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Vol. 14, Issue 5, 1913-1922, May 2003
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* Department of Cell Biology, Johns Hopkins University, School of Medicine,
Baltimore, Maryland 21205;
Laboratory of Cellular and Molecular Biology, Division of Basic Sciences,
National Cancer Institute, National Institutes of Health, Bethesda, Maryland
20892-4255; and
Section of Cell and Developmental Biology, Division of Biology, University of
California, San Diego, La Jolla, California 92093
Submitted October 31, 2002;
Revised December 23, 2002;
Accepted January 13, 2003
Monitoring Editor: Anthony Bretscher
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Jones, 2000;
Moser and Loetscher, 2001;
Carlos, 2001;
Patel and Haynes, 2001;
Thelen, 2001;
Rubel and Cramer, 2002).
Through studies in this model system and mammalian leukocytes, the signaling
events of chemotaxis are being elucidated. In both systems, the response is
mediated through seven transmembrane receptors coupled to heterotrimeric
G-proteins (Parent and Devreotes,
1999; Rickert et al.,
2000; Chung et al.,
2001, Stephens et
al., 2002; Weiner,
2002). Recruitment of pleckstrin homology (PH) domain containing
proteins, which bind to phosphoinositides (PIs) on the membrane, is a key
event downstream of G-protein activation leading to pseudopodia
production.
Studies of the distributions of signaling components in chemotaxing amoeba
and leukocytes have been particularly useful in delineating the mechanisms of
chemotaxis. In chemotaxing cells, surface receptors are uniformly distributed
on the membrane, and chemoattractant binding to the receptors mirrors the
external gradient (Xiao et al.,
1997; Servant et al.,
1999; Ueda et al.,
2001). Furthermore, the G-proteins are distributed fairly
uniformly along the membrane, and their activation likely reflects the
external gradient (Jin et al.,
2000 and unpublished observations). However, the PH domain
containing proteins bind to the membrane in sharply localized regions at the
leading edges of amoebae and neutrophils
(Parent et al., 1998;
Meili et al., 1999;
Servant et al., 2000;
Funamoto et al., 2001). PH domains that bind specifically to
phosphatidylinositol 3,4,5 trisphosphate (PI(3,4,5)P3) and
phosphatidylinositol 3,4 bisphosphate (PI(3,4)P2), mark the front
most strongly (Dormann et al.,
2002; Lemmon et al.,
2002).
Production and regulation of PI(3,4,5)P3 and
PI(3,4)P2 require most significantly PI 3-kinases (PI3Ks) and PI
3-phoshatases (PTEN; Wymann et
al., 2000; Maehama et
al., 2001; Vanhaesebroeck
et al., 2001). In D. discoideum, there are three
PI3Ks, related to the mammalian p110 PI3K, and a single orthologue of
mammalian PTEN (Zhou et al.,
1995; Iijima and Devreotes,
2002). These enzymes are coordinately regulated in the opposite
directions (Funamoto et al.,
2002; Iijima and Devreotes,
2002). In resting cells, the PI3Ks are in the cytosol, whereas
PTEN is on the membrane. With uniform stimuli, the PI3Ks transiently
redistribute from the cytosol to the membrane, whereas PTEN dissociates from
the membrane and enters the cytosol. Within a few minutes the enzymes return
to their original configurations. In chemotaxing cells, the PI3Ks are targeted
to the membrane at the front, whereas PTEN is localized on the membrane at the
rear. These movements appear to play a key role in the directional response.
In pi3k1–/pi3k2– cells
(PI3K1 and PI3K2 have been disrupted) there is a decrease in PH domains
recruited to the membrane and marking the leading edge of the chemotaxing
cells (Funamoto et al., 2001). The ability of these cells to orient
and move in chemotactic gradients is diminished. Similar results were obtained
for neutrophils lacking PI3K
(Hannigan et al.,
2002). In pten– cells, the association
of PH domains with the membrane lasts much longer than in wild-type cells, and
the region of PH domain binding and pseudopodia extension is extensively
broadened suggesting that these binding sites play a central role in
instructing the responses involved in chemotaxis, including actin
polymerization, to occur at the cell's leading edge
(Iijima and Devreotes, 2002).
These observations have led to a working hypothesis for directional sensing
and chemotactic orientation. Signals from uniformly distributed upstream
components regulate membrane association and activation of the key enzymes of
PI metabolism. Asymmetry in the response first appears as the movements of
PI3Ks and PTEN to the membrane at the front and rear of the cell,
respectively. The resulting local accumulation of specific PIs directs events
involved in pseudopodia formation.
This is a compelling model, but the alleged changes in PI levels and enzyme
activation have not been defined. The only previous report examining PI levels
in D. discoideum states that there are no changes in these lipids
during exposure of cells to cAMP (Zhou
et al., 1998). Similarly, the lipid production in
mammalian leukocytes in response to chemokines has been measured, but its
relationship to PI3K activity was not determined
(Traynor-Kaplan et al.,
1989). Previous reports have focused on the translocation pattern
of PH domains and activation of protein kinase B (PKB) as indicators of local
PI increases and PI3K activation. Although these measurements are informative,
they are indirect and, in fact, there is no evidence that PI3K is activated by
chemoattractant. It is equally possible that changes in PIs reflected in the
PH domain translocation and PKB activation are due to decreases in PTEN
activity. To address these issues, we developed assays to directly measure the
relative amounts of PI(3,4,5)P3 in wild-type,
pi3k1–/pi3k2–, and
pten– cells and to monitor the state of PI3K
activation in various cells lines during chemotactic stimulation. We further
devised a reconstitution assay that reproduces activation and inactivation of
PI3K by chemoattractant and G-protein. Thus, for the first time the initial
events leading to directional sensing can be examined in a cell free
system.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). Cell lines used included wild-type (AX3), a PH-eGFP-His
expressed in AX3 (YH7), Myr-PI3K1 and Myr-PI3K2 expressed in
pi3k1–/pi3k2– (Myr-PI3K1 and Myr-PI3K2), and
null mutants pten–,
pi3k1–/pi3k2–, g
2-(myc2),
g
–(lw6), and yakA–.
Purification of PHCrac-eGFP-His from D. discoideum
Cells were resuspended in DB at density of 2 x 107/ml.
Cells were shaken at 100 rpm for 2 h. The cells were centrifuged and
resuspended in ice cold buffer A (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, and 5 mM
immidazole). The supernatant was obtained as described
(Lilly and Devreotes, 1994)
and loaded onto a Ni2+ chelating column (Novagen,
Madison, WI). The column was washed with buffer A and washed again with buffer
A containing 25 mM immidazole. The protein was eluted at 60, 200, 350, and 500
mM immidazole in buffer A. Then each fraction was concentrated to 1 ml using
Centriplus 30 (Amicon).
Dot Blot Assay
The phosphoinositides (Matreya Inc. and Avanti Polar Lipids Inc.) at 0.1
µg/µl in chloroform:methanol:dH2O (1:1:0.3) were spotted (2
µl) onto a PVDF membrane. After drying, the PVDF membrane was blocked in
TBS-T with 3% BSA for 1 h. The purified protein (PHCrac-eGFP-His)
was added to 10 ml TBS-T, and this solution was used to incubate the PVDF
membrane for 2 h at 4°C. The membrane was processed for Western blotting
using anti-GFP antibody.
Inositol Headgroup Competition
The specified concentrations (10 µM, 1 µM, 100 nM, 10 nM, 1 nM, and
100 pM) of unlabeled competitor (Cal Biochem; 5 µl) were included in
50-µl samples (final volume) that contained 10 µl purified
PHCrac-eGFP-His, 5 µl 10x binding buffer (250 mM Tris, pH
7.4, 1 M KCl, and 10 mM EDTA), 5 µl 5 mg/ml -globulin, and 20 nCi of
[3H]Ins(1,3,4,5)P4 (NEN Life Science Products; 21
Ci/mmol). The samples were incubated on ice for 30 min. Protein was
precipitated with 35 µl of ice cold 30% (wt/vol) polyethylene glycol (PEG)
3350. Samples were vortexed and incubated on ice for additional 10 min.
Samples were centrifuged at 10,000 x g at 4°C. Pellets were
washed with 1 ml of wash buffer (50 ml 1x binding buffer and 35 ml 30%
PEG). Samples were centrifuged again at 10,000 x g for 5 min.
The final pellet was dissolved in 1 ml of 1% SDS and counted.
PI3K Activation in Response to GTPS and cAMP
The 5 h developed cells were treated with 20 mM caffeine for 20 min to
synchronize the signaling of the cells. These cells were either treated with
100 µM GTPS or 10 µM cAMP, and the cells were lysed through a
5-µm filter. The cell lysates were incubated with 20 µCi
ATP32P (NEN Life Science Products; 3000 Ci/mmol) for 3 min
or 30 s in response to GTPS or cAMP stimulation, respectively. The
kinase reaction was stopped by 1 ml 1N HCl. The lipids were extracted with 2
ml chloroform:methanol (1:1). The samples were centrifuged at 1000 rpm for 5
min. The lower phase was isolated and further extracted with 2 ml methanol:1N
HCl(1:1). The lower phase was isolated and dried under N2 gas. The
silica gel 60 TLC plate (VWR) was prerun overnight with 1.2% potassium oxalate
(Sigma) in dH2O:methanol (3:2). The next day the TLC plate was
dried and heat-activated in the oven (100°C) for 3 min. The dried lipid
samples were resuspended in 30 µl of chloroform:methanol (2:1). Ten
microliters of sample was spotted and analyzed by TLC using
chloroform:acetone:methanol:acetic acid:dH2O (30:12:10:9:6) as a
mobile phase. After the solvent front had reached the top of the plate, the
plate was taken out the tank and dried. The Kodak film was used for
autoradiograph. To calculate the specific activity of PI3K enzyme, we did the
following. We excised the bands that corresponded to PI(3,4,5)P3
and counted using scintillation counter. At 5 s, the rate of
PI(3,4,5)P3 production was at a peak. In a typical experiment, the
input of radioactivity was 20 µCi (4.44 x 107 dpm). The
PI(3,4,5)P3 produced at 5 s corresponded to 4200 dpm. We performed
an experiment to estimate a 200 µl of reaction contained 2 nm of ATP. Thus
PI(3,4,5)P3 produced was 
1 pm/107 cells/min
assuming 100% recovery through extraction and TLC. All the PI3K reactions in
this article had similar activity range.
Adaptation Experiment
The 5 h developed cells were treated with 20 mM caffeine for 20 min. During
these 20 min, the cells were either treated with buffer or with 10 µM cAMP
for every 2 min. Then the cells were lysed, and the lysates were stimulated
with 100 µM GTPS. The cell lysates were incubated with
ATP32P for 3 min as described in the previous section. The
extraction of lipids follows the previous section.
Intact Cell Lipid Labeling and Extraction
The cells were developed in MES-DB buffer (20 mM MES, pH 6.5, 2 mM
MgSO4, 0.2 mM CaCl2) for 5 h. Then the cells were
labeled with 32P (NEN Life Science Products; 8500-9120 Ci/mmol) in
which 1 mCi was added to 1 ml of cell suspension for 1 h and washed twice with
MES-DB buffer. The cells were stimulated with 10 µM cAMP, and the reaction
was stopped with 1 ml ice-cold 1N HCl. The lipids were extracted as previously
described.
Reconstitution of PIP3 Production Using Wild-type
Supernatants
The 5 h developed and caffeine-treated, wild-type and
pi3k1–/pi3k2– cells were
stimulated with 10 µM of cAMP. The cells were lysed through 5-µm filter.
The lysates were mixed with supernatants of wild-type and
pi3k1–/pi3k2– cells for 20
s. The reaction was stopped by diluting in 1 ml of ice-cold PM. For
GTPS stimulation, wild-type and
pi3k1–/pi3k2– cells were
lysed in the presence of 100 µM GTPS. The lysates were incubated
with supernatants of wild-type and
pi3k1–/pi3k2– cells for 5
min before being stopped with 1 ml of PM. In both experiments, membrane was
pelleted and blotted for anti-GFP antibody.
|
RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). Crac has a PH domain at its N-terminus, and the PH domain
is necessary and sufficient to mediate membrane localization. We used
PHCrac as a probe to investigate the binding sites for Crac on the
plasma membrane. We expressed a PHCrac-eGFP-His fusion protein in
D. discoideum and purified it using Ni2+
chelating affinity chromatography. The protein was mainly eluted in the
200–350 mM immidazole fractions; in the purified fractions it was
30% of the total protein (unpublished data). As shown by the dot blot
assay, the purified protein bound specifically to two phosphatidyinositol
lipids, PI(3,4,5)P3 and PI(3,4)P2
(Figure 1A). After serially
diluting concentrations of PI(3,4,5)P3 and PI(3,4)P2,
PHCrac bound to slightly lower amounts of PI(3,4,5)P3
than PI(3,4)P2 (Figure
1B). Similar results were obtained with unpurified protein in
high-speed supernatant.
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To examine the lipid headgroup specificity more carefully, we developed a competition assay. Four lipid headgroups, Ins(1,3,4,5)P4, Ins(1,3,4)P3, Ins(1,4,5)P3, and IP6, and PI(3,4,5)P3 were used to compete binding of [3H]Ins(1,3,4,5)P4 to PHCrac. With different concentrations of unlabeled compound ranging from 100 pM to 10 µM, each behaved as a competitive inhibitor. The concentrations that displaced 50% of bound [3H]Ins(1,3,4,5)P4 were 50, 50, and 500 nM for Ins(1,3,4,5)P4, PI(3,4,5)P3, and Ins(1,3,4)P3, respectively (Figure 1C). Ins(1,4,5)P3 and IP6 did not effectively compete [3H]Ins(1,3,4,5)P4 binding (Figure 1C).
PI(3,4,5)P3 Changes in Response to Chemoattractant
Stimulation
The dot blot assay indicates that PHCrac has high affinity for
PI(3,4,5)P3 and PI(3,4)P2 and that its relocation to the
membrane reflects changes in these PIs. We used TLC to measure changes in bulk
levels of PI(3,4,5)P3 upon chemoattractant stimulation. We
metabolically labeled the wild-type,
pi3k1–/pi3k2–, and
pten– cells with 32P and stimulated these
three cell lines with 10 µM cAMP (see
Figure 2A). We could not detect
any changes in PI(3,4,5)P3 in
pi3k1–/pi3k2– cells
(unpublished data). In wild-type cells, levels of PI(3,4,5)P3
peaked within 5 s after addition of cAMP and declined after 20 s of cAMP
stimulation (Figure 2B). In
contrast, in pten– cells levels of
PI(3,4,5)P3 peaked at 5 s, remained elevated at 20 s, and declined
but remained above prestimulus levels even after 180 s of cAMP stimulation
(Figure 2B).
|
As shown in Figure 2B, the cell labeling experiment directly measures the relative amounts of PI(3,4,5)P3. Because PHCrac is highly selective for both PI(3,4,5)P3 and PI(3,4)P2, the PHCrac translocation assay probably measures the changes in membrane levels of these two lipids (Figure 2A). Upon uniform cAMP stimulation, PHCrac associates with membrane within 5 s and dissociates from membrane within 60 s (also see Figure 6A). The PHCrac translocation profile closely tracks the increase in the level of PI(3,4,5)P3 in wild-type cells, but it does not match exactly in pten– cells. In pten– cells, the PHCrac remains associated with the membrane even after 3 min of cAMP stimulation. Yet as shown in Figure 2B, the highest levels of PI(3,4,5) P3 are sustained for only 60 s. We can offer two possible explanations for this discrepancy. First, it is possible that the combined levels of PI(3,4,5)P3 and PI(3,4)P2 are sustained at high levels for a longer time in pten– cells. Alternatively, the PHCrac-GFP binding to the membrane saturates and does not subside until PI(3,4,5)P3 decreases very substantially. Of course we cannot rule out that PHCrac may bind to something other than PI(3,4,5)P3 and PI(3,4)P2.
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Activation of PI3K in Response to Chemoattractant and GTPS
Stimulation
The steady state level of PI(3,4,5)P3 results from the balance
between PI3K and PTEN activities. We developed two assays to measure the
activation state of PI3K, reflecting chemoattractant regulation. The in vivo
assay measures the state of PI3K activation in brief intervals during
stimulation of cells with cAMP. The in vitro assay measures PI3K activation in
cell lysates treated with GTPS. The nonhydrolyzable analogue of GTP can
directly activate trimeric G proteins bypassing the need for chemoattractant
receptors.
To determine whether the pattern of the PI3K activation during
chemoattractant stimulation parallels its translocation to the membrane, we
used the in vivo assay. The wild-type,
pi3k1–/pi3k2–, and
pten– cells were persistently stimulated with
uniform cAMP. At times during stimulation, the cells were lysed and the state
of PI3K activation was monitored with a 30-s incubation with
ATP32P followed by measurement of
PI(3,4,5)P3-32P. This experiment differs from that
described above where cells were labeled with 32P because here only
newly synthesized lipids will incorporate the label. In wild-type cells, the
rate of PI(3,4,5)P3-32P synthesis peaked within 5 s and
started to decline within 20 s (Figure
3A). Significantly, the kinetics of PI3K activation was
essentially identical in pten– cells and in
wild-type cells, suggesting that the products of PI3K do not influence its
activation (Figure 3A). In
addition, the phosphatase activity of PTEN is not a significant factor in the
assay. In pi3k1–/pi3k2–
cells, the activation of PI3K was greatly reduced but not completely
abolished: its peak level was 4% of that wild-type and
pten– cells
(Figure 3A). The residual
activation in
pi3k1–/pi3k2– cells is
likely due to another PI3K, such as PI3k3.
|
Because the PI3K activation mirrors the translocation pattern of the kinases, we examined the role of membrane recruitment in the activation. We expressed N-terminal myristol tagged PI3Ks that were targeted to the membrane. In wild-type cells, the basal activities of PI3K were minimal and showed a transient activation upon cAMP stimulation (Figure 3, A and B). Similarly, when wild-type PI3K2 was transformed into pi3k1–/pi3k2– cells, the basal level of PI3K activity was not elevated (unpublished data). However, when myristolated PI3K1 and PI3K2 (Myr-PI3K1 and Myr-PI3K2) were introduced into pi3k1–/pi3k2– cells individually, the prestimulus levels of PI3K activities were significantly elevated in both cells lines (Figure 3B). Upon cAMP stimulation, there was a further activation of PI3K in both cell lines. In Myr-PIK1 cells, the increase was twofold, and the activation decayed very slowly. For Myr-PI3K2 cells, the peak PI3K activation at 5 s was higher than in wild-type cells, and the profile of Myr-PI3K2 activation was more similar to that of wild-type cells. These results indicate that this membrane localization of PI3Ks alone can increase the basal activities of the enzymes, but there may be additional activation during stimulation.
The PI3Ks were also directly activated in cell lysates treated with
GTPS. Using the in vitro assay, lysates of wild-type,
pi3k1–/pi3k2–, and
pten– cells were incubated with GTPS and
ATP32P, and the lipids were extracted and analyzed by TLC.
As shown in Figure 4A, the
intensities of PI(3,4,5)P3 bands, nearly undetectable in
unstimulated cells, were greatly increased in both wild-type and
pten– lysates during GTPS treatment. The fold
of PI3K activation by GTPS was about six- and eightfold in wild-type
and pten– cells, respectively. In
pi3k1–/pi3k2– cells, the
PI3K activation by GTPS was 4% of that of wild type. We pretreated
the wild-type cells with different concentrations of LY294002, a PI3K
inhibitor, to determine whether the treatment mimics the effect observed in
pi3k1–/pi3k2– cells
(Figure 4B). With 10 µM
LY294002, the intensity of PI(3,4,5)P3 band with GTPS
stimulation was significantly reduced, and at 100 µM there was essentially
no increase over the basal level. The intensity of PIP2 band
containing both PI(4,5)P2 and PI(3,4)P2 was also reduced
with increasing concentrations of LY294002. Although the TLC cannot separate
PI(3,4)P2 from PI(4,5)P2, we speculate that a reduced
amount of PI(3,4)P2 contributed to the weaker intensity
(Zhou et al.,
1998).
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Dependence of PI3K Activation on G Complex,
G2, and YakA
We next examined the upstream signaling components required for the
activation of PI3K. In D. discoideum, the trimeric G proteins that
coupled to cAMP receptors (cAR1s) are comprised of G2 and a unique
G complex. YakA is a member of Dyrk (dual specificity Yak-related
kinases) family. Its position in the G-protein–mediated signaling
pathway remains to be defined, but it was suggested that YakA lies downstream
of G-protein but upstream of PHCrac translocation
(van Es et al.,
2001). We measured PI3K activation in wild-type,
g2–,
g–, and yakA–
cells. We also included piaA– (pianissimo) cells,
which specifically lack adenyly cyclase activation as an additional control
(Chen et al., 1997).
In the in vivo assay with cAMP, the wild-type and
piaA– cells responded normally, whereas all of the
other cells failed to give a response. In the in vitro assay with GTPS,
PI3K activation in g2-cells was reduced
60% compared with that in wild-type cells
(Figure 5A). In
g– cells, GTPS-mediated PI3K
activation was absent. Levels of G2 and
G are unaffected by disruption of their counterparts
(Janetopoulos et al.,
2001 and unpublished observations). In
yakA– cells the reduction in PI3K activation was not
complete as in g– cells, but the
fold was greatly reduced and was lower than that in
g2– null cells
(Figure 5A). Functional levels
of receptor/G-protein interaction and PI3K activity are normal in
yakA– cells (van
Es et al., 2001 and unpublished data). These results
demonstrate that signal that triggers the activation of PI3K acts through
G complex and that G2 and
YakA also contribute to the activation of PI3K.
|
PI3K Activation Is Reduced during Adaptation
Prolonged stimulation of wild-type cells with cAMP induces adaptation, a
phenomenon in which cells, once having responded, cannot respond further to
the existing stimulus unless a higher concentration is given. Activation of
adenylyl and guanylyl cyclase, actin polymerization, and shape changes all
display this behavior (Parent and
Devreotes, 1996). FRET (fluorescence resonance energy transfer)
studies of G2 demonstrate that the 2 and subunits
remain dissociated as long as the stimulus persists
(Janetopoulos et al.,
2001). Like the other physiological responses, the recruitment of
PHCrac to the plasma membrane adapts during the persistent
treatment with cAMP (Lilly and Devreotes,
1995). These observations suggest that adaptation does not require
the G-protein subunits to reassociate but occurs upstream of
PI(3,4,5)P3. We next examined whether activation of PI3K by
GTPS was affected during adaptation. The in vitro assay was performed
on lysates of wild-type cells treated with either buffer or with persistent
cAMP. With no cAMP pretreatment, GTPS elicited an eightfold increase in
PI3K activation (Figure 5B). However, in cells that had been pretreated with 10 µM cAMP, GTPS
elicited only a threefold increase in PI3K activation, a 65% decrease in the
response (Figure 5B). These
data demonstrate that the capacity to activate PI3K adapts during persistent
stimulation.
Key Regulator Event in Chemotaxis Lies Upstream of PI3Ks
We next addressed whether these regulatory events acted directly on PI3K or
whether there are other regulators upstream of PI3Ks that predetermine the
activation and inhibition. In the latter instance, the PI3Ks may merely
transmit and amplify these upstream signals. We devised a reconstitution
assays to distinguish these possibilities. Wild-type or
pi3k1–/pi3k2– cells were
stimulated with 10 µM cAMP and lysed at specific time points as described
in the previous assays. However, in the reconstitution assays, cell lysates
were mixed with supernatants of
pi3k1–/pi3k2– or wild-type
cells. The supernatants also contained PHCrac-eGFP, which bound to
PI(3,4,5)P3 and PI(3,4)P2 produced during a brief
incubation of the mixed lysates and supernatants. Thus, we are using
PHCrac-eGFP bound to membrane as a readout for the production of
PI(3,4,5)P3 and PI(3,4)P2.
Figure 6A shows that for
wild-type cells, the capacity of mixed cell lysates and supernatants to
produce PI(3,4,5)P3 varied with the time after stimulation with
chemoattractant. Lysates taken at 5 s after stimulation were extremely active,
whereas lysates at 60 s were much less active. In the lysates of
pi3k1–/pi3k2– cells, there
was no PI(3,4,5)P3 production. These results indicate that PI3Ks
are required for PI(3,4,5)P3 production. However when the
supernatant mixed into the assay was from wild-type cells, the capacity of the
pi3k1–/pi3k2– cell lysates
to produce PI(3,4,5)P3 was restored
(Figure 6B). Similar results
were also obtained in assays where lysates were treated with GTPS
(Figure 6C). The lysates of
wild-type cells treated with GTPS produced PI(3,4,5)P3
regardless the source of supernatant that was mixed into the assay. However,
lysates of pi3k1–/pi3k2–
cells treated with GTPS only produced PI(3,4,5)P3 when
supplied with wild-type supernatant. These results demonstrate that an unknown
regulator is transient activated upon cAMP stimulation even in the absence of
PI3Ks. Addition of PI3Ks in the supernatant transmits and propagates this
signal.
|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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complex but independent
of PTEN, suggesting that PI(3,4,5)P3 and PI(3,4)P2 do
not markedly affect regulation. Membrane recruitment of PI3Ks plays a partial
role in activating the enzymes, and there may be additional activation of
membrane bound enzymes. Finally, the temporal regulation of the PI3Ks can be
reproduced in a cell free system. With this reconstitution assay, we show that
the binding/activation sites for PI3Ks are generated in the absence of the
enzymes themselves. Our data are the first demonstration that chemoattractant stimulation elicits an increase in PI(3,4,5)P3 levels in D. discoideum. The rise is transient in wild-type cells, whereas it is higher and more prolonged in pten– cells. The sustained high levels of PI(3,4,5)P3 likely contribute to the dramatic chemotactic defects observed in pten– cells. Consistent with this, the defects in pten– cells can be ameliorated by LY294002. A low dose of LY294002 reduces the prolonged PH domain association with membrane and restricts the localization of these proteins to the leading edge (Chen, personal communication). In pi3k1–/pi3k2– cells, the changes in PI(3,4,5)P3 levels in response to stimulus were below our limits of detection. This is consistent with the observation that PH domains weakly redistribute to membrane and are not localized at leading edge of chemotaxing pi3k1–/pi3k2– cells (Funamoto et al., 2001). Thus the level of PI(3,4,5)P3, a result of a balance between PI3Ks and PTEN activity, is critical for a cell to sense direction properly. Our methods using TLC cannot resolve PI(4,5)P2 and PI(3,4)P2 so we have not yet examined the changes in the level of PI(3,4)P2. It would be interesting to measure the time course of this PI and learn whether the changes of PI(3,4)P2 are primarily due to the breakdown of PI(3,4,5)P3.
Our results are the first evidence that the PI3Ks are activated upon
chemoattractant stimulation. The rise in PI(3,4,5)P3 levels
elicited by chemoattractant can be in principle due to activation of PI3Ks,
inhibition of PTEN, or combination of both mechanisms. The in vivo assay using
PI(3,4,5)P3-32P as a readout rules out the possibility
that the rise in the level of PI(3,4,5)P3 is solely due to the
inhibition of PTEN with no regulation on PI3Ks. We observed a highly regulated
pattern of PI3K activity, similar to that of wild-type cells, even in
pten– cells. Because the assay relies on endogenous
substrate and ATP32P to form
PI(3,4,5)P3-32P, it theoretically could report other
activities besides PI3Ks. However, because most of the activity is absent in
the pi3k1–/pi3k2– cells,
it is likely that the assay is specific for PI3Ks that act on
PI(4,5)P2. There also appears to be a PI3K activity that uses
PI(4)P as substrate in vitro because LY294002 decreases the amount of combined
PI(4,5)P2 and PI(3,4)P2. However, this activity does not
appear to be influenced by chemoattrantant receptor or G-protein
activation.
The finding that PI3K activation is independent of PTEN has important
consequences for feedback regulation. Recent studies using a cationic lipid
shuttling system to deliver exogenous PI(3,4,5)P3 to neutrophils
suggested that PI(3,4,5)P3 cooperates with endogenous PI3Ks (and
Rho GT-Pase) in a positive feedback loop that is necessary to establish
polarity in response to chemoattractant
(Niggli, 2000;
Weiner et al., 2002).
Our results in D. discoideum rule out feedback from
PI(3,4,5)P3 to PI3K. Even though the levels of
PI(3,4,5)P3 are higher and sustained in
pten– cells, the time course of PI3K activation is
essentially identical with that in wild-type cells: peaking at 5 s and already
declining by 20 s. Thus the decline in PI3K activation appears to be
independent of PI levels.
The PI3K activity is critically dependent on the localization of the enzymes, but the membrane targeting may not be the sole mechanism of activation. In wild-type cells activation of PI3Ks mirrors their redistribution from cystosol to the membrane in response to chemoattractant stimulation. Myristoylation targets the enzymes constitutively to the membrane and raises the basal level of activation, indicating that membrane association is sufficient for activation. Thus close proximity with substrate PI(4,5)P2 apparently can lead to increased activity. However, additional mechanisms are required to further activate the enzyme upon chemoattractant stimulation because both Myr-PI3K1 and Myr-PI3K2 can be further activated by addition of chemoattractant. These additional mechanisms might simply be the recruitment of an additional myristoylated PI3Ks in the cytosol. Alternatively, the membrane tethered PI3Ks can be further activated.
Our studies focus attention on the upstream components that recruit and
activate the PI3Ks and PTEN. Although it has been reported that Ras can
activate PI3Ks, it is not required for their membrane recruitment
(Funamoto et al.,
2002). The mammalian PI3K is activated by
subunits of G-protein in vitro, and evidence suggests that chemotactic
signaling does proceed through the complex
(Hirsch et al., 2000;
Li et al., 2000;
Sasaki et al., 2000).
However G complex cannot be the sole upstream
signaling component because the distribution and activation of
G in a chemotaxing cell are not as polarized as that of PI3Ks
(Jin et al., 2000).
Furthermore, the activation of the G-protein is persistent, whereas that of
PI3K is transient (Janetopoulos et
al., 2001). The binding sites for PTEN are not well
understood, although PTEN has a conserved N-terminal PI(4,5)P2
binding motif and its deletion renders the enzyme totally cytosolic
(Iijima and Devreotes, 2002).
This suggests that binding site for PTEN is PI(4,5)P2, raising an
interesting possibility. As reported previously, the enzymes PI3K and PTEN are
reciprocally regulated. It is intriguing to speculate based on the observation
by Weiner et al.
(2002) that
PI(3,4,5)P3 activates PI3K and that the binding site for PI3K is
PI(3,4,5)P3. If this were the case, during chemoattractant
stimulation the binding site for PTEN would be converted to that for PI3Ks,
and the binding of the two enzymes would be mutually exclusive. However, as
noted in D. discoideum the creation of binding sites for PI3Ks is
unaffected by perturbations which lower or elevate the level of
PI(3,4,5)P3. Thus this idea needs to be further studied.
Figure 7 schematically
summarizes the new findings of the mechanism of activation of PI3K by
chemoattractant receptor and G-protein. In the resting cells, there are few
binding/activation sites on the membrane for PI3Ks, and PI3Ks are in the
cytosol. Upon activation of cells with cAMP or cell lysates with GTPS,
binding/activation sites for PI3Ks rapidly appear on the inner face of the
membrane. The sites recruit and activate PI3Ks. More importantly, the creation
of binding/activation sites can take place in the absence of PI3Ks, and
cytosolic PI3Ks from unstimulated cells can be supplied to bind to the
activated membrane to restore the production of PI(3,4,5)P3.
Because the binding sites are created so transiently, we anticipate the sites
to be a modification of an existing protein by conformational change or by
phosphorylation/dephosphorylation. Persistent cAMP treatment leads to
adaptation, resulting in the loss of binding/activation sites and a diminished
capacity of GTPS to generate these sites.
|
Our data suggest that models for directional sensing should focus on the
binding sites for PI3K and PTEN. One model suggests that sensing depends on
the balance between excitation and inhibition processes
(Parent and Devreotes, 1999;
Levchenko and Iglesias, 2002).
Our results suggest that this balance controls the binding/activation sites
for PI3Ks. When a cell is exposed to a uniform increase in chemoattractant,
the binding/activation sites for PI3Ks rapidly increase and then gradually
decrease. The binding sites of PTEN behave oppositely to those of PI3Ks. When
a gradient is applied, the sites for PI3Ks form at the cell front while those
for PTEN form at the back. In wild-type cells, the net effect of the
coordinated movements of these two enzymes generates transient increase in the
membrane levels of PI(3,4,5)P3 upon uniform stimulation and leads
to a large increase in PI(3,4,5)P3 at the front of chemotaxing
cells. In pten– cells, the PI3K activation is not
affected, but elevated lipids are sustained, causing PH domains to associate
with membrane longer. In chemotaxing pten– cells,
the binding/activation sites are generated and PI3Ks move normally to the
anterior. However directional sensing is impaired because there is wider front
with PI(3,4,5)P3 accumulation, presumably because
PI(3,4,5)P3 diffusing further from its source at the front.
|
ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Footnotes |
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Corresponding author. E-mail address:
pnd{at}jhmi.edu.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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