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,1
* Unit of Physiopathology of Cell Signalling, and
Laboratory of Cellular and Molecular Endocrinology, Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, via Nazionale, 66030 Santa Maria Imbaro (Chieti), Italy;
Molecular Pharmacology Unit, Istituto di Ricerche Farmacologiche ‘Mario Negri’, via Eritrea, 62, 20157 Milan, Italy; and
§ Laboratory of Molecular Oncology, Fondazione CARIPE, Pescara, Italy
2Correspondence: Unit of Physiopathology of Cell Signalling, Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, via Nazionale, 66030 Santa Maria Imbaro (Chieti), Italy. E-mail: falasca{at}cmns.mnegri.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: antitumor agents • inositol polyphosphates • PH domain-binding antagonists • phosphatidylinositols
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Furthermore,
PtdIns(4,5)P2 is also a substrate for
phosphoinositide 3-kinase (PI3-K), which phosphorylates the 3 position
of the inositol ring, leading to the production of the lipid second
messenger phosphatidylinositol 3,4,5-trisphosphate
[PtdIns(3,4,5)P3] (2)
.
Three different classes of PI3-K have been identified, classes I, II,
and III (3)
: class I is activated by tyrosine kinase
receptors and is made up of a catalytic 110-kDa subunit (p110) and a
regulatory 85-kDa subunit (p85). Several growth factors induce the
activation of this PI3-K, which is involved in different cellular
functions, such as cell proliferation and differentiation, cytoskeletal
rearrangements, and apoptosis (4
, 5)
. The downstream
targets of this PI3-K are proteins that interact with its lipid
products through their pleckstrin homology (PH) domains, such as
protein kinase B (PKB/Akt) (6
, 7)
and phospholipase C
(PLC) (8)
. PH domains are independent protein modules
of ~120 amino acids that are homologous to two regions of pleckstrin,
the major protein kinase C substrate in platelets (9
, 10)
.
About 110 different proteins involved in signaling and cytoskeletal
organization are now thought to contain PH domains, and all PH
domain-containing proteins have a functional requirement for membrane
association (11)
. Several recent studies (5
, 12)
have shown that PH domains can recognize membrane
components, most notably phosphoinositides, in a highly specific
manner. These studies provide support for the hypothesis that some PH
domains mediate specific recruitment of their host molecules to the
plasma membrane and that this recruitment is controlled in cellular
signaling by enzymes that modify the phosphoinositides and other
membrane components. Hence, the products of PI3-K activities, the
3-phosphorylated phosphoinositides and their inositol polyphosphate
equivalents, have recently been shown to bind specifically to different
PH domains, such as those from PKB/Akt, Bruton’s tyrosine kinase
(Btk), Grp-1, PLC1, and Gap1 (8
, 12)
. For these
proteins, it appears that the following scenario occurs: growth factor
activation of PI3-K induces an increase in the levels of the
3-phosphorylated lipids in the plasma membrane; these act as second
messengers, which bind proteins containing PH domains, resulting in the
recruitment of these proteins to the plasma membrane and their
consequent activation (12)
. An increasing amount of
evidence also suggests that PI3-K may be involved in tumor formation in
animals. For instance, a transforming retrovirus that causes
hemangiosarcoma in chickens carries activated PI3-K as an oncogene
(13)
, and a mutant p85 was found to transform fibroblasts
in vitro (14)
. Furthermore, prolonged
expression of activated PI3-K can contribute to cellular changes
characteristic of cellular transformation (15)
. Finally,
PI3-K has been shown to be constitutively active in small cell lung
cancer (SCLC) cells and in ovarian cancer cell lines (16
, 17)
. It is also noteworthy that the tumor suppressor protein
PTEN, the gene that is deleted or mutated in a wide variety of human
cancers (18)
, possesses a 3-phosphoinositide-phosphatase
activity (19)
. Therefore, this has highlighted the
potential pharmacological importance of PI3-K inhibitors. Our goal has
thus been to study the inositol polyphosphates, the soluble headgroups
of these 3-phosphorylated phosphoinositides, as potential inhibitors of
PI3-K and hence as switch-off signal molecules by their competition
with the localization of PH domain-containing proteins to the plasma
membrane. Therefore, considering that extracellularly applied
inositol 1,3,4,5-tetrakisphosphate
[Ins(1,3,4,5)P4] can indeed enter
cells in culture, we have tested the activity of selected inositol
polyphosphates that are partial structural analogs of the
PtdIns(3,4,5)P3 headgroup and have
been found to bind PH domains with similar affinities to
Ins(1,3,4,5)P4. We present evidence
that two specific inositol polyphosphates that are able to interact
with certain PH domains possess in vitro antitumor activity,
and hence we propose a mechanism of action via competition with the
lipid products of PI3-K. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell culture and reagents
MCF-7 cells were cultured in DMEM/Ham’s F12 medium (1:1)
supplemented with 5% fetal calf serum (FCS), glutamine (300 µg/ml),
penicillin (100 IU/ml), and streptomycin (100 µg/ml). All SCLC cell
lines were grown in RPMI 1640 with 25 mM HEPES supplemented with 10%
heat-inactivated fetal bovine serum (FBS). The human ovarian cancer
cell lines SKOV-3 and OVCAR-432 and the human colocarcinoma cell line
SW620 were grown in RPMI 1640 supplemented with 10% FCS. COS-7 cells
were maintained in DMEM supplemented with 10% CS.
Incorporation of Ins(1,3,4,5)P4 in SCLC
cells
Ca. 1 x 107 SCLC cells, grown,
transferred, and disaggregated as described below, were pelleted by
centrifugation (50 g, 5 min) and resuspended to 1 ml in
medium containing 0.5% FCS. Following a 12-h incubation at 37°C, 50
µM cold (unlabeled) Ins(1,3,4,5)P4
and ca. 400,000 dpm
[3H]-Ins(1,3,4,5)P4
were added. After a 20-min incubation at 37°C, the cells were
pelleted by being briefly (10 s) centrifuged in a microcentrifuge, then
rapidly washed twice in phosphate-buffered salt solution (PBS)
containing 0.3% bovine serum albumin (BSA), and killed by the addition
of 750 µl cold (-20°C) methanol/1N HCl (1:1). Following the
addition of further methanol and chloroform, to form a two-phase
extraction of aqueous (upper) and organic (lower) phases as previously
demonstrated (20)
, the aqueous phase was lyophilized and
analyzed by high-performance liquid chromatography (HPLC). Parallel
extraction and HPLC analysis were also carried out on the medium from
the incorporation to determine the stability and purity of the added
[3H]-Ins(1,3,4,5)P4
through the incubation.
[3H]-Thymidine incorporation in MCF-7 cells
Assays were performed in 24-well plates. Serum-starved cells
were pretreated with inositol polyphosphates for 20 min and then
stimulated with insulin-like growth factor-1 (IGF-1). After 20 h
of incubation, [3H]-labeled thymidine (2
µCi/ml) was added. Twenty-six hours after the addition of IGF-1,
cells were fixed with 10% trichloroacetic acid, washed in water, and
lysed in 0.1 N NaOH. The levels of
[3H]-thymidine labeling were then quantified by
liquid scintillation counting.
Liquid growth assays
Five days after passage, SCLC cells were transferred to SITA
medium (RPMI 1640 medium with 25 mM HEPES supplemented with 30 nM
selenium, 5 µg/ml insulin, 10 µg/ml transferrin) and cultured for a
further 2 days. The cells were then washed twice and resuspended in
fresh SITA medium before being gently disaggregated by two passes
through a 21-gauge needle into an essentially single-cell suspension.
Cells (1x105) were seeded into 24-well plates in
SITA medium and incubated for 4 h before the addition of the
inositol polyphosphates. Cell numbers were determined on day 9, after
disaggregation into single-cell suspensions, using a Coulter Counter ZM
linked to a Coulter Channelizer 256, according to the manufacturer
instructions. The human ovarian cancer cell lines SKOV-3 and OVCAR-432
and the human colocarcinoma cells (SW620) were seeded into 96-well
plates and kept in medium with 0.5% FCS for 12 h before
treatment. The treatments with LY294002 (10 µM) and the inositol
polyphosphates were carried out in medium plus 0.5% FCS. All the
compounds to be tested were initially dissolved in DMSO with the
incubations containing a constant 0.5% DMSO.
3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT)
analysis was performed 72 h after the treatments, and the values
are described as percentages of inhibition of the control, DMSO-treated
cell growth.
Clonogenic assay
Five days after passage, SCLC cells were washed and resuspended
in SITA medium. Cells were disaggregated (as above); 1 x
104 viable cells were mixed with SITA containing
0.3% (w/v) agarose and inositol polyphosphates at the concentrations
indicated, and layered over a solid base of 0.5% (w/v) agarose in
35-mm plastic dishes. The cultures were incubated in a humidified 5%
CO2 incubator at 37°C for 21 days and then
stained with the vital stain nitroblue tetrazolium. Colonies >
120 µm were counted under a microscope.
Immunofluorescence
cDNA encoding the PKB/Akt PH domain was subcloned into the green
fluorescent protein (GFP) fusion protein expression vector pEGFP-C1
(CLONTECH, Palo Alto, Calif.), using the BglII and
EcoRI sites for expression of an EGFP-PH domain fusion
protein in mammalian cells. COS-7 cells were seeded onto 12-mm circular
glass coverslips in wells of 6-well plates and transfected with 1 µg
of the EGFP fusion protein. LipofectAMINE (Life Technologies, Grand
Island, N.Y.) was used for the transfections, according to the
manufacturer suggestions. After preincubations without or with the
selected inositol polyphosphates (50 µM), the cells were stimulated
with growth factors, washed in PBS, fixed in 4% paraformaldehyde/PBS,
and mounted for fluorescence microscopy. Microscopy was performed using
a Zeiss Axiophot fluorescence microscope or the confocal microscopy
facility of the New York University Medical Center.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, under these conditions the concentration of
[3H]-Ins(1,3,4,5)P4
inside the cells [after a 12-h starvation and a 20-min incubation at
37°C with Ins(1,3,4,5)P4] was
calculated as being ca. 60 µM. These data thus demonstrated that
Ins(1,3,4,5)P4 indeed enters the cell
to a concentration approximately equal to that of the medium
Ins(1,3,4,5)P4, indicating that all
the subsequent effects of the inositol polyphosphates seen indeed can
result from their entry into the cells. Furthermore, small but
detectable and quantifiable (see Table 1
) levels of
[3H]-inositol 1-phosphate
{[3H]-Ins(1)P},
[3H]-inositol 1,4-bisphosphate
{[3H]-Ins(1,4)P2},
[3H]-inositol 1,3,4-trisphosphate
{[3H]-Ins(1,3,4)P3},
[3H]-inositol 1,4,5-trisphosphate
{[3H]-Ins(1,4,5)P3},
and a
[3H]-InsP5
were also seen on the HPLC analysis, indicating that ca. 45% of the
total intracellular [3H]-label was present in
these
[3H]-Ins(1,3,4,5)P4
metabolic products. HPLC analysis of the medium (see Table 1
) was also
unable to show the presence of
[3H]-Ins(1)P,
[3H]-Ins(1,4)P2,
and
[3H]-InsP5,
indicating further that these
[3H]-inositol-labeled compounds must indeed
have come from the cellular entry and the subsequent metabolism of the
extracellularly applied
[3H]-Ins(1,3,4,5)P4.
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DNA synthesis of MCF-7 cells in response to IGF-1
DNA synthesis in IGF-1-stimulated MCF-7 human breast cancer cells
was determined by [3H]-thymidine incorporation.
In these cells, an active PI3-K is necessary for the transmission of
the growth-stimulatory IGF-1 signal. We therefore tested whether
preincubation of MCF-7 cells with selected inositol polyphosphates for
20 min before IGF-1 addition affected DNA synthesis. Under the same
conditions, the PI3-K inhibitors wortmannin (100 nM) and LY 294002 (10
µM) induced a complete inhibition of this
[3H]-thymidine incorporation stimulated by
IGF-1 (data not shown; 21
). Figure 1
shows the effects of 50-µM concentrations of the selected inositol
tris- to hexakisphosphates on this IGF-1-induced
[3H]-thymidine incorporation. While inositol
1,3,4,5,6-pentakisphosphate
[Ins(1,3,4,5,6)P5] and inositol
1,4,5,6-tetrakisphosphate
[Ins(1,4,5,6)P4] were almost able to
completely inhibit the response induced by IGF-1, inositol
3,4,5,6-tetrakisphosphate
[Ins(3,4,5,6)P4] showed ca. 25%
inhibition, and the other inositol polyphosphates were inactive. All
the inositol polyphosphates tested were also inactive toward the basal
levels of [3H]-thymidine incorporation in these
cells.
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Colony formation of SCLC cells in agarose semisolid medium
It is well established that the ability to form colonies in
agarose semisolid medium is a marker of anchorage-independent growth
that is a characteristic of transformed phenotypes. Previous work has
shown that PI3-K appears to play a critical role in sustaining this
anchorage-independent growth (16
, 22)
. Because SCLC cells
are characterized by a highly aggressive phenotype and contain a
constitutively active PI3-K, we examined the effects of 50-µM
concentrations of the same range of selected inositol tris- to
hexakisphosphates on H69 SCLC cell colony formation in agarose
semisolid medium. Figure 2
shows that Ins(1,4,5,6)P4 and
Ins(1,3,4,5,6)P5 inhibited basal
colony formation in this H69 SCLC cell line by up to 75% and
Ins(3,4,5,6)P4 by 10–15%. All the
other inositol polyphosphates tested were inactive. A similar level of
inhibition was obtained with LY 294002 (10 µM) (data not shown and
ref 16
).
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Liquid growth assay with SCLC cells
In addition to the semisolid growth assay above, we tested
the ability of increasing concentrations of selected inositol
polyphosphates (10–100 µM) to inhibit the liquid medium growth of
the H69 (Fig. 3
), H345 (data not shown), and H146 (data not shown) SCLC cell lines. As
shown in Fig. 3
, Ins(1,3,4,5,6)P5 and
Ins(1,4,5,6)P4 induced a
concentration-dependent reduction in the H69 SCLC cell numbers in
liquid culture, showing ca. a 50% inhibition of growth at 50 µM.
Under the same conditions, inositol hexakisphosphate
(InsP6) and
Ins(1,4,5)P3 were inactive. Similar
results were also obtained using the H345 and H146 SCLC cell lines
(data not shown), and similar levels of inhibition were obtained with
LY 294002 (10 µM) (data not shown and ref 16
).
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Liquid growth assay with SKOV-3 and SW620 cells
The two ovarian cancer cell lines, SKOV-3 (Fig. 4A
) and OVCAR-432 (data not shown), are characterized by an
increased copy number of the PIK3CA gene (17)
. The use of
PI3-K inhibitors, such as LY294002, has been demonstrated previously to
arrest the growth of these cells, while being unable to cause
significant alterations in the growth of cells with the normal PIK3CA
copy number (17)
. We initially confirmed that both of the
ovarian cancer cell lines were sensitive to LY294002 (10 µM), with a
strong inhibition of growth seen. In contrast, the SW620 human
colocarcinoma cell line (Fig. 4B
) was insensitive to this
compound and to LY 294002 (10 µM), indicating that the growth of
these cells is PI3-K independent. When we tested the ability of
Ins(1,4,5,6)P4 to alter the growth of
the SKOV-3 (Fig. 4A
), OVCAR-432 (data not shown), and SW620
(Fig. 4B
) cells in culture, we found only the two ovarian
cancer cell lines were sensitive to this treatment. At 50 µM,
Ins(1,4,5,6)P4 was able to reduce the
growth by ca. 50%. The two other inositol polyphosphates,
Ins(1,4,5)P3 and InsP6,
used as negative controls, did not show any significant activities in
any of the cell lines tested (Fig. 4A, B
).
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Growth factor-induced translocation of the PKB/Akt PH domain
One of the major downstream targets of PI3-K is PKB/Akt (23
, 24)
. This probably represents the major pathway by which PI3-K
protects cells from apoptosis, and therefore PKB/Akt may promote the
survival of transformed cells and may influence the pathology of many
human tumors. PKB/Akt activation involves the direct interaction of its
PH domain with the PI3-K-generated inositol phospholipids (6
, 7)
. To test whether selected inositol polyphosphates can affect
the interaction of the PKB/Akt PH domain with the plasma membrane on
growth-factor activation, we transfected a GFP construct of the PKB/Akt
PH domain into COS-7 cells. These cells were then preincubated without
or with (50 µM) Ins(1,4,5,6)P4 or
Ins(1,4,5)P3 for 20 min, before being
stimulated with growth factors. As shown in Fig. 5
, FCS (10%, 5 min) induced a clear membrane localization of the PKB/Akt
PH domain that was blocked by preincubation of these cells with
Ins(1,4,5,6)P4 but not with
Ins(1,4,5)P3. Similar results were
obtained in MCF-7 cells stimulated with IGF-1 (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. While
there is still a debate as to the exact target(s) of the effects of
InsP6 in different cells (see ref
26
for further discussion), one site of action of
InsP6 inhibition of intracellular pathways has
been proposed to be on the activation of PI3-K (25)
.
Therefore, in addition to the already well-documented central role for
PI3-K in signaling pathways for cell growth and transformation
(4
, 5)
, and with recent studies indicating a role for
PI3-K in tumor formation (13
14
15
16
17
18)
, PI3-K has become an
attractive target for the development of anticancer drugs. Furthermore,
an important tool for investigating the possible involvement of PI3-K
in the regulation of neoplastic cell growth could provide a potent and
highly specific inhibitor of this enzyme. Hence, the aim of this study
was to examine the consequences of the inhibition of this pathway in
human cancer cells utilizing a selection of inositol tris- to
hexakisphosphates and tumor cell growth characterized by a
hyperactivated PI3-K.
Our data show that in these cancer cell models,
Ins(1,3,4,5,6)P5 and
Ins(1,4,5,6)P4 display roughly
equipotent activities, inhibiting IGF-1-stimulated DNA synthesis in
MCF-7 cells and semisolid medium growth of SCLC cells at micromolar
concentrations. Furthermore,
Ins(1,3,4,5,6)P5 and
Ins(1,4,5,6)P4 inhibit the liquid
growth of SCLC cells, and
Ins(1,4,5,6)P4 can also inhibit SKOV-3
cell liquid growth. With
Ins(3,4,5,6)P4 showing some selective
inhibition in our in vivo assay systems as well, it is
possible to use this profile of specificities with regard to previously
published in vitro assays for the binding of a range of
inositol polyphosphates to different PH domains (27
, 28)
.
Hence, in the in vitro binding assays, it is
Ins(1,3,4,5)P4 that is the preferred
inositol polyphosphate [dissociation constant
(KD) ca. 0.03 µM] for Group 1 (see ref
28
for this nomenclature),
PtdIns(3,4,5)P3-specific, PH domains;
Ins(1,3,4,5)P3 was inactive in our
in vivo cancer cell assays. Similarly, the Group 2,
PtdIns(4,5)P2-binding, phospholipase
C-
1 PH domain is highly selective for
Ins(1,4,5)P3 (KD
ca. 0.2 µM), another inactive inositol polyphosphate in the MCF-7 and
SCLC cell growth assays. However, in the case of the other two Group 2
PH domains, diacylglycerol kinase- (DAGK-) and the amino-terminal
of pleckstrin (Plec-N), a similar rank order of
KD’s was demonstrated (calculated from Takeuchi
et al., ref 27
) for
Ins(1,3,4,5,6)P5,
Ins(1,4,5,6)P4, and
Ins(3,4,5,6)P4, with those of the
DAGK- PH domain being at least 30-fold higher. However,
Ins(1,3,4,5)P4 should also be
equipotent. Similarly, whereas the Group 3,
PtdIns(3,4)P2- and
PtdIns(3,4,5)P3-selective PH domain
encoded by EST684797 (29)
, has a low
KD toward
Ins(1,3,4,5,6)P5 and
Ins(3,4,5,6)P4 (ca. 0.04 µM; ref
28
), it also shows an equal preference for
Ins(1,3,4,5)P4. Finally, the
preferences of the PKB/Akt
and PKB/Akt PH domains appear to be
very different (28)
. Although those of the latter show the
lowest (and equal) KD’s for
Ins(1,3,4,5,6)P5 and
Ins(1,4,5,6)P4, neither of these two
inositol polyphosphates is described as having been tested for in
vitro binding to the former. Thus, this indicated that while the
PKB/Akt PH domain appeared a good candidate as a mediator of the
inhibition of cancer cell growth in the present study, there is at
present a lack of specific in vitro binding data for the
PKB/Akt PH domain. Based on this information, however, we also
decided to investigate the effects of
Ins(1,4,5,6)P4, as compared with those
of Ins(1,4,5)P3, on the serum-induced
translocation of the PKB/Akt PH domain. Indeed, despite the in
vitro binding data indicating that
Ins(1,4,5)P3 is the most potent (but
with a high KD of ca. 1.2 µM) of the inositol
polyphosphates tested, it was unable to inhibit this PH domain membrane
translocation, while Ins(1,4,5,6)P4
completely blocked this effect (with both being used at 50 µM). This
would thus indicate that
Ins(1,3,4,5,6)P5 and
Ins(1,4,5,6)P4 may indeed be selective
for all PKB/Akt PH domains (30
, 31)
and that these
inositol polyphosphates could be mediating their inhibition of cancer
cell growth in the present study via their binding to PKB/Akt PH
domains.
In addition to the earlier work with InsP6 that
demonstrated that its antitumor activity is a result of inhibition of
PI3-K (25)
, a similar effect has also previously been
attributed to Ins(1,4,5,6)P4. While
Ruschkowski et al. (32)
showed that Salmonella
invasion of epithelial cells resulted in an increase in the cellular
inositol polyphosphate turnover, more recently this response has been
shown to specifically involve increased levels of
Ins(1,4,5,6)P4, an effect that
antagonizes the EGF-stimulated PI3-K signaling pathway
(33)
. Hence these effects of InsP6
(although not in our assay systems; see also below),
Ins(1,4,5,6)P4, and now
Ins(1,3,4,5,6)P5—and, to perhaps a
lesser extent, Ins(3,4,5,6)P4—could
be a result of their being partial structural analogs of the inositol
headgroup of PtdIns(3,4,5)P3. This is
also supported by our data indicating that the effects of these
inositol polyphosphates are indeed linked to specific PI3-K
involvement; none of the inositol polyphosphates was able to inhibit
basal [3H]-thymidine incorporation in the MCF-7
cells or liquid growth of the SW620 colocarcinoma cells. Furthermore,
because we have now also demonstrated that
Ins(1,4,5,6)P4 can indeed inhibit the
growth factors-induced membrane translocation of the PKB/Akt PH domain
during serum stimulation in COS-7 and in MCF-7 cells at the same
micromolar concentration as its effects in our cancer cell growth
assays, this lends further support to this being the potential mode of
action of these inositol polyphosphates in cells.
At the same time, consideration needs to be given to the
lack of effects of InsP6 and
Ins(1,3,4,5)P4 in our systems. As indicated by
Kavran et al. (28)
, in vitro assays indicate
that all PH domains tested can indeed bind InsP6
with low micromolar KD’s, possibly indicating
the importance of the high negative charge associated with this
molecule. However, this effect is lost here in our in vivo
assays; InsP6 was unable to inhibit
IGF-1-stimulated DNA synthesis in MCF-7 cells, semisolid and liquid
medium growth of SCLC cells, and liquid medium growth of SKOV-3 cells
at micromolar concentrations. In the case of
Ins(1,3,4,5)P4, this inositol
polyphosphate might be expected to be the most potent inhibitor of
PtdIns(3,4,5)P3 interactions with PH
domains, because it is the closest structural analog to the lipid
headgroup (see Fig. 6
for a schematic representation of the interactions involved), as is the
case with the Group 1 PH domains (28)
. However,
consideration of the active inositol polyphosphates,
Ins(1,3,4,5,6)P5,
Ins(1,4,5,6)P4, and
Ins(3,4,5,6)P4, in the present study
indicates that it is the 4-, 5-, and 6-phosphates (and not the 3-, 4-,
and 5-phosphates) of the inositol ring that are important in the
recognition of the PKB/Akt PH domain in particular, thus the importance
of a specific structural recognition in this interaction. Furthermore,
and as also suggested by Takeuchi et al. (27)
, recent
evidence indicates that as well as blocking lipid-PH domain
interactions, the inositol polyphosphates are also able to directly
influence the activity of the target proteins via this binding, an
effect that has already been seen for the PKB/Akt PH domain
(34)
. Another consideration is that in the case of the Bkt
PH domain binding to its preferred headgroup structural analog,
Ins(1,3,4,5)P4, Rameh et al.
(35)
have shown that the water-soluble
dioctanoyl-PtdIns(3,4,5)P3 binds to the same PH
domain with ca. a 100-fold lower KD. Hence, these
data all indicate the potential for highly specific interactions
between specific inositol polyphosphates and specific PH domains, thus
leading to specific inhibition of polyphosphoinositide-PH domain
interactions in vivo.
|
In summary, we provide evidence in the present study that the inositol polyphosphates, Ins(1,3,4,5,6)P5 and Ins(1,4,5,6)P4, possess anticarcinogenic actions, which involve the inhibition of PI3-K as a specific target. These effects appear related to the relative in vitro affinities of these inositol polyphosphates with regard to their abilities to bind to diverse PH domains. Hence, these data provide an excellent rationale for the development of specific PI3-K inhibitors for therapeutic application.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication August 10, 1999. Accepted for publication December 22, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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by PI 3-kinase-induced PH domain-mediated membrane targeting. EMBO J 17,414-422[Medline]
subunits of G proteins. Biochem. Biophys. Res. Commun. 216,526-534[Medline]
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