PI3K/Akt signaling requires spatial
compartmentalization in plasma
Xinxin Gaoa, Pamela R. Lowrya, Xin Zhoua, Charlene Deprya, Zhikui Weib, G. William Wongb, and Jin Zhanga,c,d,1
Departments ofaPharmacology and Molecular Sciences,bPhysiology and Center for Metabolism and Obesity Research, andcOncology, anddThe Solomon
H. Snyder Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, MD 21205
Edited* by Lewis C. Cantley, Beth Israel Deaconess Medical Center, Boston, MA, and approved July 25, 2011 (received for review December 23, 2010)
Spatial compartmentalization of signaling pathway components
generally defines the specificity and enhances the efficiency of
signal transduction. The phosphatidylinositol 3-kinase (PI3K)/Akt
pathway is known to be compartmentalized within plasma mem-
brane microdomains; however, the underlying mechanisms and
functional impact of this compartmentalization are not well un-
derstood. Here, we show that phosphoinositide-dependent kinase
1 is activated in membrane rafts in response to growth factors,
whereas the negative regulator of the pathway, phosphatase and
tensin homolog deleted on chromosome 10 (PTEN), is primarily
localized in nonraft regions. Alteration of this compartmentaliza-
tion, either by genetic targeting or ceramide-induced recruitment
of PTEN to rafts, abolishes the activity of the entire pathway. These
findings reveal critical steps in raft-mediated PI3K/Akt activation
and demonstrate the essential role of membrane microdomain
compartmentalization in enabling PI3K/Akt signaling. They further
suggest that dysregulation of this compartmentalization may
underlie pathological complications such as insulin resistance.
Defects in PI3K/Akt signaling have been implicated in many
diseases, including cancer (2) and type 2 diabetes (3). The acti-
vation of this pathway is initiated at the plasma membrane,
where phosphatidylinositol (3,4,5) trisphosphate [PI(3,4,5)P3],
generated by PI3K and degraded by phosphatase and tensin
homolog deleted on chromosome 10 (PTEN), recruits Akt to the
membrane. Once at the membrane, Akt is phosphorylated in its
activation loop by phosphoinositide-dependent kinase 1 (PDK1)
and on its hydrophobic motif by mammalian TOR complex 2 (4).
After these two phosphorylation events, Akt adopts an active
conformation and proceeds to phosphorylate a variety of protein
substrates involved in diverse cellular processes. Although the
activation of the PI3K/Akt signaling pathway has been exten-
sively studied, the mechanisms by which several critical steps are
regulated in the cell are still not well understood (5). For in-
stance, it remains unclear whether growth factor stimulation
leads to activation of PDK1. A better understanding of the
complex cellular regulation of PI3K/Akt signaling may require
dissecting these molecular events in their specific cellular contexts.
Given that the plasma membrane is the site of activation for
the PI3K/Akt pathway, it is no surprise that plasma membrane
microdomains, such as sphingolipid- and cholesterol-enriched
membrane rafts (6), emerge as important regulators of this
pathway. A recent study using fluorescence correlation spec-
troscopy has indicated that raft microdomains play important
roles in recruiting Akt to the membrane after PI(3,4,5)P3pro-
duction (7). Disruption of membrane rafts was shown to inhibit
the recruitment process (7). In addition, we have observed a
preferential activation of Akt in membrane rafts by using a
fluorescence resonance energy transfer (FRET)-based Akt ac-
tivity reporter (AktAR) (8). However, the molecular mecha-
a major role in cell metabolism, growth, and apoptosis (1).
nisms by which membrane rafts control Akt recruitment and
regulate its activation are yet to be determined. Furthermore,
although spatial compartmentalization is believed to be a key
mechanism for achieving specificity and efficiency in general (9),
the role of membrane microdomain compartmentalization in
PI3K/Akt signaling remains poorly defined.
To address these questions, we used genetic targeting as a
general strategy, directing either a fluorescent biosensor or an
active enzyme to specific plasma membrane microdomains. The
use of targeted biosensors made it possible to monitor the same
molecular process in different cellular contexts, and genetic
targeting of an active form of a negative regulator allowed per-
turbation of a signaling event in specific membrane microdomains,
thereby testing the functional importance of microdomain-specific
signaling. Using this strategy, we investigated the membrane
compartmentalization of the signaling molecules dictating Akt
activation. We demonstrated that PDK1 can be activated in re-
sponse to growth factors and that this activation occurs in mem-
brane rafts. Furthermore, we found that PTEN is localized outside
of membrane rafts and that relocalizing it to rafts, instead of af-
fecting the specificity and efficiency of the signaling response,
abolished the activity of the entire pathway.
PDK1 Is Activated in Membrane Rafts. The PDK1-mediated phos-
phorylation of the activation loop of Akt is required for Akt
activity. Although the importance of PDK1 in the PI3K/Akt
pathway has been established, the regulation of PDK1 remains
controversial (5). It has been suggested that PDK1 is constitu-
tively active throughout the cell and cannot be further activated
by growth factor stimulation (10). However, based on the ob-
servation that pervanadate (PV) can increase PDK1 activity at
the plasma membrane, it has been suggested that PDK1 can be
activated by growth factor stimulation in a similar, spatially
controlled fashion (11). Thus, whether and how PDK1 is regu-
lated by growth factors remains controversial.
To address this problem, we set out to analyze the activation
of PDK1 in its native cellular context, the plasma membrane of
living cells. More specifically, we wanted to examine different
plasma membrane microdomains by generating a FRET-based
PDK1 activation reporter (PARE) that could be targeted to
these microdomains. To this end, we generated a construct in
which the full-length PDK1 was sandwiched between a pair of
fluorescent proteins, enhanced cyan fluorescent protein (ECFP)
Author contributions: X.G., Z.W., G.W.W., and J.Z. designed research; X.G., P.R.L., X.Z.,
C.D., and Z.W. performed research; X.G., P.R.L., X.Z., C.D., Z.W., and J.Z. analyzed data;
and X.G. and J.Z. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| August 30, 2011
| vol. 108
| no. 35
and citrine (Fig. 1A), to allow us to monitor the conformational
changes in PDK1 during its activation by changes in FRET. This
reporter was targeted to membrane rafts by using a targeting
motif derived from Lyn kinase, and to nonraft regions of the
plasma membrane by using a targeting motif derived from K-ras
(8) (Fig. 1A). Sucrose density gradient fractionation experiments
confirmed the localization of the two plasma membrane-targeted
PDK1 reporters to the desired microdomains (Fig. 1B).
We then used these PDK1 activation reporters to examine the
conformational changes associated with PV-induced activation
of PDK1 in serum-starved NIH 3T3 cells. No emission ratio
change was observed in cells expressing the untargeted reporter,
which also did not translocate effectively to the plasma mem-
brane. However, we saw a striking difference between the raft-
targeted and nonraft-targeted PDK1 reporters: Only the raft-
targeted PDK1 reporter showed an increase (of 20.7 ± 3.3%; n =
5) in the yellow-to-cyan emission ratio (Fig. 1C and Fig. S1),
suggesting that the PV-induced activation of PDK1 (Fig. S2),
which was reported to occur at the plasma membrane, instead
occurs primarily in raft microdomains of the plasma membrane.
We next examined growth factor-induced PDK1 activation.
Serum-starved NIH 3T3 cells expressing one of the three variants
of the PDK1 reporter were treated with 50 ng/mL platelet-
derived growth factor (PDGF) and imaged. Strikingly, the raft-
targeted PDK1 reporter showed an increase of 19 ± 4% (n = 7)
in the yellow-to-cyan emission ratio upon growth factor stimu-
lation, whereas no emission ratio change was observed in cells
expressing the untargeted or nonraft-targeted PDK1 reporter (Fig.
1 D–F). Importantly, raft disruption using methyl-β-cyclodextrin
(MCD) also abolished the response of the raft-targeted PDK1
activation reporter to 50 ng/mL PDGF (Fig. S3) but did not affect
the response of a nonraft-targeted Akt activity reporter (8). These
data suggest that growth factor stimulation can induce the acti-
vation of PDK1 and that this activation also occurs primarily in the
raft microdomains of the plasma membrane.
Using the raft-targeted PDK1 reporter, we also examined the
ability of other growth factors to activate PDK1 in live cells.
Stimulation of serum-starved HeLa cells with 100 ng/mL epi-
dermal growth factor (EGF) induced an emission ratio increase
in the case of the raft-targeted PDK1 reporter (Fig. S4A). As was
true for PDGF stimulation, the EGF-induced response was also
restricted to the membrane raft microdomains (Fig. S4A), in-
dicating that the differential activation pattern of PDK1 in the
plasma membrane microdomains was not unique to NIH 3T3
cells or PDGF receptor activation.
PDK1, like Akt, possesses a pleckstrin homology (PH) domain
that binds to 39-phosphoinositides, including PI(3,4,5)P3and PI
(3,4)P2. Studies have demonstrated that the phosphoinositide
binding of PDK1 plays a crucial role in controlling the activation of
Akt and its downstream signaling. For instance, PDK1K465E/K465E
knock-in mice expressing a mutant PDK1 that is incapable of
binding to 39-phosphoinositides are known to exhibit impaired
growth and a marked insulin resistance and glucose intolerance
(12). We therefore asked whether the observed PDK1 activation
in response to PV or PDGF depended on its binding to 39-
phosphoinositides. As shown in Fig. S4 B and C, mutation of the
two Arg residues (R472/474) responsible for phosphoinositide
binding to Ala (13) abolished PDK1 activation, as indicated by a
lack of response from the raft-targeted PDK1 reporter variant.
Furthermore, addition of the PI3K inhibitor LY294002 caused a
reversal of the response of Lyn-PARE after the PDGF-, EGF- or
PV-stimulated emission ratio change was stabilized (Fig. S4),
indicating that the response of the raft-targeted PDK1 reporter
is reversible and depends on PI3K activity.
The lack of change in the nonraft regions may reflect the in-
efficient activation of PDK1 in these regions. Alternatively,
PDK1 may be locked into conformations that correspond to high
PDK1 activity in these membrane microdomains. To directly
examine endogenous PDK1 activity in these specific micro-
domains, we studied the phosphorylation of the natural substrate
of PDK1, Akt, in raft and nonraft regions by sucrose density
gradient separation followed by Western blot analysis. Phos-
phorylation of Akt by PDK1 at T308 was found to occur pref-
erentially in the membrane rafts of insulin-stimulated HEK 293
cells, with 5.0 ± 2.5-fold stronger phosphoAkt signal (normalized
to the total Akt signal) in raft fractions than in nonraft fractions
(Fig. S5 A and B). This finding suggests that PDK1 is preferen-
tially activated in membrane rafts. At the same time, it appears
that some basal activity of PDK1 is present in nonraft micro-
domains (Fig. S5C). The Thr308 phosphorylation observed in
the nonraft fractions could be due to the basal activity of PDK1
and/or some diffusion and redistribution of phosphorylated Akt
between membrane microdomains. In fact, considering the dif-
fusion of activated PDK1 between membrane compartments,
this fivefold difference is likely an underestimation. Taken to-
gether, these studies suggest that PDK1 can be activated by a
variety of stimulating signals, and this activation occurs in the
raft microdomains of the plasma membrane.
The PDK1 activation reporter PARE was generated with full-length PDK1
sandwiched by a pair of fluorescent proteins, ECFP and citrine. PARE was
targeted to membrane rafts by using a targeting motif derived from Lyn
kinase and to nonraft regions of the plasma membrane by using a targeting
motif derived from Kras. (B) The localization of Lyn-PARE and PARE-Kras was
confirmed by sucrose density gradient fractionation. Total cell lysates from
HEK 293 cells overexpressing Lyn-PARE or PARE-Kras were subjected to su-
crose density gradient fractionation, followed by Western blotting. Cholera
toxin subunit B (CTB) was used as a raft marker. The reporters were detected
with an anti-GFP antibody. (C) Representative time courses showing the
responses of PARE (n = 5), Lyn-PARE (n = 5), and PARE-Kras (n = 4) in serum-
starved NIH 3T3 cells after stimulation with 100 μM PV. (D) Pseudocolor
images showing the response of Lyn-PARE to 50 ng/mL PDGF in NIH 3T3 cells.
The yellow fluorescence image (Left) shows the localization of Lyn-PARE. (E)
Pseudocolor images showing the null response of PARE-Kras to 50 ng/mL
PDGF in NIH 3T3 cells. The yellow fluorescence image (Left) shows the lo-
calization of PARE-Kras. (F) Stimulation of serum-starved NIH 3T3 cells with
50 ng/mL PDGF induced an emission ratio change in Lyn-PARE (n = 7), but
not PARE (n = 5) or PARE-Kras (n = 6).
Development and characterization of PDK1 activation reporters. (A)
| www.pnas.org/cgi/doi/10.1073/pnas.1019386108Gao et al.
Localization of PTEN to Nonraft Regions of the Plasma Membrane Is
Important for Proper Akt Signaling. We have shown that Akt ac-
tivation, which depends on accumulation of 39-phosphoinosi-
tides, is faster and stronger in membrane rafts (8). Our further
demonstration of raft-specific activation of PDK1, also down-
stream of 39-phosphoinositides, prompted us to examine the
regulation of 39-phosphoinositides in the context of plasma
membrane microdomains. As a lipid phosphatase, PTEN plays
an important role in restricting the accumulation of 39-phos-
phoinositides. It has been shown to be largely cytosolic, with
a small variable proportion associated with the plasma mem-
brane (14). Although membrane-associated PTEN is the actual
species that catalyzes the degradation of 39-phosphoinositides, its
microdomain-specific membrane association and the importance
of this membrane compartmentalization are not well understood.
We first examined the membrane localization of endogenous
PTEN by sucrose density gradient fractionation of crude plasma
membranes isolated from HEK 293 cells. The majority of the
membrane PTEN was found to reside in nonraft regions, with
only a small amount being present in the membrane rafts (Fig. 2
A and B). The same pattern was also observed in 3T3-L1 adi-
pocytes (Fig. 3A). These results indicated that PTEN primarily
resides in the nonraft regions of the plasma membrane, consis-
tent with previous observations (15, 16).
We then used a genetic targeting approach to determine the
role of membrane microdomain compartmentalization of PTEN
in controlling downstream signaling. An active form of PTEN,
PTEN A4 (17), has been described. In this form, four residues of
the C-terminal tail region have been mutated to Ala to abolish
the inhibitory phosphorylation. To alter the membrane micro-
domain localization of PTEN, we targeted PTEN A4 to raft
microdomains with the Lyn motif (Fig. 2C). As a control,
a nonraft-targeted PTEN was also generated by attaching the
Kras motif to PTEN A4 (Fig. 2C). The membrane localization of
Lyn-PTEN A4 and PTEN A4-Kras was verified by sucrose den-
sity gradient fractionation (Fig. 2D).
The effects of perturbing the membrane microdomain locali-
zation of PTEN were assessed at the levels of PDK1 activation,
Akt recruitment, and Akt activity by using a series of fluorescent
biosensors. First, we found that targeting of the active PTEN
to membrane rafts via Lyn-PTEN A4 prevented growth factor-
induced PDK1 activation, because no emission ratio change was
observed in growth factor-stimulated NIH 3T3 cells coexpressing
properly localized Lyn-PTEN A4 and the Lyn-PDK1 reporter
(Fig. 2E). The presence of PTEN A4-Kras, however, did not
prevent the activation of PDK1 in membrane rafts, although it
reduced the response of Lyn-PARE (Fig. 2E and Fig. S6). The
response to PDGF could be restored by treatment with 50 mM
H2O2, a condition that has been shown to inhibit cellular PTEN
(18) (Fig. S6A). This result further indicates that the suppression
of raft PDK1 activation is caused by the high PTEN activity.
The strong inhibitory effect of Lyn-PTEN A4 on PDK1 acti-
vation could also be seen at the level of the membrane re-
cruitment of Akt, as indicated by the lack of plasma membrane
translocation of the YFP-tagged PH domain of Akt in response
to growth factor stimulation (Fig. 2F). Inhibition of PTEN ac-
tivity with 50 mM H2O2restored the membrane translocation
(Fig. S7A). In contrast, active PTEN targeted to nonraft regions
had a much lower inhibitory effect, producing a 40% reduction
in Akt-PH translocation compared with cells lacking PTEN A4
expression (Fig. S7B). This nonspecific effect, presumably a re-
sult of the overexpression of an active PTEN, could also be re-
versed by H2O2treatment (Fig. S7).
Finally, we used a developed FRET-based Akt activity re-
porter, AktAR (8), to probe the effect of perturbing membrane
microdomain localization of PTEN on growth factor-stimulated
Akt activity. This biosensor serves as a surrogate substrate for
Akt and reports Akt activity by phosphorylation-dependent
increases in FRET. As shown in Fig. 2G, the presence of Lyn-
PTEN A4 abolished the response of AktAR to PDGF stimula-
tion in NIH 3T3 cells, whereas cells expressing PTEN A4-Kras
were still able to respond to PDGF stimulation (Fig. 2G and Fig.
S8). These data demonstrate that preferential localization of
PTEN outside membrane rafts has an important functional
consequence for maintaining PDK1 and Akt activity, and mis-
localizing it to raft microdomains abolished downstream signaling.
Given the importance of the specific membrane microdomain
localization of PTEN in maintaining the activity of the PI3K/Akt
pathway, we postulated that dysregulation of this compartmen-
talization can disrupt cellular functions and contribute to the
development of pathologic conditions. Ceramide, a sphingolipid
vation, Akt membrane recruitment, and Akt activity. (A) PTEN is primarily
localized to nonraft regions of the plasma membrane. Crude plasma mem-
branes from HEK 293 cells were solubilized and subjected to sucrose density
gradient fractionation, followed by Western blotting with an anti-PTEN
antibody. CTB was used as a raft marker. Anti-tubulin was used to ensure the
separation of membrane proteins from cytosolic proteins. (B) Statistically
significant differences between PTEN levels in rafts and nonraft regions
(***P < 0.001; n = 3). Densitometric analysis indicated that the majority of
the membrane PTEN resides in nonraft regions. (C) PTEN A4, fused with
a C-terminal fluorescent protein (FP), was targeted to membrane rafts and
nonraft regions with a Lyn or Kras motif. (D) The membrane localization of
Lyn-PTEN A4 or PTEN A4-Kras was verified by sucrose density gradient
fractionation of total cell lysates of HEK 293 cells expressing Lyn-PTEN A4-
YFP or PTEN A4-YFP-Kras. CTB was used as a raft marker. Targeted PTEN A4
was detected with an anti-GFP antibody. Yellow fluorescence images show
the membrane localization of PTEN. (E) Representative time courses in-
dicating that the response of Lyn-PARE was abolished by Lyn-PTEN A4 (n = 5;
PTEN fused with mCherry), but not PTEN A4-Kras (n = 7). (F) Representative
time courses demonstrating that the membrane translocation of the Akt PH
domain was abolished by Lyn-PTEN A4 (n = 4; PTEN fused with mCherry), but
not PTEN A4-Kras (n = 4). (G) Representative time courses indicating that the
response of AktAR was abolished by Lyn-PTEN A4 (n = 7; PTEN fused with
mCherry), but not PTEN A4-Kras (n = 6).
Genetic targeting of PTEN to membrane rafts abolishes PDK1 acti-
Gao et al.PNAS
| August 30, 2011
| vol. 108
| no. 35
that is known to antagonize insulin action, has been suggested to
be an important contributor to insulin resistance (19). The un-
derlying mechanism is not clearly understood, but the major
effect appears to be the inhibition of the PI3K/Akt pathway.
Interestingly, raft disruption achieved through inhibition of
ceramide synthesis also inhibited Akt signaling (7), suggesting
that both up- and down- regulation of physiological levels of
ceramide could lead to Akt inhibition. Recently, ceramide
treatment was shown to translocate PTEN to caveolin-enriched
microdomains in certain cell lines (20, 21). We confirmed this
finding by showing that preincubation of 3T3-L1 adipocytes with
C2-ceramide, a cell-permeable ceramide analog, greatly in-
creased the amount of PTEN found in raft fractions (8.8 ± 2.1-
fold; n = 3) (Fig. 3 A and B and Fig. S9). Importantly, treatment
with C2-ceramide also blocked the PDGF-induced activation of
PDK1 in membrane rafts and membrane recruitment of Akt, and
Akt activity, in 3T3 L1 preadipocytes and NIH 3T3 cells (Fig. 3
C–E, Fig. S10, and Fig. S11). These findings are most consistent
with a model in which ceramide suppresses PDK1 and Akt ac-
tivity by specifically recruiting PTEN to membrane rafts. Fur-
thermore, in agreement with previous reports (22), we found that
this treatment also blocked insulin-induced glucose uptake in
3T3 L1 adipocytes (Fig. 3F), an important function mediated by
the PI3K/Akt pathway, suggesting that ceramide-induced mis-
localization of PTEN to membrane rafts can inhibit the func-
tional output of the PI3K/Akt pathway.
Activation of the PI3K/Akt pathway involves a series of tightly
coupled molecular events occurring at the plasma membrane.
However, how these molecular events are organized in the local
signaling microdomains is not clear. By using a genetic targeting
strategy in which the signaling events in specific membrane
microdomains were monitored or perturbed, we have demon-
strated that raft microdomains are critical for organizing the
localization of active positive and negative regulators of the
pathway. Dysregulation of this membrane compartmentalization
undermined PI3K/Akt signaling transduction and may underlie
pathological complications such as insulin resistance.
As the kinase controlling the activation loop phosphorylation
of Akt, PDK1 plays a critical role in PI3K/Akt signaling. Despite
advances (23), the activation mechanisms of PDK1 are still not
well understood. Here, in contrast to the previously held belief
that PDK1 is constitutively active and cannot be further activated
by growth factor stimulation (10), we show that PDK1 can be
activated by various growth factors, and this induced activation
occurs in membrane rafts. Our genetic targeting approach
allowed us to focus on specific events in specific plasma mem-
brane domains of living cells, thereby enhancing the sensitivity of
the assay and revealing previously unrecognized mechanistic
details. This pool of further-activated PDK1 may directly con-
tribute to the preferential activation of Akt in membrane rafts
(8) and the proper functioning of the pathway. In addition, raft-
activated PDK1 may play other important roles, because PDK1,
as the master regulator of AGC kinase signal transduction (5),
activates many other critical kinases, including protein kinase C
(PKC), ribosomal S6 kinase (S6K), serum and glucocorticoid-
inducible kinase (SGK), and p21-activated kinases (PAK). In this
vein, one study has shown that raft PDK1 recruits PKC and
additional components to assemble a signaling complex that
mediates NF-κB signaling in Jurkat cells stimulated with anti-
CD3 and anti-CD28 (24).
Future studies should further elucidate the mechanisms of this
activation; for example, by examining Y373 phosphorylation,
which has been shown to contribute to PV-induced PDK1 acti-
vation in the plasma membrane (11). The membrane micro-
domain-specific dynamics of PI(3,4,5)P3, which is another
component involved in PDK1 activation, have not been sys-
tematically characterized. However, several lines of evidence
suggest that PI(3,4,5)P3may not be exclusively confined in spe-
cific membrane microdomains. For example, although PI(3,4,5)
P3exhibits structural features unfavorable for inclusion into raft
microdomains, it has been proposed that the PI(3,4,5)P3-con-
taining raft microdomains can be formed upon PH domain
binding to PI(3,4,5)P3(7). Nonetheless, we showed that PI(3,4,5)
P3production can be detected in nonraft regions (8). Augmen-
tation of this pool of PI(3,4,5)P3 by overexpression of p110-
CAAX, a potentially nonraft-targeted PI3K, can lead to elevated
Akt activity (25, 26), possibly involving elevated basal PDK1
activity in nonraft regions.
PTEN is a critical negative regulator of the PI3K/Akt pathway.
Our finding that membrane-associated PTEN is preferentially
localized to nonraft regions is consistent with previous studies
(15, 16), although in some cases raft PTEN has proved un-
detectable (27), possibly as a result of variations in the cell types
and experimental conditions applied. The mechanism underlying
this preferential localization to nonraft regions is not clear. One
potential mediator is PI(4,5)P2, a phosphoinositide that is criti-
cally involved in the membrane association of PTEN (28, 29). In
the absence of protein binding, the polyunsaturated 2’-arach-
activation, Akt membrane recruitment, and Akt activity. (A) Ceramide
recruits PTEN to membrane rafts. Crude plasma membranes from 3T3 L1
adipocytes (in the presence or absence of 50 μM C2-ceramide) were solubi-
lized and subjected to sucrose density gradient fractionation, followed by
Western blotting with an anti-PTEN antibody. CTB was used as a raft marker.
Anti-tubulin was used to ensure the separation of membrane proteins from
cytosolic proteins. (B) Densitometric analysis indicated a statistically signifi-
cant difference between the raft PTEN levels in the presence of C2-ceramide
and those in its absence (**P < 0.01; n = 3). (C) Representative time courses
showing that the response of Lyn-PARE (n = 7) in 3T3 L1 preadipocytes was
abolished by preincubation with 50 μM C2-ceramide (n = 6). (D) Represen-
tative time courses showing that membrane translocation of Akt PH domain
(n = 3) in 3T3 L1 preadipocytes was abolished by preincubation with 50 μM
C2-ceramide (n = 3). (E) Representative time courses showing that the re-
sponse of AktAR (n = 6) in 3T3 L1 preadipocytes was abolished by pre-
incubation of 50 μM C2-ceramide (n = 5). (F) Ceramide-mediated suppression
of insulin-induced glucose uptake in 3T3 L1 adipocytes. Preincubation of 50
μM C2-ceramide with 3T3 L1 adipocytes for 60 min inhibited insulin-induced
glucose uptake in these cells (****P < 0.0001; n = 3).
Ceramide recruits PTEN to membrane rafts and suppresses PDK1
| www.pnas.org/cgi/doi/10.1073/pnas.1019386108Gao et al.
idonate chain of PI(4,5)P2 does not favor partitioning into
membrane rafts (7). We further showed that the localization of
PTEN to nonraft regions is critical to preserving the activity of
PI3K/Akt pathway (Fig. 4A). Genetic targeting of PTEN A4, an
active form of PTEN, to membrane rafts completely abolished
the activation of PDK1 in rafts, and membrane recruitment of
Akt and its activity. Nonraft-targeted PTEN A4, however,
showed inhibitory effects but did not prevent this pathway from
being activated. The inhibition by nonraft-targeted PTEN A4
could have resulted from nonspecific effects of overexpressing an
active PTEN. However, there is also a possibility that the enzyme
molecules, which are localized proximal to membrane rafts,
could directly interfere with signaling in raft microdomains.
Based on these findings, we propose a model of membrane
microdomain-mediated PI3K/Akt activation (Fig. 4A). Both the
raft-specific activation of PDK1 and the lack of PTEN-mediated
down-regulation in these microdomains are believed to con-
tribute to the preferential activation of Akt in membrane rafts
(8). However, our most striking finding was that perturbing this
microdomain compartmentalization, either by genetic targeting
of active PTEN to rafts or by ceramide-induced translocation of
PTEN to the rafts, abolished the activity of the PI3K/Akt path-
way. The observed inhibition of Akt recruitment upon raft dis-
ruption (7) is in line with this finding. Thus, plasma membrane
microdomains can serve as platforms both for concentrating
active signaling components such as activated PDK1 and Akt
and for segregating them from nonraft-associated negative reg-
ulators such as PTEN, thereby enabling the activation and
functioning of the PI3K/Akt pathway. From a broader perspec-
tive, these data suggest that spatial compartmentalization not
only defines the specificity and enhances the efficiency of signal
transduction, but it also enables the activation and signaling
mediated by critical pathways.
Dysregulation of this compartmentalization may occur under
pathological conditions. Ceramide is a lipid metabolite known to
induce insulin resistance, and the underlying mechanisms are
complex and not well understood (19). For example, ceramide
has been shown to recruit the atypical PKC isoform PKCζ to
membrane rafts, where PKCζ phosphorylates the PH domain of
Akt and blocks its ability to interact with 39-phosphoinositides
(21, 30). However, ceramide has also been shown to promote the
dephosphorylation of Akt by activating the Akt phosphatase,
protein phosphatase 2A (PP2A), one of the earliest known
ceramide targets (31, 32). Here, we propose that the ceramide-
induced mislocalization of PTEN to membrane rafts, together
with the aforementioned mechanisms, critically contributes to
the inhibitory effect of ceramide on PI3K/Akt signaling (Fig. 4B).
Furthermore, our genetic targeting of PTEN to membrane rafts
recapitulated the strong inhibitory effects of ceramide on the
activation of PDK1 and Akt. Together, these data suggest that
membrane-microdomain compartmentalization is critical for
maintaining proper PI3K/Akt signaling in response to insulin,
and dysregulating raft-localized PI3K/Akt signaling by recruiting
PTEN to these microdomains may be an underlying molecular
mechanism for the insulin resistance caused by excess nutrients
such as saturated fatty acids.
Materials and Methods
Materials. PDGF, EGF, LY294002, MCD, H2O2, Na3VO4, and C2-ceramide were
purchased from Sigma-Aldrich. Anti-Akt, phospho-Akt (T308), tubulin, and
PTEN antibodies were obtained from Cell Signaling Technology, and GFP
antibody was from eBioscience. Horseradish peroxidase-conjugated cholera
toxin subunit B was purchased from Molecular Probes, catalase was pur-
chased from CalBiochem, and Lipofectamine 2000 was purchased from
Invitrogen. Complete Protease Inhibitor Mixture Tablets were obtained from
Roche Applied Science.
Preparation of Pervanadate. Pervanadate was prepared as described (33). In
brief, 25 μL of 500 mM Na3VO4and 1 μL of 30% (vol/vol) H2O2were mixed in
574 μL of PBS. After 5 min, catalase was added to release excess H2O2, which
yielded 20 mM pervanadate.
Constructs. PARE was generated by sandwiching full-length PDK1 between
a FRET pair, ECFP and citrine. Lyn-PARE and PARE-Kras were generated
by the addition of the N-terminal portion of the Lyn kinase gene
(KKKKKSKTKCVIM) of PARE, respectively. Lyn-ECFP PARE and Lyn Citrine
PARE were generated by substituting the fluorescent protein in Lyn-PARE
with ECFP or citrine, respectively. Lyn-PARE R472/474A was generated by
site-directed mutagenesis. PTEN A4 (fused with a C-terminal fluorescent
protein) was targeted to raft and nonraft regions of the plasma membrane
with the same set of targeting motifs. All of the constructs were generated
in a modified version of the mammalian expression vector pcDNA 3.
a CAAXtagat the39-end
Cell Transfection andImaging.Cells were plated on sterilized glass coverslips in
35-mm dishes and were grown to 40% confluency in medium at 37 °C with
5% CO2,then transfected with Lipofectamine 2000. In the case of NIH 3T3
and HeLa cells, the cells were serum-starved for 24 h. For imaging, cells were
washed with Hanks’ balanced salt solution (HBSS) and imaged in the dark at
room temperature. Images were acquired on a Zeiss Axiovert 200M micro-
scope with a cooled charge-coupled device camera, as described (8). Dual-
emission ratio imaging was performed with a 420DF20 excitation filter,
a 450DRLP dichroic mirror, and two emission filters. For CFP and YFP,
475DF40 and 535DF25, respectively, were used. Exposure times were 50–500
ms. Images were taken every 30 s. Imaging data were analyzed with Met-
afluor 6.2 software (Universal Imaging). Fluorescence images were back-
ground-corrected by deducting the background (from regions with no cells)
from the emission intensities of CFP or YFP. Regions of interest at the cell
periphery representing the plasma membrane were used for analysis for
Lyn-PARE and PARE-Kras. Traces were normalized by taking the emission
ratio before addition of drugs as 1.
Western Blot Analysis. Cells were washed with ice-cold PBS and then lysed in
RIPA lysis buffer containing protease inhibitor mixture, 1 mM PMSF, 1 mM
Na3VO4, 1 mM NaF, and 25 nM calyculin A. Total cell lysates were incubated
on ice for 30 min, then centrifuged at 4 °C for 20 min. Total protein was
separated with 7.5% SDS/PAGE and transferred to nitrocellulose mem-
branes. The membranes were blocked with TBS containing 0.05% Tween-20
and 1% BSA and then incubated with primary antibodies overnight at 4 °C.
After incubation with the appropriate horseradish peroxidase-conjugated sec-
ondary antibodies, the bands were visualized by enhanced chemiluminescence.
The intensity of the bands was quantified with ImageJ software.
domains is essential for proper PI3K/Akt signaling. (A) Preferential activation
of Akt in membrane rafts is mediated by activated PDK1 in rafts and lack of
PTEN-mediated down-regulation in these microdomains. (B) Dysregulation
of the raft-localized PI3K/Akt signaling as the underlying mechanism for
insulin resistance. Ceramide may inhibit Akt signaling through promoting
PKCζ phosphorylation of Akt or activating the Akt phosphatase PP2A. The
ceramide-induced mislocalization of PTEN to membrane rafts critically con-
tributes to the inhibitory effect of ceramide on PI3K/Akt signaling.
Compartmentalized PDK1 and PTEN activity in membrane micro-
Gao et al. PNAS
| August 30, 2011
| vol. 108
| no. 35
Membrane Preparation. Cells were rinsed twice with ice-cold PBS and har-
vested into 10 mM Tris (pH 7.4) with 150 mM NaCl, 5 mM EDTA, 2 mM PMSF,
2 mM Na3VO4, 2 mM NaF, and 50 nM calyculin A, with protease inhibitor
mixture. The cells then were subjected to mechanical disruption with
15 strokes of a homogenizer. Homogenates were centrifuged at 2,300 × g
for 5 min at 4 °C, and the supernatant was centrifuged at 18,000 × g for 50
min at 4 °C (34). The resulting membrane pellets were resuspended in 10 mM
Tris (pH 7.4) with 150 mM NaCl, 5 mM EDTA, 2 mM PMSF, 2 mM NaVO4,
2 mM NaF, and 50 nM calyculin A, with protease inhibitor mixture and 1%
Triton X-100, for sucrose density gradient fractionation.
Sucrose Density Gradient Fractionation. Crude plasma membranes (or total cell
lysates) were incubated in ice with periodic mixing for 1 h, then diluted 1:1
with 80% sucrose and layered on 4 mL of 35% sucrose, followed by the
addition of 1 mL of 5% sucrose solution and 4.5 mL of 10 mM Tris (pH 7.4)
at 39,000 × g for 18 h in a Beckman SW41-Ti rotor. All experimental steps
were performed at 4 °C. After ultracentrifugation, the top 3.5 mL of the
sample was discarded. Nine 880-μL fractions were then collected, starting
from the top of the gradient. The fractions were dot-blotted on nitrocellu-
lose membranes and probed with HRP-conjugated cholera toxin subunit B
antibody to identify the raft-containing fractions.
Differentiation of 3T3-L1 Adipocytes. 3T3-L1 preadipocytes were grown to
confluency in 10% calf serum/DMEM and stimulated with induction media
(DMEM containing 10% FBS, 1 μg/mL insulin, 1 μM dexamethasone, and 0.5
mM 3-isobutyl-L-methylxanthine) at 2 d after confluency. The medium was
changed to insulin medium (1 μg/mL) 2 d after induction. Two days later, the
medium was replaced with 10% FBS/DMEM and then changed every 2 d. Full
differentiation was achieved by 8 d after induction.
Glucose Uptake Assay. Adipocytes were incubated in Krebs–Ringer bi-
carbonate buffer supplemented with 30 mM Hepes at pH 7.4, with 0.5% BSA
and 2.5 mM glucose for 3 h. The cells were washed once with PBS and in-
cubated in BSA/KRH [25 mM Hepes-NaOH (pH 7.4), with 120 mM NaCl, 5 mM
KCl, 1.2 mM MgSO4, 1.3 mM CaCl2, and 1.3 mM KH2PO4] without glucose for
15 min. The cells were then incubated with 100 nM insulin for 15 min. The
assay was initiated by the addition of [14C]2-deoxyglucose (0.4 μCi per
sample) and 5 mM glucose and terminated after 15 min by washing the cells
three times with ice-cold PBS. Cells were solubilized in 1% Triton X-100, and
cell-associated radioactivity was determined by scintillation counting.
ACKNOWLEDGMENTS. We thank Qiang Ni for critical reading of the
manuscript and Deborah Ann McClellan for editorial assistance. This work
was supported by National Institutes of Health (NIH) Grants R01 DK-073368
and R21 CA-122673 (to J. Z.). C.D. is supported by NIH Predoctoral Fellowship
F31 GM087079. Z.W. is supported by American Heart Association Predoctoral
Fellowship PRE3790034. G.W.W. is supported by American Heart Association
Grant SDG2260721, Baltimore Diabetes Research and Training Center Grant
(P60DK079637), and NIH Grant DK084171.
1. Franke TF (2008) PI3K/Akt: Getting it right matters. Oncogene 27:6473e6488.
2. Wong KK, Engelman JA, Cantley LC (2010) Targeting the PI3K signaling pathway in
cancer. Curr Opin Genet Dev 20:87e90.
3. Farese RV, Sajan MP, Standaert ML (2005) Insulin-sensitive protein kinases (atypical
protein kinase C and protein kinase B/Akt): Actions and defects in obesity and type II
diabetes. Exp Biol Med (Maywood) 230:593e605.
4. Bozulic L, Hemmings BA (2009) PIKKing on PKB: Regulation of PKB activity by phos-
phorylation. Curr Opin Cell Biol 21:256e261.
5. Mora A, Komander D, van Aalten DM, Alessi DR (2004) PDK1, the master regulator of
AGC kinase signal transduction. Semin Cell Dev Biol 15:161e170.
6. Simons K, Toomre D (2000) Lipid rafts and signal transduction. Nat Rev Mol Cell Biol 1:
7. Lasserre R, et al. (2008) Raft nanodomains contribute to Akt/PKB plasma membrane
recruitment and activation. Nat Chem Biol 4:538e547.
8. Gao X, Zhang J (2008) Spatiotemporal analysis of differential Akt regulation in plasma
membrane microdomains. Mol Biol Cell 19:4366e4373.
9. Hoeller D, Volarevic S, Dikic I (2005) Compartmentalization of growth factor receptor
signalling. Curr Opin Cell Biol 17:107e111.
10. Casamayor A, Morrice NA, Alessi DR (1999) Phosphorylation of Ser-241 is essential for
the activity of 3-phosphoinositide-dependent protein kinase-1: Identification of five
sites of phosphorylation in vivo. Biochem J 342:287e292.
11. Park J, et al. (2001) Identification of tyrosine phosphorylation sites on 3-phosphoi-
nositide-dependent protein kinase-1 and their role in regulating kinase activity. J Biol
12. Bayascas JR, et al. (2008) Mutation of the PDK1 PH domain inhibits protein kinase B/
Akt, leading to small size and insulin resistance. Mol Cell Biol 28:3258e3272.
13. Komander D, et al. (2004) Structural insights into the regulation of PDK1 by phos-
phoinositides and inositol phosphates. EMBO J 23:3918e3928.
14. Tamguney T, Stokoe D (2007) New insights into PTEN. J Cell Sci 120:4071e4079.
15. Caselli A, Mazzinghi B, Camici G, Manao G, Ramponi G (2002) Some protein tyrosine
phosphatases target in part to lipid rafts and interact with caveolin-1. Biochem Bio-
phys Res Commun 296:692e697.
16. Jahn T, Leifheit E, Gooch S, Sindhu S, Weinberg K (2007) Lipid rafts are required for
Kit survival and proliferation signals. Blood 110:1739e1747.
17. Vazquez F, Ramaswamy S, Nakamura N, Sellers WR (2000) Phosphorylation of the
PTEN tail regulates protein stability and function. Mol Cell Biol 20:5010e5018.
18. Lee SR, et al. (2002) Reversible inactivation of the tumor suppressor PTEN by H2O2.
J Biol Chem 277:20336e20342.
19. Holland WL, Summers SA (2008) Sphingolipids, insulin resistance, and metabolic dis-
ease: New insights from in vivo manipulation of sphingolipid metabolism. Endocr Rev
20. Goswami R, Singh D, Phillips G, Kilkus J, Dawson G (2005) Ceramide regulation of the
tumor suppressor phosphatase PTEN in rafts isolated from neurotumor cell lines.
J Neurosci Res 81:541e550.
21. Hajduch E, et al. (2008) Targeting of PKCzeta and PKB to caveolin-enriched micro-
domains represents a crucial step underpinning the disruption in PKB-directed sig-
nalling by ceramide. Biochem J 410:369e379.
22. Summers SA, Garza LA, Zhou HL, Birnbaum MJ (1998) Regulation of insulin-stimulated
glucose transporter GLUT4 translocation and Akt kinase activity by ceramide. Mol Cell
23. Masters TA, et al. (2010) Regulation of 3-phosphoinositide-dependent protein kinase
1 activity by homodimerization in live cells. Sci Signal 3:ra78.
24. Lee KY, D’Acquisto F, Hayden MS, Shim JH, Ghosh S (2005) PDK1 nucleates T cell
receptor-induced signaling complex for NF-kappaB activation. Science 308:114e118.
25. Egawa K, et al. (1999) Membrane-targeted phosphatidylinositol 3-kinase mimics in-
sulin actions and induces a state of cellular insulin resistance. J Biol Chem 274:
26. Takano A, et al. (2001) Growth hormone induces cellular insulin resistance by un-
coupling phosphatidylinositol 3-kinase and its downstream signals in 3T3-L1 adipo-
cytes. Diabetes 50:1891e1900.
27. Odriozola L, Singh G, Hoang T, Chan AM (2007) Regulation of PTEN activity by its
carboxyl-terminal autoinhibitory domain. J Biol Chem 282:23306e23315.
28. Walker SM, Leslie NR, Perera NM, Batty IH, Downes CP (2004) The tumour-suppressor
function of PTEN requires an N-terminal lipid-binding motif. Biochem J 379:301e307.
29. Iijima M, Devreotes P (2002) Tumor suppressor PTEN mediates sensing of chemo-
attractant gradients. Cell 109:599e610.
30. Powell DJ, Hajduch E, Kular G, Hundal HS (2003) Ceramide disables 3-phosphoinosi-
tide binding to the pleckstrin homology domain of protein kinase B (PKB)/Akt by
a PKCzeta-dependent mechanism. Mol Cell Biol 23:7794e7808.
31. Chavez JA, et al. (2003) A role for ceramide, but not diacylglycerol, in the antagonism
of insulin signal transduction by saturated fatty acids. J Biol Chem 278:10297e10303.
32. Dobrowsky RT, Kamibayashi C, Mumby MC, Hannun YA (1993) Ceramide activates
heterotrimeric protein phosphatase 2A. J Biol Chem 268:15523e15530.
33. Huyer G, et al. (1997) Mechanism of inhibition of protein-tyrosine phosphatases by
vanadate and pervanadate. J Biol Chem 272:843e851.
34. Pontier SM, et al. (2008) Cholesterol-dependent separation of the beta2-adrenergic
receptor from its partners determines signaling efficacy: Insight into nanoscale or-
ganization of signal transduction. J Biol Chem 283:24659e24672.
| www.pnas.org/cgi/doi/10.1073/pnas.1019386108Gao et al.