Molecular Biology of the Cell
Vol. 18, 4772–4779, December 2007
Phospholipase C Regulation of Phosphatidylinositol
Arjan Kortholt,*†Jason S. King,†‡Ineke Keizer-Gunnink,* Adrian J. Harwood,‡
and Peter J.M. Van Haastert*
*Department of Molecular Cell Biology, University of Groningen, 9751 NN Haren, The Netherlands;
and‡School of Biosciences, Cardiff University, Cardiff CF10 3US, United Kingdom
Submitted May 4, 2007; Revised August 6, 2007; Accepted September 17, 2007
Monitoring Editor: John York
Generation of a phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] gradient within the plasma membrane is important
for cell polarization and chemotaxis in many eukaryotic cells. The gradient is produced by the combined activity of
phosphatidylinositol 3-kinase (PI3K) to increase PI(3,4,5)P3on the membrane nearest the polarizing signal and PI(3,4,5)P3
dephosphorylation by phosphatase and tensin homolog deleted on chromosome ten (PTEN) elsewhere. Common to both
of these enzymes is the lipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], which is not only the substrate of PI3K
and product of PTEN but also important for membrane binding of PTEN. Consequently, regulation of phospholipase C
(PLC) activity, which hydrolyzes PI(4,5)P2, could have important consequences for PI(3,4,5)P3localization. We investigate
the role of PLC in PI(3,4,5)P3-mediated chemotaxis in Dictyostelium. plc-null cells are resistant to the PI3K inhibitor
LY294002 and produce little PI(3,4,5)P3after cAMP stimulation, as monitored by the PI(3,4,5)P3-specific pleckstrin
homology (PH)-domain of CRAC (PHCRACGFP). In contrast, PLC overexpression elevates PI(3,4,5)P3and impairs chemo-
taxis in a similar way to loss of pten. PI3K localization at the leading edge of plc-null cells is unaltered, but dissociation
of PTEN from the membrane is strongly reduced in both gradient and uniform stimulation with cAMP. These results
indicate that local activation of PLC can control PTEN localization and suggest a novel mechanism to regulate the internal
Chemotaxis, or migration toward a concentration gradient
of chemoattractant, is an essential response of many cells. It
plays an important role in a multitude of biological pro-
cesses and organisms, such as finding nutrients in pro-
karyotes, forming multicellular structures in prokaryotes,
and tracking bacterial infections in neutrophils (Baggiolini,
1998; Campbell and Butcher, 2000; Crone and Lee, 2002).
Chemotaxis is a complex cellular process involving several
signaling pathways and molecules. The initial event is bind-
ing of chemoattractant to cell surface receptors. Receptors
convert these signals to the interior of the cell were they
activate a complex network of signaling pathways, resulting
in a gradient of cellular components. This gradient induces
coordinated remodeling of the cytoskeleton and cell adhe-
sion to the substratum, which leads to formation of new
actin filaments in the front that induce the formation or
stabilization of local pseudopodia, and acto-myosin filaments
at the back that inhibit pseudopod formation and retract the
2007). The final outcome is cellular movement up the chemoat-
Dictyostelium cells are single-celled amoeba that feed on
bacteria. On starvation, cells undergo a tightly regulated
developmental process in which they secrete and chemotax
toward cAMP, resulting in multicellular fruiting bodies. Be-
cause the mechanism of chemotaxis is essentially identical in
all eukaryotes, Dictyostelium offers a genetically tractable
model system in which to study chemotaxis (Devreotes and
Zigmond, 1988; Van Haastert and Devreotes, 2004). An im-
portant response in both the establishment of cell polarity
and chemotaxis is the formation and accumulation of phos-
phatidylinositol 3,4,5-triphosphate [PI(3,4,5)P3] at the lead-
ing edge (Parent et al., 1998; Funamoto et al., 2002; Huang et
al., 2003). PI(3,4,5)P3is produced by the phosphorylation of
phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] by phos-
phatidylinositol 3-kinases (PI3Ks), and it is reversed by the
inositol 3-phosphatase action of PTEN (Parent et al., 1998;
Funamoto et al., 2002; Huang et al., 2003). A PI(3,4,5)P3
gradient is accomplished by the reciprocal temporal and
spatial regulation of PI3K at the leading edge and phospha-
tase and tensin homolog deleted on chromosome ten (PTEN)
at the back of the cell. The accumulation of PI(3,4,5)P3at the
leading edge results in recruitment of PI(3,4,5)P3binding
signaling molecules and subsequent pseudopod extension
(Parent et al., 1998; Funamoto et al., 2002; Iijima and
It has long been known that an early event after cAMP
stimulation is the hydrolysis of PI(4,5)P2by phospholipase C
(PLC) to form two important second messengers, diacylglyc-
This article was published online ahead of print in MBC in Press
on September 26, 2007.
VThe online version of this article contains supplemental material
at MBC Online (http://www.molbiolcell.org).
†These authors contributed equally to this work.
Address correspondence to: Peter J.M. van Haastert (p.j.m.van.haastert@
4772 © 2007 by The American Society for Cell Biology
erol (DAG) and inositol-1,4,5-triphosphate [I(1,4,5)P3] (Katan,
1998). Dictyostelium contains a single plc gene, structurally sim-
ilar to mammalian PLC-? and consists of N-terminal PH and
EF hand domains followed by a split catalytic domain (all
eukaryotic PLC enzymes have an insertion in the catalytic
domain) and a C-terminal conserved C2 domain (Drayer and
van Haastert, 1992; van Haastert and Van Dijken, 1997). The
enzyme is Ca2?dependent and upon cAMP stimulation PLC
activity is increased approximately twofold. cAMP both stim-
ulates and inhibits PLC activity via G?2and G?1G protein
subunits, respectively (Bominaar and van Haastert, 1994;
Bominaar et al., 1994; Drayer et al., 1995). Previously, a plc-null
mutant was generated, and although the plc-null mutant had
no measurable PLC activity, growth, development and chemo-
taxis seemed normal (Drayer et al., 1994). Surprisingly, plc-null
cells also contain normal levels of I(1,4,5)P3generated by a
second route of synthesis via a I(1,3,4,5,6)P53/6-biphosphatase
that is up-regulated in plc-null cells (Van Dijken et al., 1997).
Previous work has generally concentrated on the roles of the
produced second messengers I(1,4,5)P3and DAG and the ob-
servation that PLC seemed to play no role in chemotaxis was
reenforced by mutation of the inositol triphosphate receptor
iplA, which also had no apparent phenotype (Traynor et al.,
2000). Here, because PI(4,5)P2is also the substrate of PI3K at
the leading edge and it is important for the membrane local-
ization of PTEN in the rear of the cell, we investigate an
alternative role for PLC in the regulation of PI(3,4,5)P3-medi-
Although pten-null mutants have a relatively strong phe-
notype in which they form multiple pseudopdia, and they
are defective in chemotaxis (Iijima and Devreotes, 2002), loss
of PI3K activity through gene knockout or use of the PI3K
inhibitor LY294002 has only mild phenotypic effects, dem-
onstrating that the PI(3,4,5)P3is not essential for direction
sensing, or cell movement (Loovers et al., 2006; Hoeller and
Kay, 2007; Takeda et al., 2007). This suggests that PI(3,4,5)P3-
mediated signaling works in conjunction with one or more
other pathways to mediate chemotaxis and that these
pathways can compensate for each other under standard
conditions. Recent studies have suggested that this sec-
ond, parallel pathway may be mediated by phospholipase
A2(Chen et al., 2007; van Haastert et al., 2007). Chen et al.
(2007) described that loss of the Ca2?-independent phos-
pholipase A2(iPLA2) homologue plaA results in cells that
are hypersensitive to LY294002 and that show dramati-
cally reduced chemotaxis (Chen et al., 2007). Van Haastert
et al. (2007) showed that inhibition of either PI3K or PLA2
has minor effects on chemotaxis, whereas inhibition of
both enzymes inhibits chemotaxis nearly completely (van
Haastert et al., 2007). Furthermore, they showed that in-
hibition of PLA2 completely blocks chemotaxis in plc-null
cells, whereas inhibition of PI3K has no effect (van Haastert et
al., 2007). To investigate whether the presence of compensa-
tory signaling processes may explain the apparent lack of
phenotype in the plc-null mutants, we investigated the role
of PLC in PI(3,4,5)P3-mediated signaling. We show here that
although plc-null cells have normal chemotaxis, they are
resistant to the effects of the PI3K inhibitor LY294002. We
further show that this does not arise through up-regulation
of PI3K signaling, but in fact it is accompanied by a dramatic
reduction. We investigated the mechanism for this change,
and we demonstrate that PLC activity regulates the localiza-
tion of PTEN, and it is important for the control of the
PI(3,4,5)P3gradient along the plasma membrane.
MATERIALS AND METHODS
To avoid effects due to specific parental strain backgrounds different plc-null
mutant strains were used in subsequent studies. The first strain 1.19 was that
described previously (Drayer et al., 1994), and it is in the DH1 strain back-
ground. The other strains, referred to as HAD236 and HAD237, were newly
generated in the AX2 strain background as described below. All experiments
were carried out with the appropriate strain controls. All cell lines were
grown in 9-cm dishes containing HG5 medium (14.3g/l peptone, 7.15 g/l
glucose, 0.49 g/l KH2PO4, and 1.36 g/l Na2HPO4?H2O). To select for trans-
formants with one of the extrachromosomal plasmids described below, cells
were grown in HG-5 supplemented with 10 ?g/ml Geneticin (G-418; Invitro-
gen, Carlsbad, CA), hygromycin B (Invitrogen), or both. For development on
filters, log-phase growing cells were washed twice in KK2 buffer (10 mM 16.5
mM KH2PO4, and 3.8 mM K2HPO4pH 6.2), serially diluted to the appropriate
densities, and then spread onto 47 mm nitrocellulose filters (Millipore, Bil-
lerica, MA) presoaked in KK2. Images were taken at 20 h.
The plasmid pWF38 expressing the pleckstrin homology (PH) domain of
CRAC fused to enhanced green fluorescent protein (GFP) (Parent et al., 1998),
and the plasmid expressing PTEN-GFP were kindly provided by Dr. Peter
Devreotes (Iijima and Devreotes, 2002). To express PLC in Dictyostelium, the
encoding DNA fragment was amplified using the forward primer: 5?-GAA-
GATCTAAAATGGATACTTTAACAAATTC-3? and reverse primer 5?-CT-
TCTAGATTCAACAAATGTAAATTTAC-3?. The BglII/XbaI fragment was
cloned in the BglII-SpeI site of a modified vector that contains a hygromycin
instead of a neomycin resistance (Bosgraaf et al., 2005). This yielded the
Generation of an AX2-derived plc Knockout
A new plc knockout construct was made by polymerase chain reaction (PCR),
and it was used to generate AX2 cells containing a 100-bp deletion (from 199
to 298 base pairs of the genomic sequence) at the plc locus, with insertion of
the loxP-flanked blasticidin cassette from pBLPBSR (Faix et al., 2001). Success-
ful recombination was screened by PCR, and the loss of protein was con-
firmed by Western blot by using a PLC-specific antibody (Drayer et al., 1994;
data not shown).
Chemotaxis was measured with the small population assay (Konijn, 1970).
Experiments were performed in a six-well plate containing 1 ml of nonnutri-
ent hydrophobic agar (11 mM KH2PO4, 2.8 mM Na2HPO4, 7 g/l hydrophobic
agar). AX3 and plc-null cells were harvested by centrifugation for 3 min at
300 g, washed in phosphate buffer (PB) (10 mM KH2PO4/Na2HPO4), and
starved in PB buffer for 5 h. Starved cells were resuspended in PB, washed
once in PB, and resuspended in PB to a final concentration of 6 ? 106cells/ml.
Droplets of ?0.1 ?l of starved cells were placed on agar. Chemotaxis toward
cAMP was tested after 30 min by placing a second 0.1-?l droplet, with the
indicated amount of cAMP, next to the droplet of cells. The distribution of the
cells in the droplet was observed about every 10 min during 90 min, and they
were scored positive when at least twice as many cells are pressed against the
side of the population closer to higher cAMP concentration as against the
other side of the droplet. The fraction of droplets scored positive, averaged
over three successive observations at and around the moment of the maximal
response, is recorded here. The data presented are the means and SE of the
means of at least three independent measurements on different days.
Chemotaxis was also measured with micropipettes containing 10?4M
cAMP with a Zeiss LSM 510 META NLO confocal laser scanning microscope
(Carl Zeiss, Jena, Germany) equipped with a Zeiss plan-apochromatic 63?
numerical aperture 1.4 objective. The chemotaxis index defined as the ratio of
the cell displacement in the direction of the gradient, and its total traveled
distance was determined for ?25 cells in a movie as follows. First, the position
of the centroid was determined with ImageJ (rsb.info.nih.gov/ij) for frames at
30-s interval, yielding a series of coordinates for that cell. Using these coor-
dinates, the chemotaxis index of each 30-s step was calculated and averaged,
yielding the chemotaxis index for that cell in the movie. The data shown are
the average and SD of the mean of the chemotaxis indices from at least three
independent experiments with ?25 cells per experiment.
Localization of GFP-tagged Proteins during Chemotactic
The same experimental setup with micropipettes was used for analyses of
cells expressing PHcrac-GFP, PI3K-GFP, and PTEN-GFP. For excitation of the
fluorochrome GFP (S65T variant) a 488-nm argon/krypton laser was used,
and the fluorescence was detected by a BP500-530 IR LP560 photo multiplier
tube. The field of observation is 206 ? 206 ?m.
To determine the localization of marker proteins during chemotaxis, we
define the front of the cell as the foremost point of the cell in the direction of
PLC Regulation of PI(3,4,5)P3-mediated Chemotaxis
Vol. 18, December 20074773
movement toward the pipette. Then, the cell was divided into three equal
regions (front, middle, and rear region) by using two lines perpendicular to
the direction of movement. For analysis of the fluorescence intensity of
cytosolic PI3K-GFP, the mean fluorescence intensity of the cytosol in the front
one-third region was compared with the fluorescence intensity of the cytosol
in the middle-third region of the same cell, taking care to exclude the nucleus
(Postma et al., 2003). For analysis of the fluorescence intensity of membrane-
associated PTEN-GFP, the fluorescence intensity at the boundary of the cell
was determined using a three-pixel wide boundary essentially as described
previously (Postma et al., 2003). The mean fluorescence intensity of the bound-
ary in front one-third region of the cell was compared with the mean fluo-
rescence intensity of the boundary in the remaining third thirds (middle and
rear part) of the cell. Data were averaged over ?20 images of the same cell
moving toward a micropipette with cAMP. The data presented are the means
and standard deviations of this value for 10 cells.
Localization of GFP-tagged Proteins during Uniform
Before stimulation, cells were washed and resuspended at 5 ? 106cells/ml in
KK2 buffer, and they were given 6-min pulses of 100 nM cAMP for 5 h. Cells
were then transferred into a final volume of 1 ml in four-well Lab-Tek
chambered coverslips (Nalge Nunc International, Rochester, NY) and left 5
min to adhere. Cells were then stimulated with 1 ?M cAMP by the addition
of 50 ?l of a 20 ?M stock solution. For analysis of the translocation of
GFP-fusion proteins, movies were taken using an IX71 inverted fluorescence
microscope (Olympus, Tokyo, Japan). Images were taken at 2-s intervals by
using a 60? objective. Membrane association of GFP-fusion proteins was
measured indirectly by measuring changes in the fluorescence intensity of the
cytoplasm relative to prestimulated levels in that cell. This was done using the
ImageJ software, by selecting oval cytosolic sections within each cell and
calculating the mean intensity at each frame. For each experiment, a mini-
mum of 20 cells was measured in this way, over four independent simula-
Protein Kinase B (PKB)/Akt Phosphorylation Assays
Before stimulation, cells were pulsed with cAMP for 5 h, with 100 ?M
LY294002 added for the last hour when required. Cells were then washed
twice, resuspended at 2 ? 107cells/ml in KK2 (including LY294002 if appro-
priate), and left for 10 min with gentle agitation. Cell suspensions were then
stimulated with a final concentration of 1 ?M cAMP, and 100-?l were samples
removed and lysed directly into an equal volume of 2? LDS gel loading
buffer (Invitrogen) supplemented with 300 mM NaF, 1.2 mM Na3VO4, and 12
mM EDTA. Samples were then fractionated on 3–8% NuPage Tris-acetate gels
(Invitrogen), blotted onto nitrocellulose membrane (GE Healthcare, Chalfont
St. Giles, United Kingdom), and probed using an anti-phosphothreonine
antibody (Cell Signaling Technologies, Beverly, MA; catalog no. 9381).
Chemotaxis of PLC-Null Cells Is Resistant to the PI3K
Previously, we used the small population assay (Konijn,
1970; van Haastert et al., 2007) to measure the effect of
different concentrations of LY294002 on the chemotactic ac-
tivity of several mutants toward different concentrations of
cAMP (Postma et al., 2004; Loovers et al., 2006). In this assay,
small droplets containing wild-type cells were placed on
hydrophobic agar very close to another small drop of che-
moattractant, setting up a gradient. Cells can freely move
within the boundary of the droplet, but they cannot move
out of the droplet. Therefore, any directional movement of
the cells leads to the accumulation of cells at the boundary of
the small population, which is easily and rapidly scored.
Any defects in the ability of the cells to either detect, or
respond to, the chemoattractant will therefore result in less
cells accumulating at the boundary. Figure 1 shows that in
wild-type cells, low concentrations of cAMP induce only a
weak chemotactic response in a small fraction of the popu-
lations, whereas 1000 nM cAMP induces a strong chemo-
taxis response in 90–100% of the populations. Disruption of
the plc gene does not influence chemotaxis in this assay, at
any cAMP concentration, consistent with previously de-
scribed experiments (Drayer et al., 1994). To investigate PI3K
signaling in these cells, both wild-type and plc-null cells
were incubated with the PI3K inhibitor LY294002 at a con-
centration of 50 ?M. The concentration of drugs used was
obtained from published dose–response curves (Loovers et
al., 2006; van Haastert et al., 2007). In wild-type cells, inhi-
bition of PI3K results in a strong reduction of chemotaxis at
low cAMP concentrations, whereas at high concentrations of
cAMP this effect is greatly reduced, indicating that the effect
of LY294002 is specific and not harmful to the cells. Further-
more, it shows that PI3K activity is important but not essen-
tial for chemotaxis of wild-type cells (Iijima and Devreotes,
2002; Funamoto et al., 2002; Postma et al., 2004; Loovers et al.,
2006). In contrast, the chemotaxis of plc-null cells was resis-
tant to LY294002 treatment at all cAMP concentrations.
PLC Regulates cAMP-dependent PI(3,4,5)P3Formation
The loss of LY294002 sensitivity in the plc-null cells could
arise through two alternative mechanisms: either a large
up-regulation of PI(3,4,5)P3synthesis or the up-regulation of
a compensatory pathway. To test these alternatives, both
wild-type and plc-null cells were transformed with the
PI(3,4,5)P3sensor PHCRACGFP, and cells were stimulated
with cAMP (Parent et al., 1998). To avoid effects due to
specific parental strain backgrounds, different plc-null mu-
tant strains were used in subsequent studies. The first strain
1.19 was that described previously (Drayer et al., 1994), and
it is in the DH1 strain background. The other strains, referred
to as HAD236 and HAD237, were newly generated in the AX2
strain background. All experiments were carried out with the
appropriate strain controls. Without cAMP stimulation, both
wild-type– and plc-null PHCRACGFP-expressing cells moved in
random directions, and they showed an evenly distributed
PHCRACGFP localization in the cytosol. As seen in previous
investigations (Parent et al., 1998; Funamoto et al., 2002; Huang
et al., 2003), introduction of a cAMP-filled pipette to one side of
wild-type cells induced a strong translocation of PHCRACGFP
from the cytosol to the membrane facing the cAMP source
(Figure 2A, see Supplemental Movie 1). Pseudopodia then
extended from PHCRACGFP-containing areas of the plasma
membrane, and cells moved persistently toward the pipette.
Although plc-null cells extend pseudopodia and move toward
the pipette, PHCRACGFP localization at the leading edge was
dramatically reduced and most fluorescence remained cytoso-
lic (Figure 2A, see Supplemental Movie 2). In addition, when
cells were globally stimulated with 1 ?M cAMP, PHCRACGFP
AX3 and plc-null cells. Chemotaxis of wild-type (open circle) and
plc-null (open triangle) cells was measured to different concentra-
tion of cAMP. To investigate the role of PI3K in chemotaxis, both
wild-type (closed circle) and plc-null (closed triangle) cells were
incubated with the PI3K inhibitor LY294002 at a concentration of 50
?M. The results show the means and SE of the means of three
Effect of the PI3K inhibitor LY294002 on chemotaxis of
A. Kortholt et al.
Molecular Biology of the Cell4774
was rapidly recruited to the plasma membrane in wild-type
cells, an effect that was again significantly reduced in plc-null
cells (Figure 3A; p ? 0.005, t test).
In Dictyostelium, the activity of Akt/PKB is transiently
stimulated in response to cAMP, in a PI(3,4,5)P3-dependent
manner, peaking at 10 s after simulation (Meili et al., 1999;
Lim et al., 2005). Dictyostelium Akt/PKB is activated by phos-
phorylation at conserved threonine residues in the kinase
domain and C terminus (Meili et al., 1999), which can be
detected as a 51-kDa protein on a Western blot by using a
phospho-threonine–specific antibody (Lim et al., 2005).
Wild-type and plc-null cells were stimulated with cAMP,
and cell lysates were analyzed by Western blotting. In ly-
sates from wild-type cells, a protein of 51 kDa was tran-
siently phosphorylated, peaking at 10 s after cAMP stimu-
lation (Figure 3B). In plc-null cells, this response was
dramatically reduced, and it was undetectable in wild-type
cells treated with LY294002 (Figure 3B).
These two experiments both indicate that plc-null cells
have substantially reduced accumulation of PI(3,4,5)P3, and
they support the second hypothesis that LY294002 resistance
arises through a loss of PI(3,4,5)P3signaling and subsequent
up-regulation of a compensatory signal pathway. Further-
more, they suggest a role for PLC as regulator of the
PI(3,4,5)P3signaling in wild-type cells.
Enhanced Levels of PI(3,4,5)P3in Cells Overexpressing
PLC Lead to Chemotaxis Defects
Based on the hypothesis that PLC activity is required for
cAMP-stimulated PI(3,4,5)P3accumulation, we would pre-
dict that cells overexpressing PLC may have an increased
PI(3,4,5)P3response after cAMP stimulation. To investigate
this hypothesis, we constructed a PLC expression plasmid
containing a hygromycin-resistance cassette and transfected
it into plc?/PHCRACGFP cells. In contrast to wild-type and
plc-null cells, which maintain a persistently focused leading
edge in the direction of a micropipette filled with cAMP,
PLCOEcells have a much broader and irregular front (Figure
2A), a significantly decreased cell speed (p ? 0.001, t test),
and they follow a less directional path toward the pipette
(p ? 0.05, t test; Figure 2B).
To examine whether the chemotaxis defect of PLCOEcells
is due to altered PI(3,4,5)P3signaling, we examined the
distribution of PHCRACGFP in PLCOEcells. Nonstimulated,
randomly moving PLCOEcells have an evenly distributed
cytosolic PHCRACGFP localization, similar to that of wild-
type and plc-null cells (Figure 2A). However, in the presence
during chemotaxis. To investigate the effect of PLC on PI(3,4,5)P3
levels, the PI(3,4,5)P3detector PHcracGFP was expressed in wild-
type, plc-null, and PLCOEcells. (A) Confocal images are shown for
cells stimulated with a micropipette, containing 10?4cAMP from
the right. The figures show a representative cell for each case. Bar,
10 ?m. (B) Chemotactic properties of cells stimulated with a mi-
cropipette, containing 10?4cAMP. The chemotaxis index (black bar)
and speed (gray bar) were calculated from three independent mov-
ies; data shown are the mean ? SD of the mean (the difference
between control and PLCOEis significant for chemotaxis, *p ? 0.05
and for speed, ***p ? 0.001, t test).
PLC regulates cAMP-mediated PI(3,4,5)P3formation
Membrane translocation of PHCRACGFP in AX2, HAD236 (plc-null),
and PLCOEcells after uniform stimulation with 1 ?M cAMP. Bar, 10
?m. Quantitation of PHCRACGFP in the cytosol is shown in the
bottom. Values are the mean of three independent experiments.
Error bars represent SD (the difference between AX2 and plc-null or
between AX2 and PLCOEis significant for ***p ? 0.005). (B) PKB
phosphorylation in wild-type and plc-null cells. Cells were cAMP
pulsed, and then they simulated with 1 ?M cAMP. Samples were
removed at the times indicated and lysed directly into SDS gel
loading buffer. Extracts were then fractionated by SDS-PAGE and
analyzed by Western blotting probing with a phospho-threonine–
specific antibody. The arrows indicate a protein of ?60 kDa that in
wild type was rapidly induced in response to stimulation, indicat-
ing, corresponding to the phosphorylation of PKB/Akt, described
by others (Lim et al., 2005). This phosphorylation was blocked by
pretreatment with the PI3K inhibitor LY294002, and it was strongly
reduced in plc-null cells. The blot shown is representative of three
PLC regulates cAMP-mediated PI(3,4,5)P3signaling. (A)
PLC Regulation of PI(3,4,5)P3-mediated Chemotaxis
Vol. 18, December 20074775
of a cAMP gradient, many cells exhibit a much broader
region along the leading edge that is associated with
PHCRACGFP. In wild-type cells, an average of 16.5 ? 3.5%
(n ? 12) of the perimeter was covered by PHCRACGFP,
whereas in PLCOE29.3 ? 4.8% (n ? 11) was occupied.
Consistently, when PLCOEcells were globally stimulated
with 1 ?M cAMP, the amount of PHCRACGFP recruited to
the plasma membrane occurred much faster, and it was
significantly more compared with that in wild-type and
plc-null cells (Figure 3A). The depletion was 15, 28, and 41%
in plc-null, wild-type, and PLCOEcells, and it was maximal
at 8, 6, and 4 s after stimulation, respectively. From these
data, we conclude that PLC activation regulates PI(3,4,5)P3
signaling and that the elevated levels of PI(3,4,5)P3in PLCOE
cells lead to chemotactic defects.
PLC Regulates the Localization of PTEN
To investigate the mechanism by which PLC regulates
cAMP-mediated PI(3,4,5)P3accumulation, the distribution
of PI3K-GFP and PTEN-GFP was analyzed during chemo-
tactic stimulation. In nonstimulated, wild-type cells, PI3K is
mainly localized in the cytosol, whereas introduction of a
pipette filled with cAMP results in the transient localization
of PI3K-GFP at the front of the cell (Figure 4A; Funamoto et
al., 2002). As shown in Figure 4A, the distribution of PI3K-
GFP in plc-null during chemotaxis is similar to that of wild-
type cells. We have quantified the fluorescence intensity of
PI3K-GFP in the cytoplasm in the front of the cell relative to
the middle of the cell, which is 192 ? 21% in wild-type and
172 ? 12% in plc-null cells (mean and SD of 10 cells; the
difference between wild-type and plc-null cells is not signif-
icant by t test). These results show that although plc-null
cells have defective PI(3,4,5)P3signaling, PI3K still localizes
at the leading edge of chemotaxing cells.
We next examined the distribution of PTEN-GFP in wild-
type and plc-null cells. Consistent with previously described
results (Funamoto et al., 2002; Iijima and Devreotes, 2002), in
chemotaxing wild-type cells PTEN localizes at the plasma
membrane along the sides and posterior of the cell, but it is
absent at the leading edge (Figure 4A). In contrast, chemo-
taxing plc-null cells show a more uniformly distributed
PTEN-GFP localization at the membrane (Figure 3A).
The fluorescence intensity of PTEN-GFP at the boundary of
the cell was quantified and expressed as the mean value of the
front one third of the cell relative to the rear two thirds of the
cell, which is 77.4 ? 3.4% in wild-type and 96.5 ? 3.6% in
When wild-type cells were uniformly stimulated with
cAMP, PTEN-GFP was rapidly dissociated from the plasma
membrane. However, when plc-null cells were stimulated
with uniform cAMP, PTEN translocation was dramatically
reduced (Figure 4B; p ? 0.01). These results indicate a mis-
localization of PTEN, and they suggest that this may explain
the reduced PI(3,4,5)P3signaling in plc-null cells and the
regulatory role for PLC in the localization of PTEN.
plc-Null Cells Are Unable to Aggregate at Low Density
Although plc-null cells have no detectable deficiency in
general chemotaxis assays by using a static, single cAMP
signal, the impaired Akt/PKB phosphorylation and inability
of these cells to effectively induce the recruitment of
PHCRACGFP and presumably other PI(3,4,5)P3-binding pro-
teins to the plasma membrane may still have significant
effects during aggregation. Indeed, pi3k1/2-null and pkbA-
null cells exhibit aggregation defects that can be detected at
low cell densities (Zhou et al., 1998; Meili et al., 1999). We
therefore investigated whether plc-null cells posses a de-
fect in cell aggregation by plating cells at different cell
densities. When cells were allowed to develop naturally
on nitrocellulose filters, although they were identical to
wild-type at high density, plc-null cells were unable to
aggregate at low density, when wild-type controls could
(Fig. 5). Thus, plc-null and pkbA-null cells exhibit aggre-
gation defects despite having normal cAMP relay (Drayer
et al., 1994; Meili et al., 1999), suggesting that although
intact PI(3,4,5)P3signaling may not be essential for che-
motaxis in a static gradient, it plays a physiological role in
the more complex conditions that occur during aggrega-
plc-null cells. To investigate the mechanism by which PLC regulates
cAMP mediated PI(3,4,5)P3accumulation, the distribution of PI3K-
GFP and PTEN-GFP were analyzed during chemotactic stimulation.
(A) Confocal images are shown for AX3 and plc-null cells expressing
PI3K-GFP (top) and PTEN-GFP (bottom). Cells were stimulated
with cAMP by a pipette that is positioned at the right. Bar, 10 ?m.
(B) Translocation of PTEN-GFP in Ax2 and HAD236 (plc-null) cells
after uniform stimulation with 1 ?M cAMP. Quantitation of the
translocation of PTEN-GFP into the cytosol upon uniform cAMP
stimulation is shown (bottom). Values are the mean of three inde-
pendent experiments, and error bars represent SD (*p ? 0.05, **p ?
0.01, t test).
Distribution of PI3K-GFP and PTEN-GFP in AX3 and
A. Kortholt et al.
Molecular Biology of the Cell 4776
A Model for PLC-regulated PI(3,4,5)P3Chemotaxis in
These experiments reveal that PLC is an important regulator
of PI3K-mediated chemotaxis. Examination of the distribu-
tion of the PI(3,4,5)P3sensor PHCRACGFP revealed that the
cAMP-mediated PI(3,4,5)P3response is strongly decreased
in plc-null cells and enhanced in PLCOEcells. Consistently,
the PKB/Akt response in plc-null cells is substantially re-
duced compared with wild type. However, both the PHcrac
and PKB responses are still detectable at a low level, sug-
gesting that the effect of plc-deletion is only partial, and this
may explain why plc-null cells do not share the defects in
growth, development, pinocytosis, and chemotaxis ob-
served in pi3k-null cells (Zhou et al., 1995; Zhou et al., 1998;
Funamoto et al., 2001). Consistently, a recent study showed
that upon decreasing level of functional PI3K in the cell, the
chemotaxis phenotypes become more severe (Takeda et al.,
Another explanation for the apparent lack of chemotactic
defects in plc-null mutants would be the up-regulation of a
second compensatory pathway. Previously, it has been
shown that plc-null cells contain normal levels of I(1,4,5)P3
generated by the up-regulation of a second route of synthe-
sis via a I(1,3,4,5,6)P53/6-biphosphatase (Van Dijken et al.,
1997). Recent studies have suggested that besides the
PI(3,4,5)P3pathway, a second, parallel pathway exists that
mediates chemotaxis (Chen et al., 2007; van Haastert et al.,
2007). Furthermore, van Haastert et al. (2007) showed that
chemotaxis in plc-null cells is sensitive to PLA2 inhibitors;
therefore, the lack of chemotaxis defects and loss of
LY294002 sensitivity in plc-null cells could be explained by
the down-regulation of PI(3,4,5)P3synthesis, accompanied
by the up-regulation of compensatory pathways.
Generation of a spatial PI(3,4,5)P3signal is regulated by
both its synthesis by PI3K and degradation by PTEN. In
wild-type cells, upon stimulation, PTEN is removed from
the plasma membrane at the leading edge, and it is permis-
sive for the accumulation of PI(3,4,5)P3from cAMP-acti-
vated PI3K there (Iijima and Devreotes, 2002). Here, we
show that in plc-null cells, this dissociation is strongly re-
duced in cells stimulated with either uniform cAMP or
within a gradient. Because PI3K is still able to localize to the
leading edge in these cells, we propose that the reduced
PI(3,4,5)P3accumulation in plc-null cells is due to retention
of PTEN at the plasma membrane rather than reduced
PI(3,4,5)P3synthesis. This mechanism also would explain
the chemotaxis defects seen when PLC is overexpressed,
because extended delocalization of PTEN would give a phe-
notype reminiscent of pten-null cells. Both pten-null and
PLC-overexpressing cells exhibit reduced chemotaxis and
elevated phosphatidylinositol (3,4,5)-trisphosphate levels at
a broad front with many protrusions.
The exact mechanism of PTEN binding to the plasma mem-
brane remains unknown, but it involves a number of protein–
lipid and protein–protein interactions, and the translocation
from the cytosol to the plasma membrane is regulated by
several mechanisms, including C-terminal phosphorylation
(Vazquez et al., 2000; Vazquez and Devreotes, 2006). It has been
demonstrated that PTEN binds PI(4,5)P2and that this binding
is essential for membrane localization and PI(3,4,5)P3-degrad-
indeed indicated that it is possible to translocate PI(4,5)P2-
specific PH domains into the cytosol upon stimulation and that
this is blocked by PLC inhibitors, but not by PI3K inhibitors
(Stauffer et al., 1998). Others have shown that calcium-activa-
tion of PLCs is also able to significantly deplete PI(4,5)P2
(Varnai and Balla, 1998), and in Dictyostelium, the PI(4,5)P2-
binding domain of Phg2 is displaced during macropinocytosis
and phagocytosis (Blanc et al., 2005). Therefore, the most direct
explanation for the regulation of PTEN localization by PLC is
by the depletion of PI(4,5,)P2, and we propose that cAMP
activation not only stimulates PI3K but also PLC, leading to a
local decrease in PI(4,5)P2and dissociation of PTEN. Indeed,
recent work by Vazquez and Devreotes (2006) has suggested
that PTEN has a low affinity for the membrane; therefore, the
combined actions of PLC and PI3K may be able to reduce
PI(4,5)P2sufficiently to stop PTEN binding, while retaining
enough substrate for PI3K to generate PI(3,4,5)P3. For the de-
tailed understanding of our model and the role for PI(4,5)P2as
a local signal, techniques to study PI(4,5)P2levels with spatial
and temporal resolution in single cells are required. Unfortu-
nately, approaches to visualize PI(4,5)P2in Dictyostelium cells
by expression of different constructs of known PI(4,5)P2-bind-
ing proteins have failed (data not shown).
Our data provide new insight into the regulation of PTEN
localization, and we propose a simple model for the PLC
regulation of PI(3,4,5)P3-mediated chemotaxis (Figure 6);
PLC is regulated via the activating G?2and inhibitory G?1
(Bominaar and van Haastert, 1994; Bominaar et al., 1994;
Drayer et al., 1995; Keizer-Gunnink et al., 2007). cAMP bind-
ing to the cAR1 receptor leads to the activation of G?2and
then PLC upgradient of the cell. Activation of PLC results in
the degradation of PI(4,5)P2at the leading edge and removal
of PTEN from the membrane. The resulting gradient of
PTEN localization and activation mediates an inverse
PI(3,4,5)P3gradient, which leads to localized actin polymer-
ization and pseudopod extension from the leading edge. The
PI(4,5)P2/PI(3,4,5)P3gradient is stabilized, because both
PI3K and PTEN are localized at sites of their products [PI3K
at PI(3,4,5)P3-induced F-actin, and PTEN at PI(4,5)P2, respec-
tively]. This mutually spatial exclusion of PI3K and PTEN
will result in symmetry breaking, by which small spatial
differences in the underlying polarity gradient can be am-
plified to the observed strong PI(3,4,5)P3gradient. In accor-
dance with our model, plc-null cells, which have stabilized
PI(4,5)P2levels, have more PTEN at the membrane and
therefore less PI(3,4,5)P3formation.
Recent observations in Dictyostelium with the antagonist
8CPT-cAMP show the importance of the regulatory role of
density. Wild-type (Ax2) and two independent plc-null clones were
allowed to develop on nitrocellulose filters at different cell densities
(cells per square centimeter). Images were taken after 20 h, and they
are representative of multiple experiments.
plc-null cells are defective in aggregation at low cell
PLC Regulation of PI(3,4,5)P3-mediated Chemotaxis
Vol. 18, December 20074777
PLC in PI(3,4,5)P3-mediated chemotaxis (Keizer-Gunnink et
al., 2007). 8CPT-cAMP inhibits PLC through G?1. Consistent
with our model, inhibition of PLC by 8CPT-cAMP at the
leading edge will lead to higher levels of PI(4,5)P2and
subsequently more membrane-bound PTEN upgradient.
The resulting gradient of PI(4,5)P2and PTEN will mediate
an opposite gradient of PI(3,4,5)P3; therefore, the cells move
downgradient and 8CPT-cAMP is a repellent. PLC, PTEN,
and PI3K are all essential components of this 8CPT-cAMP–
induced polarity switch. Although PI3K and PLC are not
essential for chemotaxis (Drayer et al., 1994; Loovers et al.,
2006; Takeda et al., 2007) these and previously described
results (Keizer-Gunnink et al., 2007; Van Haastert et al., 2007)
show they are key components of the directional sensing
pathway, especially at low cAMP concentrations. Indeed,
the localized production of PI(3,4,5)P3may be sufficient to
orientate the cell. Moreover, the defects in PKB/Akt activa-
tion described here clearly indicate a further role for PLC
and PI(3,4,5)P3in the more complex and physiological pro-
cess of aggregation.
In summary, this study shows that PLC plays an impor-
tant role in the regulation of PI3K-mediated chemotaxis and
the role of PI(4,5)P2in this process. PLC controls cAMP-
mediated PI(3,4,5)P3formation by repressing the association
of PTEN with the plasma membrane. This provides an ad-
ditional regulatory layer to the signaling pathways regulat-
ing PI(3,4,5)P3-mediated chemotaxis in Dictyostelium and
suggests a novel mechanism for the generation of PI(3,4,5)P3
gradients within the cell.
Andrew, N., and Insall, R. H. (2007). Chemotaxis in shallow gradients is
mediated independently of PtdIns 3-kinase by biased choices between ran-
dom protrusions. Nat. Cell Biol. 9, 193–200.
Baggiolini, M. (1998). Chemokines and leukocyte traffic. Nature 392, 565–568.
Blanc, C., Charette, S., Cherix, N., Lefkir, Y., Cosson, P., and Letourneur, F.
(2005). A novel phosphatidylinositol 4,5-bisphosphate-binding domain tar-
geting the Phg2 kinase to the membrane in Dictyostelium cells. Eur. J. Cell Biol.
Bominaar, A. A., Kesbeke, F., and van Haastert, P. J. (1994). Phospholipase C
in Dictyostelium discoideum. Cyclic AMP surface receptor and G-protein-reg-
ulated activity in vitro. Biochem. J. 297, 181–187.
Bominaar, A. A., and van Haastert, P. J. (1994). Phospholipase C in Dictyoste-
lium discoideum. Identification of stimulatory and inhibitory surface receptors
and G-proteins. Biochem. J. 297, 189–193.
Bosgraaf, L., Waijer, A., Engel, R., Visser, A. J., Wessels, D., Soll, D., and van
Haastert, P. J. (2005). RasGEF-containing proteins GbpC and GbpD have
differential effects on cell polarity and chemotaxis in Dictyostelium. J. Cell Sci.
Campbell, J. J., and Butcher, E. C. (2000). Chemokines in tissue-specific and
microenvironment-specific lymphocyte homing. Curr. Opin. Immunol. 12,
Chen, L., Iijima, M., Tang, M., Landree, M. A., Huang, Y. E., Xiong, Y.,
Iglesias, P. A., and Devreotes, P. N. (2007). PLA(2) and PI3K/PTEN pathways
act in parallel to mediate chemotaxis. Dev. Cell 12, 603–614.
Crone, S. A., and Lee, K. F. (2002). The bound leading the bound: target-
derived receptors act as guidance cues. Neuron 36, 333–335.
Devreotes, P. N., and Zigmond, S. H. (1988). Chemotaxis in eukaryotic
cells—a focus on leukocytes and Dictyostelium. Annu. Rev. Cell Biol. 4, 649–
Drayer, A. L., Meima, M. E., Derks, M. W., Tuik, R., and van Haastert, P. J.
(1995). Mutation of an EF-hand Ca(2?)-binding motif in phospholipase C of
Dictyostelium discoideum: inhibition of activity but no effect on Ca(2?)-depen-
dence. Biochem. J. 311, 505–510.
Drayer, A. L., Van der, K. J., Mayr, G. W., and van Haastert, P. J. (1994). Role
of phospholipase C in Dictyostelium: formation of inositol 1,4,5-trisphosphate
and normal development in cells lacking phospholipase C activity. EMBO J.
Drayer, A. L., and van Haastert, P. J. (1992). Molecular cloning and expression
of a phosphoinositide-specific phospholipase C of Dictyostelium discoideum.
J. Biol. Chem. 267, 18387–18392.
Faix, J., Weber, I., Mintert, U., Kohler, J., Lottspeich, F., and Marriott, G.
(2001). Recruitment of cortexillin into the cleavage furrow is controlled by
Rac1 and IQGAP–related proteins. EMBO J. 20, 3705–3715.
Funamoto, S., Meili, R., Lee, S., Parry, L., and Firtel, R. A. (2002). Spatial and
temporal regulation of 3-phosphoinositides by PI 3-kinase and PTEN medi-
ates chemotaxis. Cell 109, 611–623.
Funamoto, S., Milan, K., Meili, R., and Firtel, R. A. (2001). Role of phospha-
tidylinositol 3? kinase and a downstream pleckstrin homology domain-con-
taining protein in controlling chemotaxis in Dictyostelium. J. Cell Biol. 153,
Hoeller, O., and Kay, R. R. (2007). Chemotaxis in the absence of PIP3 gradi-
ents. Curr. Biol. 17, 813–817.
Huang, Y. E., Iijima, M., Parent, C. A., Funamoto, S., Firtel, R. A., and
Devreotes, P. (2003). Receptor-mediated regulation of PI3Ks confines
PI(3,4,5)P-3 to the leading edge of chemotaxing cells. Mol. Biol. Cell 14,
Iijima, M., and Devreotes, P. (2002). Tumor suppressor PTEN mediates sens-
ing of chemoattractant gradients. Cell 109, 599–610.
Iijima, M., Huang, Y. E., Luo, H. R., Vazquez, F., and Devreotes, P. N. (2004).
Novel mechanism of PTEN regulation by its phosphatidylinositol 4,5-
bisphosphate binding motif is critical for chemotaxis. J. Biol. Chem. 279,
Katan, M. (1998). Families of phosphoinositide-specific phospholipase C:
structure and function. Biochim. Biophys. Acta 1436, 5–17.
Keizer-Gunnink, I., Kortholt, A., and van Haastert, P. J. (2007). Chemoattrac-
tants and chemorepellents act by inducing opposite polarity in phospholipase
C and PI-kinase signaling. J. Cell Biol. 177, 579–585.
Konijn, T. M. (1970). Microbiological assay for cyclic 3?,5?-AMP. Experientia
Lim, C. J., Zawadzki, K. A., Khosla, M., Secko, D. M., Spiegelman, G. B., and
Weeks, G. (2005). Loss of the Dictyostelium RasC protein alters vegetative cell
size, motility and endocytosis. Exp. Cell Res. 306, 47–55.
Loovers, H. M., Postma, M., Keizer-Gunnink, I., Huang, Y. E., Devreotes,
P. N., and van Haastert, P. J. (2006). Distinct roles of PI(3,4,5)P3 during
chemoattractant signaling in Dictyostelium: a quantitative in vivo analysis by
inhibition of PI3-kinase. Mol. Biol. Cell 17, 1503–1513.
leading edge of chemotaxing cells. The model contains of two
regulatory loops: first, a PLC-regulated PI(4,5)P2/PTEN loop (indi-
cated in red) inhibiting PI(3,4,5)P3degradation; and second, a PI3K/
F-actin loop (indicated in green) providing PI(3,4,5)P3formation
and pseudopod extension. cAMP binding to the cAR1 receptor
leads to the activation of G?2and subsequently in activation of PLC
at the leading edge of the cell. Activation of PLC results in the
degradation of PI(4,5)P2at the leading edge and translocation of
PTEN to the rear of the cell. The resulting gradient of PI(4,5)P2/
PTEN mediates an opposite PI(3,4,5)P3gradient. PI3K and PTEN are
localized at sites of their effector; hence, PI(3,4,5)P3induced F-actin
and PI(4,5)P2, providing stabilization of the gradient and pseudo-
pod extension from the leading edge.
Model of PLC-mediated PI(3,4,5)P3formation at the
A. Kortholt et al.
Molecular Biology of the Cell4778
Meili, R., Ellsworth, C., Lee, S., Reddy, T. B., Ma, H., and Firtel, R. A. (1999).
Chemoattractant-mediated transient activation and membrane localization of
Akt/PKB is required for efficient chemotaxis to cAMP in Dictyostelium. EMBO
J. 18, 2092–2105.
Parent, C. A., Blacklock, B. J., Froehlich, W. M., Murphy, D. B., and Devreotes,
P. N. (1998). G protein signaling events are activated at the leading edge of
chemotactic cells. Cell 95, 81–91.
Postma, M., Roelofs, J., Goedhart, J., Gadella, T. W., Visser, A. J., and Van
Haastert, P.J.M. (2003). Uniform cAMP stimulation of Dictyostelium cells
induces localized patches of signal transduction and pseudopodia. Mol. Biol.
Cell 14, 5019–5027.
Postma, M., Roelofs, J., Goedhart, J., Loovers, H. M., Visser, A. J., and van
Haastert, P. J. (2004). Sensitization of Dictyostelium chemotaxis by phospho-
inositide-3-kinase-mediated self-organizing signalling patches. J. Cell Sci. 117,
Stauffer, T. P., Ahn, S., and Meyer, T. (1998). Receptor-induced transient
reduction in plasma membrane PtdIns(4,5)P2 concentration monitored in
living cells. Curr. Biol. 8, 343–346.
Takeda, K., Sasaki, A. T., Ha, H., Seung, H. A., and Firtel, R. A. (2007). Role
of phosphatidylinositol 3-kinases in chemotaxis in Dictyostelium. J. Biol.
Chem. 282, 11874–11884.
Traynor, D., Milne, J. L., Insall, R. H., and Kay, R. R. (2000). Ca(2?) signalling
is not required for chemotaxis in Dictyostelium. EMBO J. 19, 4846–4854.
Van Dijken, P., Bergsma, J. C., and van Haastert, P .J. (1997). Phospholipase-
C-independent inositol 1,4,5-trisphosphate formation in Dictyostelium cells.
Activation of a plasma-membrane-bound phosphatase by receptor-stimulated
Ca2? influx. Eur. J. Biochem. 244, 113–119.
van Haastert, P. J., Keizer-Gunnink, I., and Kortholt, A. (2007). Essential role
of PI3-kinase and phospholipase A2 in Dictyostelium discoideum chemotaxis.
J. Cell Biol. 177, 809–816.
van Haastert, P. J., and Van Dijken, P. (1997). Biochemistry and genetics of
inositol phosphate metabolism in Dictyostelium. FEBS Lett. 410, 39–43.
Van Haastert, P.J.M., and Devreotes, P. N. (2004). Chemotaxis: signalling the
way forward. Nat. Rev. Mol. Cell. Biol. 5, 626–634.
Varnai, P., and Balla, T. (1998). Visualization of phosphoinositides that bind
pleckstrin homology domains: calcium- and agonist-induced dynamic
changes and relationship to myo-[3H]inositol-labeled phosphoinositide pools.
J. Cell Biol. 143, 501–510.
Vazquez, F., and Devreotes, P. (2006). Regulation of PTEN function as a PIP3
gatekeeper through membrane interaction. Cell Cycle 5, 1523–1527.
Vazquez, F., Ramaswamy, S., Nakamura, N., and Sellers, W. R. (2000). Phos-
phorylation of the PTEN tail regulates protein stability and function. Mol.
Cell. Biol. 20, 5010–5018.
Zhou, K., Pandol, S., Bokoch, G., and Traynor-Kaplan, A. E. (1998). Disruption
of Dictyostelium PI3K genes reduces [32P]phosphatidylinositol 3,4 bisphos-
phate and [32P]phosphatidylinositol trisphosphate levels, alters F-actin dis-
tribution and impairs pinocytosis. J. Cell Sci. 111, 283–294.
Zhou, K., Takegawa, K., Emr, S. D., and Firtel, R. A. (1995). A phosphatidyl-
inositol (PI) kinase gene family in Dictyostelium discoideum: biological roles of
putative mammalian p110 and yeast Vps34p PI 3-kinase homologs during
growth and development. Mol. Cell. Biol. 15, 5645–5656.
PLC Regulation of PI(3,4,5)P3-mediated Chemotaxis
Vol. 18, December 20074779