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: acarrera@cnb.csic.es.

This article contains supporting information online at www.pnas.org/cgi/content/full/0812000106/DCSupplemental.

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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

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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.

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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).

7526 � www.pnas.org�cgi�doi�10.1073�pnas.0812000106 Marqués et al.

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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

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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.

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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

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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.

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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

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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.

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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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