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INTRODUCTION |
A large number of G-protein-coupled and growth factor receptors
activate phospholipase C to stimulate the hydrolysis of the phospholipid, phosphatidylinositol 4,5-bisphosphate, producing inositol
1,4,5-trisphosphate
(InsP3)1 and
diacylglycerol. InsP3 has an established second messenger role in mobilizing intracellular calcium stores (1) and can be
phosphorylated to inositol 1,3,4,5-tetrakisphosphate
(InsP4), which is proposed to play a role in capacitative
calcium entry (2) and as an activator of ras-GAP activity (3). The
sequential dephosphorylation of InsP3 and InsP4
gives rise to a host of inositol phosphate isomers in a cyclic
metabolic pathway that results in inositol returning to the
phosphoinositide pool (4).
In contrast to this well understood signaling pathway the role of
inositol hexakisphosphate (InsP6), the most abundant
cellular inositol metabolite, is very poorly understood. Present at
concentrations 10-100 times greater than InsP3 (15-60
µM) (5, 6), InsP6 has long been thought of as
metabolically rather inert with levels that do not change in response
to cell surface receptor activation. However, the discovery of higher
phosphorylated forms of InsP6 has led to the realization
that the InsP6 pool is rapidly turning over in a continual
cycle of phosphorylation and dephosphorylation (7, 8). Furthermore, a
role for InsP6 in synaptic vesicle trafficking has emerged
from studies showing that InsP6 binds with high affinity to
a number of proteins that are involved in exo/endocytosis including the
clathrin assembly proteins AP2 and AP3 and the synaptic vesicle
calcium-sensing protein synaptotagmin (9-12).
In the present study, we have investigated the possibility of the
existence of a protein kinase that is regulated by InsP6. We found that the pacsin/syndapin I, a synaptic vesicle-associated protein that acts as a molecular link coupling the endocytic machinery to the cytoskeleton, is phosphorylated by a protein kinase that is
regulated by InsP6. In addition,
InsP6-regulated phosphorylation of pacsin/syndapin I
increases the interaction between pacsin/syndapin I and dynamin I. These data provide a novel model by which InsP6 can
regulate synaptic vesicle endocytosis.
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EXPERIMENTAL PROCEDURES |
Anion Exchange Fractionation of Soluble Rat Brain
Cortex/Hippocampus Extract--
Rat brain cortex/hippocampus was
homogenized in 15 ml of TE buffer (2.5 mM Tris-HCl, 2.5 mM EDTA, pH 7.4) containing 1 mM phenylmethylsulfonyl fluoride. The homogenate was centrifuged at
50,000 × g for 10 min, and the pellet was discarded.
The supernatant was further centrifuged at 50,000 × g
for 20 min. The supernatant (13 ml at ~2.5 mg of protein/ml) was
applied to a 1-ml Resource Q (Amersham Pharmacia Biotech)
anion-exchange column (6.4 × 30 mm). The column was washed with 5 ml of TE buffer and eluted with a linear gradient of 0-1 M
NaCl in TE buffer over 20 ml (flow rate = 1 ml/min). One ml
fractions were collected and assayed for kinase activity. The entire
procedure was conducted at 4 °C.
Assay for Inositol Polyphosphate Kinase Activity in Brain
Fractions--
Aliquots of fractions (5 µl, ~5 µg of protein)
obtained from the anion exchange brain fractionation were added to
kinase buffer (20 mM Tris-HCl, 10 mM
MgCl2, 1 mM EGTA, 0.5 mM
dithiothreitol, 0.2 mM orthovanadate, 25 mM
glycerol phosphate, pH 7.5) containing 10 µM
[32P]ATP (1-4 cpm/fmol ATP), ± InsP6 (50 µM), in a final volume of 25 µl. The reactions were
continued for 10 min at 37 °C and were terminated by the addition of
10 µl (2×) SDS-PAGE sample buffer, and the proteins were resolved by
10% SDS-PAGE.
Large scale reactions that were to be fractionated on a hydroxyapatite
column were conducted using 100 µl of anion-exchange fraction in
kinase buffer containing 10 µM [32P]ATP
(1-3.0 cpm/fmol ATP), ± inositol polyphosphates (50 µM) in a final volume of 500 µl. The reactions were continued for 10 min
at 37 °C and terminated by the addition of 500 µl of buffer A (5 mM Tris-HCl, 10 mM KHPO4, pH
7.4).
Fractionation of Kinase Reactions on Hydroxyapatite--
One ml
of the above large scale kinase reaction was applied to a
hydroxyapatite column with a bed volume of 1 ml (Bio-Rad). The column
was washed with 5 ml of buffer A and eluted with a linear gradient of
0-100% buffer B (5 mM Tris-HCl, 500 mM
KHPO4, pH 7.5) over 10 ml (flow rate 1 ml/min). One-ml
fractions were collected. The proteins in 500 µl of each fraction
were concentrated by binding to 20 µl of strataclean slurry
(Stratagene). The strataclean beads were pelleted in a microcentrifuge
and washed with 1 ml of TE buffer. The beads were resuspended in 2×
SDS-PAGE sample buffer, and the proteins were resolved by 10%
SDS-PAGE.
Gels were routinely silver stained (Bio-Rad) before autoradiography to
ensure that the chromatography of each reaction was the same and that
equal amounts of protein had been loaded.
Immunoprecipitation of Pacsin/Syndapin
I--
Immunoprecipitations were carried out essentially as reported
previously (13). Large scale kinase reactions using fraction 9 (100 µl) from the anion-exchange fractionation of rat brain cytosolic
extract were carried out as described above. The reactions were stopped
by the addition of ice-cold TE buffer (500 µl). Pacsin/syndapin I
specific antiserum (14) was then added (~5 µg) and incubated for 60 min. The immune complexes were isolated on protein A-Sepharose beads.
The beads were washed 3× in TE buffer and resuspended in 20 µl of
SDS-PAGE sample buffer. Proteins were then resolved by 10% SDS-PAGE,
and an autoradiograph was obtained.
Construction and Purification of the GST-Pacsin/Syndapin I
Bacterial Fusion Protein--
Production of murine GST-pacsin/syndapin
I has been previously described (14). To produce the
GST-pacsin/syndapin I SH3 domain deletion mutant, site-directed
mutagenesis (Transformer Site-directed Mutagenesis Kit,
CLONTECH) was used to introduce two
BamHI sites, one upstream of the startcodon and the other at
position 1153 in the murine pacsin/syndapin I coding sequence. The
mutation primers used were 5'-CGCTACAGGATCCCCATGTCTGGC-3' and
5'-GATGCCAAGGGGATCCGTGTACGGGC-3', respectively. The resulting pacsin/syndapin I was subcloned into the BamHI site of the
pGEX-3X vector (Amersham Pharmacia Biotech) resulting in the production of GST-pacsin/syndapin I lacking the majority of the SH3 domain (Arg386-Ile441 was deleted).
Phosphorylation of the GST-Pacsin/Syndapin I Fusion
Protein--
Aliquots of each fraction from the anion exchange
fractionation (5 µl containing ~5 µg of protein) was added to
kinase buffer containing ~5 µg of GST-pacsin/syndapin I, 10 µM [32P]ATP (1-4 cpm/fmol ATP), ± inositol polyphosphates in a final volume of 100 µl. The reactions
were continued for 10 min at 37 °C and terminated by the addition of
ice-cold TE buffer (100 µl). GSH-Sepharose slurry (giving 20 µl of
packed beads) was added. Sepharose beads were pelleted in a
microcentrifuge and washed in TE-buffer three times. SDS-PAGE sample
buffer (20 µl) was added to the beads, and the proteins were resolved
by 10% SDS-PAGE.
Pacsin/Syndapin I Association with Dynamin I--
Kinase
reactions were performed as described above except with 2 mM ATP. The GST-pacsin/syndapin (~5 µg) bound to
glutathione-Sepharose beads was then washed in ice-cold TE buffer and
incubated with rat cytosol (1 mg protein) in a final volume of 1 ml for
60 min at 4 °C. The Sepharose beads were then washed three times in
ice-cold TE buffer, and the associated proteins were resolved by 10%
SDS-PAGE. The gels were transferred to nitrocellulose, and the
membranes were probed with anti-dynamin I polyclonal antibody (Santa
Cruz Biotechnology). To determine whether there was equal transfer and
loading of proteins, membranes were stained with Ponceau before being
probed with dynamin I antibodies. Quantification of protein bands in
the immunoblots was determined using NIH Image.
Mass Spectrometry--
Samples were separated on 10-20%
tricine/SDS-polyacrylamide gel prior to staining with GELCODE colloidal
Coomassie stain (Pierce) or a mass spectrometry-compatible silver
stain. Excised bands were destained and carbamidomethylated and
digested with trypsin prior to analysis of a portion of the digested
supernatant by MALDI-TOF mass spectrometry on a TOFSPEC-S.E. instrument
(Micromass). Nanoelectrospray mass spectrometry was performed on a
API-III triple quadruple instrument (PE-Sciex).
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RESULTS |
Identification of the Synaptic Vesicle-associated Protein,
Pacsin/Syndapin I, as a Substrate for an
InsP6-regulated Protein Kinase--
The strategy we
adopted to identify the presence of an InsP6-regulated
protein kinase was based on the fact that many signaling molecules,
including protein kinases and their substrates, form complexes with
each other that are robust enough to be purified using techniques such
as co-immunoprecipitation (15, 16). It was, therefore, considered
possible that the protein substrates for a putative inositol
polyphosphate-regulated protein kinase may form a complex with the
kinase that will allow them to co-purify over at least one
chromatographic step.
A low speed supernatant extract from rat brain cortex/hippocampus was
fractionated over an anion-exchange column. The presence of an
InsP6-regulated protein kinase was assessed by incubating an aliquot of each fraction with [P32]ATP, in the absence
or presence of InsP6 (50 µM) and resolving the phosphoproteins by SDS-PAGE. The level of phosphorylation of the
majority of phosphoproteins contained in these kinase reactions were
not affected by the presence of InsP6 (Fig.
1). However, in fractions 9 and 10, the
phosphorylation state of three proteins (Fig. 1, labeled
1-3) appeared to be altered by InsP6. Interestingly, band 3 appears to have decreased in its level of phosphorylation, whereas the other two protein bands have increased levels of
phosphorylation in the presence of InsP6. We decided to
center our attention on the identity of the ~52-kDa protein band
labeled 1 in Fig. 1.

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Fig. 1.
InsP6-dependent
phosphorylation of rat brain proteins. A soluble extract from rat
cortex/hippocampus was fractionated over an anion exchange column. An
aliquot of each fraction (~5 µg of protein) was used in a kinase
reaction ± InsP6 (50 µM). The reactions
were stopped by the addition of SDS-PAGE sample buffer, and the
proteins were resolved by 10% SDS-PAGE. The phosphorylation state of
protein bands labeled 1-3 appeared regulated by
InsP6. The results shown are are typical of at least three
experiments. The positions of molecular mass markers are shown in
kDa.
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To determine the identity of the 52-kDa phosphoprotein, large scale
kinase reactions containing vehicle, InsP3, or
InsP6, were performed. The kinase reactions were then
fractionated on a hydroxyapatite column, and the phosphoproteins were
resolved by 10% SDS-PAGE. Before the gels were exposed to film, they
were silver stained to establish the fidelity of the chromatography. The silver stain demonstrates that the chromatography of each of the
three kinase reactions on the hydroxyapatite matrix and the amount of
protein loaded on each lane were identical (Fig. 2). The autoradiograph of the
silver-stained gels revealed that the phosphorylation state of a
~52-kDa protein band eluting in fractions 14 and 15 was increased in
the presence of InsP6 but not InsP3 (Fig. 2).
By aligning the silver-stained gel with the autoradiograph, it was
possible to determine that the 52-kDa phosphoprotein in fraction
14 lined up exactly with a 52-kDa protein on the silver-stained gel
(Fig. 2).

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Fig. 2.
InsP6-regulated phosphorylation
of a 52-kDa protein. Large scale kinase reactions using
fraction 9 from the anion exchange chromatography of rat brain were
either stimulated with vehicle (C), InsP3
(3, 50 µM) or InsP6 (6,
50 µM). The reaction was stopped and fractionated on a
hydroxyapatite column. Each fraction from the hydroxyapatite column was
concentrated and resolved by 10% SDS-PAGE. The gel was first
silver-stained to establish equal protein loading and to identify the
proteins present. An autoradiograph of the gel was then obtained to
identify the position of the phosphoproteins. The position of a 52-kDa
protein that increases in its phosphorylation state in response to
InsP6 is marked with an arrow. Shown are
fractions obtained from the elution gradient of the column (fraction
14 = 90% buffer B). The results shown are of an
autoradiograph and silver stain of the same gel
and are typical of at least three experiments. The positions of
molecular mass markers are shown in kDa.
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MALDI-TOF analysis of tryptic peptide fragments of the 52-kDa protein
band excised from the SDS-PAGE gel showed 28 peptides covering 63% of
the amino acid sequence that matched exactly with the synaptic
vesicle-associated protein, pacsin/syndapin I (Fig. 3). The amino acid sequence of five
selected peptides were determined using MS/MS and shown to match
exactly with the sequence of rat pacsin/syndapin I (Fig. 3).

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Fig. 3.
Amino acid sequence analysis of the 52-kDa
protein identified to be phosphorylated in an
IP6-dependent manner. A, mass
spectra analysis (MALDI-TOF) of tryptic peptide fragments of the 52-kDa
protein shown to be phosphorylated in an
InsP6-dependent manner. 28 peptides (marked
with a diamond) matched exactly with that of pacsin/syndapin
I, covering 63% of the amino acid sequence. B, rat
pacsin/syndapin I showing the sequence of five tryptic peptides (in
bold) as determined by MS/MS.
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The identity of pacsin/syndapin I as the substrate for the putative
InsP6-regulated protein kinase was confirmed in
immunoprecipation studies using a pacsin/syndapin I-specific antiserum
(14). In these experiments, the level of phosphorylation of
pacsin/syndapin I present in kinase reactions was shown to be increased
in the presence of InsP6 (Fig.
4). We also tested the ability of a
bacterial GST fusion protein containing the full pacsin/syndapin I
sequence to act as a substrate for the InsP6-regulated
protein kinase. GST-pacsin/syndapin I was phosphorylated in an
InsP6-sensitive manner by fractions 8 to 11 from the anion
exchange chromatography of rat brain cytosolic extract (Fig.
5A). There was no
phosphorylation of the control GST protein (Fig. 5B).

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Fig. 4.
InsP6-dependent
phosphorylation of pacsin/syndapin I. Immunoprecipitation of
pacsin/syndapin I from kinase reactions using fraction 9 from the anion
exchange chromatography of rat brain ± InsP6 (50 µM). Immunoprecipitations were carried out using a 1:500
dilution of pacsin/syndapin I-specific antiserum. The data are typical
of at least four experiments. The positions of molecular mass markers
are shown in kDa.
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Fig. 5.
InsP6-dependent
phosphorylation of GST-pacsin/syndapin I. GST-pacsin/syndapin I
(A, molecular mass 76.5 kDa) or GST (B, molecular
mass 26 kDa) was employed as a substrate in kinase reactions using
fractions from the anion exchange chromatography of rat brain cytosol
(± InsP6, 50 µM). The position of
GST-pacsin/syndapin I and GST, as determined by Coomassie Blue staining
of the gels, is indicated. The positions of molecular mass markers are
shown in kDa.
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Phosphoamino acid analysis revealed that the GST-pacsin/syndapin I
fusion protein was phosphorylated in an InsP6-sensitive manner on serine (data not shown). Furthermore, a mutant of the GST-pacsin/syndapin I, where the C-terminal SH3 domain was deleted, also appeared to be a substrate for the InsP6-regulated
protein kinase (Fig. 6), indicating that
the site of phosphorylation on pacsin/syndapin I was not within the SH3
domain, which has previously been shown to be involved with coupling
pacsin/syndapin I to the synaptic vesicle endocytic machinery (17).

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Fig. 6.
InsP6-dependent
phosphorylation of SH3 domain-deficient GST-pacsin/syndapin I. A, schematic representation of the SH3 domain
deletion of pacsin/syndapin I. B, the SH3 domain deletion
mutant of pacsin/syndapin I (molecular mass 70.1 kDa) was used in
kinase reactions ± InsP6 (50 µM).
The data are typical of at least four experiments. The positions of
molecular mass markers are shown in kDa.
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Specificity of Inositol Phosphate Regulation of Pacsin/Syndapin I
Phosphorylation--
The phosphorylation of GST-pacsin/syndapin I was
not stimulated by any of the lower inositol polyphosphates indicating
that a phosphate group on each of the 6 positions on the inositol ring was essential for stimulating protein kinase activity (Fig.
7A). Importantly, inositol
hexasulfate was also unable to activate protein kinase activity,
eliminating the possibility that InsP6 may be exerting its
activity via nonspecific effects such as low affinity charge
interactions or chelation of divalent or trivalent cations. The
polyphosphoinositides, phosphatidylinositol (4,5)-bisphosphate and
phosphatidylinositol (3,4,5)-trisphosphate, were found to be unable to
stimulate the phosphorylation of GST-pacsin/syndapin I (Fig.
7B). Concentration-response analysis demonstrated that InsP6 was able to stimulate protein kinase activity in a
dose-dependent manner with maximal effects at 25-50
µM (Fig. 7C).

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Fig. 7.
Specificity of inositol phosphate-dependent
phosphorylation of pacsin/syndapin I. Analysis of phosphorylation
of GST-pacsin/syndapin I in kinase reactions stimulated with:
A, 50 µM of various inositol phosphates and
inositol hexasulfate; B, phosphatidylinositol
(4,5)-bisphosphate (PIP2, 50 µM); and phosphatidylinositol (3,4,5)-trisphosphate
(PIP3, 50 µM). C,
concentration-response of InsP6-regulated phosphorylation
of pacsin/syndapin I. The positions of molecular mass markers are shown
in kDa.
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Cellular InsP6 can be phosphorylated to produce the
inositol pyrophosphates termed diphosphoinositol pentakisphosphate
(PP-InsP5, also known as InsP7) and
bis(diphospho)inositol tetrakisphosphate (bis-PP-InsP4 or
InsP8) (7, 8). The predominant PP-InsP5 isomer
found in mammalian cells is 5-PP-InsP5, where the
pyrophosphate is in the 5-position on the inositol ring (18). We used
this isomer and an isomer that does not naturally occur
(2-PP-InsP5) to determine the ability of inositol
pyrophosphates to stimulate phosphorylation of pacsin/syndapin I. We
found that both isomers of PP-InsP5 stimulated
phosphorylation of GST-pacsin/syndapin I (Fig.
8A). Analysis of the
concentration-response curve demonstrated that the potency of
5-PP-InsP5 was similar to that of InsP6 (Fig. 8B).

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Fig. 8.
Phosphorylation of pacsin/syndapin I
stimulated by inositol pyrophosphates. A,
phosphorylation of GST-pacsin/syndapin I stimulated by
InsP6, 5-PP-InsP5, and 2-PP-InsP5
(50 µM). B, concentration response of
5-PP-InsP5-dependent phosphorylation of
GST-pacsin/syndapin I. The data presented are typical of three
experiments.
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Phosphorylation of Pacsin/Syndapin I Regulates Its Interaction with
Dynamin I--
Pacsin/syndapin I is proposed to regulate synaptic
vesicle recycling by acting as a molecular link between the
cytoskeleton and the endocytosis machinery (14, 17). In
vitro binding studies have demonstrated that one of the binding
partners for pacsin/syndapin I is dynamin I (17, 19). Consistent
with these previous studies, we show here that GST-pacsin/syndapin I
interacts with dynamin I present in a cytosolic rat brain preparation,
whereas GST alone was unable to interact (Fig.
9A). Also consistent with
earlier studies we found that this interaction appears to be via the
SH3 domain because the SH3 domain deletion mutant of pacsin/syndapin I
was unable to associate with dynamin I (data not shown).

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Fig. 9.
InsP6-dependent
phosphorylation of pacsin/syndapin I regulates its interaction with
dynamin I. A, GST or GST-pacsin/syndapin I associated
with glutathione-Sepharose beads was incubated with rat brain cytosol.
The beads were then washed and associated proteins resolved by 10%
SDS-PAGE, and the gels were transferred to nitrocellulose and
immunoblotted with anti-dynamin I antibodies. B,
GST-pacsin/syndapin I was first incubated in the presence or absence of
InsP6 (50 µM) in kinase reactions using
fraction 9 from the anion exchange fractionation of rat brain cytosol.
The GST-pacsin/syndapin I bound to glutathione beads was then washed
and incubated with rat brain cytosol (pacsin/syndapin + brain) or with vehicle (pacsin/syndapin brain)
for 60 min (4 °C). The presence of associated dynamin I was
determined by immunoblotting with anti-dynamin I antibodies.
Standard, 20 µg of rat brain cytosol. The results shown
are typical of at least four experiments. In the example shown the
-fold increase in dynamin to pacsin/syndapin I interaction following
InsP6-dependent phosphorylation of
pacsin/syndapin I was determined to be 2.43-fold. The positions of
molecular mass markers are shown in kDa.
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The possibility that phosphorylation of pacsin/syndapin I contributes
to its ability to interact with proteins involved in synaptic vesicle
endocytosis was tested by analysis of the association of the
phosphorylated form of pacsin/syndapin I and dynamin I. InsP6-dependent phosphorylation of
GST-pacsin/syndapin I increased the association between pacsin/syndapin
I and dynamin I by 3.28 ± 1.09-fold (n = 3 ± S.E.; Fig. 9B).
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DISCUSSION |
In the present study, we have shown that the synaptic
vesicle-associated protein, pacsin/syndapin I, is phosphorylated by a
novel protein kinase that is regulated by InsP6.
Furthermore, InsP6-dependent phosphorylation of
pacsin/syndapin I increases the ability of pacsin/syndapin I to
interact with dynamin I.
The strategy we adopted for the discovery of the
InsP6-regulated protein kinase and its protein substrate
was based on the hypothesis that many signaling proteins form complexes
with each other that are robust enough to allow them to be co-purified. This phenomenon has been described for the platelet-derived growth factor receptor, which forms a so-called "signal transduction particle" with PLC
, phosphatidylinositol 3-kinase, GAP, and raf (15). Other signal tranduction protein kinases, such as those of the
MAP kinase pathway, form signaling complexes that require the presence
of scaffolding proteins (16). In the present study we analyzed
chromatographic fractions from rat brain for the presence of
co-purifying InsP6-regulated protein kinase and its
substrate proteins. We found that a 52-kDa protein eluting from an
anion exchange column was phosphorylated in response to
InsP6. This protein was determined to be the synaptic
vesicle protein, pacsin/syndapin I, on the basis of MALDI-TOF and amino
acid sequence analysis and immunoprecipitation experiments. The
identification of the phosphorylation of pacsin/syndapin I is the first
demonstration of a protein phosphorylation that is regulated by
InsP6. We are currently using the
InsP6-dependent phosphorylation of the
GST-pacsin/syndapin I fusion protein in an assay to purify the
InsP6-regulated protein kinase.
Pacsin/syndapin I is a 52-kDa protein that is highly enriched in brain
and associated with presynaptic terminals where it co-localizes with
dynamin I (14, 17). In vitro binding studies have
demonstrated that the SH3 domain of pacsin/syndapin I is able to bind
to the proline-rich C terminus of dynamin I, as well as a number of
other brain-specific proteins including synaptojanin, synapsin Ia/Ib
and the neuronal Wiskott-Aldrich syndrome protein (17, 19). These
proteins are known to form part of the multicomponent protein complex
involved in clathrin-mediated synaptic vesicle recycling at nerve
terminals (20).
The assembly of this multiprotein complex has been demonstrated to be
regulated by a phosphorylation/dephosphorylation cycle that is
controlled by calcium entry at the nerve terminal (21, 22).
Interestingly, these previous studies have demonstrated that
phosphorylation of synaptic vesicle proteins such as dynamin I,
synaptojanin and amphiphysin results in a decreased ability of these
proteins to associate with other synaptic vesicle endocytic proteins.
Assembly of the endocytic machinery occurs on calcium entry via
dephosphorylation mediated by the calcium-sensitive phosphatase
calcineurin (21, 22). Our data indicate the presence of an additional
novel mechanism in the regulation of synaptic vesicle endocytsis where
InsP6-mediated phosphorylation of pacsin/syndapin I is able
to increase the ability of pacsin/syndapin I to interact with dynamin
I. This process may either contribute to the assembly of the endocytic
machinery or tether the endocytic multiprotein complex to the cytoskeleton.
Our study, therefore, adds to a growing body of evidence to suggest
that InsP6 is involved in synaptic vesicle trafficking and
membrane trafficking. The clathrin assembly proteins AP2 and AP3,
together with the visual and non-visual arrestins and synaptotagmin, have been shown to bind InsP6 with high affinity (9-12,
23). The binding of InsP6 to these proteins regulates their
interaction with the endo/exocytic pathways. The present study extends
a role for InsP6 in endocytosis by demonstrating that
InsP6-dependent phosphorylation is a mechanism
that can increase the interaction between specific endocytic proteins.
The discovery of an InsP6-regulated protein kinase supports
a signaling role for this inositol polyphosphate. Importantly, none of
the lower inositol phosphates stimulated kinase activity. Furthermore,
kinase activity was not stimulated by the polyphosphoinositides. This
is of significance because a number of high affinity
InsP6-binding proteins have also been shown to bind the
polyphosphoinositides, and it is in fact the binding of
polyphosphoinositides that is thought in some instances to be the
physiological regulators of these proteins (11, 24). We also tested the
ability of the inositol pyrophosphates to stimulate kinase activity.
Both isomers of PP-InsP5 stimulated kinase activity with a
concentration-response curve that was very similar to
InsP6. Thus, the InsP6-regulated protein kinase
is activated by inositol that contains a phosphate group on all 6 positions of the inositol ring and that addition of a further phosphate
to make a pyrophosphate at positions 5 or 2 does not change the
activation profile. It is, therefore, possible that both
InsP6 and/or 5-PP-InsP5 could be the
physiological activators of the protein kinase identified here.
However, it is interesting to note that the concentration-response
curve for InsP6 is consistent with a protein kinase that
would be expected to respond to cellular concentrations of
InsP6, which are thought to be in the µM
range (5, 6).
Early work on InsP6 demonstrated that it is ubiquitously
present at high cellular levels, and it was thought to be involved in
metal ion chelation or act as a phosphate store (25, 26). However,
studies demonstrating that InsP6 levels can change in response to receptor stimulation suggested that InsP6 may
have a signaling role (27, 28). Recently, InsP6 has been
shown to be involved in the regulation of mRNA export from the
nucleus (29), the control of phosphatase activity in pancreatic
cells (28), and to act as a key factor in DNA repair (30). Our studies demonstrating the presence of a protein kinase that is specifically activated by InsP6 adds a novel signaling pathway for this
inositol polyphosphate.