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: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| 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.
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| www.pnas.org/cgi/doi/10.1073/pnas.1019386108Gao et al.