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Specific function of phosphoinositide 3-kinase betain the
control of DNA replicationMiriam Marquésa,1, Amit Kumara,1, Ana M.
Povedab, Susana Zuluagaa, Carmen Hernándeza, Shaun
Jacksonc,Philippe Paserob, and Ana C. Carreraa,2
aDepartment of Immunology and Oncology, Centro Nacional de
Biotecnología/Consejo Superior de Investigaciones Científicas,
Universidad Autónoma deMadrid, Cantoblanco, Madrid E-28049, Spain;
bInstitute of Human Genetics, Centre National de la Recherche
Scientifique Unité Propre de Recherche 1142,141 Rue de la
Cardonille, F-34396 Montpellier, France; and cAustralian Centre for
Blood Diseases, Monash University, Melbourne, Victoria 3004,
Australia
Edited by Inder M. Verma, The Salk Institute for Biological
Studies, La Jolla, CA, and approved March 20, 2009 (received for
review November 25, 2008)
Class IA phosphoinositide 3-kinase (PI3K) are enzymes comprised
ofa p85 regulatory and a p110 catalytic subunit that induce
formationof 3-polyphosphoinositides, which activate numerous
down-stream targets. PI3K controls cell division. Of the 2
ubiquitous PI3Kisoforms, � has selective action in cell growth and
cell cycle entry,but no specific function in cell division has been
described for �. Wereport here a unique function for PI3K� in the
control of DNAreplication. PI3K� regulated DNA replication through
kinase-de-pendent and kinase-independent mechanisms. PI3K� was
found inthe nucleus, where it associated PKB. Modulation of PI3K�
activityaltered the DNA replication rate by controlling
proliferating cellnuclear antigen (PCNA) binding to chromatin and
to DNA poly-merase �. PI3K� exerted this action by regulating the
nuclearactivation of PKB in S phase, and in turn phosphorylation of
PCNAnegative regulator p21Cip. Also, p110� associated with PCNA
andcontrolled PCNA loading onto chromatin in a
kinase-independentmanner. These results show a selective function
of PI3K� in thecontrol of DNA replication.
C lass IA phosphoinositide 3-kinase (PI3K) is an enzyme
thatcontrols cell cycle entry. Mutations in this pathway areamong
the most frequent events in human cancer; a mayorobjective in
translational biology is to define PI3K isoform-specific functions.
The PI3K are comprised of a p85 regulatoryand a p110 catalytic
subunit that mediates formation of3-polyphosphoinositides (1, 2).
There are three class IA p110catalytic subunits (�, � and �), but
only p110� and � areubiquitous and essential for development (3,
4); enhanced p110�and � activity trigger cell transformation (5).
p110� regulates cellgrowth and cell cycle entry (6). In the case of
p110�, the recentdescription of p110� conditional knockout mouse
phenotypeshows that p110� activity is essential for animal growth
andtumor development (7). Nonetheless, the cellular events
selec-tively controlled by p110� remain unknown.
DNA replication controls the accurate, timely duplication of
thecell genome each time the cell divides. Preparation for
replicationrequires formation of the origin replication complex
(ORC) at theDNA replication origin. The ORC acts as a scaffold for
assemblyof the prereplicative complex that includes Cdc6 and Cdt1,
proteinsinvolved in recruitment of the minichromosome
maintenance(MCM) complex exhibiting helicase activity. When MCM is
loadedinto the ORC, the pre-RC is licensed to initiate replication
(8–12).After licensing, replication initiation involves formation
of thepreinitiation complex, which requires activation of Cdk2 and
Dbf4/Cdc7 kinases (13). These kinases phosphorylate the MCM
andinduce binding of DNA polymerase (Pol)�/primase, which
triggersprimer DNA synthesis (11). Elongation of DNA synthesis
requiressubsequent binding of the proliferating cell nuclear
antigen(PCNA), a homotrimeric factor that triggers Pol�
displacement andtethers the processive polymerases (� and �) to the
DNA templatefor rapid, accurate DNA elongation (9, 14). We examine
here thefunction of p110� and � in DNA replication.
Results and DiscussionThe p110� Controls S-Phase Progression.
p110� regulates G1 entryand cell growth (1); both p110� and �
regulate late G1 events and
accelerate G1�S transition (6); however, no p110�-specific
func-tion has been described in cell division. To examine the
potentialp110� action in this process, we compared the division
rates of NIH3T3 stable cell lines expressing p110� or � active
forms (Fig. 1A).Active p110� cells divided more rapidly (t1/2 �18
h) than activep110� cells or controls (t1/2 �24 h; Fig. 1B). In
addition, althougha small fraction of active p110� and � cells
enter cell cycle afterserum deprivation (6), only active p110�
cells escaped cell contactinhibition in confluence (Fig. S1A). We
also compared synchro-nous cell cycle progression in these cells.
Cells were first serum-deprived (G0 arrest) and released by serum
addition; using thisprotocol, NIH 3T3 cells reach S phase at �9 to
12 h postrelease(15). Active p110� cells were faster in terminating
S phase thancontrol or active p110� cells (Fig. 1C; Fig. S1B), as
confirmed bycalculation of S phase duration (4 � 0.5 h for active
p110� cells vs.5.5–6 h for active p110� cells and �6 h for control
cells); threedistinct clones behaved similarly.
We also examined the consequences of reducing endogenousp110�
and � activity using inactive K802R-p110� and K805R-p110� mutants
(KR hereafter) (6). Expression of KR mutants inexponentially
growing NIH 3T3 cells reduced PKB phosphoryla-tion (pPKB, Fig. 1D)
and affected cell division; we were unable toprepare stable
KR-p110� or � lines. We expressed KR mutants byretroviral infection
(95% efficiency), which yielded levels similar toendogenous p110
proteins (Fig. 1D). Cell division was significantlyslower in
KR-p110� cells (Fig. 1E), which remained in S phase forprolonged
periods (Fig. 1F; Fig. S1C) and showed a longer S phase (�6h
control cells; 6–6.5 h KR-p110� cells, �8 h for KR-p110�
cells).
p110� expression did not vary appreciably throughout the
cellcycle. We examined the consequences of reducing p110�
expressionusing various shRNA and protocols in NIH 3T3 cells and
humanU2OS cells (Methods). Whereas efficient protocols for
p110�deletion interfered with cell viability, partial p110�
reductionpermitted cell cycle progression studies. To reduce p110�
expres-sion in U2OS cells, we stably transfected pTER-shRNA
vectors,which allow inducible shRNA expression (16). shRNA
reducedp110� and � levels even before induction, but reduction was
greaterafter doxycycline treatment (Fig. 1G). U2OS cells were
synchro-nized at G1/S boundary by double thymidine block and
examinedS phase progression after release. We confirmed slower cell
cycleentry in cells with reduced p110� or � levels (6); in
addition, onlythe cells with reduced p110� levels remained in S
phase forprolonged periods, showing a Gaussian peak at mid-S phase
DNAcontent at 6–7 h postrelease (Fig. 1G).
Author contributions: A.C.C. designed research; M.M., A.K.,
A.M.P., S.Z., and C.H. per-formed research; S.J. contributed new
reagents/analytic tools; M.M., A.K., P.P., and A.C.C.analyzed data;
and A.C.C. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1M.M. and A.K. contributed equally to this work.
2To whom correspondence should be addressed. E-mail:
[email protected].
This article contains supporting information online at
www.pnas.org/cgi/content/full/0812000106/DCSupplemental.
www.pnas.org�cgi�doi�10.1073�pnas.0812000106 PNAS � May 5, 2009
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We used the selective inhibitors PIK75 and TGX221 to
inhibitp110� and �, respectively (17, 18). We confirmed inhibitor
selec-tivity in NIH 3T3 cells (Fig. S2 A–D). Inhibition using 0.5
�M PIKresulted in complete blockade of S phase entry and
triggeredapoptosis (Fig. S2E), showing that p110� is needed for
cell survival(19). p110� inhibition (0.08 �M PIK) near S phase
permitted cellcycle entry (Fig. S2F) although it impaired G2/M
entry, suggestingthat p110� could be the isoform that acts in
mitosis (1). Thistreatment nonetheless allowed S phase progression,
as indicated bythe increased proportion of S phase cells and
displacement of theS phase population from near-G1 to near-G2 DNA
content overthe time course (Fig. S2F). In contrast, selective
inhibition of p110�permitted G2/M entry but extended S phase
compared withcontrols (Fig. S2F).
The p110� Activity Controls DNA Elongation. To compare S
phaseprogression rates more accurately we BrdU-labeled (1 h
pulse)newly synthesized DNA in exponentially growing cells
andcollected cells at various times after BrdU deprivation.
Whilemost BrdU� control and KR-p110� cells reached G2/M at 3 to5 h,
the majority of BrdU� KR-p110� cells remained in S phaseat 5 h
(Fig. S3A). We examined the consequences of impairedp110� function
on DNA elongation with the DNA combingassay (20, 21). We used PI3K
inhibitors, as they permit p110�
or � blockade in late G1 without affecting prior
events.G0-synchronized NIH 3T3 cells were serum-released,
treatedwith PIK 75 (0.08 �M) or TGX 221 (30 �M) at 7 h,
BrdU-labeled (20 min) at 12 h, then collected to examine
thereplication profile (Fig. 2A). For each sample, we analyzed�30
MB of individual DNA fibers (�250 kb). TGX-treatedcells showed 43%
reduction in the length of BrdU tracksrelative to controls,
suggesting that p110� is required fornormal replication fork
progression; in contrast, elongationwas not significantly affected
by p110� inhibition (Fig. 2 A).Median center-to-center distance
between adjacent BrdUtracks, indicative of the initiation rate, was
shorter in TGX-than in PIK-treated cells or in controls (Fig. 2B),
consistentwith cell activation of additional replication origins to
com-pensate slow fork progression (21). The percentage of
repli-cation of individual DNA fibers was lower in TGX- (20.7%)than
in control or PIK-treated cells (33.0 and 32.3%). These
0 1 432
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ControlActivep110α
Activep110β
Activep110β
Activep110α
KRp110α
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% S
% G0/G1
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shRNACtr shRNAα_+ _+ Dox
p110α
p110β
PKBpPKB
PKBpPKB
Ctr Activep110βActivep110α
NIH3T3
U2OS
Ctr
PKB
pPKB
p110β p110α
CtrActivep110β
Activep110αCtr
0h3h
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5h7h
Time G1/Srelease
% G2/M
(*)
shRNA control
shRNA p110α
shRNA p110β
shRNACtr shRNAα_+ _+ Dox
Fig. 1. Interference with p110� alters S phase progression. (A)
NIH 3T3 stablecell clones expressing active-p110� or � were
examined in Western blotting (WB).(B) Control NIH 3T3 cells, active
p110� and active p110� cells were seeded atsimilar densities and
counted at 24 h intervals (mean � SD, n � 6). (C) Percentageof
cells with an S phase DNA cell content at different times after
release from G0arrest (mean � SD; n � 5). (D) NIH 3T3 cells
transfected with KR-p110� or � wereexamined in Western blot (WB) at
24 h posttransfection. (E) Cell division time forcontrol or
KR-p110�- and KR-p110�-infected NIH 3T3 cells (mean � SD, n � 3)
asin B. (F) Percentage of S phase cells (mean � SD, n � 5) of
control, KR-p110�- and�-infected cells, as in C. (G) U2OS clones
expressing control, -�, or -� shRNA wereinduced with doxycycline
for 48 or 120 h, respectively; p110� or � expression wasexamined in
WB. Cells were subjected to thymidine block and released
fordifferent times, the profiles show cell cycle distribution. *, P
� 0.05.
Control
shRNA
A
B
AN
D +
UdrB
300
Center-to-center distance (kb)
Control
BrdU track length (kb)
2001000150100500
Ctr
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PIK
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011pα011pβ
p110β
cDNA
Histone
Tubulin
p110α
C
p110α
DAPI shRNAα
p110β
shRNA
Control
DAPI shRNAβ
Fig. 2. p110� is a nuclear protein and regulates DNA elongation.
(A) Single-molecule analysis of DNA replication in synchronized NIH
3T3 cells treated withPIK75 or TGX221 inhibitors at 7 h postserum
addition, pulse-labeled (20 min)
withBrdUat12hpostserumaddition,andcollectedimmediatelyforanalysis.GenomicDNA
fibers were stretched by DNA combing. Newly replicated DNA was
detectedby immunofluorescence with an anti-BrdU Ab (green); DNA
fibers were
coun-terstainedwithanti-DNAAb(red).Representativefibersare shown.
(Scalebar,50Kb.) (B) Distribution of BrdU track length and
center-to-center distances betweenadjacent BrdU tracks. Box: 25–75
percentile range. Whiskers: 10–90-percentilerange. [Vertical bar,
median value (kb).] ***, Mann–Whitney rank sum test P �0.0001. (C)
NIH 3T3 cells were cotransfected with red fluorescence protein
(RFP)and control, p110�, or -� shRNA; p110 localization was
examined by immuno-fluorescence. DAPI nuclear staining is shown in
Insets. (Scale bar, 10 �m.) NIH 3T3cells or WT-p110� or -�
transfected cells were fractionated and examined in WB(Right).
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results suggest that p110� activity controls replication
forkprogression.
The p110� Is Located in the Nucleus and Controls PCNA Binding
toChromatin. Since DNA replication occurs in the nucleus, we
exam-ined p110� localization. Subcellular fractionation (Methods)
andimmunofluorescence analysis showed that the majority of
endog-enous p110�, but not of �, concentrated in the NIH 3T3 cell
nucleus(Fig. 2C). Both the nuclear p110� signal and the mainly
cytosolicp110� signal decreased with selective shRNA (Fig. 2C; Fig.
S3B).A similar distribution was observed in MEF, COS-7, HeLa
andU2OS cells. These results indicate that p110� concentrates in
thenucleus.
We examined the mechanisms by which p110� regulates
repli-cation. One of the first events required to initiate
replication isMCM complex loading on origins (replication
licensing, 8,13,22).We compared MCM loading to chromatin by cell
fractionation onnuclear and chromatin extracts (23). Whereas in
control cells,MCM 2/4 appeared on chromatin fractions in
exponential growth,but not after GF starvation or in confluence,
active p110� or �expression induced a similar and moderate
enhancement of MCM2/4 loading onto chromatin in starving and
confluence conditions(Fig. S3C). Accordingly, KR-p110� or �
expression induced a slightreduction in late G1 MCM loading (Fig.
3A). MCM loading ontochromatin is thus modulated to some extent by
p110� and �, but isnot selectively controlled by p110�.
The p110� Activity Regulates PCNA Loading onto Chromatin.
Afterreplication origin activation, Pol� binding to the ORC
triggersprimer DNA synthesis; elongation of DNA synthesis
requiressubsequent binding of PCNA that tethers the processive
poly-merases Pol� and � to the DNA template (9, 14). In
controlsynchronized NIH 3T3 cells, we observed PCNA appearance
inchromatin extracts (22) as well as PCNA-Pol� association at �12
hafter GF addition, at the onset of S phase (Fig. 3B). Active
p110�cells behaved similarly; in contrast, active p110� expression
accel-erated PCNA binding to chromatin and PCNA-Pol�
association(Fig. 3B). Moreover, expression of KR-p110� (Fig. 3C),
reductionof p110� levels with shRNA (Fig. 3D; Fig. S3D) and
p110�inhibition (Fig. S3 E and F) diminished PCNA loading onto
chromatin as well as PCNA-Pol� association; interference
withp110� only had a modest inhibitory effect. These data show
thatp110� controls PCNA binding to chromatin and to Pol�,
providinga potential mechanism for DNA elongation impairment
afterinterference with p110� function.
The p110� Activity Regulates p21Cip Phosphorylation. PCNA
loadsPol � and � to the DNA template for efficient elongation;
PCNAalso binds p21Cip through the same region, p21Cip thus
impairsPCNA association to Pol�/� (22, 24). We examined
PCNA-p21Cipcomplex formation in cells with altered p110� activity.
Whereasinterference with p110� did not appreciably affect
PCNA-p21Cipcomplexes, active-p110� reduced (Fig. 3E) and inactive
p110� (orp110� inhibition) increased PCNA-p21Cip association (Fig.
3F, Fig.S4A). Phosphorylation of p21Cip on T145 and Ser-146
phosphor-ylation (by PKB and PKC) regulates its dissociation from
PCNA(25–28), nonetheless, in vivo T145 appears to be the critical
residue(27, 28). We confirmed that T145 phosphorylation induced
PCNA-p21Cip dissociation in U2OS cells and NIH 3T3 cells (Fig.
4A);expression of the phosphomimetic D145-p21Cip mutant
reducedPCNA-p21Cip association increasing PCNA binding to
chromatin(Fig. 4A).
We also examined whether p110� regulates T145 phosphoryla-tion.
Whereas in control cells T145 was phosphorylated near Sphase entry,
both p110� shRNA and KR-p110� expression reducedpT145-p21Cip levels
(Fig. 4B; Fig. S4 B–D). In these assays weobserved that
interference with p110� activity also resulted ingreater p21Cip
expression levels. p21Cip is degraded after its releasefrom PCNA
(29); the higher p21Cip levels in cells with impairedp110� function
might be due to stabilization of p21Cip in complexwith PCNA. Both
KR-p110� and p110� shRNA expression in-creased p21Cip protein
stability (Fig. S5A), whereas active p110�reduced p21cip stability
(Fig. S5B). p110� activity is thus needed forp21Cip phosphorylation
and dissociation from PCNA.
The p110� Regulates Nuclear PKB. The PI3K effector PKB
phos-phorylates T145-p21Cip (27, 28). We confirmed that PKB
phos-phorylates T145-p21Cip in vitro (Fig. S6A) and examined
whetherp110� regulates PKB-mediated T145-p21Cip phosphorylation.
Wefound that expression of KR-p110� (or p110� inhibition)
reduced
PCNA
PolδPCNA in Chr
Time, h 0 9 12 0 9 12 0 9 12
shRNACtr shRNAα2 shRNAβ2
%S Phase141010 18 45 12 15 49 29
DC
PCNA%S Phase2212710 19 28 7 10 18
PCNA in Polδ IP
PolδPCNA in Chr
0 9 12
Control
0 9 12 0 9 12
KRp110α KRp110βTime, h
E FKRp110α KRp110βControl
2212 710 19
28 7 10 18
PCNAin p21Cip IP
PCNA
p21Cip in IP
%S phase
Time, h 0
9 12 0 9 12 0 9 12
43291314 17 28 13 24 34
0 9 12 0 9 12
Activep110α
Activep110β
Time, h
PCNAin p21Cip IP
PCNA
p21Cip in IP
%S phase
Control
0 9 12
A CYTOSOL NUCLEUS CHROMATIN
MCM2MCM4
MCM2MCM4
0 9 12 14 Time, h
Con
trol
MCM2MCM4KR
-p1
10α
KR
-p1
10β
Activep110α
Activep110β
0 9 12
Control
0 9 12 0 9 12
PCNA
PolδPCNA in Chr
Time, h
%S Phase50381813 16 25 14 23 31
B0 9 12 140 9 12 14
PCNA in Polδ IP
PCNA in Polδ IP
Fig. 3. p110� controls PCNA binding to chromatin andto DNA Pol�.
(A) NIH 3T3 cells were infected with KR-p110�- or -�-encoding
viruses, synchronized and
col-lectedatdifferenttimes.MCM2/MCM4levels incytosolic,nuclear and
chromatin fractions were examined in WB.(B) Active p110�- and -�
cells and control NIH 3T3 cellsweresynchronized
inG0andreleasedfordifferenttimes.PCNA in Pol� immunoprecipitates
and PCNA levels wereexamined in the chromatin fraction, total Pol�
and PCNAwere also examined in WB. (C and D) NIH 3T3 transfectedwith
KR-p110� or -� (C) or with control, -�, or -� shRNA(D) were
collected and examined at the indicated times.Analyses were as in B
(n � 3). (E) Active p110�- and�-expressing NIH 3T3 cells were
synchronized in G0 andreleased for different times. PCNA and p21Cip
levels inp21Cip immunoprecipitates and total PCNA levels in
chro-matin-free extracts were examined in WB. (F) NIH 3T3cells
transfectedwithKR-p110�or-�wereexaminedas inE. Chr, chromatin.
Percentage cells in S phase indicatedbelow gels. The circles show
the time for S phase entry.(A–F) One representative experiment of
at least threewith similar results.*, P � 0.05.
Marqués et al. PNAS � May 5, 2009 � vol. 106 � no. 18 �
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S phase PKB kinase activity in vitro (Fig. S6 A and B). Western
blotanalysis of pPKB in extracts from synchronized NIH 3T3
cellsexpressing KR-p110� or treated near S phase with
TGX221confirmed that p110� regulates S phase pPKB, whereas
p110�
inhibition had a lesser effect (Fig. 4C); results were similar
in U2OScells (Fig. S6C). As an alternative approach, we examined
pPKB byimmunofluorescence. At 1 h postserum addition (G1 phase)
pPKBconcentrated at the cell membrane and was reduced by
KR-p110�(Fig. S7A), whereas in S phase pPKB concentrated in the
nucleusand was notably reduced by KR-p110� and p110� inhibition
(Fig.4D; Fig. S7B). Cell fractionation confirmed TGX inhibition of
Sphase nuclear pPKB (Fig. S7C).
We examined other PKB substrates in S phase; GSK3�
phos-phorylation was reduced by p110� inhibition, whereas
FKHRL1phosphorylation was p110� activity-dependent (Fig. S7D), as
is thecase in G1 phase (6). WB using anti-pPKB substrate Ab
showedthat p110� inhibition reduced phosphorylation of some
PKBsubstrates in S phase cells (such as p21Cip, Fig. S7C), while
otherswere p110�-regulated (Fig. S7D). Results were similar using
Sphase U2OS cells treated with PI3K inhibitors and then
fraction-ated (Fig. 4E); this assay also showed that p110�
inhibition affectedmainly cytosolic substrates and p110� nuclear
substrates, suggestingthat p110� and � control distinct PKB pools.
p110� thus governsnuclear S phase PKB activity. Since p110� is
activated at the G1/Sboundary (6), the early timing of
phosphorylation of some PKBsubstrates or their cytosolic
localization might determine a p110�activity requirement for
phosphorylation.
Based on p110� regulation of S phase nuclear pPKB-mediatedp21Cip
phosphorylation, expression of the phosphomimetic D145-p21Cip
mutant in cells with impaired p110� activity could replacep110�
activity in S phase. BrdU labeling of newly-synthesized DNAin
exponentially growing cells expressing KR-p110� alone or
incombination with D145-p21Cip showed that D145-p21Cip
expressionaccelerated S phase progression in KR-p110� cells (Fig.
4F).D145-p21Cip expression also increased PCNA-Pol� association
andreduced PCNA-p21Cip complexes in KR-p110� cells (Fig.
S8A).Accordingly, A145-p21Cip expression corrected PCNA-Pol�
com-plexes in active p110� cells (Fig. S8B). Thus, expression of
phos-phomimetic p21Cip mutants corrects the S phase defects of
cells withaltered p110� activity.
PI3K� Protein Regulates PCNA Loading onto Chromatin. The
recentlydescribed conditional p110��/� mouse phenotype and that
ofinactive p110� knock-in mice (7, 30) indicate that p110�
kinaseactivity regulates mouse growth and tumor development and
alsothat p110� has a kinase-independent function in embryonic
devel-opment. Kinase-independent functions often reflect the
ability of aprotein to associate a necessary partner, as is the
case for PI3K� inthe control of cardiac stress response (31). We
examined whetherp110� expression (independent of its kinase
activity) regulatesDNA elongation, studying the extent of PCNA
binding to chroma-tin after p110� inhibition or p110� knockdown. To
improve p110�deletion, we transfected cells with
puromycin-shRNA-encodingvectors, selected them for 48 h and
immediately analyzed theseasynchronous cultures (synchronization
requires longer culturetimes) before reduction of cell viability.
Pulse–chase BrdU analysisin exponentially growing NIH 3T3 cells
showed that p110� inhi-bition reduced S phase progression, but
p110� knockdown had agreater effect in decelerating S phase (Fig.
5A). PCNA loading ontochromatin was also reduced by p110� or PKB
inhibition, but wasdrastically diminished by p110� knockdown (Fig.
5B).
We also analyzed asynchronous cultures of p110��/� immortal-ized
mouse embryonic fibroblasts (MEF) reconstituted with WT orKR-p110�
(7). KR-p110� MEF progressed through S phase moreslowly than WT
p110� MEF, although p110��/� MEF showed theslowest S phase
progression (Fig. 5A). KR-p110� MEF had lesschromatin-bound PCNA
than controls, but PCNA loading waslowest in p110��/� MEF (Fig.
5B). These results suggest that PCNAloading onto chromatin and in
turn S phase progression rate isfurther regulated via a
kinase-independent p110� function.
pPKB was little affected by p110� deletion in
asynchronouscultures (7). To define whether p110� controls nuclear
PKB in S
0 3 975
D
Cell 2 KRp110α
Cell 1Control
KR-p110α KR-p110β
Cell 3 KRp110β
S ni gni nia
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Chasetime, h
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Ctr A146A145 D146D145PCNA in Myc-p21Cip IP PCNA in Chr
PCNA
Myc-p21Cip
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541A
641D
541D
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Chasetime, h
S ni gni nia
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KRp110αKRp110β
ControlKRp110α + D145p21CipKRp110β + D145p21Cip
Control + D145p21Cip
ecnecseroulF
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Cell 1 Control
0
150
0 10 20Distance, µm
0 10 200 10 20
Cell 2 KRp110α Cell 3 KRp110β
F
B0 9 0 9 0 9
shRNACtr shRNAα shRNAβTime, h
Actin
pThr145in p21Cip IP
p21Cip in IP
%S phase19913 35 12 24
0
100
0 9
50
0 9 0 9
12p541Tppi
C12p /
piC
Control shRNAα shRNAβ
(*)rtCANRhs
ANRhs
αANRhs
β
Actin
p110αp110β
Time, hG1/S
21 21 21
NUCLEUS
Control TGX221PIK 75
21 21 21
CYTOSOL
Control TGX221PIK 75E
WB: pPKB substrateWB: pPKB substrate
37
25
MW
50
75
37
25
50
75
MW
Fig. 4. p110� controls nuclear PKB. (A) NIH 3T3 cells
transfected with A145,A146, D145, or D146 p21Cip mutants were
fractionated. PCNA levels were mea-sured in p21Cip
immunoprecipitates, chromatin-containing and -free fractions;p21Cip
expression was also examined in chromatin-free fractions. Graphs
showthe percentage PCNA signal (mean � SD) in p21Cip
immunoprecipitates and thatof PCNA in chromatin fractions, compared
with the maximum PCNA signal ineach case (n � 3). (B) NIH 3T3
transfected with control, p110�, or � shRNA weresynchronized in G0
and released (9 h). WB shows pT145-p21Cip in p21Cip
immu-noprecipitates from chromatin extracts; graphs show the
pT145-p21Cip signal(percentage�SD)normalizedtop21Cip
levelsandcomparedwiththesignalat9hin controls (100%; n � 3). WB
(bottom left) shows p110 expression levels. (C) NIH3T3 cells
transfected with KR-p110 mutants were synchronized after 24 h
andother cells were treated at 7 h with TGX221 (30 �M) or PIK75
(0.08 �M); cells werecollected at 9 h. pPKB levels were measured in
WB. (D) pPKB localization exam-ined by immunofluorescence in cells
cotransfected with KR-p110 mutants andRFP, fixed 9 h after G0
release. Graphs show fluorescence intensity in arbitraryunits (AU)
examined along the line in the images. Insets show expression of
KRmutants. (Scale bar, 50 �m.) (E) Phosphorylation of PKB
substrates was examinedby WB in fractionated extracts of U2OS cells
that were thymidine-arrested, thenreleased (1 and 2 h). (F) NIH 3T3
cells expressing KR-p110� or � mutants alone orin combination with
D145-p21Cip were BrdU-labeled and chased at differenttimes. Graph
shows the cell percentage remaining in S phase (mean � SD, n �
3).
*, P � 0.05.
7528 � www.pnas.org�cgi�doi�10.1073�pnas.0812000106 Marqués et
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phase in these MEF, we synchronized cells at the G1/S border
andexamined them after release. In WT p110�-reconstituted MEF,pPKB
was found mainly in the nuclear fraction in S phase;KR-p110� MEF
behaved similarly but had lower nuclear activepPKB levels (Fig.
5C). Both nuclear pPKB and PKB were unde-tectable in p110��/� MEF
(Fig. 5C), indicating that p110� expres-sion might control PKB
nuclear entry. Cytosolic pPKB was moreabundant in p110��/� MEF, but
they expressed lower levels ofPTEN (Fig. 5C); this might represent
a compensatory mechanismfor p110� deletion. We also analyzed
nuclear/cytoplasmic distribu-tion of pPKB and PKB in NIH 3T3 cells
to further examine whetherp110� deletion reduces not only nuclear
phospho-PKB but alsonuclear PKB, as in MEF. p110� shRNA diminished
but did notcompletely eliminate nuclear PKB (Fig. S8C). These
results do notdemonstrate, but suggest that PKB nuclear entry is
facilitated byp110� expression, an aspect that requires further
study. In contrast,both p110� inhibition and p110� shRNA expression
clearly re-duced S phase nuclear pPKB (Fig. S8C), further
confirming thefunction of p110� in control of nuclear PKB activity
in S phase.
PI3K� Protein Associates PKB and PCNA. To determine
whetherp110�-dependent PKB nuclear activity is due to direct
association,we studied PKB-p110� complex formation in cytosolic and
nuclearfractions. Cells were fractionated as described (32), since
themethod used earlier (Fig. 2C) (33) destroys protein–protein
inter-actions. NIH 3T3 cells were cotransfected with HA-gagAKT
andWT-p110� or -�, collected at 12 h post-G0 release, and
examined.Although PKB and p110� associated in cytosol, this
association waslower than that of PKB and p110�, and was not found
in thenucleus, where only PKB-p110� complexes were observed
(Fig.S9A). We also analyzed association of endogenous proteins
insynchronized NIH 3T3 cells collected at 12 h postserum
addition.WB analysis of the fractions confirmed that p110� was
mainlycytosolic and p110� was more abundant in the nucleus (Fig.
5D).Although immunoprecipitation concentrated the scarce
nuclearp110� protein, endogenous PKB associated mainly with p110�
inthe nuclear fraction (Fig. 5D).
To identify other nuclear proteins that regulate DNA
replicationand associate to p110�, we performed a pull-down assay
usingmammalian GST-p110�; we obtained a number of candidateproteins
including PCNA. Immunoprecipitates of endogenousPCNA from nuclear
extracts contained associated endogenousp110� but not p110� (Fig.
5E); results were similar in a reciprocalassay (Fig. S9B). To
determine whether the selective association ofPCNA with p110� was
due to a p110�-specific structural feature orto its subcellular
distribution, we inserted a nuclear localizationsignal (NLS) in p85
and cotransfected it with myc-WT-p110� or -�,which increased their
nuclear localization. Both nuclear p110� and� associated with PCNA,
although p110� association to PCNA wasgreater than that of nuclear
p110� (Fig. S9C). Therefore, inaddition to its subcellular
distribution, p110� has a structuraladvantage for association to
PCNA.
Here, we describe a role for p110� in replication fork
elongationin mammalian cells, providing an example of elongation
control byextracellular signal-regulated molecules. The nuclear
localizationand function of p110� resembles that of class IV PI3K,
which arerecruited to DNA damage sites and mediate cell responses
as DNArepair (34). Although some cell cycle phenotypes were
moderate(Fig. 1), complete p110� elimination interfered with cell
survival,and p110� function was studied in partial p110� deletion
condi-tions. p110� regulated DNA replication through
kinase-dependentand -independent mechanisms. p110� associated with
PKB, andp110� activity regulated nuclear PKB-mediated p21Cip
phosphor-ylation, PCNA release, PCNA binding to Pol� and
replicationelongation. Interference with p110� activity had a
slight inhibitoryeffect on p21Cip phosphorylation, and might
partially compensatefor p110� activity-dependent functions. In
addition, p110� associ-ated with PCNA and controlled PCNA loading
onto chromatin in
0 6420 642
(*)
(*)
Chase time, h
WTp110βKRp110βp110β−/−
MEF
0
100
50
S ni gninia
meR
%
(*)
(*)
Control
shRNA βTGX
0
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S ni gniniame
R %
A
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NUCLEI CYTOSOL
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3T3 MEF3T3 MEF011p
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β
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β 011pR
Kβ
3 0
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PKB
1 3 01 30 1Time, hG1/S
WTp110β KRp110β p110β−/−
PTEN
3 01 3 01 30 1
WTp110β KRp110β p110β−/−
PKB
0
50
100)U
A( B
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3 01 3 01 30 1 3 01 3 01 30 1Time, hG1/S
WTp110β KRp110β p110β−/−WTp110β KRp110β p110β−/−
(*) (*)
0
50
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A( B
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p R
AE
LC
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CU
N
PKB
011pβ
IP
1 lortnoC
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3 lortnoC
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β 011pα
BK
P
PKBlongexposure
NUCLEICYTOSOL
PKB
Tubulin
Histones
p110β
p110α
Extracts
20
Chase time, h
IPIP
NUCLEI CYTOSOL
NUCLEICYTOSOL
Fig. 5. p110� associates with PKB and PCNA. (A) NIH 3T3
transfected withcontrol or p110� shRNA were selected with puromycin
(2 �g/mL, 48 h), thenexamined. Other samples were treated with
TGX221 or PKB inhibitors for 12 hbefore collection. Immortalized
p110��/� mouse MEF, and p110��/� MEFreconstituted with WT- or
KR-p110� were cultured in exponential growth. Afraction of the
cells were pulsed-labeled with BrdU (1 h). Graphs show
thepercentage of cells remaining in S phase at each chase time
(mean � SD, n �3). (B) Lysates of cells treated as in A were
analyzed in WB to determine PCNAin the chromatin fraction, as well
as PCNA and p110� in the chromatin-freefraction. Graphs show the
percentage of chromatin-bound PCNA normalizedto total PCNA and
compared with maximum signal in control NIH 3T3 or inMEF. (C)
Immortalized MEF as in A were arrested by thymidine treatment,
thenreleased for different times. Cell fractions were examined in
WB to test forpPKB and PKB levels; the latter was then reprobed for
PTEN. The graphs shownuclear pPKB or PKB signal in arbitrary units
(AU) (mean � SD, n � 3). (D)Synchronized NIH 3T3 cell cultures
collected at 12 h postserum addition werefractionated. The levels
of PKB, p110� and � in these fractions were examinedby WB (Left).
Endogenous p110� or � from cytosolic (1500 �g) and nuclearextracts
(600 �g), or PKB from cytosolic (300 �g) and nuclear extracts (200
�g)were immunoprecipitated. We tested for PKB and p85 in p110
immunopre-cipitates by WB. Controls 1–3, protein A plus each of the
antibodies. Graphshows the percentage of p110-associated PKB
signal, compared with maximalPKB signal (in PKB immunoprecipitates
from an equivalent protein amount).(E) Nuclear fractions were
obtained from synchronized NIH 3T3 cells (at 12 h).PCNA (800 �g) or
p110 (200 �g) immunoprecipitates were tested in WB forp110. For
control 1, protein A was incubated with Ab; control 2, protein A
wasincubated with lysate. Graphs show the percentage of p110 signal
in PCNAimmunoprecipitates compared with maximal p110 signal (p110
immunopre-cipitated from an equivalent protein amount). *, P �
0.05.
Marqués et al. PNAS � May 5, 2009 � vol. 106 � no. 18 �
7529
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a kinase-independent manner. Since PCNA loading onto chroma-tin
is essential for DNA duplication, this kinase-independent func-tion
explains the greater division defects in cells with reduced
p110�expression. The role of p110� in DNA replication could
contributeto cause the early lethality (E2–3, ref.4) of
p110�-deficient mice.
Materials and MethodsComplementary DNA and shRNA. pSG5-p110�CAAX
(active p110�), pSG5-HA-wt-PKB and -gag-PKB were described (5, 35).
pCEF2-hp110�CAAX (active p110�)was a gift of Dr. Murga (Centro de
Biología Molecular/CSIC, Madrid, Spain).PcDNA-Myc -WT and p21Cip
mutants were donated by Dr. Rössig (28). pcDNAMyc-S146A/T145A
double mutant was generated using Quick Change Site-Directed
mutagenesis (Stratagene). Myc-K802R-hp110� and
myc-K805R-hp110�mutants were subcloned into pSG5 and pRV-IRES-GFP
for retroviral infection (6).We used several specific short hairpin
RNA (shRNA) directed to human or murinep110 sequences, each assay
was performed at least with two shRNA, with similarresults. These
shRNA (6) were subcloned in pBluescript/U6 or in pTER vector;
weused control shRNA that did not reduce p110� or � expression. We
also usedPik3cb shRNA (Origene; Fig. 5). To prepare NLS-p85, the
PKKKRKV sequence wasinserted 3� of the p85 sequence.
Cell Lines, Cell Culture, and Retroviral Transduction. Active
p110� and activep110� NIH 3T3 cells lines were described (6).
KR-p110� and � mutations weretransduced by transient transfection
or retroviral infection. We generated pTER-p110� or pTER-p110� U2OS
clones according to manufacturer’s protocol (Invitro-gen); shRNA
expression was induced for 2 days (p110�) or 5 days (p110�)
inmedium plus doxycycline (6 �g/mL, Sigma). NIH 3T3 murine
fibroblasts, U20S andCOS7 cells were cultured as described (6). For
retrovirus production, Phoenix cellswere transfected using
JetPei-NaCl (Qbiogene). MEF were donated by Drs. Zhaoand Roberts
(7) (Dana Farber Cancer Institute, Boston, MA).
Cell Cycle, BrdU Labeling, Immunofluorescence, and Dynamic
Molecular Comb-ing. Immunofluorescence and NIH 3T3 G0
synchronization were as reported (15).Briefly, cells were incubated
in serum-free medium (19 h) and released by serumaddition. Cell
cycle distribution was examined by DNA staining with propidium
iodide and analyzed by flow cytometry (Beckman-Coulter) using
Multicycle AV(Phoenix Flow Systems). Cells were synchronized at
G1/S by double thymidineblock (6) or using aphidicolin (22). To
determine cell division time (t1/2), cells wereseeded at similar
densities and counted at 24 h intervals. S phase duration
wascalculated considering t1/2 (mean of n � 6) and the proportion
of cells in S phasein exponential growth (mean of n � 12). S phase
progression rates were exam-ined in exponentially growing cultures
incubated with 20 �M bromodeoxyuri-dine (BrdU; 1 h), chased at
different times and stained with BrdU-FITC Ab (BDBiosciences), then
examined by three-dimensional FACS.
For dynamic molecular combing, synchronized NIH 3T3 cells were
treated with0.08 �M PIK75 or 30 �M TGX221 at 7 h postserum
addition; 20 min before harvest(12hpostserumaddition), cellswere
treatedwith20 �MBrdU.Afterharvest, cellswere embedded in LMP
agarose plugs (3 106 cells/plug) and DNA fibers werepurified and
stretched on silanized coverslips as described (21). BrdU tracks
weredetected with rat monoclonal Ab (clone BU1/75; AbCys) and an
Alexa 488-conjugated secondary Ab (Molecular Probes). DNA fibers
were counterstainedwith mouse anti-ssDNA (MAB3034, Chemicon) and
Alexa 546-secondary Ab(Molecular Probes). Signals were analyzed
with MetaMorph.
Statistical analyses were performed using StatView 512�
(Calabasas, CA). Gelbands and fluorescence intensity were
quantitated with ImageJ software. Sta-tistical significance was
calculated using Student’s t test. For DNA combing,statistical
analysis was performed with GraphPad Prism 5.0 (GraphPad
Software).
For description of antibodies and reagents, cell lysis,
subcellular fractionation,Western blotting, immunoprecipitation,
and kinase assays, see SI Methods.
ACKNOWLEDGMENTS. We thank Drs. Roberts and Zhao for sharing
p110��/�immortal MEF, M. White for the myc-p110 plasmid, C. Murga
for pCEFL2-p110�-CAAX, B. Vanhaesebroeck for His-p110�, M. van de
Wetering for the pTer vector,Y. Shi for the pBlue/U6 plasmid, A.
Klippel for anti-p110�, J. Méndez for help inchromatin
purification, as well as E. Schwob and the DNA combing
facility(Montpellier) for silanized coverslips, and C. Mark for
editorial assistance. M.M.has a predoctoral Formacion de
Profesorado Universitario fellowship from theSpanish Ministry of
Science and Innovation, and A.M.P. a postdoctoral fellowshipfrom
the Fondation Recherche Medicale. This work was supported in part
bygrants from the American Institute for Cancer Research
Foundation, the Funda-ción Ramón Areces, the Asociacion Española
de la Lucha Contra el Cancer, theCentre National de la Recherche
Scientifique, and the Spanish Dirección Generalde Ciencia y
Desarrollo Tecnologico Grants SAF2004-05955 and SAF2007-63624.
1. García Z, Kumar A, Marques M, Cortes I, Carrera AC (2006)
PI3K controls early and lateevents in mammalian cell division. EMBO
J 25:655–661.
2. Fruman DA, Meyers RE, Cantley LA (1998) Phosphoinositide
kinases. Annu Rev Biochem67:481–507.
3. Bi L, Okabe I, Bernard DJ, Wynshaw-Boris JA, Nussbaum RL
(1999) Proliferative defect andembryonic lethality in mice
homozygous for a deletion in the p110-alpha subunit of PI3K.J Biol
Chem 274:10963–10968.
4. Bi L, Okabe I, Bernard DJ, Nussbaum RL (2002) Early embryonic
lethality in mice deficientin the p110beta catalytic subunit of
PI3K. Mamm Genome 13:169–172.
5. Kang S, Denley A, Vanhaesebroeck B, Vogt PK (2006) Oncogenic
transformation inducedby the p110b, g and d isoforms of class I
PI3K. Proc Natl Acad Sci USA 103:1289–1294.
6. Marqués M, et al. (2008) PI3K p110alpha and p110beta
regulate cell cycle entry, exhibitingdistinct activation kinetics
in G1 phase. Mol Cell Biol 28:2803–2814.
7. Jia S, et al. (2008) Essential roles of PI(3)K-p110beta in
cell growth, metabolism andtumorigenesis. Nature 454:776–779.
8. Cvetic C, Walter JC (2006) Getting a grip on licensing:
Mechanism of stable Mcm2–7loading onto replication origins. Mol
Cell 21:143–144.
9. Frouin I, et al. (2002) Cell cycle-dependent dynamic
association of cyclin/Cdk complexeswith human DNA replication
proteins. EMBO J 21:2485–2495.
10. Sasaki T, Gilbert DM (2007) The many faces of the origin
recognition complex. Curr OpinCell Biol 19:337–343.
11. Hübscher U, Maga G, Spadari S (2002) Eukaryotic DNA
polymerases. Annu Rev Biochem71:133–163.
12. NishitaniH,LygerouZ(2002)ControlofDNAreplication licensing
inacell cycle.GenesCells7:523–534.
13. Sclafani RA, Tecklenburg M, Pierce A (2002) The mcm5-bob1
bypass of Cdc7p/Dbf4p inDNA replication depends on both
Cdk1-independent and Cdk1-dependent steps in Sac-charomyces
cerevisiae. Genetics 161:47–57.
14. Waga S, Stillman B (1994) Anatomy of a DNA replication fork
revealed by reconstitutionof SV40 DNA replication in vitro. Nature
369:207–212.
15. Martínez-Gac L, Marqués M, García Z, Campanero M, Carrera
AC (2004) Control of cyclinG2 mRNA expression by forkhead
transcription factors: A novel mechanism for cell cyclecontrol by
PI3K and forkhead. Mol Cell Biol 24:2181–2189.
16. Van de Wetering M, et al. (2003) Specific inhibition of gene
expression using a stablyintegrated, inducible
small-interfering-RNA vector. EMBO Rep 4:609–615.
17. Jackson S, et al. (2005) PI 3-kinase p110beta: A new target
for antithrombotic therapy. NatMed 11:507–514.
18. Knight ZA, et al. (2006) A pharmacological map of the PI3-K
family defines a role forp110alpha in insulin signaling. Cell
125:733–747.
19. Downward J (2004) PI 3-kinase, Akt and cell survival. Semin
Cell Dev Biol 15:177–182.20. Michalet X, et al. (1997) Dynamic
molecular combing: Stretching the whole human
genome for high- resolution studies. Science 277:1518–1523.21.
Tourriere H, Versini G, Cordon-Preciado V, Alabert C, Pasero P
(2005) Mrc1 and tof1
promote replication fork progression and recovery independently
of Rad53. Mol Cell19:699–706.
22. Riva F, et al. (2004) Distinct pools of proliferating cell
nuclear antigen associated to DNAreplication sites interact with
the p125 subunit of DNA polymerase delta or DNA ligase I.Exp Cell
Res 293:357–367.
23. Mendez J, Stillman B (2000) Chromatin Association of Human
Origin Recognition Com-plex, Cdc6, and Minichromosome Maintenance
Proteins during the Cell Cycle: Assembly ofPrereplication Complexes
in Late Mitosis. Mol Cell Biol 20:8602–8612.
24. Cazzalini O, et al. (2003) p21CDKN1A does not interfere with
loading of PCNA at DNAreplication sites, but inhibits subsequent
binding of DNA polymerase delta at the G1/Sphase transition. Cell
Cycle 2:596–603.
25. Scott MT, Morrice N, Ball KL (2000) Reversible
phosphorylation at the C-terminal regula-tory domain of
p21Waf1/Cip1 modulates proliferating cell nuclear antigen binding.
J BiolChem 275:11529–11537.
26. Walker JL, Castagnino P, Chung BM, Kazanietz MG, Assoian RK
(2006) Post-transcriptionaldestabilization of p21cip1 by protein
kinase C in fibroblasts. J Biol Chem 281:38127–38132.
27. Zhou BP, et al. (2001) Cytoplasmic localization of
p21Cip1/WAF1 by Akt-induced phos-phorylation in
HER-2/neu-overexpressing cells. Nat Cell Biol 3:245–252.
28. Rössig L, et al. (2001) Akt-dependent phosphorylation of
p21Cip1 regulates PCNA bindingand proliferation of endothelial
cells. Mol Cell Biol 21:5644–5657.
29. Touitou R, et al. (2001) A degradation signal located in the
C-terminus of p21CIP1 is abinding site for the C8 alpha-subunit of
the 20S proteasome. EMBO J 20:2367–2375.
30. Ciraolo E, et al. (2008) PI3-kinase p110beta activity: Key
role in metabolism and mammarygland cancer but not development. Sci
Signal 1:ra3.
31. Patrucco E, et al. (2004) PI3Kg modulates the cardiac
response to chronic pressure overloadby distinct kinase-dependent
and -independent effects. Cell 118:375–387.
32. Carrera AC, Li P, Roberts TM (1991) Characterization of an
active, non myristylated,cytoplasmic form of the lymphoid protein
Tyr kinase pp56lck. Int Immunol 3:673–682.
33. Qu L, et al. (2004) Endoplasmic reticulum stress induces p53
cytoplasmic localization andprevents p53-dependent apoptosis by a
pathway involving glycogen synthase kinase-3beta. Genes Dev 18:
261–277.
34. Bakkenist CJ, Kastan MB (2004) Initiating cellular stress
responses. Cell 118:9–17.35. Álvarez B, Martinez AC, Burgering BM,
Carrera AC (2001) Forkhead TFs contribute to
execution of the mitotic programme in mammals. Nature
413:744–747.
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