MOLECULAR AND CELLULAR BIOLOGY, May 2011, p. 2122–2133
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 31, No. 10
Nuclear but Not Cytosolic Phosphoinositide 3-Kinase Beta
Has an Essential Function in Cell Survival?
Amit Kumar,1† Javier Redondo-Mun ˜oz,1† Vicente Perez-García,1†
Isabel Cortes,1Monica Chagoyen,2and Ana C. Carrera1*
Department of Immunology and Oncology1and Computational Systems Biology Group,2
Centro Nacional de Biotecnología/CSIC, Cantoblanco, Madrid, Spain
Received 17 November 2010/Returned for modification 17 December 2010/Accepted 18 February 2011
catalytic subunit that induce the formation of 3-polyphosphoinositides, which mediate cell survival, division, and
migration. There are two ubiquitous PI3K isoforms p110? and p110? that have nonredundant functions in
embryonic development and cell division. However, whereas p110? concentrates in the cytoplasm, p110? localizes
to the nucleus and modulates nuclear processes such as DNA replication and repair. At present, the structural
features that determine p110? nuclear localization remain unknown. We describe here that association with the
p85? regulatory subunit controls p110? nuclear localization. We identified a nuclear localization signal (NLS) in
p110? C2 domain that mediates its nuclear entry, as well as a nuclear export sequence (NES) in p85?. Deletion of
p110? induced apoptosis, and complementation with the cytoplasmic C2-NLS p110? mutant was unable to restore
cell survival. These studies show that p110? NLS and p85? NES regulate p85?/p110? nuclear localization,
supporting the idea that nuclear, but not cytoplasmic, p110? controls cell survival.
The phosphoinositide 3-kinase (PI3K) family is divided into
four groups (IA, IB, II, and III) according to structural features
and substrate specificity. Of these, only class I enzymes catalyze
the production of PI(3,4,5)P3and PI(3,4)P2in vivo. Class IA
PI3Ks are heterodimeric proteins consisting of a p110 cat-
alytic subunit (p110?, p110?, and p110?) and an associated
p85 regulatory subunit (p85?, p85?, and p55?) (14, 18, 21,
22, 53). p110? (class IBPI3K) is structurally similar but
associates with a distinct class of regulatory subunits. The
catalytic subunits p110? and p110? are expressed ubiqui-
tously, whereas p110? and p110? are more abundant in
hematopoietic cells (14, 44, 53).
Despite the similarity in sequence, expression patterns, and
regulatory subunits, p110? and p110? have distinct functions
in cell proliferation, cell cycle progression, and development
(5, 6, 12, 26, 32–35, 47). p110? has a key role in insulin action
and cell cycle entry (12, 13), whereas p110? is reported to play
a pivotal role in DNA replication, S phase progression, and
DNA repair (32, 34, 35). Activating mutations of p110?, but
not of p110?, have been found in human cancer; nonetheless,
p110? drives tumorigenesis in PTEN-defective cells and in-
duces focus formation in fibroblasts (8, 9, 26, 29). Moreover,
overexpression of p110? is found in specific tumor types (7, 54,
58). Previous studies showed that part of the specific functions
of p110? and p110? result from their distinct subcellular lo-
calization and activation requirements (34, 35), highlighting
the emergence of subcellular localization as a major mecha-
nism to govern cell responses (30). Previous reports showed
that p85/p110 complex can translocate to the nucleus regulat-
ing cell survival, particularly in neuronal cell lines (37). In
addition, p110?, but not p110?, localizes to the nucleus in
several cell types. The mechanisms controlling p110? intracel-
lular localization nonetheless remain elusive. We studied here
the mechanism by which p110? localizes to the nucleus. p110?
is unable to enter the nucleus as a monomer and requires
association with the p85? regulatory subunit. We identified a
nuclear localization signal (NLS) in the p110? C2 domain that
controls the translocation of p85?/p110? complexes to the
nucleus. Conversely, the export of the p85?/p110? het-
erodimer from the nucleus is regulated by a nuclear export
sequence (NES) in p85?. We show that nuclear, but not cyto-
plasmic, p110? regulates cell viability.
MATERIALS AND METHODS
Cell lines and cell culture. Murine embryonic fibroblasts (MEFs) were pre-
pared as reported elsewhere (15). The cells were maintained in Dulbecco mod-
ified Eagle medium (Gibco-BRL, Auckland, New Zealand) supplemented with
10% fetal bovine serum, 2 mM glutamine, 10 mM HEPES, 100 U of penicillin/
ml, and 100 ?g of streptomycin/ml. PC12, U2OS, NIH 3T3, SAOS-2, and HeLa
cell lines were maintained as described previously (35).
Plasmids. Untagged wild-type (WT) p110? was donated by B. Vanhaese-
broeck (Institute of Cancer, London, United Kingdom). pSG5-myc-p110?,
pSG5-myc-p110?, and mutant myc-K805R-hp110? have been described in an-
other study (34). NLS-myc-p110?-mutant1, -mutant2, and -mutant3, as well as
NESmut rp85?, were generated by using a QuikChange site-directed mutagen-
esis kit (Stratagene, La Jolla, CA) with appropriate oligonucleotides. pSG5-p85?
and -HA-p85? are described elsewhere (2). The p85?-? chimera was prepared by
replacing p85? residues 77 to 351 with the corresponding p85? sequence. Short
hairpin RNA (shRNA) against murine PI3K subunits and control-scrambled
shRNA were custom-made (Origene Technologies, Rockville, MD). shRNA-
resistant WT and mutant p110? were human cDNA.
Antibodies and reagents. Blots were probed with the following antibodies
(Abs): anti-Myc tag (9B11), anti-p-PKB Ser473, and anti-p-PKB Thr308 (Cell
Signaling, Beverly, MA); anti-pan-p85, anti-p85?, and anti-histones (Upstate
Biotechnology; Millipore, Billerica, MA); and anti-tubulin (GTU-88; Sigma, St.
Louis, MO). anti-p110? was donated by A. Klippel (Merck, Boston, MA). Anti-
cytochrome c was purchased from Santa Cruz (Santa Cruz, CA), anti-HA was
* Corresponding author. Mailing address: Department of Immunol-
ogy and Oncology, Centro Nacional de Biotecnología/CSIC, Darwin 3,
Campus de Cantoblanco, Madrid E-28049, Spain. Phone: (34) 91 585-
4846. Fax: (34) 91 372-0493. E-mail: email@example.com.
† A.K., J.R.-M., and V.P.-G. contributed equally to this study.
?Published ahead of print on 7 March 2011.
from Covance (Emeryville, CA), and anti-p85? is described elsewhere (I. Corte ´s
and A. C. Carrera, unpublished data). Alexa 488- and Cy3-labeled Abs were
from Molecular Probes (Eugene, OR), horseradish peroxidase-conjugated sec-
ondary Abs were from Dako (Glostrup, Denmark), and ECL was from GE
Healthcare (Buckinghamshire, United Kingdom). Leptomycin B and cyclohexi-
mide were from Sigma. Platelet-derived growth factor (PDGF) and nerve growth
factor (NGF) were purchased from PeproTech (Rocky Hill, NJ).
Immunofluorescence, WB, and immunoprecipitation. Western blotting (WB)
and immunoprecipitation were performed as described previously (39). For
immunofluorescence (IF), cells were plated on coverslips and fixed with 4%
formaldehyde (10 min, room temperature [RT]), permeabilized with 0.3% Triton
X-100 in phosphate-buffered saline (PBS) staining buffer (10 min), and incu-
bated with blocking buffer (0.1% Triton X-100–3% bovine serum albumin in
PBS; 30 min), followed by incubation with primary antibody (1 h, RT, with
end-to-end rocking). Cells were washed three times with blocking buffer to
remove unbound antibody and incubated with the appropriate secondary anti-
body (1:500, 1 h, RT). Samples were washed three times with blocking buffer,
followed by incubation with the mounting medium Vectashield (Vector Labo-
ratories, Inc., Burlingame, CA). DAPI (4?,6?-diamidino-2-phenylindole) was
used to stain the DNA. Images were captured in a Leica Leitz DMRB micro-
scope (Wetzlar, Germany) using an Olympus DP70 charge-coupled device cam-
era or by using a confocal fluorescence microscope with an Olympus FluoView
(Olympus, Tokyo, Japan).
In vitro transcription translation and PI3K assay. Human myc-p110? WT or
mutant 1 (C2 domain) and mouse HA-p85? cDNA were transcribed and trans-
lated in vitro in the presence of [35S]methionine using the TNT T7-coupled
reticulocyte lysate system (Promega, Southampton, United Kingdom). In vitro
binding of proteins was analyzed by immunoprecipitation of hemagglutinin (HA)
or myc tags. The kinase assays were performed as described previously (27).
Transfection, subcellular fractionation, and apoptosis analysis. Transfection
assays were performed by using JetPei-NaCl according to the manufacturer’s
protocols (Qbiogene, Irvine, CA). Transfected cells were cultured 48 h prior to
analysis. For subcellular fractionation (see Fig. 1 and 4), cells were cultured in
exponential growth and then collected. Cytoplasmic, nuclear, and chromatin
fractions were isolated as described previously (40). Buffer A, used for cytoplas-
mic extraction, consisted of 10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM
MgCl2, 0.34 M sucrose, 10% glycerol, and 1 mM dithiothreitol (DTT). The
nonsalt buffer for nuclear extraction was composed of 3 mM EDTA, 0.2 mM
EGTA, and 1 mM DTT; for chromatin, proteins were extracted after boiling and
sonicating samples in Laemmli buffer. In all chases, samples were quantified with
a BCA protein assay kit (Pierce, Rockford, IL), and the same amount of protein
was analyzed by WB. For apoptosis and cytochrome c release, we transfected
cells with different shRNAs in combination with rp85? and either WT p110? or
NLS-p110?-mutant1 (24 h). Cells were gamma-irradiated (MARK 1; Shephard,
Louisville, KY) using a137Cs probe, collected after 24 h, and analyzed by flow
cytometry in a Cytomics FC500 (Beckman-Coulter, Fullerton, CA) using annexin
V and propidium iodide. Cytochrome c release was examined by using WB.
Modeling of the p85?/p110? complex. Models of the full-length p110? asso-
ciated with the p85? fragment containing nSH2 and iSH2 domains were inde-
pendently created by using I-TASSER (60), and their qualities were evaluated
with the Swiss-MODEL server (3). The two models were structurally aligned to
the corresponding chains in the crystal structure of the p85?(nSH2-iSH2)/p110?
complex (PDB 3hhm ) in order to generate a draft model of the complex.
This structural alignment was generated with the Dali system (19). Finally,
the model of the complex was refined by molecular dynamics to remove
clashes between chains, etc. Molecular dynamics analysis was performed
using GROMACS (52).
Statistical analyses and databases. The fluorescence intensity was quantitated
using ImageJ software; to determine the nuclear signal, we selected the area and
calculate the pixels referred to those found in the entire cell. Error bars represent
the standard deviations of the mean values compared. Statistical significance was
evaluated with a Student t test and the chi-square test calculated using
Prism5V.5.0 software. For NES and NLS sequence identification, we used online
databases (one at http://www.cbs.dtu.dk/databases/NESbase, CBS [Technical
University of Denmark], and one at http://cubic.bioc.columbia.edu/db/NLSdb,
Columbia University, respectively).
p110? concentrates in the nucleus. Most of the research on
inositide-dependent signal transduction pathways has focused
on events that take place at the plasma membrane. Nonethe-
less, PI3K is also found in the nucleus (36, 38, 43); we previ-
ously reported that p110?, but not p110?, localizes at the cell
nucleus concentrating at this site in S phase in NIH 3T3 cells
(35). HeLa cells, primary MEFs, and SAOS-2 cells also con-
tained nuclear p110?, as determined by IF analysis (Fig. 1A).
We also analyzed the localization of the other endogenous
ubiquitous PI3K subunits in NIH 3T3 cells. Whereas p110?
was predominantly nuclear, p110? localized mainly in the cy-
toplasm (Fig. 1B), as reported previously (35). The Abs used
were shown to be specific, since the p110? or p110? IF signal
decreased following depletion of the corresponding isoform
(35). As for the p85 ubiquitous regulatory subunits, the major-
ity of the p85? localized in the cytoplasm but p85? was more
abundant in the nuclear compartment (Fig. 1B). To control
antibody specificity, we cotransfected NIH 3T3 cells with
shRNA for p85? or p85? and a green fluorescent protein
(GFP) transfection reporter; cells transfected with p85?
shRNA showed reduction of the p85? signal, whereas p85?-
specific shRNA reduced the p85? signal (Fig. 1C).
In a complementary experiment, we confirmed intracellular
localization for p85 and p110 subunits by cell fractionation and
WB. We tested whether the distinct ubiquitous class IAPI3K
subunits appeared in cytoplasmic, nuclear, or chromatin frac-
tions (MEFs, HeLa cells, and NIH 3T3 cells) (Fig. 1D). Al-
though a proportion of the different subunits appeared in
the nucleus and the cytoplasm (visible in long exposures
[data not shown]), p110? and p85? concentrated in the
nucleus, in contrast to the cytoplasmic localization of p110?
and p85? (Fig. 1D).
To determine the contribution of cell activation for p110?
translocation to the nucleus, we examined various cell types
(PC12, U2OS, and NIH 3T3) upon serum deprivation or
after stimulation with growth factors (NGF, serum, and
PDGF, respectively). Whereas p110? localization showed
minor changes after cell stimulation in the three cell types,
p110? was mainly nuclear even in quiescence and stimula-
tion of PC12 and NIH 3T3 cells increased p110? fraction
bound to chromatin (Fig. 2A).
p110? overexpression results in cytoplasmic retention. To
elucidate the structural features that determine p110? nuclear
localization, we transfected full-length p110? into NIH 3T3
cells. Recombinant (r)p110? overexpression resulted in cyto-
plasmic accumulation of this protein (Fig. 2B). Transfection of
Myc-tagged-rp110? yielded a similar result using anti-tag Ab
for IF (data not shown). We confirmed that the entire se-
quence of the rp110? cDNA clones was correct. We also ex-
amined the localization of recombinant p110?, p85?, and p85?
(rp110?, rp85?, and rp85?) in NIH 3T3 cells; rp110? and
rp85? concentrated in the cytoplasm (Fig. 2C), similar to their
endogenous counterparts (Fig. 1B). Overexpressed p85?
showed diffuse cytoplasmic and nuclear staining (Fig. 2C), with
a larger proportion of cytoplasmic protein compared to the
endogenous protein (Fig. 1).
The ectopic cytoplasmic localization of recombinant p110?
in cells could result from the accumulation of newly translated
protein in the endoplasmic reticulum prior to translocation to
the nuclei. To exclude this possibility, we transfected the
rp110? and tested whether inhibition of de novo protein syn-
thesis by cycloheximide treatment (5 h prior to IF analysis)
facilitated the accumulation of rp110? to the nucleus. This was
VOL. 31, 2011NUCLEAR PI3K? MEDIATES CELL SURVIVAL2123
not the case; rp110? remained cytoplasmic after cycloheximide
treatment, excluding that this fraction represents newly trans-
lated protein (Fig. 2C).
p85? promotes p110? nuclear localization. Class IAcata-
lytic and regulatory subunits normally form heterodimers (16).
We confirmed biochemically that p85? and p85? form com-
plexes with either p110? or p110? (data not shown), as re-
ported earlier (16). We examined the possibility that cytoplas-
mic accumulation of rp110? might result from the lack of
sufficient associated regulatory subunit. To determine whether
the ubiquitous regulatory subunits (p85? or p85?) were nec-
essary for p110? nuclear localization, we cotransfected combi-
nations of the ubiquitous catalytic and regulatory subunits.
Cotransfection of rp110? with rp85? or rp85? did not alter
the cytoplasmic localization of rp110?, although rp85? was
cytoplasmic and rp85? was cytoplasmic and nuclear (Fig. 3A).
In contrast, cotransfection of rp110? with rp85?, but not with
rp85?, yielded a significant proportion of nuclear rp110? (Fig.
3A). To analyze the contribution of endogenous p85 regulatory
subunits in the nuclear localization of p110?, we analyzed its
localization in WT or p85?-deficient MEFs. A moderate re-
duction in nuclear p110? was seen in p85??/?MEFs (Fig. 3B),
suggesting that other p110?-associated nuclear proteins (such
as PCNA or Nbs1 [32, 35]) might facilitate p110? translocation
to the nucleus in p85?-deficient cells. In contrast, acute reduc-
tion of p85? levels with shRNA (as in Fig. 1C) induced a
significant decrease in p110? nuclear levels (Fig. 3C). Endog-
enous p85? thus regulates the nuclear entry of p110?.
A polybasic region of p85? does not act as an NLS. The
finding that p85? expression, but not that of p85?, induced
p110? nuclear localization led us to examine the primary se-
quence of p85? and p85? to search for potential nuclear lo-
calization sequences (NLSs). We found a polybasic region be-
tween the BCR (Bcr homologous region) and the N-SH2
region of p85? (residues 77 to 351); this sequence was not
present in p85?. To establish the contribution of this p85?
region in the nuclear localization of p85?/p110? complexes, we
constructed a p85?-? chimera, replacing p85? amino acids 77
FIG. 1. Class IAPI3K subunits p85? and p110? concentrate in the nucleus. (A) HeLa and SAOS-2 cells and freshly isolated MEFs were
cultured in exponential growth and analyzed by IF using anti-p110? Ab. DNA was stained with DAPI (insets). The graph shows the percentage
of cells with predominant p110? nuclear staining (n ? 30). (B) Endogenous p110?, p110?, p85?, and p85? localization in NIH 3T3 cells was
analyzed by IF using specific Ab; DNA was stained with DAPI (insets). The graph is as described in panel A. (C) NIH 3T3 cells were cotransfected
with GFP plus p85?- or p85?-specific shRNA (48 h), and the cells were fixed and analyzed by IF using specific Abs. WB shows the downregulation
of p85? and p85? after shRNA transfection. Insets show transfected (GFP?) cells. (D) HeLa cells, MEFs, and NIH 3T3 cells were fractionated
into cytoplasmic nuclear and chromatin extracts, which were analyzed by WB using the indicated Abs. Tubulin and histone were used as
cytoplasmic and nuclear/chromatin controls. Bar, 10 ?m. Dashed lines depict cell membrane. n.s., not statistically significant;**, P ? 0.001
(Student t test).
2124KUMAR ET AL.MOL. CELL. BIOL.
FIG. 2. Overexpressed p110? localizes in cytoplasm. (A) PC12, U2OS, and NIH 3T3 cells were cultured alone or treated with NGF (100 ng/ml),
fetal bovine serum (20%), or PDGF (50 ng/ml), respectively (30 min). Cells were fractionated, and cytoplasmic nuclear and chromatin fractions
were analyzed by WB using the indicated Abs. Graphs show the p110? and p110? signal intensities in arbitrary units (AU). (B) NIH 3T3, HeLa,
and SAOS-2 cells were transfected with rp110? (48 h). rp110? localization was examined by IF using anti-p110? Ab. (C) Myc-tagged rp110?,
HA-rp85?, and HA-rp85? were transfected individually into NIH 3T3 cells and processed for indirect IF with appropriate tag-specific antibodies.
(D) rp110?-transfected NIH 3T3, HeLa, and SAOS-2 cells were treated with cycloheximide (10 ?g/ml, 5 h) before IF staining as described in panel
A. Bar, 10 ?m.
VOL. 31, 2011NUCLEAR PI3K? MEDIATES CELL SURVIVAL2125
FIG. 3. The p85? regulatory subunit controls p110? nuclear translocation. (A) NIH 3T3 cells cotransfected with Myc-rp110? or -rp110? in
combination with HA-rp85? or -rp85? (48 h) were fixed and analyzed by IF. Catalytic subunits were stained with anti-Myc-tag Ab and p85 with
2126KUMAR ET AL.MOL. CELL. BIOL.
to 351 with the corresponding residues in the p85? sequence
(amino acids 77 to 363; see Fig. 3D). We cotransfected the
rp85?-? chimera with rp110? and examined rp110? subcellu-
lar distribution. No difference was observed in rp110? nuclear
localization when cotransfected with the p85?-? chimera or
WT p85?; the p85?-? chimera continued to localize with
p110? in the nucleus (Fig. 3D), similar to WT-rp85? (Fig. 3A).
Quantification of the proportion of p110? and p110? nuclear
signal (Fig. 3E) confirmed that rp110? can transit to the nu-
cleus when cotransfected with rp85?; nonetheless, the polyba-
sic sequence located between BCR and N-SH2 domains in
p85? is not a NLS.
The p110? C2 domain contains an NLS. We sought poten-
tial NLSs in p110? that could explain the nuclear localization
of p85?/p110? complexes and identified three putative NLS
polybasic motifs in p110?, one in the C2 domain (residues 310
to 318; KVKTKKSTK), one in the Ras-binding domain (RBD;
residues 149 to 154; RRKMRK), and one at the C terminus
(residues 994 to 996; RRH) (Fig. 4A). To establish which of
these motifs might be functional, we generated a structural
model of the p85?(nSH2iSH2)/p110? complex (Fig. 4B) based
on the p85? (nSH2iSH2)/p110? structure (33). This model
showed that the basic motif in the C2 domain is located in a
loop in close proximity to p85?; only the residues at the be-
ginning and the end of the NLS are resolved in this structure
(Fig. 4B). Alignment of this region in p110? and p110? pri-
mary structure (Fig. 4C), as well as examination of p85?/p110?
structure (24, 33, 42), showed that most of this motif is lost in
p110?. The other candidate motifs are not found near p85?
and seem less likely to be affected by interaction with this
protein (Fig. 4B).
We replaced several basic residues in each of the three
motifs with nonbasic residues to generate the C2 domain NLS-
p110?-mutant1 (KVNTTKSTK), RBD NLS-p110?-mutant2,
and C-terminal NLS-p110?-mutant3 (RGH) (Fig. 4A). The
expression levels of these constructs were similar (Fig. 4D). We
tested whether any of these mutants, in combination with
rp85?, was excluded from the nucleus. NIH 3T3 cells trans-
fected with rp85? and the rp110?NLS mutants in the RBD and
C-terminal domain showed minor differences compared to
WT-rp110?; in contrast, the C2 domain NLS-p110?-mutant1
was cytoplasmic (Fig. 4E and F). This suggested that the KV
KTKRSTK motif in the C2 domain acts as an NLS for p110?.
Separation of cells expressing the NLS-p110?-mutant1 plus
rp85? into cytoplasmic, nuclear, and chromatin fractions
showed that WT-rp110? localized in nuclear and chromatin
fractions and confirmed that mutation of the NLS-p110?-mu-
tant1 is mainly cytoplasmic, similar to p110? (Fig. 4G).
We confirmed that mutation in the C2 domain does not
affect association of in vitro-transcribed translated purified
p110? to purified p85? (Fig. 4H). A similar association of
rp110? or NLS-p110?-mutant1 with rp85? was confirmed in
transfected NIH 3T3 cells (not shown). Moreover, there was
no difference in kinase activity between WT or mutant1-
rp110? (Fig. 4I). Thus, the C2 mutant associates with p85?
similarly to WT-p110? and shows kinase activity but does not
translocate to the nucleus.
p85? regulates p110? nuclear exit. We previously observed
changes in the relative amount of nuclear p110? during cell
cycle progression, suggesting that this molecule shuttles in and
out of the nucleus (35). We studied the mechanism that con-
trols p110? nuclear export. Various means of nuclear export
have been documented (28); the most common mechanism is
a conserved leucine-rich NES that binds the nuclear export
protein Crm1 (11, 31). We used leptomycin B to inhibit Crm1
binding to the cargo proteins; this treatment results in reten-
tion of NES-containing proteins in the nucleus (11). NIH 3T3
cells were transfected with rp110?, rp110?, rp85?, or rp85?
constructs and, after 24 h, the cells were treated with leptomy-
cin B (5 ng/ml, 2 h). After leptomycin B treatment, only rp85?
showed a notable increase in the amount of nuclear protein
(Fig. 5A). This suggests that nuclear exit of p85?/p110? com-
plex is mediated by an NES located in p85? via Crm1. The
moderate enhancement of p110? nuclear localization after
leptomycin B treatment might result from association to en-
To define the putative region containing the NES in p85?,
we transfected the rp85?-? chimera described above and
tested whether leptomycin B treatment affected its intracellu-
lar localization. Overexpressed p85?-? chimera localized to
the cytoplasm and nucleus and responded to leptomycin B
treatment by increasing its nuclear localization (Fig. 5B), sim-
ilar to rp85? (Fig. 5A). A C-terminal deletion mutant in p85?
(p65?) behaves as an oncogene (27); a similar deletion in p85?
was reported in a tumor cell line (25). We prepared a similar
C-terminal deletion mutant in p85? (p65?) lacking residues
562 to 723 of the C terminus and tested the effect of leptomycin
B treatment on its subcellular localization; p65? behaved as
did WT p85? (Fig. 5A and B). Indeed, transfection of rp110?
plus rp85?, the p85?-? chimera, or rp65?, followed by lepto-
mycin B treatment of cells, led to a comparable increase in
p110? nuclear localization (Fig. 5A and C).
Thus, p85? regulates p110? nuclear import and export; how-
ever, neither p85? residues 77 to 351 nor the p85? C-terminal
region (amino acids 562 to 723) control p85?/p110? nuclear
The p85? N-terminal region has an NES. To determine the
p85? region involved in nuclear export, we used specific NES
databases to search for conserved leucine-rich regions; this
search rendered three potential NES motifs (Fig. 6A). One of
anti-HA Ab; square brackets indicate channels from the same image. Graphs show the percentage of p110 nuclear signal relative to the total
(100%) (n ? 30). (B) WT or p85?-deficient MEFs were cotransfected with GFP plus scrambled or p85? shRNA (48 h), and the cells were fixed
and analyzed by IF using specific Abs. Insets show transfected (GFP?) cells. The graph shows the percentage of cells with predominant p110?
nuclear staining (n ? 30). (C) NIH 3T3 cells were cotransfected with GFP plus p85?- or p85?-specific shRNA or both (48 h), the cells were fixed,
and nuclear p110? was analyzed by IF using specific Abs. Insets show transfected (GFP?) cells. Dashed lines depict the cell nuclei. Graphs are
as described in panel A. (D) Scheme of the rp85?-? chimera. The p85? region between amino acids 78 to 351 was replaced with amino acids 77
to 363 from p85?. NIH 3T3 cells cotransfected with the HA-rp85?-? chimera plus rp110? were stained with anti-p110? and -HA Ab. (E) Graphs
are as described in panel A. Bar, 10 ?m.**, P ? 0.001.
VOL. 31, 2011NUCLEAR PI3K? MEDIATES CELL SURVIVAL2127
FIG. 4. p110? contains an NLS motif in the C2 domain. (A) Domain structure of p110?, potential NLS sequences, and replacement of basic
with nonbasic residues in mutants 1 to 3. (B) Computational model of the p85?/p110? complex. The p85? fragment (containing the nSH2 and iSH2
domains) is indicated in blue. The following p110? domains are indicated: the p85-binding domain (brown), the Ras-binding domain (purple), the
catalytic domain (yellow), the C2 domain (green), and the helical domain (cyan). NLS sequences (located in the RBD, C2 domain, and C terminus)
are shown as gray spheres; intermediate sequences are indicated in red. (C) Alignment of human and mouse p110? and p110? sequences at the
region surrounding the C2 domain NLS of p110? (boxed). (D) NIH 3T3 cells were transfected with rp85? and WT or rp110? mutants 1 to 3.
Expression levels were examined by WB. (E) The cellular localization of rp110? mutants was analyzed by IF using anti-Myc tag Ab. (F) Percent
cells with the indicated phenotypes (n ? 30). (G) NIH 3T3 cells were cotransfected with rp85? with WT-rp110? or NLS-p110?-mutant1.
2128KUMAR ET AL.MOL. CELL. BIOL.
these was found at residues 683 to 688, although these residues
are absent in rp65?, a mutant that behaves like WT p85? after
leptomycin B treatment. An alternative high score region was
found at residues 214 to 229, which are absent in the rp85?-?
chimera; since this chimera remains sensitive to leptomycin B
treatment (Fig. 5), this motif is not a functional NES for p85?.
Finally, a potential motif was indicated at residues 25 to 32. We
generated a 100-amino-acid N-terminal deletion mutant of
p85? (?100Np85?), as well as a double point mutation in this
Leu-rich motif (L25 and L30; NESmut-p85?). Deletion or
mutation of this region rendered p85? predominantly at the
nucleus and unaffected by leptomycin B treatment (Fig. 6B),
confirming that this region contains a functional Crm1-regu-
lated NES sequence.
We examined the role of this region in p110? nuclear export.
NIH 3T3 cells transfected with rp110? in combination with
?100NT-p85? or with NESmut-p85? showed an increase in
rp110? nuclear localization (Fig. 6C), confirming a contribu-
tion of p85? residues 25 to 32 in the regulation of p85?/p110?
Reconstitution of p110?-deficient cells with nuclear but not
cytoplasmic p110? restores cell survival. Mice deficient in
p110? die at embryonic days 2 to 3 (5). We previously showed
that p110? is mainly nuclear and controls DNA replication and
repair (32, 34, 35); in the course of these studies, we observed
that efficient p110? knockdown reduced cell survival (34). To
test whether p110? nuclear localization influences cell sur-
vival, we depleted NIH 3T3 cells of p110? using shRNA and
reconstituted p110? expression with WT-rp110? or cyto-
plasmic NLS-p110?-mutant1. WB was used to confirm
p110? silencing with specific shRNA, as well as the expres-
sion of WT or mutant rp110? (Fig. 7A). We cotransfected
cells with p110?-specific shRNA and shRNA-resistant hu-
manWT-p110? or shRNA-resistant human NLS-p110?-mu-
tant1; the second combination was more sensitive to spon-
taneous and gamma-irradiation-induced apoptosis than
untransfected cells or rp110? WT-expressing cells (Fig. 7B).
As an alternative method to examine apoptosis, we moni-
tored cytochrome c release in WB. Cytochrome c was present
in the cytoplasmic fractions of apoptotic positive control cells
(H2O2treated), as well as in cells lacking p110? expression, but
not in controls (Fig. 7C). Expression of shRNA-resistant WT-
p110? nonetheless rescued cell death, since it decreased cyto-
chrome c release; in contrast, expression of the shRNA-resistant
cytoplasmic C2-domain NLS-p110?-mutant1 did not reduce cy-
tochrome c release (Fig. 7C). The results indicate that nuclear
localization of p110? is necessary for cell viability and that its
expression in the cytoplasm does not prevent apoptotic events.
We examined the structural features that determine p110?
nuclear localization. Whereas overexpressed recombinant
p110? remains mainly cytoplasmic, transfected rp110? in com-
bination with rp85?, but not rp85?, localizes to the nucleus.
Although the ubiquitous catalytic and regulatory subunits form
all possible heterodimeric combinations (16), only p85?/p110?
complexes localize efficiently in the nucleus. The search for
nuclear localization motifs in p85? and p110? yielded several
candidate sequences, but only mutation of the NLS located
within the p110? C2 domain significantly reduced p110? nu-
clear localization. The fact that p110? alone does not enter the
nucleus suggests that p110? must associate with p85? for its
NLS motif to be functional; the predicted quaternary structure
of p85?/p110? reported here supports this possibility (see be-
low). p110? nuclear PI3K activity is maximal in S phase (35),
suggesting that p85?/p110? complexes shuttle in and out of the
nucleus. We identify here a functional NES in p85? which,
when deleted, increases p85?/p110? nuclear localization.
Proteins enter the nucleus through nuclear pores, large mac-
romolecular complexes composed of nucleoporins. Under-
standing of macromolecular transport processes across the nu-
clear envelope has increased in recent years, and many
transport receptors have been identified. Most of these recep-
tors are similar to the import receptor importin ? (karyopherin
?). Members of this family have been classified as importins or
exportins, and both types are regulated by the GTPase Ran.
Importins recognize their substrates in cytoplasm and trans-
port them to the nucleus; once in the nucleus, RanGTP binds
to importins, inducing the release of import cargoes. In con-
trast, exportins interact with their substrates only in the nu-
cleus in the presence of RanGTP and release them after GTP
hydrolysis in the cytoplasm (reviewed in reference 49). Nuclear
import and export are multistep processes initiated by the
recognition of NLSs or NESs. The most thoroughly examined
import signal (“classical” and bipartite NLS) contains multiple
basic residues. Their transport is mediated by importin ?,
which directly associates these NLS via the adaptor protein
importin ? (49). The functional NLS in p110? is homologous
to that found in class II PI3KC2?, which also transits to the
nucleus (10), suggesting potential conservation of structural
features for nuclear import between PI3K classes.
The best-studied exportins are Crm1/Xpo1, which recog-
nizes leucine-rich NES. Crm1 forms a stable ternary complex
with Ran-GTP and with NES cargoes that can exit the nucleus.
Studies of Crm1-mediated export were aided by the discovery
of the antifungal agent leptomycin B, a highly specific and
potent inhibitor of Crm1 function (11). Of the three potential
NES sequences in p85?, only the one located at the N terminus
Cytoplasmic, nuclear, and chromatin fractions were examined by WB. Tubulin and histones were used as controls. Bar, 10 ?m. (H) cDNA encoding
mouse rp85? and WT- or C2 mutant1-rp110? were transcribed or translated in vitro in the presence of [35S]methionine. The association of rp85?
with WT- or mutant1-rp110? was analyzed by HA- or Myc-tag IP. The extract composition (TT extracts) and p85/p110 complex formation were
examined by SDS-PAGE and autoradiography. (I) cDNA encoding mouse rp85? and WT- or mutant1-rp110? were transcribed or translated as
in panel A. rp85?/rp110? complexes were purified with anti-pan-p85 Ab and tested in an in vitro kinase assay using PtdIns(4,5)P2as a substrate.
*, P ? 0.01 (Student t test).
VOL. 31, 2011NUCLEAR PI3K? MEDIATES CELL SURVIVAL2129
regulated the nuclear localization of p85?/p110?. In the p85?/
p110? complex, p110? therefore contributes by providing the
NLS, whereas p85? supplies a functional NES, showing that
this complex acts as a single entity for nuclear transport. In-
deed, the predicted structure of p85?/p110? described here
(based on that in reference 33) shows that the NLS sequence
in the C2 domain is in close proximity to p85?, supporting the
possibility that p110? association to p85? alters p110? struc-
ture in this region to yield a functional NLS.
Neither p110? nor p85? is exclusively nuclear; the cytoplas-
mic forms might represent complexes with p85? and p110?,
respectively, or p85?/p110? complexes in transit from both
compartments. In the case of p85?, its overexpression renders
a fraction of this protein nuclear, suggesting that it associates
with other NLS-containing proteins. In support of this possi-
bility, p85? and, to a greater extent, p85?, associates with
X-box binding protein 1 (XBP1), modulating the nuclear lo-
calization of this transcription factor (which contains an NLS)
FIG. 5. p85? regulates p110? nuclear export. (A) NIH 3T3 cells were transfected with HA-rp85?, HA-rp85?, Myc-rp110?, or Myc-rp110? (48
h). Transfected cells were untreated or leptomycin B treated (5 ng/ml; 2 h) before fixing. Samples were stained for IF using anti-HA or -Myc tag
Ab. (B) NIH 3T3 cells were transfected with the HA-rp85?-? chimera or HA-rp65? (48 h). Cells were untreated or pretreated with leptomycin
B 2 h prior to fixing, and then stained as described above. The graph shows the percentage of nuclear signal relative to total cell signal (100%)
(n ? 30). (C) rp85?/rp110? or p65?/p110? were expressed in NIH 3T3 cells (48 h). The cells were treated with leptomycin B as described above
and examined by IF; square brackets indicate channels from the same image. The graph is as described in panel B. Bar, 10 ?m.*, P ? 0.01;**,
P ? 0.001.
2130KUMAR ET AL.MOL. CELL. BIOL.
(46, 59). Similarly, in the case of p110?, association with p85?
is critical for its translocation to the nucleus; however, other
p110?-associated nuclear proteins (such as PCNA or Nbs1 [32,
35]) might facilitate the translocation of p110? to the nucleus
in p85?-deficient cells.
We focused on a comparison of the class IAPI3K isoforms
p110? and p110?; there is nonetheless an additional class IA
isoform, p110?, as well as the closely related class IBp110?
isoform, which associates with p101 and p84 regulatory sub-
units (50). When overexpressed in HepG2 cells, p110? local-
izes to the nucleus after serum treatment; in this case, inter-
ference of p110? association with p101 increases p110? nuclear
localization (41). There is no region homologous to that of the
p110? C2 domain in p110? (45), although we found polybasic
motifs in the N terminus, in the helical domain, and at the
beginning of the C2 domain (data not shown). Alignment of
the NLS region in p110? and p110? (Fig. 4) shows that most of
this basic motif in p110? is lost in p110?. Comparison of the
p85?/p110? structure (24, 33, 42) to the p85?/p110? structural
prediction described here (Fig. 4) also shows that the loop in
which p110? NLS localizes is much shorter in p110?. These
observations might explain why a large proportion of p110?,
but not of p110?, localizes to the nucleus. An in silico search
for p110? NLS homologues in p110?, as well as p110? struc-
ture (4), showed a similar polybasic region in p110? and p110?
C2 domains; further study is needed to define whether the
p110? motif is a functional NLS.
The first report of nuclear PIP3showed rapid translocation
of a PIP3-binding protein (PIP3BP), which is abundant in
brain, to the nuclei of the rat pheochromocytoma PC12 cell
line after NGF treatment, as well as in PDGF-treated NIH 3T3
cells (51). In human promyelocytic HL60 cells, both retinol and
vitamin D3induced differentiation to granulocytes or mono-
cytes, respectively, and triggered an increase in nuclear p85
staining (reviewed in references 36 and 37). In all of these
cases, the authors defined the specific isoform localizing to the
nucleus. The negative regulator of PI3K, PTEN, is also re-
ported to transit to the nucleus and regulate cell survival (17).
FIG. 6. The p85? subunit has a Crm1-dependent nuclear export signal. (A) Scheme of Leu-rich regions in the p85? sequence. (B) NIH 3T3
cells were transfected with cDNA encoding ?100Np85? or NESmut-rp85? (48 h). Transfected cells were untreated or leptomycin B treated (5
ng/ml, 2 h) and processed for IF using anti-HA Ab. The graph shows the percentage of nuclear signal relative to total cell signal (100%) (n ? 30).
(C) NIH 3T3 cells were cotransfected with Myc-rp110? and either ?100Np85? or NESmut-rp85? (48 h); protein localization was analyzed by IF
using anti-HA or -p110? Ab. The graph is as described in panel B. Bar, 10 ?m.**, P ? 0.001.
VOL. 31, 2011NUCLEAR PI3K? MEDIATES CELL SURVIVAL2131
In the case of nuclear PI3K in PC12 cells, a nucleus-specific
phospholipase C activates a neuron-specific GTPase, PIKE
(phosphoinositide 3-kinase enhancer), which is able to increase
nuclear PI3K activity (56, 57). Isolated nuclei from PC12 cells
treated with NGF or transfected with active PI3K were resis-
tant to DNA fragmentation factor caspase-activated DNase
(DFF40/CAD); interference with p110? diminished NGF pro-
tection from apoptosis, supporting p110? control of nuclear
PIP3in PC12 cells (1).
The antiapoptotic function of nuclear PIP3in PC12 cells is
proposed to result from PIP3binding to B23 nucleophosmin, a
protein that inhibits DFF40/CAD (20, 55). Other authors have
suggested that nuclear PKB function in NGF-treated PC12
cells is mediated by PKB phosphorylation of acinus, resulting
in acinus inhibition of apoptotic chromatin condensation (23).
A third mechanism has been reported for the function of
nuclear PI3K in NGF-treated PC12 cells, PCK?-PI3K-depen-
dent nuclear translocation, which mediates phosphorylation of
nucleolin, a stabilizing agent for the antiapoptotic protein
Bcl-2 (48). These results indicate that in some cell types (PC12
cells), the neurotrophin NGF activates nuclear PI3K, which in
turn induces cell survival.
We report here that the p85?/p110? complex localizes to the
nucleus in several cell types. This translocation is regulated by
an NLS sequence in the p110? C2 domain and by an NES in
the p85? N-terminal domain. Our results demonstrate that the
p85?/p110? complex regulates cell viability only when it is
correctly localized at the cell nucleus.
We thank M. White for the myc-p110? plasmid, B. Vanhaesebroeck
for the p110? plasmid, A. Klippel for anti-p110 Ab, F. Pazos for
support in p85?/p110? structure prediction, and C. Mark for editorial
A.K. held a predoctoral fellowship associated with a project financed
by the Fundacio ´n Ramo ´n Areces. J.R.-M. has a JAE postdoctoral
fellowship from the Spanish National Research Council (CSIC).
V.P.-G received a predoctoral FPI fellowship associated with a
project financed by the Spanish Ministry of Science and Innovation
(MICINN). This study was financed by grants from the Spanish As-
sociation Against Cancer (AECC), the MICINN (SAF2007-63624 and
SAF2010-21019 and Network of Cooperative Research in Cancer
RD07/0020/2020), the Madrid regional government (S-BIO-0189/06),
the Sandra Ibarra Foundation, and Genoma Espan ˜a.
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