From the
The pathway of synthesis and metabolism of bis-diphosphoinositol
tetrakisphosphate (PP-InsP
The extensive literature on the synthesis, metabolism, and
functions of inositol phosphates is largely focused on
Ins
(1, 4, 5) P
The search for the physiological significance of
PP-InsP
Other
studies have also provided important information about the metabolic
characteristics of PP-InsP
In
contrast to the catalogue of exciting observations concerning
PP-InsP
The
discovery of diphosphoinositol polyphosphates has also provided us with
the opportunity to gain further insight into a puzzling enzyme activity
which was originally identified as an
Ins
(1, 3, 4, 5) P
The metabolism of inositol
polyphosphates by tobacco ``pyrophosphatase''
(17) was performed by a modification of earlier procedures
(3) . Trace amounts of
In cells treated with 10 mM F
We have established that PP-InsP
In [
The differential sensitivity to F
Vesicle trafficking seems likely to be
one area of cell biology that may be relevant to the function of
diphosphoinositol polyphosphates. AP-2, AP-3, and coatomer all help
regulate different aspects of vesicle traffic
(7, 8) .
These proteins share the ability to bind PP-InsP
MIPP, which attacks
Ins
(1, 3, 4, 5) P
In
conclusion, our characterization of the pathway of synthesis and
metabolism of PP-InsP
Homogenates, and 100,000
-PP) was elucidated by high
performance liquid chromatography using newly available
H-
and
P-labeled substrates. Metabolites were also identified
by using two purified phosphatases in a structurally diagnostic manner:
tobacco ``pyrophosphatase'' (Shinshi, H., Miwa, M., Kato, K.,
Noguchi, M. Matsushima, T., and Sugimura, T. (1976) Biochemistry 15, 2185-2190) and rat hepatic multiple inositol
polyphosphate phosphatase (MIPP; Craxton, A., Ali, N., and
Shears, S. B. (1995) Biochem. J. 305, 491-498). The
demonstration that diphosphoinositol polyphosphates were hydrolyzed by
MIPP provides new information on its substrate specificity, although
MIPP did not metabolize significant amounts of these polyphosphates in
either rat liver homogenates or intact AR4-2J cells. In liver
homogenates, inositol hexakisphosphate (InsP
) was
phosphorylated first to a diphosphoinositol pentakisphosphate
(PP-InsP
) and then to PP-InsP
-PP. These kinase
reactions were reversed by phosphatases, establishing two coupled
substrate cycles. The two dephosphorylations were probably performed by
distinct phosphatases that were distinguished by their separate
positional specificities, and their different sensitivities to
inhibition by F
(IC
values of 0.03
mM and 1.4 mM against PP-InsP
and
PP-InsP
-PP, respectively). In
[
H]inositol-labeled AR4-2J cells, the
steady-state levels of PP-[
H]InsP
and
PP-[
H]InsP
-PP were, respectively,
2-3 and 0.6% of the level of
[
H]InsP
. The ongoing turnover of
these polyphosphates was revealed by treatment of cells with 0.8
mM NaF for 40 min, which reduced levels of
[
H]InsP
by 50%, increased the levels
of PP-[
H]InsP
16-fold, and increased
levels of PP-[
H]InsP
-PP 5-fold. A
large increase in levels of PP-[
H]InsP
also occurred in cells treated with 10 mM NaF, but then
no significant change to levels of
PP-[
H]InsP
-PP were observed; there
may be important differences in the control of the turnover of these
two compounds.
,
(
)
Ins
(1, 3, 4, 5) P
,
and the pathways by which these compounds are recycled to inositol
(1, 2) . There is a substantially smaller body of
information on the more highly phosphorylated inositol phosphates such
as InsP
. Interest in this particular inositol polyphosphate
has now increased following the discovery that it is further
phosphorylated to a diphosphoinositol pentakisphosphate
(PP-InsP
)
(3, 4) , a widely distributed
cellular constituent of organisms as phylogenetically diverse as
Dictyostelium discoideum(4) and rat
(3) .
PP-InsP
has added a new dimension to the study of the many
aspects of cell biology that are regulated by a variety of inositol
derivatives.
is currently taking several divergent lines of
enquiry. One area of interest in our laboratory is to understand the
consequences of high affinity binding of PP-InsP
to
AP-2,
(
)
AP-3
(5) and coatomer
(6) . These three proteins are functionally related by virtue of
their participation in the control of vesicle traffic
(7, 8) ; this important cellular activity is therefore a
candidate for a site of action of PP-InsP
. Indeed,
PP-InsP
is the most potent, known inhibitor of
AP-3-dependent clathrin assembly
(5) . This raises the
possibility that polyphosphates such as PP-InsP
could
represent one of the series of ``fusion clamps'' which may
need to be released before the synaptic vesicle can proceed rapidly
through its exocytic-endocytic cycle
(7, 8) .
; it is particularly striking
that in intact mammalian cells there is an ongoing, rapid metabolic
flux through the kinase-phosphatase cycle that interconverts InsP
with PP-InsP
(3) . The high turnover of
PP-InsP
was one of the factors which led to the suggestion
that it might be an important energy donor
(4) . Another
interesting finding is that, in intact cells, levels of PP-InsP
decreased following treatment with thapsigargin
(9) . It
appears that the InsP
kinase was inhibited by
thapsigargin-mediated changes in the status of cellular Ca
pools
(9, 10) . The regulation of PP-InsP
turnover by second messengers such as Ca
is
further testimony to the likely importance of this compound.
, there is another diphosphate derivative of
InsP
in mammalian cells
(3, 4) that has not
yet been studied in any detail. It is possible that this is a
bis-diphosphoinositol tetrakisphosphate (PP-InsP
-PP); such
material has been isolated from Dictyostelium(4) , and
it has HPLC elution properties very similar to the mammalian compound.
We are unlikely to appreciate the physiological significance of these
diphosphoinositol polyphosphates until ( a) we determine how
many members of this class of compounds are present in cells, and
( b) we understand all their metabolic characteristics and
inter-relationships. With these objectives in mind, we have conducted
the first detailed study of the synthesis and metabolism of
PP-InsP
-PP in vitro and in vivo.
3-phosphatase
(11) . The demonstration that this enzyme
also hydrolyzed
Ins
(1, 3, 4, 5, 6) P
and InsP
has led to its redesignation as a multiple
inositol polyphosphate phosphatase (MIPP)
(12, 13) . We
have now sought further information on the specificity of this
intriguing enzyme by investigating whether diphosphoinositol
polyphosphates may also be substrates. Additionally, these experiments
have provided further confirmation of the structures of the
diphosphoinositol polyphosphates.
Materials
MIPP, originally designated as
``Ins
(1, 3, 4, 5) P 3-phosphatase,'' was purified from rat liver
(13) .
[
H]Inositol was obtained from American
Radiolabeled Chemicals (St. Louis, MO). A
H-labeled
diphosphoinositol tetrakisphosphate was prepared as described
previously
(3) . The following were obtained from DuPont NEN
(catalogue numbers are in parentheses): NENSORB
preparative deproteinizing cartridges (NLP 028),
[
H]InsP
(NET 1023),
PP-[
H]InsP
(NET 1093),
PP-[
H]InsP
-PP (NET 1098),
[
-
P]PP-InsP
(NEG 214), and
[
-
P]PP-InsP
-PP (NEG 215). For
some experiments, PP-[
H]InsP
and
PP-[
H]InsP
-PP were prepared by
phosphorylation of [
H]InsP
and
PP-[
H]InsP
respectively, using liver
homogenate incubated as described under ``Experimental
Procedures.'' There were no differences in the results obtained
using substrates provided by NEN, as compared to those synthesized at
NIEHS. Non-radioactive InsP
was from Aldrich. Bovine serum
albumin, Triton X-100, Na
EDTA, and tobacco pyrophosphatase
were obtained from Sigma. NaF was obtained from either Sigma or Fluka.
Preparation of Liver Homogenate
Sprague-Dawley
rats (200-250 g) were asphyxiated with CO, and blood
was removed from the liver by perfusion with ice-cold buffer containing
300 mM sucrose, 1 mM EGTA, 1 mM
dithiothreitol, and 10 mM HEPES (pH 7.2 with KOH). The liver
was placed in 25 ml of perfusion buffer supplemented with 15 µg/ml
leupeptin, 0.2 µg/ml aprotinin, and 15 µM trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane.
The liver was chopped with scissors, gently homogenized with a
motor-driven Teflon pestle, and filtered through cheesecloth; protein
concentration was measured
(14) with bovine serum albumin as a
standard. Homogenates were fractionated into soluble and particulate
fractions by centrifugation for 1 h at 100,000
g; each
fraction was adjusted to the same volume as the parent homogenate with
homogenization buffer.
Inositol Polyphosphate Metabolism
For the study of
the phosphorylation of inositol polyphosphates, trace amounts of
radiolabeled substrate were incubated at 37 °C in 0.5-1-ml
aliquots of assay buffer 1 (100 mM KCl, 7 mM
MgSO, 5 mM ATP, 10 mM phosphocreatine, 10
mM NaF, 10 mM HEPES (pH 7.2 with KOH), 1 mM
Na
EDTA, 1 mM dithiothreitol, and 0.05 mg/ml
phosphocreatine kinase). Dephosphorylation of inositol polyphosphates
was studied using assay buffer 2 (50 mM KCl, 50 mM
HEPES (pH 7.2 with KOH), 4 mM CHAPS, 0.05 mg/ml bovine serum
albumin, 1 mM Na
EDTA, 2 mM
MgSO
). The reactions were initiated by the addition of
aliquots of appropriately diluted tissue and quenched with 4 ml of
ice-cold medium comprising 0.1 M Tris (pH 7.7 with HCl at 25
°C), 10 mM triethylamine, 2 mM
Na
EDTA. Protein was subsequently removed in the following
manner: a NENSORB
preparative deproteinizing
cartridge
(15) was first primed with 10 ml of methanol,
followed by 10 ml of quench buffer. The quenched reaction was added to
the column, which was then washed with 2
4-ml aliquots of
quench medium. The flow-rate was
3 ml/min. Recoveries of inositol
polyphosphates exceeded 95% in the ``flow-through'' from the
column, which was analyzed by HPLC (see below) either immediately, or
following storage at -20 °C. Alternately, when metabolism was
to be analyzed by gravity-fed anion-exchange columns (see below),
assays were quenched with 0.2 ml of 2 M HClO
and
then neutralized with KOH
(16) .
H-labeled substrate were
incubated at 37 °C with 100-300 µl of medium containing
30 mM sodium acetate buffer (pH 5.5), 10 mM
Na
EDTA, 2 mM dithiothreitol, 0.01% (v/v) Triton
X-100, 0.4 mg/ml bovine serum albumin. Immediately prior to use, the
stock solution of pyrophosphatase was diluted 1:1 with 50 mM
Na
EDTA (pH 7.0) and added to the incubation buffer to a
final concentration of 150-170 Sigma Units/ml. After 16-22
h, incubations were quenched with 4 volumes of ice-cold water, followed
by 2 volumes of 0.2 ml of 2 M perchloric acid plus 1 mg/ml
InsP
. Samples were neutralized with freon-octylamine
(16) . There was no hydrolysis of substrate in control samples
to which enzyme was not added.
Analysis of Inositol Polyphosphate Metabolism by
Gravity-fed Anion-exchange Columns
The release of
[P]P
from
P-labeled
diphosphoinositol polyphosphates was conveniently assayed by loading
acid-quenched, KOH-neutralized samples (see above) in 10 ml of water,
onto 0.8 ml of gravity-fed anion-exchange columns (Bio-Rad, AG1-X8
200-400 mesh, formate form). [
P]P
was eluted with 2
10-ml aliquots of 0.4 M
ammonium formate, 0.1 M formic acid. The diphosphoinositol
polyphosphate (recovery = 83 ± 1%, n =
11) was then eluted with 2
10-ml aliquots of 2 M
ammonium formate, 0.1 M formic acid. Radioactivity was
assessed from the Cerenkov radiation. Culture and Incubation of AR4-2J Cells-AR4-2J cells
were cultured in 35-mm wells in plastic culture dishes as described
previously
(3) . Cells were labeled with 100 µCi/ml
[
H]inositol for 96 h. The culture medium was
changed (and fresh [
H]inositol was added) at 48
h. One h before each experiment, the medium was removed and replaced
with HEPES-buffered KREBS Ringer (pH 7.4 with NaOH) containing 11
mM glucose, but without added
[
H]inositol. Cultures were maintained at 37
°C, and then NaF was added as described in the figure legends.
Incubations were terminated by aspiration of the incubation medium, and
then cells were immediately lysed by addition of 1.2 ml of ice-cold
medium containing 10 mM NaF, 5 mM Na
EDTA,
1 mg/ml InsP
, 0.1% (v/v) Triton X-100. Extracts were kept
on ice for 15 min prior to their centrifugation to remove cell debris.
The resultant supernatants were saved and applied to
NENSORB
columns to remove soluble protein as
described above.
HPLC Analysis of Inositol
Polyphosphates
Deproteinized samples were loaded onto a
Partisphere 5-µm SAX HPLC column at a flow-rate of 1 ml/min using a
WISP model 712 (Waters Associates), which injected each sample in
aliquots interspersed with HPLC buffer A (1 mM
NaEDTA) in a ratio of sample: buffer A of approximately
1:10. When required, fractions of 2.5 ml were collected during this
loading procedure. The following gradient was then generated from a
mixture of buffer A (1 mM Na
EDTA, disodium salt)
and buffer B (buffer A plus 1.3 M
(NH
)
HPO
(pH 3.8) with
H
PO
) at a flow-rate of 1 ml/min (unless
otherwise indicated): 0-2 min, B = 0%; 2-10 min, B
increased linearly to 44%; 10-100 min, B increased linearly to
100% and was retained at this level until either 101 min (for older
columns) or 110 min (new columns). Finally, B then returned to 0%.
Either 1 or 2 min fractions were collected. A slightly different
gradient was used to resolve inositol polyphosphates in extracts of
intact cells: 0-10 min, B = 0%; 10-30 min, B
increased linearly to 40%; 30 to 130 min, B increased linearly to 75%;
130-131, B increased linearly to 100%; 131-157 B =
100%; 157 min, B returned to 0%. One-ml fractions were collected. All
HPLC fractions were mixed with 4 volumes of Monoflow 4 scintillant
(National Diagnostics, Manville, NJ), and radioactivity was quantified
by liquid scintillation spectrometry.
Phosphorylation of InsP
It was recently shown
that extracts of both Dictyostelium, and some mammalian
cultured cell types, can phosphorylate InsP to
PP-InsP
-PP by Liver Homogenate
to
PP-InsP
(3, 4) . The initial goal of our
current study was to identify a mammalian tissue that could be a useful
in vitro model for the study of InsP
phosphorylation. We found that liver homogenate readily converted
InsP
( peak A, Fig. 1, upper panel)
to PP-InsP
( peak B, Fig. 1, upper
panel) when 10 mM NaF was employed to inhibit
PP-InsP
phosphatase (
(3) , and see below). A novel
observation in our present experiments was the phosphorylation of
[
H]InsP
to substantial amounts of an
additional
H-labeled product which was more polar than
PP-InsP
. Approximately 25% of InsP
/h/(mg
protein) was converted to peak C (in Fig. 1, upper
panel), greatly exceeding the tiny amounts observed in some
earlier experiments with extracts of cultured cells
(3, 4) . We have never observed any further products
which were more polar than peak C, even when the HPLC column was
extensively washed with 1.5 M of
[NH
]
HPO
for 30 min (data
not shown).
Figure 1:
Phosphorylation of InsP and
PP-InsP
by liver homogenate. Liver homogenates were
incubated at a final concentration of 0.9 mg protein/ml for 40 min in
0.5 ml of assay buffer 1 (see ``Experimental
Procedures'') that contained either
10,000 dpm
[
H]InsP
( peak A, upper
panel) or
5,000 dpm PP-[
H]InsP
( peak B, lower panel). Reactions were quenched
and extracted as described under ``Experimental Procedures,''
spiked with
600 dpm
[
-
P]PP-InsP
-PP, and
analyzed by HPLC as described under ``Experimental
Procedures''; one-min fractions were collected. The
circles depict
H dpm/fraction, and triangles depict
P dpm/fraction for the internal standard. The
data are representative of five experiments ( upper panel) or
seven experiments ( lower panel). The difference in absolute
elution times of the HPLC runs described in the upper and
lower panels arises from differences in the age of the column;
this is compensated for by the internal standard of
[
-
P]PP-InsP
-PP.
A likely structure for peak C is a bis-diphosphoinositol
tetrakisphosphate, PP-InsP-PP (see below). However, two
different structures for the material in peak C were considered:
1) a second isomer of PP-InsP
, formed either by
phosphorylation of InsP
or by isomerization of the
PP-InsP
in peak B; 2) a PPP-InsP
,
i.e. a triphosphoinositol pentakisphosphate. Both of these
alternative structures were ultimately excluded, on the basis of the
data described below. Indeed, one informative experiment was the
determination of the immediate precursor of the putative
PP-InsP
-PP. Here, PP-InsP
was incubated with
liver homogenate in the presence of ATP and NaF ( Fig. 1 lower
panel). Little PP-InsP
was dephosphorylated during 1-h
incubations; instead, about 50% of the PP-InsP
was itself
converted to PP-InsP
-PP (Fig. 1, lower
panel). In other words, PP-InsP
, and not
InsP
, is the immediate precursor of PP-InsP
-PP.
Indeed, the [
-
P]PP-InsP
-PP
supplied by DuPont NEN is produced from
[
-
P]PP-InsP
in an ATP-dependent
enzymatic reaction, and when this material was used as an internal
standard during HPLC, it co-eluted with peak C (Fig. 1).
A Substrate Cycle for InsP
In the absence of ATP and NaF,
PP-InsP and PP-InsP
in Liver Homogenate
was rapidly dephosphorylated by liver homogenate,
and no PP-InsP
-PP was formed (compare Fig. 1,
lower panel, with Fig. 2). We determined which phosphate
group(s) was hydrolyzed by HPLC analysis of the products formed from
the metabolism of a mixture of PP-[
H]InsP
and [
-
P]PP-InsP
. The
initial
H-labeled product did not contain any
P-label (Fig. 2); thus, the only significant route
of dephosphorylation of PP-InsP
was to InsP
by
removal of the
-phosphate in the diphosphate group, all of which
was recovered as [
P]P
(Fig. 2). This phosphatase activity completes the substrate
cycle between InsP
and PP-InsP
. Note that the
extract from each assay was loaded onto the HPLC in a relatively large
volume (see ``Experimental Procedures''); much of
the [
P]P
did not bind to the HPLC
column, and instead was eluted in the load ( inset to
Fig. 2
).
Figure 2:
Dephosphorylation of PP-InsP
by liver homogenate. Liver homogenates were incubated at a final
concentration of 3.5 µg protein/ml for 30 min in 1 ml of assay
buffer 2 (see ``Experimental Procedures'') that
contained
2500 dpm PP-[
H]InsP
and
1500 dpm
[
-
P]PP-InsP
. Reactions were
quenched, extracted, and analyzed by HPLC as described under
``Experimental Procedures.'' 5-min fractions were
collected while the sample was loaded onto the HPLC (see
inset), and 2-min fractions were collected during the
gradient. Closed circles depict
H dpm/fraction,
and open circles depict
P dpm/fraction. The
arrow indicates the elution position of PP-InsP
-PP
(determined in a separate HPLC run). The data are representative of
four experiments.
Diphosphoinositol Polyphosphates Are Hydrolyzed by
MIPP
Mammalian tissues contain a MIPP that has particularly high
affinity toward
Ins
(1, 3, 4, 5, 6) P and InsP
(12, 13) . We have now
discovered that MIPP also dephosphorylated PP-InsP
(Fig. 3): in these experiments MIPP was incubated with
PP-[
H]InsP
and
[
-
P]PP-InsP
. The immediate
products formed were [
H]InsP
and
[
P]P
; no
[
-
P]PP-InsP
was detected
(Fig. 3). Thus, MIPP was highly specific in its expression of
monoesterase activity toward the diphosphate group in
PP-InsP
. This was an unexpected observation, because MIPP
nonspecifically removes every phosphate from InsP
, and then
further hydrolyzes several of the InsP
isomers that are
formed
(12) . Clearly, the addition of a single diphosphate
group to InsP
has a dramatic effect upon the specificity of
this intriguing enzyme. Therefore, we next investigated if MIPP could
also hydrolyze PP-InsP
-PP.
Figure 3:
PP-InsP metabolism by MIPP.
MIPP was incubated at a final concentration of 0.02 µg protein/ml
for 35 min in 0.5 ml of assay buffer 2 (see ``Experimental
Procedures'') that contained
5000 dpm
PP-[
H]InsP
and
3000 dpm
[
-
P]PP-InsP
. Reactions were
quenched, extracted, and analyzed by HPLC as described under
``Experimental Procedures''; one-min fractions were
collected. The closed circles depict
H
dpm/fraction, and the open circles depict
P
dpm/fraction. The data are representative of seven
experiments.
MIPP was found to slowly
dephosphorylate a mixture of
[-
P]PP-InsP
-PP and
PP-[
H]InsP
-PP (Fig. 4). The
immediate
H-labeled reaction product was designated as
InsP
-PP, since it had lost the
P-label
(Fig. 4), which was recovered as
[
P]P
(data not shown). The
designation of InsP
-PP also has the advantage of
emphasizing that it is a different isomer from PP-InsP
.
Indeed, upon HPLC, the peak fraction of
[
H]InsP
-PP eluted prior to
[
-
P]PP-InsP
(Fig. 5).
Thus, MIPP attacks the same diphosphate group of both PP-InsP
and PP-InsP
-PP. It therefore became important to
determine the extent to which MIPP is responsible for the metabolism of
diphosphoinositol derivatives both in vitro and in vivo (see below).
Figure 4:
PP-InsP-PP metabolism by MIPP.
MIPP was incubated at a final concentration of 1.5 µg protein/ml
for 120 min in 1 ml of assay buffer 2 (see ``Experimental
Procedures'') that contained
2500 dpm
PP-[
H]InsP
-PP and
750 dpm
[
-
P]PP-InsP
-PP. Reactions were
quenched, extracted, and analyzed by HPLC as described under
``Experimental Procedures''; one-min fractions were
collected. The closed circles depict
H
dpm/fraction (no other dpm were present in the remainder of the
gradient). The open circles depict
P
dpm/fraction. The data are representative of three
experiments.
Figure 5:
Separation of
[-
P]PP-InsP
from
[
H]InsP
-PP by HPLC.
PP-[
H]InsP
-PP was dephosphorylated by
MIPP to [
H]InsP
-PP as described in
the legend to Fig. 4. The [
H]InsP
-PP
was desalted (3) and
600 dpm were mixed with
500 dpm of an
internal standard of [
-
P]PP-InsP
which was then analyzed by HPLC as described under
``Experimental Procedures.'' Fraction size was 2 min.
Closed circles depict
H dpm/fraction, and
triangles depict
P dpm/fraction. The data are
representative of seven experiments.
These data are also useful in that they confirm
the actual structure of the
[-
P]PP-InsP
-PP: recall that
this is synthesized by phosphorylation of
[
-
P]PP-InsP
using
non-radiolabeled ATP (see above). Therefore, an alternative
triphosphate structure ( i.e. [
-
P]PPP-InsP
) is
eliminated because the
P-labeled phosphate could not then
have been removed by the monoesterase activity of MIPP. Furthermore,
the demonstration that InsP
-PP (rather than
InsP
) is formed by MIPP-catalyzed dephosphorylation of
PP-InsP
-PP, proves that the latter is indeed a more highly
phosphorylated derivative of PP-InsP
, rather than another
isomer of PP-InsP
.
Dephosphorylation of PP-InsP
The pathway of PP-InsP-PP by Liver
Homogenate
-PP metabolism has
not previously been studied in any cell type. To address this question,
liver homogenate was incubated with a mixture of
[
-
P]PP-InsP
-PP and
PP-[
H]InsP
-PP ( peak C in
Fig. 6
); reaction products were analyzed by HPLC; there were two
major
H-labeled downstream metabolites, peaks D and E (Fig. 6). In five experiments (including that
described by Fig. 6), the value of the
[
P]/[
H] ratio of peak D
was 93 ± 2% of the value of the
[
P]/[
H] ratio for the
PP-InsP
-PP (Fig. 6). Thus, in liver homogenate the
initial step in the dephosphorylation of
[
-
P]PP-InsP
-PP was near
exclusively by removal of an unlabeled phosphate. Thus, peak D was
either PP-InsP
or PP-InsP
-PP, depending upon
whether a monophosphate or the unlabeled diphosphate group of
[
-
P]PP-InsP
-PP was hydrolyzed.
Peak E (Fig. 6) did not contain any
P-label.
Therefore, the dephosphorylation of peak D to peak E occurred by
specific removal of the
P-labeled phosphate. Some
PP-InsP
-PP could also have been directly converted to peak
E. In other words, three catabolic pathways were possible: 1)
PP-InsP
-PP
PP-InsP
InsP
; 2) PP-InsP
-PP
PP-InsP
-PP
InsP
-PP; 3)
PP-InsP
-PP
InsP
-PP.
Figure 6:
PP-InsP-PP metabolism by liver
homogenate. Aliquots of liver homogenate were incubated at a final
concentration of 0.8 mg protein/ml for 60 min in 0.5 ml of assay buffer
2 (see ``Experimental Procedures'') containing
5000 dpm PP-[
H]InsP
-PP and
4000 dpm [
-
P]PP-InsP
-PP.
Reactions were quenched, extracted, and analyzed by HPLC as described
under ``Experimental Procedures.'' One-min fractions
were collected. Closed circles depict
H
dpm/fraction, and open circles depict
P
dpm/fraction. The data are representative of five
experiments.
To help
distinguish between these alternative metabolic pathways, we prepared
individual, desalted
(3) samples of peaks D and E that were
only H-labeled ( i.e. by dephosphorylation of
PP-[
H]InsP
-PP). Upon HPLC, peak D
coeluted with a PP-InsP
standard (Fig. 7, upper
panel), and peak E coeluted with InsP
(Fig. 7, lower panel). These data are
consistent with PP-InsP
-PP being metabolized to
PP-InsP
and then to InsP
( i.e. the
first of the three options described above). Nevertheless, we sought an
additional means of discriminating between the three alternative
pathways. Note that two of them (options 2 and 3) predict that peak E
would contain a diphosphoinositol tetrakisphosphate
(InsP
-PP). Only option 1 predicts that peak E is
InsP
. This distinction is important because we have
previously demonstrated
(3) that a tobacco
``pyrophosphatase''
(17) will attack
diphosphoinositols but not monophosphoinositols such as
InsP
. We therefore incubated
800 dpm of peak E with
``pyrophosphatase'' (see ``Experimental
Procedures''). In control experiments, pyrophosphatase was also
incubated with 2000 dpm of
H-labeled standards of both
InsP
and a diphosphoinositol tetrakisphosphate. Up to 77%
of the latter was dephosphorylated by the pyrophosphatase in four
experiments. In contrast, no detectable hydrolysis of InsP
was ever observed and, importantly, pyrophosphatase did not
hydrolyze peak E (data not shown). Thus, peak E contained only
InsP
, so peak D must be PP-InsP
, itself formed
by dephosphorylation of PP-InsP
-PP. This pathway represents
a complete reversal of the route of PP-InsP
-PP synthesis,
establishing two coupled substrate cycles (Fig. 8). Note also the
different positional specificities of the phosphatase activities toward
PP-InsP
-PP as compared to PP-InsP
; this may
reflect the participation of two distinct enzymes.
Figure 7:
HPLC analysis of the products of
PP-InsP-PP metabolism.
H-Labeled peaks Dand Ewere prepared by dephosphorylation of
PP-[
H]InsP
-PP (see text and Fig. 6).
In the upper panel,
600 dpm of peak D( circles) was mixed with
600 dpm of
[
-
P]PP-InsP
( triangles) and analyzed by HPLC (see
``Experimental Procedures''). One-min fractions were
collected. The data are representative of three experiments. In the
lower panel,
750 dpm of peak E ( circles) were
mixed with
2000 dpm [
-
P]PP-InsP
( triangles, peak at 74 min) and analyzed by HPLC (as
described under ``Experimental Procedures,'' except
that buffer B increased from 44 to 90% between 10 and 120 min). One-min
fractions were collected. Immediately before the latter HPLC run, the
elution positions were determined for standards of diphosphoinositol
tetrakisphosphate and InsP
( triangles, peaks at 46
and 56 min, respectively); the standards were themselves spiked with
[
-
P]PP-InsP
(not shown, peak at
74 min; therefore, the column did not significantly differ in its
elution characteristics between the two runs). Data are typical of four
experiments.
Figure 8:
Interconversion of InsP,
PP-InsP
and
PP-InsP
-PP.
MIPP selectively
removed the P-labeled phosphate from
[
-
P]PP-InsP
-PP, but this
reaction was not detected in experiments with liver homogenate (see
above). Further evidence that MIPP cannot have made a significant
contribution to the metabolism of PP-InsP
-PP in vitro arose when liver homogenates were fractionated into 100,000
g particulate and soluble fractions; it was the latter
which contained the bulk (70-90%) of total phosphatase activities
toward PP-InsP
and PP-InsP
-PP (),
whereas MIPP is predominantly associated with 100,000
g particulate fractions
(12, 13) . Another discerning
feature of PP-InsP
and PP-InsP
-PP
dephosphorylation by MIPP was its relative insensitivity to
F
: in a typical experiment, the rate of metabolism of
PP-InsP
by MIPP (% dephosphorylation/ng protein/min) was
0.66 ± 0.04 in the absence of F
and 0.55
± 0.02 in the presence of 10 mM F
( n = 3). Similarly, the rate of metabolism of
PP-InsP
-PP was 0.014 ± 0.001 in the absence of
F
and 0.018 ± 0.001 in the presence of 10
mM F
( n = 4). In complete
contrast, F
strongly inhibited dephosphorylation by
homogenate of both PP-InsP
and PP-InsP
-PP
(IC
0.03 mM and 1.4 mM respectively,
Fig. 9
). It is worth emphasizing the 40-fold difference in the
sensitivities of the PP-InsP
phosphatase and the
PP-InsP
-PP phosphatase to inhibition by
F
. These data strengthen the idea that distinct
enzymes perform these two reactions.
Figure 9:
Effect of F upon
metabolism of PP-InsP
and PP-InsP
-PP by liver
homogenate. The metabolism of trace amounts of substrate was assayed as
described under ``Experimental Procedures'' using
assay buffer 2 with the indicated concentration of NaF.
[
-
P]PP-InsP
(1 nM) was
incubated with liver homogenate (4 µg protein/ml), and metabolism
was followed by assaying [
P]-P
release on gravity-fed columns (circles).
PP-[
H]InsP
-PP (0.2 nM) was
incubated with liver homogenate (80 µg protein/ml) and metabolism
was followed by HPLC ( squares). Data are representative of two
experiments.
Diphosphoinositol Polyphosphates in Intact
Cells
Levels of PP-[H]InsP
in
intact cells are about 2-3% those of
[
H]InsP
(Fig. 10, and see Refs.
3, 4, 9). In several earlier experiments PP-InsP
-PP was not
detected, possibly because of difficulties in recovering this material
from cell extracts
(3, 9) . Our improved methods (see
``Experimental Procedures'') now reproducibly show
(Fig. 10) that AR4-2J cells contain small quantities of
PP-[
H]InsP
-PP (0.6% of the
[
H]InsP
peak). This compound was
identified by its co-elution with a
P-labeled standard
(Fig. 10) and its metabolism by AR4-2J cell homogenates to
compounds which, upon HPLC, were identified as PP-InsP
and
InsP
(data not shown). Another new observation was that the
PP-[
H]InsP
isolated from intact cells
co-eluted with a [
-
P]PP-InsP
standard (Fig. 10). Thus, in AR4-2J cells, there was
no indication of the presence of significant amounts of
[
H]InsP
-PP, which elutes 2 min prior
to PP-InsP
(Figs. 5 and 10).
Figure 10:
Levels of
[H]InsP
,
PP-[
H]InsP
, and
PP-[
H]InsP
-PP in
[
H]inositol-labeled AR4-2J cells and the
effect of NaF. AR4-2J cells were cultured and labeled with
[
H]inositol as described under
``Experimental Procedures.'' Cells were incubated
for 40 min either without NaF ( panel A) or with 0.8
mM NaF ( panel B) or with 10 mM NaF
( panel C). Cells were then quenched, extracted, spiked with
standards of [
-
P]PP-InsP
plus
[
-
P]PP-InsP
-PP, and analyzed by
HPLC as described under ``Experimental Procedures.''
Note the difference in scale between the x axes depicting
H dpm/fraction ( circles) and
P
dpm/fraction ( triangles). The arrows in panel A depicts the individual peak elution positions of a mixture of
standards of [
H]InsP
-PP and
[
-
P]PP-InsP
, determined by HPLC
immediately before the sample was analyzed. The sizes of the peaks (in
dpm) were as follows (from left to right): panel A, InsP
= 64,729; PP-InsP
= 1958;
PP-InsP
-PP= 402. Total
[
H]InsP
(not shown) was 13,268.
Panel B, InsP
= 31,657; PP-InsP
= 32244; PP-InsP
-PP= 1947.
Total [
H]InsP
(not shown) was 14,925.
Panel C, InsP
= 42260; PP-InsP
= 24788; PP-InsP
-PP= 451.
Total [
H]InsP
(not shown) was 53,686.
Data are representative of three
experiments.
We have previously shown
that the ability of F to inhibit PP-InsP
dephosphorylation may be exploited as a metabolic trap to expose
the ongoing rate of turnover of PP-InsP
and InsP
(3) . We have now compared the effects of F
upon both PP-InsP
and PP-InsP
-PP.
AR4-2J cells were incubated for 40 min with 0.8 mM
F
, which is below the level that stimulates
phospholipase C through an AlF
-dependent activation of
GTP-binding proteins
(3) . Indeed,
[
H]InsP
levels were not significantly
affected by this protocol (see legend to Fig. 10). On the other
hand, F
mediated inhibition of PP-InsP
phosphatase caused levels of
PP-[
H]InsP
to increase 16-fold, at
the expense of a 50% decrease in the amount of
[
H]InsP
(Fig. 10). Presumably
because of the differential sensitivities of the PP-InsP
phosphatase and the PP-InsP
-PP phosphatase to
inhibition by F
(Fig. 9), the anion only
elevated levels of PP-[
H]InsP
-PP
5-fold.
,
phospholipase C was activated and total
[
H]InsP
increased 4-fold (legend to
Fig. 10
). Levels of PP-[
H]InsP
were still greatly elevated (13-fold), but levels of
PP-[
H]InsP
-PP were then similar to
those of control cells (Fig. 10). Thus, increasing
[F
] from 0.8 to 10 mM had
disparate effects upon the amounts of these two diphosphoinositol
polyphosphates in intact cells, indicating that there are some
important quantitative and perhaps qualitative differences in the
processes that control their turnover.
-PP is formed by
a mammalian tissue; we have also delineated the pathway by which
PP-InsP
-PP is synthesized and metabolized in vitro and in vivo. This novel information was obtained with the
aid of newly available
H- and
P-labeled
substrates, the metabolism of which was analyzed by anion-exchange
HPLC. We also utilized two purified enzyme preparations (MIPP
(13) and tobacco pyrophosphatase
(17) ) in structurally
diagnostic experiments. A fundamental conclusion of our study is that
two coupled substrate cycles participate in the interconversion of
PP-InsP
-PP and InsP
(Fig. 8). It will be
important to determine how these reactions are regulated, since there
are many precedents for the activities of substrate cycles being
targets of intracellular control processes. Indeed, the importance of
the substrate cycles that closely control the levels of individual
inositol lipids are well recognized to be key elements in the
regulation of cell-signaling itself. Thus, our new data represent a
solid foundation for further exploration into the mechanisms by which
the metabolism of PP-InsP
and PP-InsP
-PP might
be regulated and what the physiological significance of these compounds
might be.
H]inositol-labeled AR4-2J
cells, only small levels of PP-[
H]InsP
and PP-[
H]InsP
-PP were detected
(Fig. 10), even in comparison with total
[
H]InsP
(
1 µM(18) ). However, the absolute levels of both diphosphoinositols
may not be fully represented by the data in Fig. 10because
InsP
, with which these compounds are in isotopic
equilibrium
(3, 9) , is not itself completely labeled in
our studies (see Ref. 18). In any case, it is the rate of turnover of
PP-InsP
and PP-InsP
-PP that is so intriguing.
Here, F
is a useful analytical tool; this anion
strongly inhibited PP-InsP
phosphatase ( Fig. 9and
Ref. 3), exposing the ongoing rate of PP-InsP
synthesis
(Fig. 10). PP-InsP
-PP phosphatase is inhibited less
potently by F
(Fig. 9), which may at least
partly explain why treatment of intact cells with 0.8 mM
F
only elevated levels of PP-InsP
-PP
5-fold, well below the 16-fold increase in levels of PP-InsP
(Fig. 10). Thus, we are very likely to be underestimating
the true extent of PP-InsP
-PP turnover in intact cells.
Despite this, it was an important observation that further increasing
the [F
] to 10 mM prevented the
rise in PP-InsP
-PP levels from being ob-served
(Fig. 10). In contrast, the levels of PP-InsP
were
similar when cells were treated with either 0.8 or 10 mM
F
(Fig. 10). Experiments with cell-free systems
showed that the PP-InsP
kinase was not directly inhibited
by 10 mM F
(data not shown). However,
F
could have acted on the kinase indirectly, perhaps
secondarily to the activation of phospholipase C. Indeed,
Ca
mobilization has already been reported to inhibit
the InsP
kinase
(9) , and it is possible that the
PP-InsP
kinase is a more sensitive target. Further studies
into this aspect of the regulation of diphosphoinositol polyphosphate
metabolism will be an important future direction, aided by our
characterization of the pathway of PP-InsP
-PP synthesis and
metabolism.
of
PP-InsP
phosphatase and PP-InsP
-PP phosphatase
(Fig. 9) implies that these two dephosphorylations may be
performed by distinct enzymes. This possibility is consistent with the
different positional specificity of the two reactions, which,
incidentally, contrasts with the reactivity of MIPP toward the same
diphosphate group on both compounds. The purification of the
phosphatases that participate in the metabolism of the
diphosphoinositols will provide more definite information on their
number and their specificity.
tighter
than any other known ligand
(5, 6) .
In the case of coatomer, binding of PP-InsP
was
associated with gating of the inherant K
-channel
activity of this protein complex
(6) . PP-InsP
binding to AP-3 inhibited this protein's ability to promote
clathrin assembly
(5) . Our understanding of the structural
specificity of both ligand binding and the ensuing physiological
effects would now be improved if we investigated the interactions of
PP-InsP
-PP with these proteins and the cellular processes
that they regulate. The relative cellular concentrations of
PP-InsP
and PP-InsP
-PP (described in
Fig. 10
) will also help evaluate the physiological significance
of any effects of these polyphosphates that we observe in
vitro.
,
Ins
(1, 3, 4, 5, 6) P
,
and InsP
(12) , has now been shown to specifically
attack the same diphosphate group of PP-InsP
and
PP-InsP
-PP (Figs. 3 and 4). Since MIPP is nonspecific in
its removal of every phosphate of InsP
, our data
demonstrate that the diphosphate groups confer a profound increase in
the specificity of this enzyme. This will likely prove to be a
significant observation in the elucidation of the tertiary structure of
the active site. On the other hand, experiments with both homogenates
and intact cells indicated that MIPP did not significantly contribute
to the metabolism of either PP-InsP
-PP or
PP-InsP
. As it happens, MIPP resides inside the endoplasmic
reticulum (at least in liver)
(19) . We have found that both
PP-InsP
and PP-InsP
-PP are unable to gain
access to MIPP in hepatic microsomes unless they are permeabilized with
detergent (data not shown). This segregation of MIPP from its potential
substrates probably explains why intact AR4-2J cells do not
contain significant levels of InsP
-PP, which is the unique
product of MIPP-dependent attack on PP-InsP
-PP. However,
the mammalian erythrocyte is an example of a cell that contains MIPP in
its plasma membranes
(13) . Perhaps MIPP has greater access to
diphosphoinositol polyphosphates in this and maybe some other cell
types; our data show that one investigative approach to this issue
would be to search for InsP
-PP in intact cells.
-PP establishes a framework for
further studies into the physiological significance of
diphosphoinositol polyphosphates. We can surely be confident that the
cell reaps a substantial reward for investing considerable amounts of
cellular energy into the ongoing turnover of these compounds.
Table:
Phosphatase activities towards PP-InsP and PP-InsP
-PP in the 100,000
g
supernatant and particulate fractions of liver homogenate
g soluble and particulate
fractions, were prepared and assayed for phosphatase activities as
described under ``Experimental Procedures'' using
4
µg protein ml (for PP-InsP
phosphatase) or
40
µg protein/ml (for PP-InsP
-PP phosphatase).
PP-InsP
phosphatase activity was assayed by measuring
[
P]-P
release from
[
P]PP-InsP
using gravity fed
columns. Dephosphorylation of
PP-[
H]InsP
-PP was assayed by HPLC.
Reaction rates were calculated using the first-order rate equation,
i.e. [ S]
=
[ S]
e
, where
k is the first-order rate constant in units of
min
, and [ S] is the concentration
of substrate at times 0 and t. Data are means ±
standard errors from the number of experiments indicated in
parentheses.
represents inositol
1,4,5-triphosphate); InsP
-PP, diphosphoinositol
tetrakisphosphate (a diesterphosphate derivative of InsP
);
PP-InsP
(formerly InsP
P (3)) and
InsP
-PP, diphosphoinositol pentakisphosphate isomers
(diesterphosphate derivatives of InsP
) which differ in the
position of the diphosphate group on the inositol ring;
PP-InsP
-PP, bis-diphosphoinositol tetrakisphosphate.
MIPP, multiple inositol polyphosphate phosphatase (formerly
Ins(1,3,4,5)P
3-phosphatase (see Ref. 13)); HPLC, high
performance liquid chromatography; CHAPS,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic
acid; dpm, disintegrations/min.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.