurin can dephosphorylate and activate purified GAD in vitro further supports the above notion. Since calcineurin
is the major Ca
-dependent phosphatase in the brain (29) and has been suggested to be localized in GABA-ergic
neurons(30) , it seems reasonable to suggest that calcineurin
plays an important role in the GABA synthesis through its activation of
GAD activity by dephosphorylation.
In addition to in vitro studies, evidence obtained from in vivo studies as
reported in this paper showing that GAD activity in cultured neurons is
enhanced by high K stimulation in the presence of
Ca
, but not in Ca
-free media, is
also compatible with the above notion. All of the observations
discussed above are consistent with our working hypothesis that when
GABA-ergic neurons are stimulated, Ca
ions enter the
nerve terminals through voltage-dependent Ca
channels, followed by the release of GABA. The influx of
Ca
then triggers activation of
Ca
-dependent phosphatase (one of the candidates is
calcineurin) which in turn activates GAD by dephosphorylation,
resulting in an increase in GABA synthesis to replenish GABA depletion
due to the release.
In summary, phosphorylation by PKA inhibits GAD activity, and dephosphorylation by calcineurin activates it. However, more investigations are required to identify definitely the endogenous kinase and phosphatase responsible for GAD regulation in the brain.