Membrane-Permeant Phosphoinositide Derivatives as Modulators of Growth Factor Signaling and Neurite Outgrowth

Article abstract of DOI:10.1016/j.chembiol.2009.10.005

Phosphoinositides are important signaling molecules that govern a large number of cellular processes such as proliferation, differentiation, membrane remodeling, and survival. Here we introduce a fully synthetic membrane-permeant derivative of a novel, easily accessible, and very potent phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P₃] mimic: phosphatidylinositol 3,4,5,6-tetrakisphosphate [PtdIns(3,4,5,6)P₄]. The membrane-permeant PtdIns(3,4,5,6)P₄ derivative activated pathways downstream of phosphatidylinositol 3-kinase (PI3K), including protein kinase B, p70S6K, mitogen-activated protein kinase, and protein kinase C, more potently than similar membrane-permeant PtdIns(3,4,5)P₃ and PtdIns(3,4)P₂ derivatives in the absence of receptor stimulation. In addition, we demonstrate that treatment of PC12 cells with the membrane-permeant PtdIns(3,4)P₂, PtdIns(3,4,5)P₃, and PtdIns(3,4,5,6)P₄ derivatives increases the number of neurites per cell in the presence of NGF. This work establishes membrane-permeant phosphoinositides as powerful tools to study PI3K signaling and directly demonstrates that 3-phosphorylated phosphoinositides are instrumental for neurite initiation.

Full text of DOI:10.1016/j.chembiol.2009.10.005

Chemistry & Biology
Article
Membrane-Permeant Phosphoinositide
Derivatives as Modulators of Growth
Factor Signaling and Neurite Outgrowth
Vibor Laketa,1,3 Sirus Zarbakhsh,1,3 Eva Morbier,2 Devaraj Subramanian,1 Carlo Dinkel,1 Justin Brumbaugh,1
Pascale Zimmermann,2 Rainer Pepperkok,1 and Carsten Schultz1,
*
1Cell Biology and Cell Biophysics Unit, European Molecular Biology Laboratory, Meyerhofstraße 1, 69117 Heidelberg, Germany
2Department of Human Genetics, K.U. Leuven, B-3000 Leuven, Belgium
3These authors contributed equally to this work
*Correspondence: schultz@embl.de
DOI 10.1016/j.chembiol.2009.10.005
SUMMARY
phorylate various signaling proteins including p70S6K, protein
kinase B (also known as Akt), and protein kinase C (PKC)
Phosphoinositides are important signaling mole- (Figure 1A) (Cantley, 2002).
So far an effective approach to manipulate phosphoinositide
cules that govern a large number of cellular pro-
cesses such as proliferation, differentiation, mem-
brane remodeling, and survival. Here we introduce
a fully synthetic membrane-permeant derivative of
a novel, easily accessible, and very potent phospha-
tidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3]
mimic: phosphatidylinositol 3,4,5,6-tetrakisphos-
phate [PtdIns(3,4,5,6)P4]. The membrane-permeant
PtdIns(3,4,5,6)P4 derivative activated pathways
downstream of phosphatidylinositol 3-kinase (PI3K),
levels for investigating signaling is to overexpress or knock
down phosphoinositide-forming or metabolizing enzymes. How-
ever, these methods require hours or days to develop an effect
and often the response is wiped out by a compensation mecha-
nism. Therefore, there is a necessity for methods that alter phos-
phoinositide levels within minutes, resulting in a better control of
the experiment, a more immediate onset of the signal, and there-
fore more physiological mimicking of the growth factor signaling.
One way to alter intracellular phosphoinositide levels would be to
modulate the activity of engineered phosphoinositide metabo-
including protein kinase B, p70S6K, mitogen- lizing enzymes with a ‘‘chemical dimerizer’’ such as rapamycin
(Suh et al., 2006). Another, more direct, possibility uses synthetic
membrane-permeant derivatives of these phospholipids (Dinkel
et al., 2001; Jiang et al., 1998). This involves masking the
activated protein kinase, and protein kinase C,
more potently than similar membrane-permeant
PtdIns(3,4,5)P3 and PtdIns(3,4)P2 derivatives in the
absence of receptor stimulation. In addition, we
demonstrate that treatment of PC12 cells with the
membrane-permeant PtdIns(3,4)P2, PtdIns(3,4,5)P3,
and PtdIns(3,4,5,6)P4 derivatives increases the
number of neurites per cell in the presence of NGF.
charged phosphate groups by bioactivatable acetoxymethyl
esters (AM esters). The uncharged AM esters are sufficiently
stable outside cells to be applied in regular buffers. Once the
derivatives enter cells, enzymatic cleavage of the bioactivatable
esters leads to unmasking of phosphates and the reconstitution
of negative charges and biological function (Schultz, 2003). In
This work establishes membrane-permeant phos-
phoinositides as powerful tools to study PI3K
addition, butyrates are frequently used to mask the hydroxy
groups (Vajanaphanich et al., 1994). Their enzymatic hydrolysis
signaling and directly demonstrates that 3-phos- is slower than AM ester hydrolysis (Bartsch et al., 2003), thereby
preventing phosphates from scrambling during bioactivation.
phorylated phosphoinositides are instrumental for
Upon addition to cells, the lipophilic derivatives pass the plasma
membrane by diffusion (Figure 1B). Once reaching the cytosol,
endogenous unspecific carboxyhydrolases are expected to first
neurite initiation.
INTRODUCTION
remove the AM esters and subsequently the butyrates, thereby
releasing the phosphoinositide of interest or an active metabo-
Intracellular signaling mediated by growth factors activate lite. We previously applied the AM ester technique to show that
various phosphoinositide 3-kinase (PI3K) isoforms (Cantley, a membrane-permeant derivative of PtdIns(3,4,5)P3 (5; Fig-
2002), which phosphorylate phosphoinositides like phosphati- ure 1C), but not PtdIns(3,4)P2 (7; Figure 1C), reduced chloride
dylinositol 4,5-bisphosphate [PtdIns(4,5)P2] at the 3-hydroxy secretion of epithelial cells (Dinkel et al., 2001). An alternative
group of the inositol ring, giving rise to phospholipids such to bioactivatable masking groups are cationic polyanions for
as phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3; 2] permitting phospholipid entrance to living cells (Ozaki et al.,
and phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P2]. 2000).
PtdIns(3,4,5)P3 and PtdIns(3,4)P2 are recognized as second
The total synthesis of membrane-permeant PtdIns(3,4,5)P3 (5)
messengers that govern many downstream events by activating and PtdIns(3,4)P2 (7) derivatives required an enormous synthetic
target protein kinases such as PDK1, which subsequently phos- effort (Dinkel et al., 2001). A derivative with an additional
1190 Chemistry & Biology 16, 1190–1196, November 25, 2009 ª2009 Elsevier Ltd All rights reserved

Chemistry & Biology
Membrane-Permeant Phosphoinositides
Figure 1. Growth Factor Signaling, Mem-
brane-Permeant Phosphoinositide Deriva-
tives, and Their Mode of Cell Delivery
(A) A simplified EGF/PI3K signaling pathway. PIP2,
PtdIns(4,5)P2; PIP3, PtdIns(3,4,5)P3.
(B) Mechanism of membrane-permeant phosphoi-
nositide derivative cell entry and activation. The
negatively charged phosphate groups of the phos-
pholipid are masked with enzyme cleavable AM
ester groups (green) while hydroxy groups are
masked by butyrates (blue). The uncharged mole-
cule diffuses across the plasma membrane. Inside
the cell the protecting groups are cleaved by
endogenous enzymes and the active compound
is liberated.
(C) Phosphoinositides and their membrane-per-
meant derivatives. The naturally occurring phos-
phoinositides PtdIns(4,5)P2 (1) and PtdIns(3,4,5)P3
(2), the artificial tetrakisphosphate PtdIns(3,4,
5,6)P4 (3), and the respective membrane-perme-
ant derivatives 4 to 6 are shown. A similar deriva-
tive of PtdIns(3,4)P2 (7) is also shown. Please
note that all lipid moieties have unnatural octanoic
acid esters unless stated otherwise. Bt, butyrate;
Ac, acetate.
phosphate in the 6 position would be much easier to prepare. over the 1-OH position (85:15) via formation of an intermediate
Furthermore, very little is known about structure-activity relation- orthoester, 1,2-di-O-octanoyl-sn-glycerol 3-(benzyl N,N-diiso-
ships of phosphoinositides, mainly due to a lack of synthetic propyl)phosphoramidite (10) was used to introduce of the lipid
chemistry efforts and solubility problems of the phospho- moiety. The fully protected PtdIns(3,4,5,6)P4 derivative 11 was
inositides in physiologically relevant buffers. To facilitate the quantitatively deprotected by catalytic hydrogenolysis. The
preparation of membrane-permeant PtdIns(3,4,5)P3 (5) and resulting free acid was alkylated to the final nonakis(acetoxy-
PtdIns(3,4)P2 (7) derivatives and to study relationship between methyl) ester 6 by using 27 equivalents of acetoxymethyl
phosphoinositide structure and their activity, we extended the bromide and an equimolar amount of N,N-diisopropylethylamine
masking approach to a previously unknown phosphoinositide, in acetonitrile. PIP4/AM (6) was purified to homogeneity by
PtdIns(3,4,5,6)P4 (3). We performed a comprehensive biochem- preparative HPLC. This synthetic pathway involved only six
ical and live cell analysis of membrane-permeant phosphoinosi- synthetic steps plus the formation of the lipid phosphoramidite
tide derivative effects on intracellular signaling pathways and provides the simplest access to a highly phosphorylated
downstream of PI3K. This demonstrated that membrane-perme- membrane-permeant phosphoinositide derivative known so
ant phosphoinositides are able to stimulate many aspects of far. The overall yield from 8 was 19%. In a similar way, a di-O-
PI3K-dependent signaling. Remarkably, PtdIns(3,4,5,6)P4/AM myristylglyceryl derivative was prepared (data not shown).
behaves like a much more potent activator of the PI3K pathway
than membrane-permeant PtdIns(3,4)P2 and PtdIns(3,4,5)P3
derivatives. In addition, we examined the role of membrane-per-
Phosphoinositide Derivatives Activate Signaling
Networks Downstream of PI3K
First, we tested how all membrane-permeant phosphoinositide
meant phosphoinositides in a more complex PI3K-dependent
process such as neurite outgrowth, where we show that addition
derivatives affect different aspects of PI3K-dependent intracel-
of membrane-permeant PI3K products induces an increase in
lular signaling. Mitogen-activated protein kinases (MAPKs) pro-
the number of neurites per cell, indicating their role in neurite
vide a functional signaling ‘‘hub’’ where signaling inputs are
initiation.
amplified and integrated (Cuevas et al., 2007) and thus MAPK
phosphorylation levels are a good marker of intracellular sig-
RESULTS
naling activity. Therefore, we first investigated p42/p44 MAPK
phosphorylation after treatment with different membrane-per-
meant phosphoinositide derivatives. All 3-phosphorylated phos-
phoinositides tested induced p42/p44 MAPK phosphorylation
Synthesis of Membrane-Permeant PtdIns(3,4,5,6)P4
Derivative
We prepared 2-O-butyryl-phosphatidylinositol 3,4,5,6-tetrakis- (Figure 3A), while the corresponding membrane-permeant
phosphate nonakis(acetoxymethyl) ester (PtdIns(3,4,5,6)P4/AM, PtdIns(4,5)P2 derivative was inactive (see Figure S1A available
PIP4/AM, 6), starting from commercially available enantio- online). Remarkably, the membrane-permeant PtdIns(3,4,5)P3
merically pure 1,2-O-cyclohexylidene-myo-inositol (8; Figure 2). (PIP3/AM, 5) and PtdIns(3,4)P2 (PI(3,4)P2/AM) derivatives were
Phosphorylation was achieved using standard dibenzyl phos- significantly less potent than the PtdIns(3,4,5,6)P4 derivative
phoramidite chemistry in excellent yield. Deprotection of the (PIP4/AM, 6) (Figure 3A and see the quantification in Fig-
ketal under acidic conditions gave the tetrakisphosphate deriva- ure S1B). The induction of p42/p44 MAPK phosphorylation
tive 9. After regioselective butyrylation of the axial 2-OH group was unchanged in the presence of PI3K inhibitor wortmannin
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Chemistry & Biology
Membrane-Permeant Phosphoinositides
Akt, GSK3b, p70S6K, Rac1, Bad, IRS1, FAK, JNK, etc., as well
as mediators of MAPK signaling, such as ERK1/2, MEK1/2,
MAPKAP2, etc. To confirm these results, we used two different
Kinetworks multi-immunoblot assays (KPSS 10.1 and KPSS
11.0), a more accurate western blot-based assay, which uses
30–40 phosphospecific antibodies and 20 lane multichannel
blotters. Again, the results showed an increase in the phosphor-
ylation levels of several PI3K signaling mediators such as
p70S6K, GSK3b, RSK1/2, JNK, and MAPK signaling members
such as ERK1/2 and MEK1/2 (Figures S2A and S2B; lists of all
the phosphoproteins tested are shown in Tables S2 and S3).
Moreover the phosphorylation pattern induced by PIP4/AM stim-
ulation resembled that induced by EGF stimulation both qualita-
tively and quantitatively (Figures S2A and S2B).
To investigate other aspects of PI3K signaling, such as PKC
activation, we used KCP-1, a fluorescent sensor that monitors
atypical PKC activity in real time (Schleifenbaum et al., 2004),
simultaneously with Fura red, a calcium-sensitive sensor.
Addition of PIP4/AM led to an increase in PKC-dependent phos-
phorylation, but had no effect on intracellular calcium levels
(Figure 3E). The lack of calcium response suggested that a non-
classic, calcium-independent PKC was triggered by PIP4/AM,
which is exactly the type of PKCs that are known to be activated
downstream of PI3K (Toker, 2000). In addition, the PKC sensor
Figure 2. Synthesis of the Membrane-Permeant Phosphoinositide
PIP4/AM (6)
(A) (BnO)2PNiPr2, DCI, MeCN, 1d, then À40ꢀC, CH3CO3H, 84%.
(B) TFA/CHCl3, 78%.
(C) C3H7C(OMe)3, PPTS-resin, CH2Cl2, H2O, 80%.
(D) 10, DCI, DCM, then À40ꢀC, CH3CO3H, 80%.
(E) Pd/C, H2, 99%.
(F) AcOCH2Br, iPr2NEt, MeCN, 48%.
(data not shown). The MAPK phosphorylation caused by 3-phos- used here is predominantly responding to atypical PKC phos-
phorylated phosphoinositides lasted much longer than the one phorylation (Schleifenbaum et al., 2004). Interestingly, other
caused by EGF. However in a wash-out experiment, the pulse 3-phosphorylated phosphoinositides failed to stimulate PKC
application of PIP4/AM shortened the duration of MAPK phos- activity. Possibly, the more powerful trigger of PIP4/AM is
phorylation, leading to a transient signaling pattern typical for required to allow KCP-1 to respond. From the above data,
EGF treatment (Figure S1C and see the quantification in Fig- PIP4/AM appears to trigger a significant subset of growth factor
ure S1D). This is significant because it indicates that the cells signaling without the need of growth factor receptor stimulation.
are capable of metabolizing PIP4/AM and deal with increased
signaling activity.
Phosphoinositide Derivatives Induce PH Domain
Since p42/p44 MAPK phosphorylation levels are a measure of Translocation in Live Cells
signaling events several steps downstream from PI3K, we tested Phosphoinositides achieve direct signaling effects through the
the phosphorylation status of Akt, which is a more direct down- binding of their head groups to various signaling proteins pos-
stream target of PI3K/PDK1 signaling (Figure 1A) (Cantley, 2002). sessing phosphoinositide binding domain (Di Paolo and De
Similarly to p42/p44 MAPK phosphorylation experiments, PIP4/ Camilli, 2006). Interaction between 3-phosphorylated phosphoi-
AM induced Akt phosphorylation with remarkable potency nositides and signaling proteins is often achieved via pleckstrin
(Figure 3B). The highest Akt phosphorylation was induced by homology (PH) domains. This interaction regularly results in
PDGF (Figure 3B), which was used as a positive control in these protein translocation to the plasma membrane (Di Paolo and
experiments. Similar phosphorylation dynamics were observed De Camilli, 2006). We used the GFP-tagged PH domain of
for p70S6K (Figure S1E), another direct target of PI3K/PDK1 Grp1 (Grp1-PH-GFP), a commonly used PtdIns(3,4,5)P3 sensor
signaling (Figure 1A) (Cantley, 2002). This demonstrates that (Halet, 2005), and imaged its dynamics after stimulation with
membrane-permeant 3-phosphorylated phosphoinositides are PIP4/AM using live cell confocal microscopy. Stimulation of
able to induce phosphorylation of proteins downstream of NRK cells with PIP4/AM resulted in Grp1-PH-GFP domain trans-
PI3K/PDK1, with PIP4/AM being the most potent derivative.
location to the plasma membrane. Translocation started to be
Given the strong responses of the PIP4/AM derivative, we noticeable 3 min after stimulation (Figures 3C and 3D and Movie
wanted to investigate its effects on intracellular signaling in a S1). The translocation was more complete and almost instanta-
more global fashion. We measured the phosphorylation state neous when the cell were stimulated with PDGF (Figures 3C and
of a large number of cellular proteins using commercial Kinex 3D and Movie S2), which was used as a positive control. The
antibody microarray and Kinetworks multi-immunoblot phos- same was observed using other PIP3 sensors such as GFP-
phoprotein-screening platforms (KPSS). Lysates from non- tagged PH domain of PDK1 (Figure S3) and Akt (data not shown).
stimulated (control) and PIP4/AM stimulated HeLa cells were Inhibition of endogenous PI3K by wortmannin did not have an
subjected to the Kinex antibody microarray assay, a screen effect on PIP4/AM-induced PH domain translocation while it
that uses 650 different antibodies to monitor protein expression completely inhibited growth factor-induced translocation
and phosphorylation levels. Over 40 proteins were found to (Figure S3). This is in agreement with the aforementioned protein
significantly change phosphorylation levels after PIP4/AM stimu- phosphorylation results and suggests that PIP4/AM is able to
lation (Table S1), many being mediators of PI3K signaling such as translocate relevant PH domain-bearing proteins to the correct
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Chemistry & Biology
Membrane-Permeant Phosphoinositides
Figure 3. Effects of Membrane-Permeant
Phosphoinositide Stimulation on Intracel-
lular Signaling Downstream of PI3K
(A) Time course of p42/44 MAPK phosphorylation
after stimulation of HeLa cells with 10 mM
PI(3,4)P2/AM, PIP3/AM, PIP4/AM, and EGF
(100 ng/ml). p42/44 MAPK phosphorylation on
residues T202/Y204 is shown in green and
a-tubulin control is shown in red. Duration of stim-
ulation in minutes is indicated on the top of the
panel.
(B) Time course of Akt phosphorylation after stim-
ulation of NRK cells with PIP3/AM (10 mM), PIP4/
AM (10 mM), and PDGF (100 ng/ml). Akt phosphor-
ylation on S473 is shown in green and a-tubulin
control is shown in red. The time course is shown
on top of the panel.
(C) Translocation of the PH-GFP domain of Grp1
(PIP3 sensor) to the plasma membrane after
PDGF (100 ng/ml) or PIP4/AM (10 mM) treatment
of NRK cells. Arrows point to areas where the
translocation is the most pronounced. Scale
bars, 15 mm.
(D) Quantification of PH-GFP Grp1 domain trans-
location. Shown is the loss of cytosolic fluores-
cence as the GFP fusion translocates to the
plasma membrane. Error bars represent the SD
of the mean of seven cells from three independent
experiments.
(E) Multiparameter imaging of events downstream
from PI3K. PKC activity is monitored by the FRET-
based sensor KCP-1 (black) and calcium levels are
measured by the indicator Fura Red (red) (as
described in Experimental Procedures).
need to be removed for exhibiting biolog-
ical activity in cells. We therefore incu-
bated the 2-O-butyrylated PIP4 derivative
(BtPIP4) and the 2,6-di-O-butyrylated
PIP3 derivative (Bt2PIP3) with the two
most prominent metabolizing enzymes
for PIP3, PTEN and SHIP2. Both enzymes
membrane and activate signaling downstream of PI3K in the were very weakly dephosphorylating the compounds (Figure S5),
presence of PI3K inhibitor. The delay of 3 min in PH domain suggesting that the butyrate is required to be removed for rapid
translocation between growth factor and PIP4/AM stimulations dephosphorylation after the washout. In addition, we investi-
(Figures 3C and 3D) probably reflects the time needed for the gated the recognition of BtPIP4 by the Grb-1 PH domain using
enzymatic reaction required to convert PIP4/AM to a biologically surface plasmon resonance experiments. As is shown in Fig-
active compound. We were not able to detect significant PH ure S6, only weak binding of the Grp-1 PH domain to BtPIP4-
domain translocation after stimulation with PIP3/AM, which containing vesicles was observed. Again, this implies that the
might explain its lower potency.
butyrate needs to be removed for the substantial Grp-1 PH
To furthercharacterizetheonsetofPIP4/AM-induced signaling, domain translocation observed in NRK cells after incubation
we used YFP-tagged p42 MAPK and observed its translocation with PIP4/AM (Figure 3C).
from the cytosol to the nucleus. In HeLa cells, PIP4/AM led to
YFP-p42 MAPK translocation from the cytosol to the nucleus Regulation of Neurite Formation by Different
within several minutes (Figure S4A and Movie S3). Similar to PH Phosphoinositides
domain translocation experiments, YFP-p42 MAPK translocation After investigating the effects of membrane-permeant phosphoi-
started 5 min after the stimulation (Figure S4B). EGF stimulation, nositides 5, 6, and 7 in rapidly occurring PI3K-dependent
used as a control, induced almost instantaneous YFP-p42 events such as protein phosphorylation and translocation, we
MAPK translocation (Figures S4A and S4B and Movie S4).
From the present data, it is difficult to determine whether the physiological processes, such as neurite formation. By using
butyrates of membrane-permeant phosphoinositide derivatives PI3K inhibitors and by overexpressing constitutively active
were interested in observing more complex, PI3K-regulated
Chemistry & Biology 16, 1190–1196, November 25, 2009 ª2009 Elsevier Ltd All rights reserved 1193

Chemistry & Biology
Membrane-Permeant Phosphoinositides
Figure 4. Effects of Membrane-Permeant
Phosphoinositides on NGF-Induced Neurite
Outgrowth in PC12 Cells
(A) Phenotypes of PC12 cells after 24 hr of stimu-
lation with NGF alone (control) and NGF together
with PI(3,4)P2/AM (50 mM), PI(3,4,5)P3/AM (50 mM),
PIP4/AM (10 mM), PI(3)P/AM (100 mM), and
PI(4,5)P2/AM (100 mM), respectively. Arrows point
to individual neurites. Note that PI(3,4)P2/AM,
PI(3,4,5)P3/AM, and PIP4/AM induced a dramatic
increase in the number of neurites per cell, albeit
with differences in the required doses, whereas
PI(3)P/AM and PI(4,5)P2/AM had no effect on neu-
rite number. Scale bar, 10 mm.
(B) Quantification of the effect of synthetic phos-
phoinositide derivatives on neurite numbers.
Shown are changes in the percentage of cells
with 1, 2, 3, and 4 or more neurites per cell in
response to treatment with the derivative indi-
cated. Error bars represent SD of the mean of at
least 300 cells from two or more independent
experiments.
(C) PC12 cell phenotypes after stimulation with
serum, PIP4/AM (10 mM), or NGF (100 ng/ml).
Note the appearance of short neurite-like struc-
tures (arrows) on PIP4/AM-stimulated PC12 cells.
Scale bar, 10 mm.
PI3K, it has been very well established that neurite outgrowth is indicators. First, suppression of proliferation was measured by
a PI3K-dependent process (Kimura et al., 1994; Kobayashi et al., the extent of BrdU incorporation into DNA of proliferating cells.
1997). Furthermore, it has been suggested that the PI3K product, Both NGF and PIP4/AM almost completely abolished BrdU
PtdIns(3,4,5)P3, plays a crucial role in neurite initiation (Aoki et al., incorporation, indicating suppression of PC12 cell proliferation
2005, 2007), a very early step during neurite outgrowth. (Figure S7A). Second, we used a MAPK phosphorylation profile
However, this has not been directly demonstrated in living cells. as an indicator of differentiation. It was previously shown that
To test this directly, we chose PC12 cells stably transfected with sustained MAPK phosphorylation leads to PC12 cell differentia-
human NGF receptor (PC12 6–15), a model cell line for studying tion, while transient MAPK phosphorylation results in cell prolif-
early events during neurite outgrowth including neurite initiation eration (Marshall, 1995). Both NGF and derivative PIP4/AM
(Laketa et al., 2007). This cell line shows rapid responses to NGF induced a sustained MAPK phosphorylation over 2 hr, which is
and develops neurites on the time scale of hours rather than typical for differentiation processes, while EGF stimulation pro-
days, as is the case for standard PC12 cells (Hempstead et al., duced only transient MAPK phosphorylation and failed to trigger
1992). When these cells were incubated with 10 mM PIP4/AM neurite initiation (Figure S7B). Taken together, both morpholog-
(6) in the presence of NGF, they produced dramatically more ical and biochemical observations indicate that cells undergo
neurites per cell compared to cells treated with NGF alone a differentiation process in response to the PtdIns(3,4,5)P3-
(Figures 4A and 4B). While NGF-treated control cells produced mimicking derivatives such as PIP4/AM. However, the neurites
an average of two neurites per cell, cells treated with PIP4/AM induced by PIP4/AM remained short even after 48 hr, suggesting
(6) produced mostly four or more neurites per cell (Figures 3A that longitudinal neurite growth depends on additional sig-
and 3B). Ten micromoles of PIP3/AM and PI(3,4)P2/AM had no nals, not triggered by the 3-phosphorylated phosphoinositide
effect on neurite numbers, again confirming their lower potency derivatives.
(data not shown). However, when the cells were treated with five
times higher concentration of PIP3/AM and PI(3,4)P2/AM, we DISCUSSION
observed similar effects on neurite number as with PIP4/AM
(Figures 4A and 4B). No effect on the number of neurites per cell We have demonstrated, in a comprehensive set of biochemical
was observed when the cells were treated with membrane- and live cell assays, that membrane-permeant phosphoinositi-
permeant PtdIns(4,5)P2 (4) or membrane-permeant PtdIns(3)P des are able to stimulate many aspects of PI3K-dependent
derivatives (Figures 4A and 4B), demonstrating specificity. This signaling. Remarkably, the ‘‘unnatural’’ PtdIns(3,4,5)P3 deriva-
suggests that the number of neurites is under the direct control tive with a phosphate on 6-OH position, PIP4/AM (6), is a much
of 3-O-phosphorylated lipids as products of PI3K.
more powerful activator of PI3K signaling than the other 3-O
When cells were incubated with PIP4/AM in the absence of phosphorylated membrane-permeant compounds, also in the
NGF, a large number of short extensions resembling neurite initi- presence of PI3K inhibitor. Although our results suggest that
ation events were formed within 24 hr (Figure 4C). To be sure endogenous PtdIns(3,4,5)P3 levels are not altered, the precise
that this morphological observation resembles the beginning of nature of the biologically active entity produced in cells and the
a differentiation process we used two additional biochemical mechanism of action remains to be elucidated. As estimated
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Chemistry & Biology
Membrane-Permeant Phosphoinositides
by reversed-phase chromatography the hydrophobicity of SIGNIFICANCE
all membrane-permeant derivatives seems to be very sim-
ilar. Therefore, large differences in cell entry between the In order to facilitate the investigation of PI3K signaling and
compounds are not expected. Very little is known about struc- the function of the various phosphoinositides, methods
ture-activity relationships of phosphoinositides; however, one are needed that artificially, acutely, but nondisruptively,
explanation for higher PIP4/AM activity comes from closer exam- increase the concentration of a specific phospholipid. This
ination of the PDK1 PH domain crystal structures. The structure work demonstrates that membrane-permeable phosphoi-
shows that the phosphoinositide-binding site of PDK1 is unusu- nositide derivatives are suitable for rapidly activating intra-
ally spacious (Komander et al., 2004). In contrast to other cellular signaling pathways downstream of PI3K. This
PtdIns(3,4,5)P3-binding PH domains such as those from Akt or makes them important tools to investigate the contribution
Grp1 where the 6-OH position is exposed to the solvent (Lietzke of phosphoinositide signaling in living cells. We have synthe-
et al., 2000), in the PDK1 PH domain additional binding sites are sized a phosphoinositide analog with a phosphate on the
present around the 6-OH position, which could interact with 6-OH position, which showed remarkable potency in acti-
another phosphate group. In fact, it was demonstrated that vating PI3K-dependent signaling. Although phosphoinositi-
PDK1 binds 6-OH phosphorylated inositol phosphates, such des with a phosphate on this position have not been
as Ins(1,3,4,5,6)P5 and InsP6, with submicromolar affinity, detected in vivo, we cannot entirely exclude that PtdIns(3,4,
whereas binding to inositol phosphates without the phosphate 5,6)P4 occurs in living cells as a putative messenger lipid that
on the 6-OH position, such as Ins(1,4,5)P3, is virtually undetect- escaped detection due to its very low abundance. In addi-
able (Komander et al., 2004). It is therefore possible that PIP4/ tion, we show that membrane-permeant phosphoinositides
AM-derived phosphoinositides, by having an additional phos- are able to modulate complex PI3K-dependent processes
phate on 6-OH position, may bind to PDK1 with higher affinity such as neurite initiation and outgrowth. Previous work
than those from the similar compound PIP3/AM and conse- has suggested that the PI3K product PtdIns(3,4,5)P3 plays
quently induce the PI3K signaling pathways more effectively. a crucial role in neurite initiation but the hypothesis that
Alternatively, the higher activity could be assigned to a slower more PtdIns(3,4,5)P3 will lead to more neurites in vivo was
metabolism of the biologically active product in cells by endog- never directly tested. The increase in the number of neurites
enous phosphatases and/or the lack of butyrate hydrolysis per cell after application of membrane-permeant PI3K prod-
required in the 6-OH position. Finally, we cannot entirely exclude ucts is complementing earlier neurite initiation studies from
that PtdIns(3,4,5,6)P4 occurs in living cells as a putative mes- other labs and provides a direct demonstration of the
senger lipid and that its very low abundance has prevented involvement of these phosphoinositides in neurite initiation.
detection so far. Despite our current lack of understanding of In the future, membrane-permeant derivatives that are
the precise mode of action, PIP4/AM will likely become an attrac- limited in their metabolism or photoactivatable varieties
tive tool to study growth factor signaling in the future, especially might pave the way for even more exciting possibilities.
considering its comparably facile synthesis and potency. A tech-
nical problem to be considered in this respect is the low shelf-life
of about six months for this type of compound.
EXPERIMENTAL PROCEDURES
Cell Lines and Antibodies
Application of PI(3,4)P2/AM, PIP3/AM, and PIP4/AM together
with NGF induced a significant increase in the number of neurites
per cell, which is consistent with their role in neurite initiation dis-
cussed in the literature. Aoki et al. (2005, 2007), using FRET
based PtdIns(3,4,5)P3 sensors, showed that local accumulation
of PtdIns(3,4,5)P3 initiates neurite protrusion at the plasma mem-
brane and constructed a model for neurite initiation in silico
based on regulation of PIP3 levels. Furthermore, Fivaz et al.
(2008) have shown that the local PI3K-HRas-positive feedback
loop is instrumental for breaking the initial neuronal symmetry
and for neurite initiation. These studies failed to directly test
the hypothesis that more PtdIns(3,4,5)P3 will lead to more neu-
rites in vivo. Our results are thus complementing these studies
by showing that application of membrane-permeant PI3K prod-
ucts leads to a dramatic increase in the number of neurites per
cell. Also, we provide a direct demonstration of the involvement
of these phosphoinositides in neurite initiation.
PC12 6–15 cells (stably transfected with NGF receptor TrkA) were a gift from
D. Martin Zanca (University of Salamanca). NRK cells were a gift from J. Ellen-
berg (European Molecular Biology Laboratory). U2OS cells were a gift from
A. Dinarina (Spanish National Cancer Research Centre). HeLa CCL-2 cells
were purchased from LGC Promochem GmbH. Anti-phosphoERK1/2 (T202/
Y204), anti-phosphoAkt (S473), and anti-phosphop70S6K (T389) were
purchased from Cell Signaling Technology. Anti-a-tubulin antibody was
obtained from NeoMarkers. Secondary antibodies for western blots were
goat anti-rabbit IRDye 800CW (Rockland Immunologicals) and goat anti-
mouse Alexa-680 (Molecular Probes).
Imaging of PH Domain and MAPK Translocation
NRK or HeLa cells were grown on 35 mm glass-bottom dishes (MatTek). The
NRK were transfected with GFP-tagged PH domain of Grp1 while HeLa cells
were transfected with YFP-tagged ERK1 using Fugene6 (Roche) according
to the manufacturer’s protocol. After the 12–16 hr starvation in serum-free
DMEM, the cells were imaged using a Leica SP2 AOBS confocal microscope
with a 633 oil objective in PH domain translocation experiments or using a
Leica AF6000 LX widefield microscope with a 603 glycerol objective for
studying MAPK translocation.
Together with the biochemical and live cell data on signaling
network activation downstream of PI3K/PDK1, we show that
membrane-permeant phosphoinositide derivatives are useful
to study both immediate PI3K-dependent events such as protein
phosphorylation and translocation, as well as complex PI3K-
dependent cellular processes, which take many hours to
develop, such as neurite outgrowth.
Ca2+ and PKC Activity Imaging
HeLa CCL-2 cells (LGC Promochem GmbH) were transfected with KCP-1
using Fugene6 (Roche). After 12 hr the cells were incubated for 30 min with
15 mM Fura Red/AM (Molecular Probes) and imaged using a Leica SP2 AOBS
confocal microscope with a 633 oil immersion objective (Leica). FRET
Chemistry & Biology 16, 1190–1196, November 25, 2009 ª2009 Elsevier Ltd All rights reserved 1195

Chemistry & Biology
Membrane-Permeant Phosphoinositides
measurements with KCP-1 were carried out as previously described (Schlei-
fenbaum et al., 2004).
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Neurite Outgrowth Assay
1655–1657.
Neurite outgrowth assay was performed as previously described (Laketa et al.,
2007; also available in the Supplemental Experimental Procedures).
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protein kinase kinase kinases in signal integration. Oncogene 26, 3159–3171.
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Western Blot Assay
membrane dynamics. Nature 443, 651–657.
HeLa or NRK cells were grown in a 6 well dish in DMEM and 10% fetal calf
serum. After 12–16 hr starvation in serum-free DMEM, the cells were treated
with EGF (100 ng/ml) and PDGF (100 ng/ml) (Sigma-Aldrich) or with various
concentrations of membrane-permeant phosphoinositide derivatives 4–7.
Obtained lysates were analyzed using standard western blot protocols. Anti-
body signal detection was performed with an Odyssey Infrared Imaging
System (LI-Cor Biosystems).
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Protein Phosphorylation Screens
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Detergent-solubilized extracts from non-stimulated (control) and PIP4/AM
(10 mM)-stimulated HeLa cells were subjected to Kinex antibody microarray
screen. Also, extracts from non-stimulated (control), PIP4/AM (10 mM)-, and
EGF (100 ng/ml)-stimulated HeLa cells were subjected to KPSS 10.1 and
KPSS 11.0 as described on the Kinexus Bioinformatics Corp. website
(http://www.kinexus.ca). Kinex antibody microarray screen uses a panel of
650 antibodies to track the differential binding of dye-labeled proteins in
lysates prepared from stimulated versus non-stimulated cells. This screen
was performed twice. KPSS 10.1 and KPSS 11.0 screens use panels of
30–40 highly validated commercial phosphosite-specific antibodies and 20
lane multichannel blotters. The intensity of the ECL signals for the target
protein bands on the Kinetworks immunoblots were quantified with a FluorS
Max Multi-Imager and Quantity One software (Bio-Rad).
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materials is available in the Supplemental Experimental Procedures.
Komander, D., Fairservice, A., Deak, M., Kular, G.S., Prescott, A.R., Peter
Downes, C., Safrany, S.T., Alessi, D.R., and van Aalten, D.M. (2004). Structural
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SUPPLEMENTAL DATA
Supplemental Data include Supplemental Experimental Procedures, seven fig-
ures, three tables, and four movies and can be found with this article online at
http://www.cell.com/chemistry-biology/supplemental/S1074-5521(09)00332-9.
Laketa, V., Simpson, J.C., Bechtel, S., Wiemann, S., and Pepperkok, R. (2007).
High-content microscopy identifies new neurite outgrowth regulators. Mol.
Biol. Cell 18, 242–252.
ACKNOWLEDGMENTS
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Lambright, D.G. (2000). Structural basis of 3-phosphoinositide recognition
by pleckstrin homology domains. Mol. Cell 6, 385–394.
We thank H. Stichnoth for cultured cells and J. Gross (University of Heidelberg)
for high resolution mass determination. Funding was provided by the VW foun-
dation (I/81 597) and the Helmholtz Initiative for Systems Biology (SBCancer)
to C.S. R.P. is supported by a grant from the German Federal Ministry of
Education and Research within the framework of NGFN2 SMP Cell
(01GR0423).
Marshall, C.J. (1995). Specificity of receptor tyrosine kinase signaling: tran-
sient versus sustained extracellular signal-regulated kinase activation. Cell
80, 179–185.
Ozaki, S., DeWald, D.B., Shope, J.C., Chen, J., and Prestwich, G.D. (2000).
Intracellular delivery of phosphoinositides and inositol phosphates using poly-
amine carriers. Proc. Natl. Acad. Sci. USA 97, 11286–11291.
Received: July 6, 2009
Revised: October 6, 2009
Accepted: October 7, 2009
Published: November 24, 2009
aum, A., Stier, G., Gasch, A., Sattler, M., and Schultz, C. (2004).
Schleifenb
Genetically encoded FRET probe for PKC activity based on pleckstrin.
J. Am. Chem. Soc. 126, 11786–11787.
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1196 Chemistry & Biology 16, 1190–1196, November 25, 2009 ª2009 Elsevier Ltd All rights reserved