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J Biol Chem, Vol. 274, Issue 27, 18973-18980, July 2, 1999
From the Ca2+-activated
Cl There is considerable interest in the idea that
Ins(1,4,5)P3 1
and Ins(1,3,4,5)P4 (Fig. 1) act in a co-ordinated manner as
mediators of stimulus-dependent Ca2+
mobilization (1, 2). This has naturally led us to consider that the
5-phosphatases that degrade Ins(1,4,5)P3 and
Ins(1,3,4,5)P4 (3) are signaling "off-switches." This
in turn has created the impression that the pathway by which these two
inositol phosphates are dephosphorylated serves only as a conduit that
replenishes the free inositol pool. In contrast, we have recently
suggested that one of these downstream products, namely
Ins(1,3,4)P3, should be viewed in an important
cell-signaling context (4). This new hypothesis comes from the
observation that a rat hepatic Ins(3,4,5,6)P4 1-kinase was inhibited in vitro by Ins(1,3,4)P3
(4, 5). The reason that this effect of Ins(1,3,4)P3 upon
Ins(3,4,5,6)P4 metabolism is of such interest is that
Ins(3,4,5,6)P4 is an inhibitor of the conductance of
the calcium-activated Cl The cellular accumulation of Ins(3,4,5,6)P4 is known to
correlate well with receptor-dependent changes in PLC
activity, but the molecular mechanisms that link these two events have
not been fully elucidated (14). Our current hypothesis (15, 16) is that
cellular levels of Ins(3,4,5,6)P4 depend upon a dynamic
balance between two competing enzyme activities acting in a closed
substrate cycle: Ins(1,3,4,5,6)P5 1-phosphatase and
Ins(3,4,5,6)P4 1-kinase (Fig. 1). The poise of this cycle
is proposed to be regulated in such a manner that it can shift in favor
of Ins(3,4,5,6)P4 accumulation whenever PLC is activated,
perhaps through inhibition of the Ins(3,4,5,6)P4 1-kinase
by Ins(1,3,4)P3 (Fig. 1). However, to date such inhibition
has only been observed in studies with the purified rat hepatic kinase
(4, 5). No direct evidence has previously been published that indicates
Ins(1,3,4)P3 can regulate Ins(3,4,5,6)P4
1-kinase activity in intact cells; it was a goal of the current study
to explore this issue.
In order to investigate if Ins(1,3,4)P3 can regulate
Ins(3,4,5,6)P4 levels in intact cells, it was necessary to
increase cellular levels of Ins(1,3,4)P3 specifically,
under conditions where PLC activity was not activated. In this way, we
could avoid the possibility of Ins(3,4,5,6)P4 metabolism
also being regulated by the many additional signal transduction
processes that are activated downstream of PLC. To this end, the
development of cell-permeant, bioactivatable analogues of inositol
phosphates (7, 17, 18) has provided us with new opportunities to
examine the functions of inositol polyphosphates in intact cells. The
charge-masking groups that enable these derivatives to permeate into
cells are hydrolyzed by intracellular esterases, releasing the native
isomer (7, 17). In this study we used a new cell-permeant analogue,
2,5,6-tri-O-butyryl-myo-inositol 1,3,4-trisphosphate hexakis(acetoxymethyl) ester
(Bt3Ins(1,3,4)P3/AM; Ref. 19), to elevate the
cellular concentration of Ins(1,3,4)P3 inside rat
pancreatoma (AR4-2J) cells.
This experimental approach was important for another reason. Rather
than Ins(1,3,4)P3 regulating an enzyme of
Ins(3,4,5,6)P4 metabolism (Fig.
1), in principle,
Ins(1,3,4)P3 could instead elevate
Ins(3,4,5,6)P4 concentration simply by being metabolized to
it (i.e. a mass action effect). For example,
Ins(1,3,4)P3 can be converted to Ins(3,4,5,6)P4
by the sequential actions of Ins(1,3,4)P3 6-kinase,
Ins(1,3,4,6)P4 5-kinase, and Ins(1,3,4,5,6)P5
1-phosphatase (15, 16, 20). Others have proposed an alternative pathway for de novo Ins(3,4,5,6)P4 synthesis, which
requires the sequential actions of Ins(1,3,4)P3 6-kinase,
Ins(1,3,4,6)P4 1-phosphatase, and Ins(3,4,6)P3
5-kinase (21-23). We have now used AR4-2J cells to examine whether
Ins(1,3,4)P3 alters Ins(3,4,5,6)P4 levels by mass action effects. Our strategy was based upon first prelabeling the
metabolic pool of Ins(3,4,5,6)P4 to steady-state with
[3H]inositol. These cells were then treated with
non-radiolabeled Bt3Ins(1,3,4)P3/AM. We
investigated if there was any significant metabolic flux from
Ins(1,3,4)P3 to Ins(3,4,5,6)P4, which would have revealed itself by tending to decrease the amount of
3H label in the
[3H]Ins(3,4,5,6)P4 pool, due to a pulse-chase
effect (15).
Another feature of an effective signal transduction process relates to
its specificity. If the biological effects of a signaling compound
cannot be imitated by its products and precursors, this provides
sensitivity in the signaling "on" and "off" switches. In the
case of signaling by Ins(1,3,4)P3, the "on-switch" is
dephosphorylation of Ins(1,3,4,5)P4 (3). This process is
particularly sensitive, as Ins(1,3,4,5)P4 is a 290-fold
weaker inhibitor of the 1-kinase than is Ins(1,3,4)P3 (4).
We have now turned our attention to considering how effective is the
dephosphorylation of Ins(1,3,4)P3 as a signaling
off-switch. In vivo, both 4- and 1-phosphatases actively
degrade Ins(1,3,4)P3 to Ins(1,3)P2 and
Ins(3,4)P2, respectively (24-26). We have therefore
determined the potency with which these bisphosphate degradation
products inhibit the 1-kinase.
There was one further aspect to this study that is relevant to the
development of Ins(3,4,5,6)P4 agonists and antagonists for
pharmacological intervention in the signaling actions of
Ins(3,4,5,6)P4 (18, 27). This goal is directed at diseases
that might be treated by either up-regulating or down-regulating
Ca2+-activated Cl Materials--
[3H]Inositol was purchased from
American Radiolabeled Chemicals Inc. or NEN Life Science Products.
[3H]Ins(1,3,4)P3 was prepared by
dephosphorylating [3H]Ins(1,3,4,5)P4 (20 Ci/mmol, NEN Life Science Products) with recombinant
Ins(1,3,4,5)P4 5-phosphatase, which was kindly provided by
Dr. C. Erneux (29). Ins(1,4)P2 was purchased from Sigma. D/L-2,5,6-Tri-O-butyryl-myo-inositol
1,3,4-trisphosphate-hexakis(acetoxymethyl) ester
(D/L-Bt3Ins(1,3,4)P3/AM) was
synthesized as described previously (19). In some experiments, we used
enantiomerically pure
D-Bt3Ins(1,3,4)P3/AM. This compound
was prepared from the enantiomerically pure precursor, 4-O-benzyl-1,2:5,6-di-O-cyclohexylidene-myo-inositol
(30). Alkylation of the hydroxy group with benzyl bromide in dimethyl
formamide at 50 °C for 20 h and in the presence of an excess of
sodium hydride and tetrabutyl ammonium iodide afforded
3,4-di-O-benzyl-1,2:5,6-di-O-cyclohexylidene-myo-inositol. Purification by preparative HPLC (92% MeOH, RP-18, 10 µm, 50 × 250 mm, 40 ml/min) gave 55% yield as an oil
[
Ins(1,3)P2, D/L-Ins(3,4)P2,
D/L-Ins(1,2,4)P3, Ins(1,2,3)P3,
D/L-Ins(1,2,4,6)P4, and
D/L-Ins(1,2,3,4)P4 were prepared as described previously (31-33). Ins(1,3,6)P3 and
Ins(1,4,6)P3 were prepared as described previously (34,
35). Sources of other inositol phosphates are given elsewhere (4,
8).
Bombesin, bovine serum albumin, phosphocreatine, phosphocreatine
kinase, heparin agarose resin (type II and IIIs), and protease inhibitors were purchased from Sigma. The
calmodulin-dependent protein kinase (CaM KII) was obtained
from New England Biolabs. Protein kinases A and C, and their assay kits
(SpinZyme Format), were the products of Pierce. The UNO Q12 anion
exchange column was acquired from Bio-Rad Laboratories. Polyethylene
glycol 4000 was purchased from Fluka. Frozen bovine aorta were
purchased from Pel-Freez Biological.
Assay of Ins(3,4,5,6)P4 1-Kinase--
The 1-kinase
activity was assayed as described before (4). Briefly, 10-20 µl of
enzyme was incubated at 37 °C in a final volume of 100 µl
containing about 4000 dpm [3H]Ins(3,4,5,6)P4,
which was adjusted to a concentration of 0.25 µM with
non-radioactive substrate, 20 mM HEPES (pH 7.2), 6 mM MgSO4, 0.4 mg/ml saponin, 100 mM
KCl, 0.3 mg/ml bovine serum albumin, 2 µM
InsP6, 5 mM ATP, 10 mM
phosphocreatine, and 2.5 Sigma units of phosphocreatine kinase. After
30 min., the reaction was stopped by quenching with 1 ml of ice-cold
medium containing 1 mg/ml InsP6, 0.2 M ammonium
formate, and 0.1 M formic acid. The quenched reactions were
diluted to 10 ml with deionized water, and chromatographed on Bio-Rad
gravity-fed columns using AG 1-X8 ion exchange resin.
For some assays, the 1-kinase was preincubated at 30 °C for 10 min
with (a) 125 units of the catalytic subunit of protein kinase A, (b) 0.2 unit of protein kinase C, (c)
600 units of calmodulin, or (d) 500 units of CaM KII,
preactivated with calmodulin/Ca2+ (New England Biolabs).
The protein kinases used in these experiments were all shown to be
active in control experiments (assay kits for protein kinases A and C
were supplied by Pierce; the CaM KII was checked using a kit purchased
from Upstate Biochemicals).
The 1-kinase was also used as a diagnostic tool to verify the nature of
HPLC-purified [3H]Ins(3,4,5,6)P4. In these
incubations, 45 µl of purified 1-kinase was added to 225 µl of
medium containing 67 mM HEPES (pH 8.0 with KOH), 0.7 mM EDTA, 8.7 mM MgSO4, 6.7 mM ATP, 13.3 mM phosphocreatine, 1.33 µM InsP6, and 6 Sigma units of
phosphocreatine kinase. Then, 30 µl of the appropriate HPLC fraction
was added (which brought the final pH to approximately 6.5). Reactions
(at 37 °C) were allowed to proceed to completion (over a 3-h
period), and then the amount of [3H]InsP5
formed was determined using gravity-fed ion-exchange columns, as
described above.
Purification of Ins(3,4,5,6)P4 1-Kinase--
Frozen
bovine aortas were thawed on ice, the attached fat was removed, and
then the aorta were pulverized in a meat grinder. In a typical
preparation, 300-350 g of ground aortas were homogenized in two
volumes of 50 mM bis-Tris (pH 7.0), 1 mM EGTA,
1 µg/ml leupeptin, 1 µg/ml pepstatin A, and 1 mM
phenylmethylsulfonyl fluoride in a tissue blender.
The homogenate was filtered through four layers of cheesecloth, and a
10-30% (w/v) polyethylene glycol 4000 precipitate was prepared. The
resultant pellet was resuspended in 100 ml of Buffer A containing 50 mM bis-Tris (pH 7.0), 1 mM EGTA, 1 µg/ml
leupeptin, and 1 µg/ml pepstatin A. The suspension was filtered and
loaded at a flow rate of 1 ml/min onto a heparin-agarose type II column (3.2 × 24 cm). After washing with 300 ml of Buffer A at flow rate of 1.5 ml/min, the bound protein was eluted with a linear gradient of
0-30 mM of sodium pyrophosphate in Buffer A.
The peak fractions of enzyme activity eluted from the heparin column
were pooled, then frozen and stored at
Peak fractions of enzyme activity eluted from the UNO Q12 column were
pooled, dialyzed against 2 liters of 25 mM bis-Tris (pH
7.0) at 4 °C for 3 h, and loaded on to heparin-agarose type IIIs (1.1 × 13.5 cm), which was pre-equilibrated with 50 ml of Buffer A. A constant flow rate of 0.5 ml/min was maintained throughout. After washing with 60 ml of Buffer A, the bound protein was eluted with
a linear gradient of 0-300 mM NaCl in Buffer A, followed by 60 ml of 1 M NaCl in Buffer A.
The protein concentration of the 1-kinase preparation was determined
using Bio-Rad Protein Assay Dye Reagent with bovine serum albumin as
standard. Final enzyme preparations were stored in 10% glycerol plus 1 mg/ml bovine serum albumin at Gel Filtration--
A 1-ml aliquot of a resuspension of a
10-30% PEG precipitation was loaded at a flow rate of 0.25 ml/min to
Sephacryl S100 column (2.0 × 86 cm), which was pre-equilibrated
with 600 ml of bis-Tris buffer containing 50 mM bis-Tris
(pH 7.0), 1 mM EGTA, 1 µg/ml leupeptin, 1 µg/ml
pepstatin A, and 100 mM NaCl. The protein was then
chromatographed using the same buffer at a constant 0.25 ml/min flow
rate. Fractions (5ml) were collected and assayed for enzyme activity.
The column was calibrated under the exactly same conditions using
bovine serum albumin, chicken ovalbumin, equine myoglobin, and vitamin
B-12.
Culturing and Incubation of AR4-2J Cells--
The AR4-2J
pancreatoma cells were cultured in Dulbecco's modified Eagle's medium
containing 4.5 g/liter glucose, 10% fetal bovine serum, 2 mM glutamine, 500 units/ml penicillin, and 500 µg/ml
streptomycin, with 10% conditioned medium, and harvested by brief
trypsinization. Either 2.0 × 105 or 1.2 × 106 cells were seeded in 24-well or 6-well tissue culture
plates, respectively. Cells were labeled with 75-150 µCi/ml
[3H]myo-inositol for 4 days (medium was
replaced on the 3rd day) in 700 µl (for 24-well plates) or 3 ml (for
6-well plates) of the above culture medium. After completion of the
labeling protocol, the culture medium was aspirated and the cells were
washed twice with Krebs/Ringer/HEPES solution (15). Cells were then
incubated in 300 µl (for 24-well) or 1 ml (for 6-well) of
Krebs/Ringer/HEPES solution for 2 h. Then 20 mM LiCl
was added, and 20 min later cells were treated for the indicated time
with (i) a cell-permeant inositol phosphate, (ii) vehicle, or (iii) bombesin.
Cells were quenched and neutralized, and the inositol phosphates were
separated by HPLC as described elsewhere (36). Radioactivity was either
counted on-line, using a Radiomatic Flo-1, or recovered in 1-ml
fractions. The levels of 3H-labeled inositol phosphates
were normalized as a ratio to cellular levels of
[3H]InsP6; the latter were unaffected by any
of the experimental protocols performed in this study.
For some experiments, after the AR4-2J cells were harvested, cells were
collected by centrifugation in serum-containing culture medium. The
pellet was washed in HEPES-buffered saline, and then a lysate was
prepared by resuspending the packed cells in an equal volume of
ice-cold lysis buffer comprising: 50 mM KCl, 50 mM HEPES (pH 7.2), 1 mM EDTA, 5 mM
ATP, 4 mM CHAPS, 0.4 mM phenylmethylsulfonyl fluoride, 40 µM E-64, 10 µM leupeptin, 3 µM pepstatin.
Assay of Mass Levels of Ins(1,3,4)P3 inside
AR4-2J Cells--
Following the HPLC fractionation of extracts
of [3H]inositol-labeled cells (see above), 1-ml fractions
were saved, from which 25-µl aliquots were counted for radioactivity
so as to identify the Ins(1,3,4)P3 peak (because of the low
levels of endogenous [3H]Ins(1,3,4)P3,
samples were "spiked" with 4000 dpm
[3H]Ins(1,3,4)P3 (20 Ci/mmol) before they
were applied to the HPLC column). The Ins(1,3,4)P3 peak was
then desalted (37) and resuspended in 60 µl Ins(1,3,4)P3
6-kinase assay buffer: 50 mM KCl, 50 mM HEPES,
pH 7.2, 10 mM phosphocreatine, 6 mM ATP, 8 mM MgSO4, 25 Sigma units/ml phosphocreatine
kinase, 0.5 mg/ml bovine serum albumin. Recovery of
[3H]Ins(1,3,4)P3 from the cell extract was
typically 70-75%. Each Ins(1,3,4)P3 sample was then
divided into two equal portions, named A and B. The
Ins(1,3,4)P3 was depleted from portion B by its incubation
for 60 min at 37 °C with 0.1 µg of recombinant Ins(1,3,4)P3 1-phosphatase (kindly supplied by Dr. J. York,
Duke University, Durham, NC). Control experiments showed that the
extent of Ins(1,3,4)P3 hydrolysis exceeded 95%. The
Ins(1,3,4)P3 1-phosphatase was then heat-inactivated (3 min
at 100 °C). Aliquots (9 µl) of either portion A or B were then
incubated in triplicate for 20 min in a total volume of 50 µl
containing purified Ins(1,3,4)P3 6-kinase, its assay buffer
(see above), plus approximately 4000 dpm
[3H]Ins(1,3,4)P3. Other incubations (in
quadruplicate) were performed that, in place of the
Ins(1,3,4)P3 from a cell extract, contained between 0.1 and
50 pmol of non-radiolabeled Ins(1,3,4)P3. A standard curve
was constructed from the decrease in the phosphorylation of
[3H]Ins(1,3,4)P3 to
[3H]Ins(1,3,4,6)P4 that was observed as the
Ins(1,3,4)P3 mass was increased. The difference in
Ins(1,3,4)P3 mass values between portions A and B
(i.e. the amounts hydrolyzed by the Ins(1,3,4)P3 1-phosphatase) were taken to represent the quantity of
Ins(1,3,4)P3 in the original HPLC-purified sample.
Purification and Properties of the Ins(3,4,5,6)P4
1-Kinase from Bovine Aorta--
In our earlier study with the
Ins(3,4,5,6)P4 1-kinase in rat liver, the enzyme was found
to be extremely labile, and we were only able to elicit a 1600-fold
purification with a 1% yield (4). No other laboratory has published a
purification protocol for this enzyme. We developed a new strategy for
the current study, the most notable aspect of which was the
efficiency of an affinity purification
step using heparin IIIS (Fig. 2; Table
I). Thus, using homogenates of bovine
aortas as starting material, we purified the 1-kinase 22,500-fold with
a 13% yield (Fig. 2; Table I). Our preparations of 1-kinase have an
affinity for Ins(3,4,5,6)P4 (0.1-0.2 µM,
data not shown) that is very similar to the substrate affinity of the
rat hepatic enzyme (4). Gel filtration indicated the size of the enzyme
to be 46 kDa (Fig. 3), which is also
similar to that of the rat liver enzyme (4). The 1-kinase was strongly inhibited by Ins(1,3,4)P3 (IC50 = 0.17 µM, Fig. 4A).
The enantiomer of Ins(1,3,4)P3, namely
Ins(1,3,6)P3, was a 100-fold weaker inhibitor of the
Ins(3,4,5,6)P4 1-kinase (Table
II). The activity of the purified
Ins(3,4,5,6)P4 1-kinase was unaffected when 20 mM KCl in the incubation buffer was substituted with 20 mM LiCl (data not shown).
The purified 1-kinase was reconstituted with either protein kinase A,
protein kinase C, Ca2+/calmodulin, or CaM KII. In no case
was there any modification to 1-kinase activity, nor was there any
effect upon the potency of inhibition by Ins(1,3,4)P3 (data
not shown). Positive controls for each of these protein kinases were
obtained by verifying their activities using appropriate assay kits
(see "Experimental Procedures").
The Effect of Bt3Ins(1,3,4)P3/AM upon
[3H]Inositol Phosphates in AR4-2J Cells--
One
aim of this study was to use intact cells to examine the physiological
relevance of the inhibition of the Ins(3,4,5,6)P4 1-kinase
by Ins(1,3,4)P3, since this effect has only previously been
observed with the isolated enzyme (4, 5). For these experiments we used
a cell-permeant and bioactivatable analogue of
Ins(1,3,4)P3. There are several important aspects of the
experimental protocol that should be emphasized. First, we chose to
perform these studies with AR4-2J pancreatoma cells. This decision
reflects the fact that, in these cells, Ins(1,4,5)P3 is
primarily metabolized by the 5-phosphatase pathway; the
Ins(1,4,5)P3 3-kinase is a quantitatively minor metabolic
route (38, 39), so the ensuing downstream products, including
Ins(1,3,4)P3, are present at unusually low levels in
"resting" cells (15). This low base line improves the
"signal-to-noise" ratio, making our experiments more sensitive to
small experimental manipulations of Ins(1,3,4)P3 levels. We also checked that the Ins(3,4,5,6)P4 1-kinase in these
cells was sensitive to inhibition by Ins(1,3,4)P3. In cell
lysates, 10 µM Ins(1,3,4)P3 inhibited the
rate of phosphorylation of Ins(3,4,5,6)P4 by greater than
95% (Fig. 4B).
A second aspect of our experimental protocol that is worth emphasizing
is that we preincubated cells with [3H]inositol for 4 days. At this point the cellular pool of Ins(3,4,5,6)P4 was
radiolabeled to steady state (16, 39). Thus, any increases in
[3H]Ins(3,4,5,6)P4 that we observed truly
reflect elevated mass levels of this polyphosphate. Third, we were
concerned that cell-permeant analogues of inositol phosphates are
typically de-esterified relatively slowly (17), such that a rapid rate
of Ins(1,3,4)P3 dephosphorylation would act to prevent the
accumulation of this compound inside cells. Our cell incubation medium
was therefore supplemented with lithium, so as to inhibit the
Ins(1,3,4)P3 1-phosphatase (40). However, it should be
noted that this is only a partial solution of this particular problem,
since lithium does not inhibit the less active, alternative pathway of
Ins(1,3,4)P3 dephosphorylation by a 4-phosphatase (41).
Control experiments indicated that this lithium treatment did not
affect levels of [3H]Ins(3,4,5,6)P4 (data not shown).
Extracts of [3H]inositol-labeled control cells were
resolved by HPLC, and the various 3H-labeled inositol
phosphates were assayed using an on-line scintillation counter (Fig.
5, upper panel). We also
analyzed extracts from cells treated with 200 µM
D/L-Bt3Ins(1,3,4)P3/AM (Fig. 5,
lower panel). In these experiments, any metabolic conversion
of Ins(1,3,4)P3 to Ins(3,4,5,6)P4 would, by a
pulse-chase effect, tend to decrease the amount of
[3H]label in the Ins(3,4,5,6)P4 pool (15). In
fact, the opposite result was obtained; the size of the
[3H]Ins(3,4,5,6)P4 peak increased
about 2-fold (Fig. 5, Table III). The
elevation in [3H]Ins(3,4,5,6)P4 levels in our
experiments (Fig. 5) cannot be caused by an increased flux of
non-radiolabeled Ins(1,3,4)P3 into the
[3H]Ins(3,4,5,6)P4 metabolic pool. Note also
that there were no significant changes in the sizes of other
[3H]inositol-labeled peaks after treatment with
D/L-Bt3Ins(1,3,4)P3/AM. To control
for the possibility of nonspecific effects of cell-permeant inositol
phosphates, we also incubated AR4-2J cells with 200 µM D-Bt2Ins(1,4,5,6)P4/AM (which does
not affect 1-kinase activity, see below), and this had no effect upon
the [3H]inositol-polyphosphate profiles (data not
shown).
We were only able to synthesize limited amounts of
Bt3Ins(1,3,4)P3/AM, and so we did not have
sufficient material to perform detailed dose-response curves or time
courses. However, we did observe that the treatment of cells with 400 µM
D/L-Bt3Ins(1,3,4)P3/AM approximately doubled the elevation in
[3H]Ins(3,4,5,6)P4 levels, compared with the
effect of 200 µM
D/L-Bt3Ins(1,3,4)P3/AM; 20 µM
D/L-Bt3Ins(1,3,4)P3/AM did not
affect [3H]Ins(3,4,5,6)P4 levels (data not
shown). One factor that must be taken into account when using these
types of cell-permeant analogues is the relatively slow rate of their
activation by intracellular esterases (17).
The Ins(3,4,5,6)P4 1-kinase was used as a diagnostic tool
so as to confirm the identity of the
[3H]Ins(3,4,5,6)P4 peak that eluted from the
HPLC. For these experiments, we did not assay
[3H]inositol phosphates by on-line scintillation
counting. Instead, individual HPLC fractions were collected. The amount
of material loaded onto the HPLC column was increased by culturing
greater numbers of cells in larger wells (see "Experimental
Procedures"). Thus, the quantity of
[3H]Ins(3,4,5,6)P4 was larger than in the
experiments described by Fig. 5. Aliquots of the putative
[3H]Ins(3,4,5,6)P4 peak were incubated with
the purified 1-kinase (see "Experimental Procedures"). The
[3H]InsP5 formed, after the assays had been
allowed to proceed to completion, was used to identify the amounts of
[3H]Ins(3,4,5,6)P4 in the original cell
extracts: 6964 ± 35 dpm from control cells, and 13,322 ± 131 dpm after treatment with D/L-Bt3Ins(1,3,4)P3/AM. These
experiments confirm that levels of
[3H]Ins(3,4,5,6)P4 in intact cells were
elevated 2-fold by Ins(1,3,4)P3.
We next investigated if the treatment of AR4-2J cells with
Bt3Ins(1,3,4)P3/AM elicited increases in levels
of Ins(1,3,4)P3 and Ins(3,4,5,6)P4 that were
physiologically relevant. For these experiments, we compared the
effects of the cell-permeant analogue with those of
receptor-dependent activation of PLC, using bombesin as the
agonist. [3H]Inositol-labeled AR4-2J cells were treated
for 20 min with 200 nM bombesin, or for 60 min with either
100 µM
D-Bt3Ins(1,3,4)P3/AM, or vehicle.
The cell-permeant derivative used in these experiments was from a batch
that was different from that used in the experiments described above.
This particular batch of the analogue was also enantiomerically pure,
and therefore it was used at half the concentration of the
D/L-Bt3Ins(1,3,4)P3/AM used in the
experiments described above. In three experiments, 60-min treatment of
AR4-2J cells with 100 µM
D-Bt3Ins(1,3,4)P3/AM elevated
[3H]Ins(3,4,5,6)P4 levels 1.9 ± 0.3-fold (Fig. 6), which is not significantly different from the results obtained with 200 µM D/L-Bt3Ins(1,3,4)P3/AM (Table III).
The fact that these changes in Ins(3,4,5,6)P4 levels were
within a physiologically relevant range was confirmed by
comparison with the effects of bombesin, which led to a nearly 5-fold
increase in [3H]Ins(3,4,5,6)P4 levels (Fig.
6).
We also compared the effects of
D-Bt3Ins(1,3,4)P3/AM and bombesin
upon cellular Ins(1,3,4)P3 levels (see under
"Experimental Procedures"). Stimulation of AR4-2J cells with
bombesin elicited a 13-fold increase in levels of
Ins(1,3,4)P3 (Fig. 6); treatment with
D-Bt3Ins(1,3,4)P3/AM elevated
Ins(1,3,4)P3 levels 2.7-fold, an effect that may therefore
be considered to be within the physiologically relevant range (Fig.
6).
Effects of InsP2 isomers on Ins(3,4,5,6)P4
1-Kinase Activity--
There is another important issue that is
relevant to our evaluation of the significance of
Ins(1,3,4)P3 as an intracellular signal. The extent to
which metabolites of Ins(1,3,4)P3 also inhibit the 1-kinase
contributes to the efficiency with which the cell "switches
off" the Ins(1,3,4)P3 signal. The major route of
Ins(1,3,4)P3 metabolism in vivo is by its
dephosphorylation, by separate 4- and 1-phosphatases, to
Ins(1,3)P2 and Ins(3,4)P2, respectively (24-26). We therefore determined the potency with which these
bisphosphate degradation products inhibited the 1-kinase, relative to
Ins(1,3,4)P3. The IC50 for
Ins(1,3,4)P3 was 0.17 µM (Table II and Fig.
4A). In contrast, both Ins(1,3)P2 and
D/L-Ins(3,4)P2 did not significantly affect
1-kinase activity until their concentration exceeded 3 µM
(Fig. 7A); therefore, these
bisphosphates were at least 100-fold weaker inhibitors. Thus,
dephosphorylation of Ins(1,3,4)P3 is a very effective
off-switch for relieving inhibition of 1-kinase activity.
The product of 5-phosphatase attack upon Ins(1,4,5)P3,
namely Ins(1,4)P2, was found to be a poor inhibitor of the
1-kinase (Fig. 7A). This is also an important observation
that demonstrates that it specifically requires
Ins(1,4,5)P3 metabolism through the 3-kinase pathway to
yield an inhibitor of the Ins(3,4,5,6)P4 1-kinase. In
addition, this result shows that all three phosphates of
Ins(1,3,4)P3 contribute substantially to the specificity of inhibition of the 1-kinase.
The Contribution of the 2-Phosphate Group to Inhibition of the
1-Kinase--
We found that, in an appropriate context, the addition
of a 2-phosphate to the inositol ring could make a positive
contribution to inhibitory potency; this is illustrated by the
observation that Ins(1,2,3)P3 was a more potent inhibitor
of the 1-kinase (IC50 = 4.2 µM, Table II)
than was Ins(1,3)P2 (IC50 > 30 µM, Fig. 7A).
D/L-Ins(1,2,3,4)P4 was also a relatively potent
1-kinase inhibitor (IC50 = 1.6 µM, Table II).
D- and/or L-Ins(1,2,3,4)P4 and
Ins(1,2,3)P3 are found in mammalian cells, the latter at
concentrations of up to 10 µM (15, 42, 43). These
polyphosphates may be formed by dephosphorylation of InsP6
(15, 42, 43). Among the inositol phosphates that we tested that contain
a 2-phosphate (Table II), D/L-Ins(1,2,4)P3 was
the most potent inhibitor of the 1-kinase (IC50 = 0.7 µM, Fig. 7B). Neither D- nor
L-Ins(1,2,4)P3 have been detected in mammalian
cells, even under circumstances where, had they been present, they
should have revealed themselves to some detailed structural analyses
(43). As the inhibitory action of Ins(1,3,4)P3 upon the
1-kinase was so effectively imitated by
D/L-Ins(1,2,4)P3 (Table II), the latter could
be a useful new starting point for developing drugs that might
intervene in the 1-kinase/1-phosphatase cycle with therapeutic benefit.
Two aspects of this study are of particular importance to
improving our understanding of the role of Ins(1,3,4)P3 as
an intracellular signal. First, by using a new, cell-permeant,
bioactivatable form of Ins(1,3,4)P3, we have shown that
Ins(1,3,4)P3 elevates levels of Ins(3,4,5,6)P4
inside intact cells, without Ins(1,3,4)P3 being metabolized
to Ins(3,4,5,6)P4. Second, we have discovered that Ins(1,3,4)P3-mediated inhibition of the
Ins(3,4,5,6)P4 1-kinase is very efficiently switched off by
dephosphorylation of Ins(1,3,4)P3. Prior to this study,
there had not been a consensus of opinion concerning the mechanism by
which PLC activation is coupled to increases in
Ins(3,4,5,6)P4 levels. This is an important issue to
resolve, if we are to understand how the cell regulates
Ins(3,4,5,6)P4-mediated inhibition of conductance through
Ca2+-activated Cl D/L-Bt3Ins(1,3,4)P3/AM provided a
means of delivering a pulse of non-radiolabeled
Ins(1,3,4)P3 into intact [3H]inositol-labeled
cells. If, as a consequence of this treatment, there had been a
significant metabolic flux from Ins(1,3,4)P3 into the
Ins(3,4,5,6)P4 pool, this would have been expected to decrease the [3H]label in this
Ins(3,4,5,6)P4 pool by a pulse-chase effect (15). In fact,
the opposite result was obtained;
D/L-Bt3Ins(1,3,4)P3/AM elevated intracellular levels of
[3H]Ins(3,4,5,6)P4 (Table III). Thus, for the
first time, we have shown that Ins(1,3,4)P3 regulates
levels of Ins(3,4,5,6)P4 inside cells, without
Ins(1,3,4)P3 being metabolized to
Ins(3,4,5,6)P4.
An Ins(1,3,4)P3-mediated inhibition of
Ins(3,4,5,6)P4 1-kinase in intact cells would be
expected to alter the poise of the substrate cycle that
interconverts Ins(3,4,5,6)P4 with
Ins(1,3,4,5,6)P5 (Fig. 1). There was not a
statistically significant decrease in the levels of
[3H]Ins(1,3,4,5,6)P5 after treatment with
D/L-Bt3Ins(1,3,4)P3/AM (Table III),
but this is not unexpected. The
[3H]Ins(1,3,4,5,6)P5 pool is relatively
large, and it is difficult to detect the small changes in its size that
are sufficient to support a 2-fold increase in the size of the much
smaller [3H]Ins(3,4,5,6)P4 pool.
Ins(1,3,4)P3 would seem to be particularly well suited to
its task as an intracellular mediator that links
Ins(3,4,5,6)P4 levels to changes in PLC activity, since
cellular levels of Ins(1,3,4)P3 quite closely follow
both the extent and the duration of PLC activation (45, 46). In
addition, we have shown the relative ineffectiveness with which the
1-kinase is inhibited by both of the InsP2 products of
Ins(1,3,4)P3 metabolism, namely Ins(1,3)P2 and
Ins(3,4)P2 (Fig. 7). Thus, the dephosphorylation of
Ins(1,3,4)P3 comprises an efficient signaling off-switch.
In this context, it now seems more significant that both the 1- and
4-phosphatases that attack Ins(1,3,4)P3 may be regulated.
This is indicated first by the amino acid sequence of the 4-phosphatase
containing consensus motifs for phosphorylation by protein kinases
(47). Second, and more directly, Ca2+ activates the
1-phosphatase (40).
As a result of this study, we are wiser as to the structural
determinants of Ins(1,3,4)P3-mediated inhibition of the
1-kinase (Table II). We also made the intriguing observation that a
2-phosphate could increase potency of 1-kinase inhibition, depending
upon which other phosphate groups were also present. A practical
outcome of this increased information concerning specificity is that
the non-physiological material,
D/L-Ins(1,2,4)P3, proved to be a particularly potent 1-kinase inhibitor; this could be a productive starting point
for the rational design of therapeutically useful drugs that might
inhibit the Ins(3,4,5,6)P4 1-kinase in vivo.
This provides an alternative to the approach of designing drugs that
act at the site of action of Ins(3,4,5,6)P4 (27).
The very existence of the Ins(1,4,5)P3 3-kinase, but more
so its complex regulation through cross-talk from other signaling pathways, are observations that have been used to bolster the teleological argument that Ins(1,3,4,5)P4 must be
functionally significant (2). Indeed, there is a large body of evidence that Ins(1,3,4,5)P4 does indeed perform a valuable role
inside cells (2). The 3-kinase also has the role of inactivating
Ca2+ signaling by Ins(1,4,5)P3. Our new data
assign additional significance to this metabolic pathway: control over
the production of Ins(1,3,4)P3, which in turn regulates
cellular levels of Ins(3,4,5,6)P4 (an inhibitor of
Ca2+-activated Cl *
The work in Pohang was supported by the Korean Science and
Engineering Foundation/Center for Biofunctional Molecules and Ministry of Education Grant BSRI 97-3437), studies at Bath were supported by
Wellcome Trust Program Grant 045491 (to B. V. L. P), and work in Bremen was supported by Deutsche Forschungsgemeinschaft Schu 943/1-6
(to C. S.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed. Tel.: 919-541-3308;
Fax: 919-541-0559; E-mail: yang3{at}niehs.nih.gov.
The abbreviations used are:
InsPn, inositol polyphosphate, where
n is the number of phosphates (e.g.
InsP3);
PEG, polyethylene glycol;
PLC, phospholipase C;
HPLC, high pressure liquid chromatography;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
bis-Tris, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane;
CaM KII, calmodulin-dependent protein kinase;
Bt3Ins(1,3,4)P3/AM, 2,5,6-tri-O-butyryl-myo-inositol
1,3,4-trisphosphate hexakis (acetoxymethyl) ester.
Inositol 1,3,4-Trisphosphate Acts in Vivo as a
Specific Regulator of Cellular Signaling by Inositol
3,4,5,6-Tetrakisphosphate*
§,,,
,,,
Laboratory of Signal Transduction, NIEHS,
National Institutes of Health, Research Triangle Park, North
Carolina 27709, the ¶ Institut für Organische Chemie,
Universität Bremen, 28359 Bremen, Germany, the Wolfson
Laboratory for Medicinal Chemistry, Department of Pharmacy and
Pharmacology, University of Bath, Claverton Down, Bath BA2 7AY, United
Kingdom, the Department of Medicinal
Chemistry and Pharmacognosy, College of Pharmacy, Chicago, Illinois
60612-7231, ** Department of Chemistry, Pohang University of Science and
Technology, San 31 Hyoja Dong, Pohang 790-784, Korea
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
channels are inhibited by inositol
3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P4) (Xie, W.,
Kaetzel, M. A., Bruzik, K. S., Dedman, J. R., Shears,
S. B., and Nelson, D. J. (1996) J. Biol.
Chem. 271, 14092-14097), a novel second messenger that is formed
after stimulus-dependent activation of phospholipase C
(PLC). In this study, we show that inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) is the specific signal that ties increased
cellular levels of Ins(3,4,5,6)P4 to changes in PLC
activity. We first demonstrated that Ins(1,3,4)P3 inhibited
Ins(3,4,5,6)P4 1-kinase activity that was either (i) in
lysates of AR4-2J pancreatoma cells or (ii) purified 22,500-fold
(yield = 13%) from bovine aorta. Next, we incubated
[3H]inositol-labeled AR4-2J cells with cell permeant and
non-radiolabeled 2,5,6-tri-O-butyryl-myo-inositol
1,3,4-trisphosphate-hexakis(acetoxymethyl) ester. This treatment
increased cellular levels of Ins(1,3,4)P3 2.7-fold, while
[3H]Ins(3,4,5,6)P4 levels increased 2-fold;
there were no changes to levels of other 3H-labeled
inositol phosphates. This experiment provides the first direct evidence
that levels of Ins(3,4,5,6)P4 are regulated by Ins(1,3,4)P3 in vivo, independently of
Ins(1,3,4)P3 being metabolized to
Ins(3,4,5,6)P4. In addition, we found that the
Ins(1,3,4)P3 metabolites, namely Ins(1,3)P2 and
Ins(3,4)P2, were >100-fold weaker inhibitors of the
1-kinase compared with Ins(1,3,4)P3 itself (IC50 = 0.17 µM). This result shows that
dephosphorylation of Ins(1,3,4)P3 in vivo is an
efficient mechanism to "switch-off" the cellular regulation
of Ins(3,4,5,6)P4 levels that comes from Ins(1,3,4)P3-mediated inhibition of the 1-kinase. We also
found that Ins(1,3,6)P3 and Ins(1,4,6)P3 were
poor inhibitors of the 1-kinase (IC50 = 17 and >30
µM, respectively). The non-physiological trisphosphates,
D/L-Ins(1,2,4)P3, inhibited 1-kinase relatively potently (IC50 = 0.7 µM), thereby suggesting
a new strategy for the rational design of therapeutically useful
kinase inhibitors. Overall, our data provide new information to support
the idea that Ins(1,3,4)P3 acts in an important signaling cascade.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
channels in the plasma membrane
(6-9). These ion channels make important contributions to salt and
fluid secretion, and in addition they may participate in
osmoregulation, pH balance, and smooth muscle excitability
(10-13).
View larger version (19K):
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Fig. 1.
Proposed mechanism for the regulation of
cellular Ins(3,4,5,6)P4 levels by
Ins(1,3,4)P3. The proposed link between
Ins(1,3,4)P3 levels and the interconversion of
Ins(1,3,4,5,6)P5 and Ins(3,4,5,6)P4 (15, 16) is
described. The broken line illustrates the inhibitory effect of
Ins(1,3,4)P3 upon Ins(3,4,5,6)P4
phosphorylation by the 1-kinase.
secretion (18, 27). A major
challenge to pharmacological intervention at the effector site for
Ins(3,4,5,6)P4 comes from the exquisite specificity with
which it blocks Cl channel conductance;
Ins(1,3,4)P3, Ins(1,3,4,5)P4,
Ins(1,3,4,6)P4, Ins(1,4,5,6)P4, and
Ins(1,3,4,5,6)P5 are all ineffective (6, 8, 9). Moreover,
at least one, and possibly both of the OH groups on
Ins(3,4,5,6)P4, are also critical determinants of substrate
specificity (18, 28). This tight specificity may make it difficult to
rationally design a functional analogue of Ins(3,4,5,6)P4.
A possible alternative therapeutic strategy might be to target
Ins(3,4,5,6)P4 synthesis, rather than its site of action.
To this end, we examined the impact on the 1-kinase of some analogues
of Ins(1,3,4)P3 that contain a phosphate group in the
2-position.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
]D20 was +23°; c = 0.75, chloroform). The more labile ketal was removed by a 2-h treatment
with acetyl chloride (5%) in a mixture of acetonitrile and methanol
(4:5, v/v). The solution was neutralized with triethylamine and
evaporated to dryness. The crude material was purified by preparative
HPLC (90% MeOH, RP-18, 10 µm, 50 × 250 mm, 40 ml/min) to give
3,4-di-O-benzyl-1,2-O-cyclohexylidene-myo-inositol
in 58% yield as a clear oil []D20
was 
11.1°; c = 0.72, chloroform). The latter was
finally converted to
D-Bt3Ins(1,3,4)P3/AM, as described
previously for the racemic precursor (19). Analytical data for
D-Bt3Ins(1,3,4)P3/AM were as
follows: []D20 was 7.3°
(c = 0.87, toluene). Direct chemical ionization high resolution mass spectroscopy [M-CH2OAc]-
(C33H52O28P3) gave a
calculated m/z of 989.1858; found m/z was 989.1868. 1H and 31P NMR data were in
accordance with those of the racemic compound. Enantiomerically pure
D-Bt2Ins(1,4,5,6)P4/AM was
synthesized as described previously (7). All bioactivatable esters were
dissolved in Me2SO/Pluronic (5%, v/v) as described
previously (7).
70 °C. Either two or three
preparations were subsequently thawed and combined, dialyzed against 2 liters of 25 mM bis-Tris (pH 7.0) at 4 °C for 3 h,
and loaded onto a UNO Q12 anion exchange column (1.5 × 6.8 cm),
which was pre-equilibrated with 100 ml of Buffer A. A constant flow
rate of 0.5 ml/min was maintained throughout the chromatography. After
washing with 60 ml of Buffer A, the bound protein was eluted with a
linear gradient of Buffer A plus 0-300 mM NaCl, followed by 60 ml of Buffer A plus1 M NaCl.
70 °C.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 2.
Purification of Ins(3,4,5,6)P4
1-kinase. The 1-kinase was purified as described under
"Experimental Procedures" by subjecting a 10-30% PEG precipitate
to Heparin II-agarose affinity chromatography (panel
A), followed by UNO Q12 anion exchange chromatography
(panel B) and Heparin IIIS affinity
chromatography (panel C). Data are representative
of three experiments.
Purification of Ins(3,4,5,6)P4 1-kinase
View larger version (16K):
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Fig. 3.
Molecular size of the
Ins(3,4,5,6)P4 1-kinase. The 1-kinase was analyzed by
size-exclusion chromatography as described under "Experimental
Procedures". Enzyme activity is indicated by the circles;
the squares depict the elution positions of standards: serum
albumin albumin (66 kDa), chicken ovalbumin (44 kDa), equine myoglobin
(17 kDa), and vitamin B-12 (1.35 kDa).
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Fig. 4.
Inhibition, by Ins(1,3,4)P3,
of the Ins(3,4,5,6)P4 1-kinase from bovine aorta and
AR4-2J pancreatoma cells. Panel A,
Ins(3,4,5,6)P4 1-kinase activity was assayed as described
under "Experimental Procedures," using either the enzyme purified
from bovine aorta incubated with the indicated concentrations of
Ins(1,3,4)P3 (n = 3, vertical
bars denote standard errors) (panel
A), or lysates of AR4-2J cells in medium containing either
10 µM Ins(1,3,4)P3 (open
circles) or no Ins(1,3,4)P3 (closed
circles) (panel B); a single
experiment is shown, representative of two.
Relative potencies of inositol phosphates as inhibitors of the
Ins(3,4,5,6)P4 1-kinase
View larger version (20K):
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Fig. 5.
The effect of
D/L-Bt3
Ins(1,3,4)P3/AM upon
3H-labeled inositol phosphates in AR4-2J cells.
[3H]Inositol-labeled AR4-2J cells were incubated with
D/L-Bt3Ins(1,3,4)P3/AM or vehicle
for 60 min, and then the cells were quenched and the inositol
phosphates resolved by HPLC, as described under "Experimental
Procedures." The data are from one of four representative
experiments. The identities of the various peaks (see Ref. 36) are as
follows: A, GroPIns; B, Ins1P; C,
Ins4P; D, InsP2; E,
Ins(1,4,5)P3; F, Ins(1,3,4,6)P4;
G, Ins(3,4,5,6)P4; H,
InsP5; I, InsP6. The integrated
value of the Ins(3,4,5,6)P4 peaks were 640 dpm
(upper panel, vehicle) and 1505 dpm
(lower panel,
D/L-Bt3Ins(1,3,4)P3/AM-treated).
The effect of D/L-Bt3Ins(1,3,4)P3/AM upon
levels of 3H-labeled inositol phosphates in AR4-2J cells
View larger version (16K):
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Fig. 6.
Effect of bombesin and
D-Bt3
Ins(1,3,4)P3/AM upon levels of
[3H]Ins(3,4,5,6)P4 and non-radiolabeled
Ins(1,3,4)P3 in intact AR4-2J cells.
[3H]Inositol-labeled AR4-2J cells were incubated with
either (i) 200 nM bombesin (BBS) or vehicle for
20 min, or (ii) 100 µM
D-Bt3Ins(1,3,4)P3/AM or vehicle for
60 min. The cells were quenched and the inositol phosphates resolved by
HPLC, as described under "Experimental Procedures." The changes in
levels of [3H]Ins(3,4,5,6)P4 (gray
bars) and non-radiolabeled Ins(1,3,4)P3
(black bars) were then determined as described
under "Experimental Procedures." Means and standard errors are from
three experiments.
View larger version (13K):
[in a new window]
Fig. 7.
Effects of InsP2 isomers and
D/L-Ins(1,2,4)P3 upon
Ins(3,4,5,6)P4 1-kinase activity. 1-Kinase activity
was assayed as described under "Experimental Procedures," in the
presence of the following non-radiolabeled inositol phosphates:
D/L-Ins(3,4)P2 (panel A,
open squares), Ins(1,3)P2
(panel A, triangles),
Ins(1,4)P2 (panel A,
circles), D/L-Ins(1,2,4)P3
(panel B). Enzyme activity in each case is
presented as a percentage of activity in the absence of any inhibitor.
Data are derived from three or four experiments, and
vertical bars denote standard errors (omitted
from the experiments with InsP2 isomers for the sake of
clarity).
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
channels (6, 8). We (15)
have previously suggested that Ins(3,4,5,6)P4 levels are
controlled by PLC-initiated regulation of the activities of a
Ins(1,3,4,5,6)P5 1-phosphatase/Ins(3,4,5,6)P4 1-kinase substrate cycle (Fig. 1). Others have argued that
Ins(1,3,4)P3 is converted to Ins(3,4,5,6)P4 by
the sequential actions of Ins(1,3,4)P3 6-kinase,
Ins(1,3,4,6)P4 1-phosphatase, and Ins(3,4,6)P3
5-kinase (22, 23, 44). An increased metabolic flux from
Ins(1,3,4)P3 to Ins(3,4,5,6)P4 could elevate
Ins(3,4,5,6)P4 levels by mass action effects.
D/L-Bt3Ins(1,3,4)P3/AM enabled us
to test this idea.
channels) (8). The
acknowledgment that the Ins(1,4,5)P3 3-kinase has several
important roles provides us with a better appreciation of why so many
cellular control processes converge on the regulation of this enzyme's
activity (48).
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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