(Received for publication, July 10, 1995; and in revised form, August 30, 1995)
From the
Wortmannin and its structural analogue demethoxyviridin (DMV)
have been reported to be specific inhibitors of phosphatidylinositol
3-kinase activity. Here we report that these compounds are not as
selective as assumed and demonstrate inhibition of bombesin-stimulated
phospholipase A activity by both wortmannin and DMV with an
IC
(2 nM) which is slightly more potent than the
inhibition of insulin-stimulated phosphatidylinositol
3,4,5-trisphosphate generation in these cells (
10 nM).
While it has not been possible to fully block in vitro phospholipase A
activity with wortmannin, inhibition
cannot be a consequence of inhibition of PI 3-kinase activity since
bombesin fails to generate 3-phosphorylated lipids in the intact cell.
Therefore, while wortmannin is indeed a PI 3-kinase inhibitor, it is
not as specific as previously reported, and experimental conclusions
based solely on its use should be treated with caution.
The fungal metabolite wortmannin and its structural analogue
demethoxyviridin (DMV) ()have been demonstrated to have an
inhibitory effect upon phosphatidylinositol 3-kinase (PI 3-kinase)
activity at nanomolar concentrations(1) . Inhibition has been
demonstrated upon both PI 3-kinase activity in anti-p85
immunoprecipitates and the stimulation of phosphatidylinositol
3,4,5-trisphosphate (PIP
) generation in N-formylmethionylleucylphenylalanine (fMLP)-stimulated
neutrophils(1) . The binding of
17-[
H]hydroxywortmannin to a number of neutrophil
proteins has been demonstrated, and one of these has been identified as
the 110-kDa subunit of PI 3-kinase(2) . On the basis of these
findings, it has been proposed that wortmannin and DMV are specific
inhibitors of PI 3-kinase, and, thus, addition of these compounds to
cells will result in the specific inhibition of the PI 3-kinase
pathway. Consequently, an increasing number of papers have described
the use of wortmannin and, on the sole basis of such experiments,
assigned a role for PI 3-kinase in a number of physiological responses (e.g. (3, 4, 5) ).
Wortmannin has
also been reported to inhibit phospholipase D (PLD)(6) , myosin
light chain kinase, and plekstrin phosphorylation (see (1) ).
Although these results could cast doubt upon the specificity of
wortmannin, these effects have been shown to occur at concentrations
greater than those reported to inhibit PI 3-kinase. However, the
specificity of wortmannin for other lipid-metabolizing enzymes has not
been examined. In this paper, we have examined the specificity of
wortmannin and DMV and show that they inhibit stimulated
PIP-phospholipase C (PI-PLC), PLD, phospholipase A
(PLA
) as well as PI 3-kinase in Swiss 3T3 cells and,
in addition, in vitro PI 3-kinase and PLA
activities. We also demonstrate that both compounds are more
potent inhibitors of PLA
than PI 3-kinase.
In the Swiss 3T3 fibroblast cell line, bombesin stimulates
the hydrolysis of phosphatidylinositol 4,5-bisphosphate by
phospholipase C (PI-PLC) and phosphatidylcholine by phospholipase D
(PLD) and phospholipase A (PLA
) (12) .
Bombesin has been reported to have no effect upon the activation of PI
3-kinase in Swiss 3T3 cells(10) ; therefore, insulin was used
to stimulate PI 3-kinase activity.
DMV inhibited bombesin-stimulated
PI-PLC activity, as determined by inositol phosphate accumulation, in
Swiss 3T3 cells with an IC value of approximately 30
nM (Table 1). The wortmannin analogue also inhibited
phosphatidylcholine-PLD activity, as measured by phosphatidylalcohol
generation, with an IC
of approximately 50 nM,
although maximum inhibition was never greater than 60% (Table 1).
Similar potency values were reported for fMLP-stimulated neutrophils (6) except that the sensitivity of the PLC response to DMV was
greater in the 3T3 cells, and, in the neutrophil, the PLD response was
completely inhibited. These differences may reflect different
phospholipase isoforms in the different cell types.
DMV was an
extremely potent inhibitor of bombesin-stimulated
phosphatidylcholine-PLA activity in Swiss 3T3 cells. Fig. 1shows that inhibition was observable at concentrations as
low as 0.1 nM, and that the IC
was approximately
2 nM. This inhibition of PLA
was also observed
with wortmannin (Fig. 1), demonstrating the potency of this
class of compounds as PLA
inhibitors. Inhibition of
PLA
was also observed in an in vitro assay.
However, inhibition was only partial with approximately 30% inhibition
being detected at 0.1 nM (Table 2). This inhibition was
dependent upon the inclusion of PIP
in the assay, which has
been reported to enhance phospholipase A
activity(13) . Incubation of a Swiss 3T3 cell lysate or
an anti-PLA
immunoprecipitate with wortmannin for 10 min
prior to addition of the lipid substrate failed to induce any PLA
inhibition in vitro.
Figure 1:
Inhibition of
bombesin-stimulated phospholipase A in Swiss 3T3 cells by
increasing concentrations of DMV (A) and wortmannin (B). Phospholipase A
was determined as described
under ``Experimental Procedures,'' the results are means
± S.D. where n = 3 and from single experiments
typical of three. For the experiments shown, basal and maximum
stimulated disintegrations/min in arachidonate values were 190 ±
60 and 600 ± 40 (A) and 860 ± 93 and 2354
± 252 (B).
Bombesin has been reported not
to stimulate PI 3-kinase activity in Swiss 3T3 cells(10) , and
we have confirmed this observation both by assaying the activity of
immunoprecipitated kinase (Fig. 2) and by determining changes in
PIP levels (see Fig. 3). Therefore, we examined the
effects of wortmannin and DMV in insulin-stimulated cells. Fig. 2shows that insulin and platelet-derived growth factor, but
not bombesin, increase the PI 3-kinase activity in anti-p85
immunoprecipitates. In the in vitro PI 3-kinase assay, where
the insulin-stimulated kinase was immunoprecipitated using an anti-p85
subunit antibody, DMV inhibited enzyme activity with an IC
value between 1 and 2 nM (Fig. 2). This is
similar to the value of 3.4 nM previously reported (14) and compares with reported IC
values between
1 and 10 nM for PI 3-kinase inhibition by
wortmannin(14) .
Figure 2:
Inhibition of PI 3-kinase activity by DMV.
PI 3-kinase activity was determined in anti-p85 immunoprecipitates
prepared from insulin-stimulated cells. Increasing concentrations of
DMV were added prior to the addition of the
[P]ATP. Results are presented as percent
inhibition ± S.D. of the activity in the absence of DMV which
was 0.0129 ± 0.0005 pmol of phosphate incorporated into PI
3-P/min/mg of cellular protein. The inset shows the generation
of PI 3-P in immunoprecipitates prepared from cells stimulated with
insulin, plateletderived growth factor (PDGF) or
bombesin.
Figure 3:
The generation of PIP in Swiss
3T3 cells.
P-Labeled Swiss 3T3 cells were stimulated with
insulin or bombesin ± DMV or ± wortmannin, the lipids
were isolated, deacylated, and deglycerated, and the
Ins(1,3,4,5)P
fraction which is generated from PIP
was isolated by HPLC. A, generation of PIP
in response to insulin (
) and bombesin (
). B, generation of PIP
in response to insulin
- DMV (
) or + 1 µM DMV (
). C, generation of PIP
in response to insulin
- wortmannin (
) or + 1 µM wortmannin
(
).
Inhibition of PI 3-kinase activity in
anti-p85 immunoprecipitates where phosphatidylinositol rather than
phosphatidylinositol 4,5-bisphosphate is presented as the substrate can
only give an indication of the effects of a compound in the whole cell.
The only definitive measure of effects upon in vivo PI
3-kinase activity is to examine changes in the cellular levels of the
3-phosphorylated lipids. Therefore, lipids were isolated from control
and stimulated [P]P
-labeled cells,
deacylated, and deglycerated, and the generated water-soluble
phosphates were separated by ion exchange HPLC. Fig. 3shows
that insulin stimulates PIP
generation in Swiss 3T3 cells,
the figure also shows that bombesin did not stimulate PIP
generation in agreement with Jackson et
al.(10) . Insulin-stimulated PIP
generation
was inhibited by both DMV and wortmannin (Fig. 3) with DMV being
slightly more potent with an approximate IC
of 10 nM compared to a value greater than 10 nM for wortmannin (Fig. 4). The more potent effect of DMV compared to wortmannin
upon 3-phosphorylated lipid generation is similar to that reported for
effects upon PI 3-kinase activity in immunoprecipitates(14) .
Figure 4:
Inhibition of PIP generation
by DMV and wortmannin. Cells were treated as in Fig. 3but with
increasing concentrations of DMV or wortmannin. Results are expressed
as percent insulin-stimulated PIP
generation.
The data presented in Fig. 1clearly define wortmannin and
DMV as potent and complete inhibitors of stimulated phospholipase
A activity in Swiss 3T3 cells. However, in vitro inhibition was incomplete when the enzyme was activated by
PIP
, diacylglycerol, and calcium (Table 2). The
contrast between the in vitro and in vivo inhibition
of phospholipase A
suggests a complex mode of action for
wortmannin and DMV. It is possible that they interact directly with the
phospholipase and that the incomplete effect was due to a
nonphysiological conformation taken up by the protein in the test tube.
An alternative possibility is that inhibition may be due to an
interaction with a protein which regulates phospholipase A
activity in the stimulated cell. Should this be the case, it is
unlikely that the protein involved is PI 3-kinase since in Swiss 3T3
cells bombesin activates phospholipase A
without
stimulating PI 3-kinase. Additionally, when stimulated phospholipase
A
and PI 3-kinase activities are inhibited in vivo by wortmannin and DMV, both compounds are more potent against the
release of arachidonate than the generation of 3-phosphorylated lipids.
The potency of DMV was greater than that of wortmannin for each of the enzymes examined. However, this difference is offset by the reduced stability of DMV when compared to wortmannin. Indeed, when DMV was incubated in a physiological buffer at 37 °C for 10 min prior to addition to cells, it was rendered inactive (results not shown). In view of the instability of these compounds in aqueous solution, their experimental and therapeutic use will remain limited until the development of more stable analogues.
The results presented in this
paper demonstrate that wortmannin and its structural analogue DMV are
not specific or selective inhibitors of PI 3-kinase in intact cells.
Inhibition of other enzymes by wortmannin, e.g. phospholipase
D, myosin light chain kinase, has been reported previously; however,
the concentrations required were much greater than those reported to
inhibit PI 3-kinase. The inhibition of bombesin-stimulated
phospholipase A activity in Swiss 3T3 cells is apparent at
subnanomolar concentrations and has an IC
(approximately 5
nM) which is similar to that previously reported for the
inhibition of PI 3-kinase in vitro and lower than that
determined for the inhibition of insulin-stimulated PIP
generation (Fig. 4). This value is also similar to that
measured for the inhibition of PI 3-kinase activity in anti-p85
immunoprecipitates from Swiss 3T3 cells (Fig. 2). The proposed
specificity of action of wortmannin upon PI 3-kinase has been supported
by the demonstration of wortmannin binding to a 110-kDa protein in an
SDS-polyacrylamide gel and to the 110-kDa subunit of PI
3-kinase(2) . However, the 85-kDa phospholipase A
migrates at 110 kDa on SDS-polyacrylamide gel electrophoresis,
highlighting the possibility that one of the bands shown to bind
[
H]wortmannin could be phospholipase
A
.
Accordingly, where a role for PI 3-kinase in a
particular pathway or cellular function has been assigned on the basis
purely of its inhibition by wortmannin, the conclusions may be
incorrect and a role for other signaling pathways such as phospholipase
A must be considered. A clear example of this is in the
regulation of neutrophil responses where a central role for PI 3-kinase
has been proposed on the basis of wortmannin sensitivity(1) .
Other studies have suggested a role for the phospholipase A
product arachidonate in the activation of
neutrophils(15) , and, thus, the nonselective action of
wortmannin would suggest a need for a reassessment. Many papers
reporting the use of wortmannin have utilized concentrations of 100
nM and above; however, at these levels, as the results
presented here demonstrate, there will be inhibition of PLC, PLD,
PLA
, and PI 3-kinase. Despite these caveats, it is clear
that in many cases where wortmannin has been used to identify a role
for PI 3-kinase the conclusions are correct, but these studies have
utilized further experimental evidence, e.g. dominant negative
p85 subunit transfections(16) , to confirm their findings. In
other cases, it could be phospholipase A
that is the enzyme
of importance.