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INTRODUCTION |
Nitric oxide, derived from neuronal nitric-oxide synthase
(nNOS),1 has important
physiological functions in the nervous system (1, 2). In the peripheral
nervous system, NO mediates certain actions of autonomic motor neurons
on smooth muscle and thereby NO regulates intestinal peristalsis and
penile erection. In the central nervous system, NO does not primarily
act on smooth muscle but instead modulates synaptic transmission
associated with N-methyl-D-aspartate (NMDA) type
glutamate receptors. By regulating synaptic plasticity, NO can
influence hippocampal long term potentiation (3) as well as aspects of
learning (4) and memory (5).
Although small amounts of NO mediate physiological signaling, excess NO
production can cause tissue injury (6). The primary stimulus for NO
synthesis in central neurons is activation of NMDA receptors (2).
Overactivity of NMDA receptors is implicated in numerous
neurodegenerative processes including stroke, Huntington's chorea, and
amyotrophic lateral sclerosis (7). A role for NO in NMDA-mediated
degeneration was first suggested by experiments showing neuroprotection
by NO synthase antagonists (8). Definitive evidence that nNOS mediates
brain injury derives from studies of nNOS knockout mice, which are
strikingly resistant to excitotoxicity following focal cerebral
ischemia (9).
NO can mediate neurotoxicity by inhibiting metabolic pathways and
causing cellular energy depletion, which is a hallmark of ischemic
injury-induced neuronal death. NO erodes energy stores by reacting with
certain metabolic enzymes that contain heme-iron prosthetic groups,
iron-sulfur clusters, or reactive thiols (10). Through these reactions,
NO can inhibit glycolysis by reacting with cis-aconitase and
can attenuate oxidative phosphorylation by inhibiting mitochondrial
iron-sulfur enzymes and by competing with oxygen at cytochrome oxidase
(11, 12). Interestingly, nNOS-containing neurons are themselves
relatively resistant to excitotoxic injury, suggesting that nNOS
neurons have specific mechanisms that protect them from energy
depletion associated with NO toxicity (13).
Because NO mediates critical physiological functions but can also be
toxic, nNOS activity must be tightly regulated. Selective regulation of
calmodulin-dependent nNOS by specific calcium pools is
mediated by the targeting of nNOS to subcellular sites (14). In brain,
nNOS is targeted to the synaptic membranes through interactions with
postsynaptic density-95 (PSD-95) and PSD-93 (15, 16). PSD-95 and PSD-93
are major proteins of the postsynaptic density and bind to nNOS via PDZ
(PSD-95, discs-large, ZO-1) domains, consensus sequences of
approximately 100 amino acids found in some cytoskeletal proteins and
enzymes that mediate protein-protein interactions (17). The association
of nNOS and PSD-95 is mediated by a direct PDZ-PDZ interaction (15).
PDZ domains from nNOS and PSD-95 can also interact with specific
sequences at the C termini of target proteins (18-21). PSD-95 and
PSD-93 also synaptically cluster NMDA receptors, which are
glutamate-activated calcium channels (22, 23). This macromolecular
signaling complex is poised to regulate the synaptic activity of nNOS
(14).
As nNOS occurs at both synaptic and nonsynaptic sites, we wondered
whether other protein complexes might also bind the PDZ domain of nNOS.
To address this, we performed affinity chromatography using a fusion
protein column containing the PDZ domain of nNOS. Purification of brain
lysates on this column yielded a protein band of approximately 80 kDa
that was identified as an isoform of phosphofructokinase (PFK-M).
Endogenous PFK-M is indeed associated with nNOS, and PFK-M and nNOS are
both concentrated in a synaptic vesicle fraction from brain
homogenates. At the cellular level, PFK-M is selectively enriched
in cortical neurons that express nNOS. These data identify PFK as a
major nNOS-interacting protein in brain cytosol. As PFK catalyzes the
rate-limiting step in glycolysis and the product of this reaction,
fructose-1,6-bisphosphate, is neuroprotective (24-26), PFK is a
candidate enzyme for mediating neuroprotection of nNOS neurons.
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EXPERIMENTAL PROCEDURES |
Materials--
Peptides were from Research Genetics (Birmingham,
AL). Glutathione-Sepharose and pGEX4T-1 vector were from Amersham
Pharmacia Biotech. Affi-Gel 10 beads were from Bio-Rad. Horseradish
peroxidase-coupled donkey anti-guinea pig antibody was from Sigma. ECL
reagents were from Amersham Pharmacia Biotech. Mouse antibody raised
against nNOS was from Transduction Laboratories (Lexington, KY). Guinea pig antibody raised against rabbit PFK-M was generously provided by Dr.
Robert G. Kemp, University of Chicago. All other reagents were from Sigma.
Expression and Purification of GST and GST-nNOS1-195
Proteins--
A glutathione S-transferase fusion protein
encoding the first 195 amino acids of nNOS was constructed by
polymerase chain reaction and was expressed and purified from
Escherichia coli as described previously (16). For protein
affinity chromatography studies, GST fusion proteins were not eluted
from the beads.
Protein Affinity Chromatography--
Purified GST fusion
proteins were dialyzed against PBS at 4 °C for 16 h. Five mg of
protein was cross-linked to 1 ml of Affi-Gel 10 by incubating in PBS + 50 mM HEPES, pH 7.4, at 4 °C for 16 h on a shaking
platform. Affinity chromatography was performed as described previously
(27) with modifications. GST- and GST-nNOS-(nNOS) Affi-Gel beads were
incubated in 0.1 M Tris-HCl, pH 8.0, 350 mM NaCl on ice for 1 h. The affinity resin was then washed
consecutively with Buffer C (50 mM HEPES, pH 7.6, 125 mM NaCl, 20% glycerol) containing 1 mM DTT,
Buffer C containing 1 mM DTT and 2.5 M urea, Buffer C containing 1 mM DTT and 4 M urea, and
Buffer D (20 mM HEPES, 125 mM NaCl, 10%
glycerol, 1 mM DTT, PMSF, 1 mM EDTA, 1 mM EGTA). These washes precleared the columns of any
unlinked GST fusion protein that might leach off of the Affi-Gel beads during incubation with brain extract. Bovine serum albumin-Affi-Gel 10 beads were constructed in a similar manner and were used to clear the
extract prior to its application to the GST- and nNOS-Affi-Gel 10 to
reduce the background binding of nonspecific proteins.
Twenty-five adult rat brains were homogenized in ice-cold Buffer D,
Triton X-100 (TX) was added to 1%, and proteins were extracted for
1 h at 4 °C. The homogenate was spun at 12,000 × g for 10 min, and the supernatant was precleared by
incubating with 2 ml of bovine serum albumin-Affi-Gel 10 beads that
were pre-washed with Buffer D at 4 °C for 2 h. The bovine serum
albumin beads were removed, and 100 ml of extract was incubated with
0.5 ml of nNOS-Affi-Gel 10 at 4 °C for 3 h. As controls, 10 ml
of extract was incubated with 50 µl of GST-Affi-Gel 10, and 10 ml of
Buffer D containing 1% TX was incubated with 50 µl of nNOS-Affi-Gel
10. All incubations were done in batch. Extract/beads were then loaded into the appropriate size columns and were washed with 200 ml of Buffer
D containing 0.5% Nonidet P-40, 1% TX, and 1 mM PMSF, and
100 ml Buffer D containing 1% TX (per 2 ml of beads). Proteins were
eluted consecutively with 2.5 ml of Buffer D containing 1% TX, 2.5 M urea twice and 2.5 ml Buffer D containing 1% TX and 4 M urea, and finally 2.5 ml Buffer D containing 1% SDS.
Each fraction was precipitated with 20% trichloroacetic acid, washed twice with 1:1 ether ethanol, and resuspended in 50 µl 100 mM Tris 8.8, 1% SDS. Proteins were resolved by 8%
SDS-polyacrylamide gel electrophoresis and visualized using silver.
Western Transfer and Protein Sequencing--
The 2.5 M urea and 4 M urea fractions were combined,
and proteins were separated on an 8% polyacrylamide gel and
transferred to polyvinylidene difluoride membrane (Immobilon P,
Millipore). The membrane was stained with Coomassie to visualize bands,
the desired protein bands were excised, and protein sequencing was performed by ProSeq (Salem, MA). Proteins were cleaved with CNBr and
were then coupled with O-phthalaldehyde as described
previously (28). O-Phthalaldehyde treatment blocks all
peptides that do not contain an N-terminal proline. Proteins were then
sequenced by Edman degradation. The single peptide sequence was used to search the National Center for Biotechnology Information using the
BLAST programs.
GST Fusion Protein Binding--
One adult rat brain or 2.2 g or skeletal muscle was homogenized in 5 or 7.5 ml of TEE (25 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF), respectively. TX was added
to a final concentration of 1%, and the homogenates were incubated
on a rocking platform at 4 °C for 1 h. The extract was spun at
12,000 × g for 15 min, and the resulting brain
supernatant was diluted 1:10 in TEE. 1 ml of brain or skeletal muscle
extract was incubated with 50 µl of glutathione-epharose bound to 50 µg of GST or GST-nNOS1-195 at 4 °C for 1 h. Extract/beads
were loaded into the appropriate sized column and were washed three
times with 20 ml TEE containing 0.2% TX. Bound proteins were eluted
with 200 µl of 0.5% SDS and 100 mM NaCl, resolved on
10% polyacrylamide gels, and transferred to polyvinylidene difluoride
membrane as described above. Blots were probed using a guinea pig
antibody raised against rabbit PFK-M and visualized using a secondary
antibody coupled to horseradish peroxidase and ECL reagents.
Immunodepletion--
Skeletal muscle extract, prepared as
described above, was precleared with 50 µl of protein A-Sepharose/ml
at 4 °C for 1 h. The protein A-Sepharose was removed, and the
extract was diluted 1:10 in TEE containing 1% TX and 1 mM
PMSF. 5 µl of guinea pig anti-PFK-M or guinea pig preimmune serum was
added to 1 ml of the diluted extract, and the extract was incubated at
4 °C for 1 h. Protein A-Sepharose (50 µl/ml extract) was
added, and the extract was incubated at 4 °C for 1 h. The
protein A-antibody complex was pelleted, and the resulting supernatant
was separated on a 10% SDS-polyacrylamide gel. Protein was transferred
to polyvinylidene difluoride as described above, and the blot was
probed with a monoclonal antibody raised against nNOS.
Synaptosomal Fractionation--
Synaptosomes were prepared as
described by Li et al. (29) with modification. Four rat
cortices were homogenized in 36 ml of homogenization buffer (320 mM sucrose, 4 mM HEPES, pH 7.4, 1 mM EGTA, 1 mM PMSF) using 10 strokes at 900 rpm
of a loose fitting glass-Teflon homogenizer (Kontes; size 22). The
homogenate was centrifuged at 1000 × g for 10 min. The
supernatant (S1) was collected and centrifuged at 12,000 × g for 15 min, and the pellet (P2) was resuspended in 24 ml
of homogenization buffer and centrifuged at 13,000 × g
for 15 min. The resulting pellet (P2'), representing a crude
synaptosomal fraction, was lysed by osmotic shock and homogenized by
three strokes of the glass-Teflon homogenizer at 2000 rpm, and the
homogenate was spun at 33,000 × g for 20 min to yield
supernatant (LS1) and pellet (LP1, heavy membranes). LS1 was spun at
251,000 × gmax for 2 h. The resulting
supernatant (LS2) contained soluble proteins, and the pellet (LP2)
contained synaptic vesicle proteins. Proteins were resolved on a 10%
SDS-polyacrylamide gel, and Western blotting was performed as described above.
Immunohistochemistry--
Adult rats were perfused with 4%
paraformaldehyde, and brains were harvested, postfixed at 4 °C for
3 h, and cryoprotected in 20% sucrose overnight. Brains were
sectioned sagitally at 20 µm with a cryostat, and sections were
blocked for 1 h in PBS containing 0.1% Triton X-100 and 2%
normal goat serum and then incubated in PBS containing 0.1% Triton
X-100 containing anti-PFK-M (guinea pig, 1:1000) and anti-nNOS (rabbit,
1:500) for 1 h at room temperature. Immunoreactivity was
visualized using Cy3-conjugated anti-guinea pig and Cy2-conjugated
anti-rabbit antibodies. Nuclei of all cells in the section were stained
with 4',6'-diamidino-2-phenylindole. Sections were mounted on slides
(Frost Plus slides; Fisher), and photographs were taken under
fluorescence microscopy at 200×. Control experiments lacking
anti-PFK-M or anti-nNOS antibodies yielded no staining.
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RESULTS |
Isolation of an 80-kDa Protein That Binds to the PDZ Domain of
nNOS--
To identify proteins that bind to the PDZ domain of nNOS in
brain, we covalently coupled a nNOS PDZ domain GST fusion protein to
Affi-Gel 10 (nNOS-Affi-Gel) and incubated this column with brain
extract (nNOS-extract in Fig. 1). The
column was washed with the buffer used to solubilize the brain extract,
and proteins tightly bound to the column were eluted off sequentially
with 2.5 and 4 M urea in buffer. Eluted proteins were
compared with proteins that eluted from two control columns: first, a
column of GST coupled to Affi-Gel 10 (GST-Affi-Gel) that was incubated with brain extract (GST-extract) to identify nonspecific binding proteins, and second, a column of the PDZ domain protein coupled to
Affi-Gel 10 incubated with buffer (nNOS-buffer) to identify nNOS fusion
protein that leached off of the column. As seen in Fig. 1, proteins of
45, 53, 60, and 65 kDa leached from the nNOS-Affi-Gel buffer control
column (lanes 1 and 4). Additionally, a number of
nonspecific proteins (31, 33, 35, 39, and 50 kDa) eluted from both the
nNOS- and GST-Affi-Gel columns (lanes 2, 3,
5, and 6). These proteins either bind to GST or
nonspecifically bind to Affi-Gel beads. The most abundant protein
eluted specifically from the nNOS-column is a protein doublet at
approximately 80 kDa (lanes 3 and 6). This
doublet does not bind to a control GST column and is not a product of
proteolysis of the linked fusion protein. This doublet as present in
both the 2.5 and 4 M urea eluates (lanes 3 and
6). Fractions containing this doublet were combined, the more abundant upper protein was isolated, and a single peptide was
sequenced by Edman degradation as described under "Experimental Procedures." The sequence obtained (Fig.
2) showed high homology to the previously
sequenced rat muscle phosphofructokinase (PFK-M, Fig. 2).

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Fig. 1.
Affinity chromatography with the PDZ domain
of nNOS. The PDZ domain of nNOS fused to GST was coupled to
Affi-Gel 10 beads. Brain extract was incubated with nNOS-Affi-Gel 10 or
GST-Affi-Gel 10. Buffer incubated with nNOS-Affi-Gel 10 served as a
control. Proteins were eluted consecutively with 2.5 M urea
twice and 4 M urea. Proteins were resolved by 8%
SDS-polyacrylamide gel electrophoresis, and the gel was visualized by
silver staining. The arrow points to the 80-kDa doublet
specifically eluted from the nNOS beads but not from the nNOS beads
incubated in buffer or the GST control beads.
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Fig. 2.
Sequence identity of the muscle isoform of
rat PFK and the peptide obtained from the 80-kDa nNOS-binding
protein. Fractions eluted from the nNOS column were resolved on
8% SDS-polyacrylamide gel electrophoresis above and were blotted onto
polyvinylidene difluoride paper. The upper band of the 80-kDa doublet
was excised and digested with CNBr, and peptides lacking an N-terminal
proline were blocked with O-phthalaldehyde. Microsequencing
then yielded the single major sequence shown. X represents
ambiguous amino acids in the sequenced peptide.
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nNOS Binds PFK at Its Peptide-binding Site--
It has been
previously shown that the PDZ domain of nNOS preferentially binds to
peptides ending with the sequence G(D/E)AV (19, 30) or EIAV (20).
Interestingly, the final four amino acids of rat PFK-M are EAAV, a
sequence similar to that of CAPON, a brain-specific protein isolated as
a nNOS PDZ-binding partner (20), which ends with EIAV. To determine
whether PFK binds to the peptide-binding site in the nNOS PDZ domain,
we performed binding experiments with the PDZ domain of nNOS fused to
GST. Fig. 3 (top panel) shows
that PFK-M from both brain and skeletal muscle binds to the PDZ domain
of nNOS. Furthermore, this binding can be competed with the peptide
VSPDFGDAV, suggesting that PFK-M binding is at the peptide-binding site
in nNOS-PDZ. Neither of the peptides, KLSSIESDV, which binds to the
second PDZ domain of PSD-95, and KLSSIEADA, a control peptide that does
not bind to any PDZ domain, compete the binding of PFK-M to the PDZ
domain of nNOS (Fig. 3, middle panel). Experiments using the
final nine amino acids of PFK-M fused to GST show that nNOS can bind to
this C-terminal protein sequence (data not shown). In addition,
VSPDFGDAV also competes the binding of CAPON to nNOS PDZ (Fig. 3,
bottom panel). These results suggest that PFK-M binds to the
nNOS-PDZ peptide-binding site, as does CAPON.

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Fig. 3.
PFK-M binds competitively to the
peptide-binding site in the nNOS PDZ domain. Brain or skeletal
muscle extract was incubated with glutathione-Sepharose bound to GST or
GST-nNOS1-195 in the presence of increasing concentrations
(µM) of the peptide VSPDFGDAV, which binds to the nNOS
PDZ peptide-binding site, KLSSIESDV, which binds to the second PDZ
domain of PSD-95, or KLSSIEADA, which does not bind to PDZ domains.
Columns were washed and eluted with 0.5% SDS/100 mM NaCl.
Top, Western blotting of eluates demonstrates that PFK-M
binds specifically to nNOS PDZ and that binding is blocked by the
peptide VSPDFGDAV with an IC50 of ~5 µM.
Middle, Western blotting shows that neither the peptide
KLSSIESDV nor KLSSIEADA blocks PFK-M binding to nNOS PDZ.
LD, load. Bottom, Western blotting shows that
CAPON interaction with nNOS-PDZ beads is also specifically blocked by
the nNOS-binding peptide.
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Immunodepletion of nNOS by PFK-M--
To determine whether PFK-M
and nNOS interact in vivo, we immunoprecipitated PFK-M from
skeletal muscle extract. Skeletal muscle was used rather than brain
because PFK-M is the only PFK isoform expressed in muscle (31). As
shown in Fig. 4, immunoprecipitation of
PFK-M depleted nNOS from the extract, whereas incubation of the extract
with preimmune serum did not. These data suggest that PFK-M and nNOS
interact in vivo and that a large fraction of nNOS in
skeletal muscle cytosol is associated with PFK-M.

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Fig. 4.
nNOS is immunodepleted from cytosolic
skeletal muscle extract by immunoprecipitation of PFK-M. Guinea
pig anti-PFK-M or guinea pig preimmune serum was added to cytosolic
muscle extract, antibody complexes were collected with
protein-A-Sepharose, and the immunoprecipitated extracts were separated
on a 10% SDS-polyacrylamide gel. Western blotting for nNOS reveals an
nNOS doublet of approximately 140-160 kDa; bands below the doublet are
proteins that cross-react with the antibody. Note that the only protein
that is immunodepleted by the antibody against PFK-M is the nNOS
doublet.
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nNOS and PFK-M Are Both Concentrated in the Synaptic Vesicle
Fraction--
We also evaluated possible association of nNOS and PFK
in brain. In order for these two proteins to interact, they must be co-localized. To determine whether nNOS and PFK-M are co-localized in
brain, we performed subcellular fractionation (Fig.
5). In this preparation (29), cortical
homogenate is centrifuged at low speed yielding a crude nuclear
fraction (P1). The supernatant (S1) is centrifuged providing a P2
pellet and S1 supernatant. The P2 pellet is washed to yield P2', a
crude synaptosome fraction also containing mitochondria. After
hypotonic lysis of P2', a plasma membrane fraction (LP1) containing
pre- and postsynaptic membranes and the postsynaptic density was
obtained. The soluble fraction was spun to obtain synaptic vesicles
(LP2) and soluble cellular components (LS2). As seen in Fig. 5, nNOS is
found in both soluble and particulate fractions. nNOS is enriched in
synaptosomes (P2') and in the synaptic vesicle fraction (LP2), which is
enriched in synaptophysin, a synaptic vesicle marker. Similarly, PFK is found in both soluble and particulate fractions. PFK is present in
synaptosomes (P2'); however, unlike nNOS, PFK is found only in the
synaptic vesicle fraction (LP2) when P2' is fractionated. It is
important to note that PSD-95, a protein enriched in the postsynaptic
density, is concentrated in LP1 (Fig. 5). Because both nNOS and PFK-M
are enriched in the LP2 fraction, or synaptic vesicle fraction,
synaptic vesicles serve as a cellular site where the two proteins can
interact.

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Fig. 5.
nNOS and PFK-M are enriched in the synaptic
vesicle fraction of synaptosomes. Subcellular fractionation of
brain was performed as described under "Experimental Procedures."
nNOS is concentrated in synaptosomes (P2') and in the synaptic vesicle
fraction (LP2), which is enriched in synaptophysin, a synaptic vesicle
marker. PFK is also present in synaptosomes (P2') and in the synaptic
vesicle fraction (LP2). In contrast, PSD-95 is concentrated in the
synaptic membrane and PSD fraction (LP1).
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PFK-M Is Enriched in Neurons That Express nNOS--
nNOS is
expressed in a discrete subpopulation of interneurons in the corpus
striatum and cerebral cortex. To determine whether PFK-M is present in
these neurons, we immunostained for both nNOS and PFK-M in adult rat
brain sections. As seen in Fig. 6,
cortical interneurons that express nNOS are enriched in PFK-M. This was seen in 15 of 15 cortical neurons and 12 of 12 striatal interneurons examined.

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Fig. 6.
PFK-M is enriched in cortical neurons that
express nNOS. 20-µm sections from adult rat brain were stained
for nNOS (top) and PFK-M (middle), and nuclei
were visualized by staining with 4',6'-diamidino-2-phenylindole
(DAPI, bottom). Sections were examined by
fluorescence microscopy using a 20× objective. Arrows point
to cells that are enriched in both nNOS and PFK-M. Control experiments
lacking anti-PFK-M and anti-nNOS yielded no staining. The scale
bar is equal to 10 µm.
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DISCUSSION |
The primary finding of this study is that PFK-M is associated with
nNOS in brain and skeletal muscle. PFK-M binds to the PDZ domain of
nNOS, and this binding can be competed with a peptide, VSPDFGDAV, that
also specifically binds to the PDZ domain of nNOS. Like the binding of
PFK-M, the binding of CAPON (20) to the PDZ domain of nNOS can also be
competed by the VSPDFGDAV peptide, suggesting that the two proteins
bind to the same site in the nNOS PDZ domain. We have also shown that
PFK-M and nNOS associate in vivo because immunoprecipitation
of PFK-M from cytosolic muscle extract depletes nNOS. Furthermore, many
cortical neurons that are enriched in nNOS are also enriched in
PFK-M.
There are three main isoforms of PFK: PFK-M (alternatively known as
PFK-A), PFK-L (alternatively known as PFK-B), and PFK-C. PFK-M the only
isoform of PFK found in skeletal muscle and diaphragm (31). PFK-L is
the major isoform of PFK expressed in liver (31). PFK-C was originally
isolated from brain (32) and is co-expressed with the two other
isoforms of PFK in the brain where these three isoforms form
heterotetramers (31, 33, 34). PFK-M is the only isoform that ends in
the consensus for nNOS PDZ binding (20), and hence is likely to be the
only isoform that can bind to nNOS directly. We have shown that PFK-M
binds to nNOS at a peptide-binding site in the PDZ domain of nNOS (Fig.
3) as PFK-M can be competed off with a peptide that binds to this
peptide-binding site and that competes with the binding of CAPON.
Because PFK-M is the only isoform found in muscle, it serves as a good
tissue in which to study the association of PFK-M and nNOS in
vivo. Our data demonstrate that PFK-M binds to nNOS from skeletal
muscle extracts and that a large fraction of nNOS in muscle cytosol
associates with PFK (Figs. 3 and 4). This interaction may be
functionally important because NO can regulate energy metabolism in
normal muscle. In rodent myofibers, nNOS is specifically enriched at
neuromuscular endplates (16) and in fast twitch fibers, which are known
to be glycolytic (35). Exercise-stimulated glucose transport (36) and
cytokine-modulated glucose transport (37) are dependent on NO. It is
possible that in glycolytic fast twitch fibers, nNOS regulates both
glucose transport and PFK function.
We wondered whether the nNOS-PFK-M interaction might directly regulate
enzyme activities. However, we found that PFK activity of skeletal
muscle from nNOS
/
mice is not different from that of
wild type mice, nor does addition of a glutathione
S-transferase protein encoding the PDZ domain of nNOS to
skeletal muscle extracts change PFK
activity.2 PFK activity does
not affect nNOS activity because adding brain lysate containing PFK
activators has no effect on nNOS activity.2 With this in
mind, we suspect that PFK-M interactions help determine the disposition
of cytosolic nNOS rather than directly regulate enzyme activity.
In brain, nNOS is targeted to the plasma membrane and postsynaptic
density by PSD-95 and PSD-93 proteins via PDZ-PDZ interactions (15,
16), whereas PFK-M is a cytosolic protein and may target nNOS to the
cytosol. In fact, a significant fraction nNOS in brain is soluble (38).
In addition, the catalytic subunit of protein phosphatase-1 is targeted
to PFK-M via a protein-protein interaction (39), and thus, the binding
of PFK-M to the PDZ domain of nNOS may target nNOS to a cytosolic
complex of enzymes.
By subcellular fractionation of brain homogenates, we find that both
nNOS and PFK-M are enriched in synaptosomes and, more specifically, in
a synaptic vesicle fraction (Fig. 5). These data are consistent with
electron microscopic data that nNOS is present at nerve terminals in
the cerebral cortex (40) and the nucleus accumbens (41) of the rat
where NO can serve to modulate synaptic vesicle docking (42, 43).
Similarly, phosphofructokinase activity is detected in synaptosomes
(44), and phosphofructokinase enzyme protein occurs in presynaptic
terminals (45) where glycolysis generates ATP after synaptic
transmission (46). Thus, the binding of PFK-M to the PDZ domain of nNOS
might serve to localize these proteins to presynaptic terminals where
they are involved in different phases of synaptic transmission.
A striking result of our work is that PFK-M is enriched in many
nNOS-expressing neurons in the cerebral cortex (Fig. 6). This is an
important concept because neurons that express nNOS are resistant to
NO-mediated neurotoxicity. However, actual mechanisms responsible for
this neuroprotection are uncertain. Very recently, Dawson and
colleagues (47) reported that manganese superoxide dismutase (MnSOD)
protects neurons that express nNOS. The group found that MnSOD is
enriched in cortical neurons that express nNOS and that antisense to
MnSOD renders nNOS-expressing neurons susceptible to NMDA
receptor-induced neurotoxicity. Overexpression of MnSOD results in
protection of cortical neurons from NMDA- or NO-induced neurotoxicity,
and cortical neurons expressing nNOS protein cultured from MnSOD
knockout mice are sensitive to NMDA exposure. It is possible that MnSOD
is not the only protective protein that confers resistance to
glutamate-induced neurotoxicity because MnSOD is not exclusively
enriched in nNOS containing neurons.
We believe that we have provided an additional mechanism for
neuroprotection of nNOS containing neurons: the binding of PFK-M to
nNOS and its enrichment in nNOS protein-expressing neurons. There are a
number of reports that fructose-1,6-bisphosphate (FBP), the glycolytic
intermediate produced via the reaction catalyzed by PFK, is
neuroprotective. FBP administered systemically after hypoxia-ischemia
reduces central nervous system injury in neonatal rats (24). The
mechanism by which FBP acts is unknown, but Sola and colleagues suggest
three alternative models (24). First, FBP may enhance anaerobic
carbohydrate metabolism (48). FBP can also increase intracellular pH
(49, 50) and reduce intracellular calcium by binding it (51).
Similarly, FBP preserves ATP levels during hypoxia in neonatal brain
slices (25). In aging rats, ischemic-induced memory dysfunction can be
reversed by dimethyl sulfoxide-FBP, which serves to scavenge free
radicals (26). Thus, nNOS-expressing neurons may express high levels of
FBP because they are enriched in PFK-M. Furthermore, FBP activates PFK
via a feed-forward mechanism, resulting in amplification of FBP
production. Thus, nNOS positive neurons may be protected from
NO-induced neurotoxicity via a FBP-dependent mechanism.