Specific function of phosphoinositide 3- beta in the control of DNA replication

Miriam Marque´ sa,1, Amit Kumara,1, Ana M. Povedab, Susana Zuluagaa, Carmen Herna´ ndeza, Shaun Jacksonc, Philippe Paserob, and Ana C. Carreraa,2

aDepartment of Immunology and Oncology, Centro Nacional de Biotecnología/Consejo Superior de Investigaciones Científicas, Universidad Auto´noma de Madrid, Cantoblanco, Madrid E-28049, Spain; bInstitute of Human Genetics, Centre National de la Recherche Scientifique Unite´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 comprised of accelerate G1ϾS transition (6); however, no p110␤-specific func- a p85 regulatory and a p110 catalytic subunit that induce formation tion has been described in cell division. To examine the potential of 3-polyphosphoinositides, which activate numerous down- p110␤ action in this process, we compared the division rates of NIH stream targets. PI3K controls cell division. Of the 2 ubiquitous PI3K 3T3 stable cell lines expressing p110␣ or ␤ active forms (Fig. 1A). ␣ isoforms, has selective action in cell growth and cell cycle entry, Active p110␤ cells divided more rapidly (t1/2 Ϸ18 h) than active but no specific function in cell division has been described for ␤.We p110␣ cells or controls (t1/2 Ϸ24 h; Fig. 1B). In addition, although report here a unique function for PI3K␤ in the control of DNA a small fraction of active p110␣ and ␤ cells enter cell cycle after replication. PI3K␤ regulated DNA replication through kinase-de- serum deprivation (6), only active p110␤ cells escaped cell contact ␤ pendent and kinase-independent mechanisms. PI3K was found in inhibition in confluence (Fig. S1A). We also compared synchro- ␤ the nucleus, where it associated PKB. Modulation of PI3K activity nous cell cycle progression in these cells. Cells were first serum- altered the DNA replication rate by controlling proliferating cell deprived (G0 arrest) and released by serum addition; using this nuclear antigen (PCNA) binding to and to DNA poly- Ϸ ␦ ␤ protocol, NIH 3T3 cells reach S phase at 9 to 12 h postrelease merase . PI3K exerted this action by regulating the nuclear (15). Active p110␤ cells were faster in terminating S phase than activation of PKB in S phase, and in turn phosphorylation of PCNA control or active p110␣ cells (Fig. 1C; Fig. S1B), as confirmed by negative regulator p21Cip. Also, p110␤ associated with PCNA and calculation of S phase duration (4 Ϯ 0.5 h for active p110␤ cells vs. controlled PCNA loading onto chromatin in a kinase-independent 5.5–6 h for active p110␣ cells and Ϸ6 h for control cells); three manner. These results show a selective function of PI3K␤ in the distinct clones behaved similarly. control of DNA replication. We also examined the consequences of reducing endogenous p110␣ and ␤ activity using inactive K802R-p110␣ and K805R- lass IA phosphoinositide 3-kinase (PI3K) is an that p110␤ mutants (KR hereafter) (6). Expression of KR mutants in Ccontrols cell cycle entry. Mutations in this pathway are exponentially growing NIH 3T3 cells reduced PKB phosphoryla- among the most frequent events in human cancer; a mayor tion (pPKB, Fig. 1D) and affected cell division; we were unable to objective in translational biology is to define PI3K isoform- prepare stable KR-p110␣ or ␤ lines. We expressed KR mutants by specific functions. The PI3K are comprised of a p85 regulatory retroviral infection (95% efficiency), which yielded levels similar to and a p110 catalytic subunit that mediates formation of endogenous p110 proteins (Fig. 1D). Cell division was significantly 3-polyphosphoinositides (1, 2). There are three class I p110 A slower in KR-p110␤ cells (Fig. 1E), which remained in S phase for catalytic subunits (␣, ␤ and ␦), but only p110␣ and ␤ are ␣ prolonged periods (Fig. 1F; Fig. S1C) and showed a longer S phase (Ϸ6 ubiquitous and essential for development (3, 4); enhanced p110 ␣ Ϸ ␤ and ␤ activity trigger cell transformation (5). p110␣ regulates cell h control cells; 6–6.5 h KR-p110 cells, 8 h for KR-p110 cells). ␤ p110␤ expression did not vary appreciably throughout the cell growth and cell cycle entry (6). In the case of p110 , the recent ␤ description of p110␤ conditional knockout mouse phenotype cycle. We examined the consequences of reducing p110 expression shows that p110␤ activity is essential for animal growth and using various shRNA and protocols in NIH 3T3 cells and human ␤ tumor development (7). Nonetheless, the cellular events selec- U2OS cells (Methods). Whereas efficient protocols for p110 ␤ tively controlled by p110␤ remain unknown. deletion interfered with cell viability, partial p110 reduction DNA replication controls the accurate, timely duplication of the permitted cell cycle progression studies. To reduce p110␤ expres- cell genome each time the cell divides. Preparation for replication sion in U2OS cells, we stably transfected pTER-shRNA vectors, requires formation of the origin replication complex (ORC) at the which allow inducible shRNA expression (16). shRNA reduced DNA replication origin. The ORC acts as a scaffold for assembly p110␣ and ␤ levels even before induction, but reduction was greater of the prereplicative complex that includes Cdc6 and Cdt1, proteins after doxycycline treatment (Fig. 1G). U2OS cells were synchro- involved in recruitment of the minichromosome maintenance nized at G1/S boundary by double thymidine block and examined (MCM) complex exhibiting activity. When MCM is loaded S phase progression after release. We confirmed slower cell cycle into the ORC, the pre-RC is licensed to initiate replication (8–12). entry in cells with reduced p110␣ or ␤ levels (6); in addition, only After licensing, replication initiation involves formation of the the cells with reduced p110␤ levels remained in S phase for preinitiation complex, which requires activation of Cdk2 and Dbf4/ prolonged periods, showing a Gaussian peak at mid-S phase DNA Cdc7 (13). These kinases phosphorylate the MCM and content at 6–7 h postrelease (Fig. 1G). induce binding of DNA (Pol)␣/, which triggers primer DNA synthesis (11). Elongation of DNA synthesis requires subsequent binding of the proliferating cell nuclear antigen Author contributions: A.C.C. designed research; M.M., A.K., A.M.P., S.Z., and C.H. per- MEDICAL SCIENCES (PCNA), a homotrimeric factor that triggers Pol␣ displacement and formed research; S.J. contributed new reagents/analytic tools; M.M., A.K., P.P., and A.C.C. tethers the processive (␦ and ␧) to the DNA template analyzed data; and A.C.C. wrote the paper. for rapid, accurate DNA elongation (9, 14). We examine here the The authors declare no conflict of interest. function of p110␣ and ␤ in DNA replication. This article is a PNAS Direct Submission. 1M.M. and A.K. contributed equally to this work. Results and Discussion 2To whom correspondence should be addressed. E-mail: [email protected]. ␤ ␣ The p110 Controls S-Phase Progression. p110 regulates G1 entry This article contains supporting information online at www.pnas.org/cgi/content/full/ and cell growth (1); both p110␣ and ␤ regulate late G1 events and 0812000106/DCSupplemental.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0812000106 PNAS ͉ May 5, 2009 ͉ vol. 106 ͉ no. 18 ͉ 7525–7530 Downloaded by guest on October 5, 2021 B

A BCActive Active A Control Udr NIH3T3 p110β p110α Control Active -5 Active 5 β 50 AND + Active Ctr p110 Ctr p110β p110α 40 BU 4 (*) r Ud p110β p110α 3 Control 30 Active BA Active Active 2 20 dr Ctr p110β Ctr p110α p110α PIK U

pPKB Cell numberx 10 1 10 Percent S phase cells PKB 0 0 AND + 0 3 6121518219 24 0132 4 BU r Time, d Time from G0, h d drB DE F TGX U KR KR 4 NIH3T3 Control 50 Control p110α p110β ND + Mock KR KR -5

KR B Ctr p110α p110β 3 α 40 p110 dr α (*) p110 30 2 KR p110β p110β 20 pPKB 1

Cell numberx 10 10 PKB Percent S phase cells B 50.1 0 0 Ctr Ctr 111.3 01 32 4 0 3 69 1215182124 Time, d Time from G0, h G PIK 53.9 PIK 113.6 U2OS % G0/G1 α shRNA shRNACtr 32 34 19 TGX 28.4 TGX 79.9 + _ + _ Dox 37 % S 40 22 41 55 43 p110α 50 55 50 40 59 0 50 100 150 0 100 200 300 pPKB 55 29 65 48 % G2/M 34 45 PKB 40 25 36 BrdU track length (kb) Center-to-center distance (kb) 19 21 27 16 shRNAα shRNACtr 11 21 10 .godnE + _ + _ Dox 9 7h 21 7h 10 7h .godnE β β α 9 5h 21 5h 9 5h DAPI shRNAα DAPI shRNAβ α 011p 011p 011p p110β 3h 3h 3h C 011p 0h 0h 0h Time pPKB cDNA shRNA shRNA shRNA G1/S PKB release control p110α p110β p110α p110α p110β p110β Fig. 1. Interference with p110␤ alters S phase progression. (A) NIH 3T3 stable cell clones expressing active-p110␣ or ␤ were examined in Western blotting (WB). Histone (B) Control NIH 3T3 cells, active p110␣ and active p110␤ cells were seeded at shRNA Tubulin similar densities and counted at 24 h intervals (mean Ϯ SD, n ϭ 6). (C) Percentage shRNA Control CYTOSOL NUCLEUS of cells with an S phase DNA cell content at different times after release from G0 Control arrest (mean Ϯ SD; n ϭ 5). (D) NIH 3T3 cells transfected with KR-p110␣ or ␤ were Fig. 2. p110␤ is a nuclear protein and regulates DNA elongation. (A) Single- examined in Western blot (WB) at 24 h posttransfection. (E) Cell division time for molecule analysis of DNA replication in synchronized NIH 3T3 cells treated with control or KR-p110␣- and KR-p110␤-infected NIH 3T3 cells (mean Ϯ SD, n ϭ 3) as PIK75 or TGX221 inhibitors at 7 h postserum addition, pulse-labeled (20 min) with in B.(F) Percentage of S phase cells (mean Ϯ SD, n ϭ 5) of control, KR-p110␣- and BrdU at 12 h postserum addition, and collected immediately for analysis. Genomic ␤-infected cells, as in C.(G) U2OS clones expressing control, -␣,or-␤ shRNA were DNA fibers were stretched by DNA combing. Newly replicated DNA was detected induced with doxycycline for 48 or 120 h, respectively; p110␣ or ␤ expression was by immunofluorescence with an anti-BrdU Ab (green); DNA fibers were coun- examined in WB. Cells were subjected to thymidine block and released for terstained with anti-DNA Ab (red). Representative fibers are shown. (Scale bar, 50 different times, the profiles show cell cycle distribution. , P Ͻ 0.05. * Kb.) (B) Distribution of BrdU track length and center-to-center distances between adjacent BrdU tracks. Box: 25–75 percentile range. Whiskers: 10–90-percentile range. [Vertical bar, median value (kb).] , Mann–Whitney rank sum test P Ͻ We used the selective inhibitors PIK75 and TGX221 to inhibit *** ␣ ␤ 0.0001. (C) NIH 3T3 cells were cotransfected with red fluorescence protein (RFP) p110 and , respectively (17, 18). We confirmed inhibitor selec- and control, p110␣,or-␤ shRNA; p110 localization was examined by immuno- tivity in NIH 3T3 cells (Fig. S2 A–D). Inhibition using 0.5 ␮M PIK fluorescence. DAPI nuclear staining is shown in Insets. (Scale bar, 10 ␮m.) NIH 3T3 resulted in complete blockade of S phase entry and triggered cells or WT-p110␣ or -␤ transfected cells were fractionated and examined in WB apoptosis (Fig. S2E), showing that p110␣ is needed for cell survival (Right). (19). p110␣ inhibition (0.08 ␮M PIK) near S phase permitted cell cycle entry (Fig. S2F) although it impaired G2/M entry, suggesting ␤ that p110␣ could be the isoform that acts in mitosis (1). This or blockade in late G1 without affecting prior events. treatment nonetheless allowed S phase progression, as indicated by G0-synchronized NIH 3T3 cells were serum-released, treated the increased proportion of S phase cells and displacement of the with PIK 75 (0.08 ␮M) or TGX 221 (30 ␮M) at 7 h, BrdU- S phase population from near-G1 to near-G2 DNA content over labeled (20 min) at 12 h, then collected to examine the the time course (Fig. S2F). In contrast, selective inhibition of p110␤ replication profile (Fig. 2A). For each sample, we analyzed permitted G2/M entry but extended S phase compared with Ϸ30 MB of individual DNA fibers (Ͼ250 kb). TGX-treated controls (Fig. S2F). cells showed 43% reduction in the length of BrdU tracks relative to controls, suggesting that p110␤ is required for The p110␤ Activity Controls DNA Elongation. To compare S phase normal replication fork progression; in contrast, elongation progression rates more accurately we BrdU-labeled (1 h pulse) was not significantly affected by p110␣ inhibition (Fig. 2A). newly synthesized DNA in exponentially growing cells and Median center-to-center distance between adjacent BrdU collected cells at various times after BrdU deprivation. While tracks, indicative of the initiation rate, was shorter in TGX- most BrdUϩ control and KR-p110␣ cells reached G2/M at 3 to than in PIK-treated cells or in controls (Fig. 2B), consistent 5 h, the majority of BrdUϩ KR-p110␤ cells remained in S phase with cell activation of additional replication origins to com- at5h(Fig. S3A). We examined the consequences of impaired pensate slow fork progression (21). The percentage of repli- p110␤ function on DNA elongation with the DNA combing cation of individual DNA fibers was lower in TGX- (20.7%) assay (20, 21). We used PI3K inhibitors, as they permit p110␣ than in control or PIK-treated cells (33.0 and 32.3%). These

7526 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0812000106 Marque´s et al. Downloaded by guest on October 5, 2021 Active Active A CYTOSOL NUCLEUS CHROMATIN B Control p110α p110β 0 9 12 14 091214 0 9 12 14 Time, h 09120 9 12 0 9 12 Time, h MCM2 PCNA MCM4 δ

Control in Pol IP α MCM2 PCNA in Chr δ KR- p110 MCM4 Pol ␤ β MCM2 Fig. 3. p110 controls PCNA binding to chromatin and PCNA to DNA Pol␦.(A) NIH 3T3 cells were infected with KR-

KR- p110 MCM4 13 16 25 14 23 31 503818 %S Phase p110␣-or-␤-encoding viruses, synchronized and col- lected at different times. MCM2/MCM4 levels in cytosolic, nuclear and chromatin fractions were examined in WB. C α β D α2 β2 Control KRp110 KRp110 shRNACtr shRNA shRNA (B) Active p110␣- and -␤ cells and control NIH 3T3 cells 0 912 0912 0912Time, h 09120912 0912Time, h were synchronized in G0 and released for different times. PCNA PCNA PCNA in Pol␦ immunoprecipitates and PCNA levels were in Polδ IP in Polδ IP ␦ PCNA in Chr PCNA in Chr examined in the chromatin fraction, total Pol and PCNA were also examined in WB. (C and D) NIH 3T3 transfected Polδ Polδ with KR-p110␣ or -␤ (C) or with control, -␣,or-␤ shRNA PCNA PCNA (D) were collected and examined at the indicated times. 10 19 28 7 10 18 7 2212 %S Phase 10 18 45 12 15 49 1410 29 %S Phase Analyses were as in B (n ϭ 3). (E) Active p110␣- and ␤-expressing NIH 3T3 cells were synchronized in G0 and E F released for different times. PCNA and p21Cip levels in Active Active Control Control p110α p110β KRp110α KRp110β p21Cip immunoprecipitates and total PCNA levels in chro- 0 09120 9 12 0 9 12 Time, h 912 0912 0912Time, h matin-free extracts were examined in WB. (F) NIH 3T3 PCNA PCNA cells transfected with KR-p110␣or -␤were examined as in in p21Cip IP in p21Cip IP E. Chr, chromatin. Percentage cells in S phase indicated Cip Cip p21 in IP p21 in IP below gels. The circles show the time for S phase entry. PCNA PCNA 10 19 (A–F) One representative experiment of at least three 14 17 28 13 24 34 432913 %S phase 28 7 10 18 7 2212 %S phase with similar results.*, P Ͻ 0.05.

results suggest that p110␤ activity controls replication fork chromatin as well as PCNA-Pol␦ association; interference with progression. p110␣ only had a modest inhibitory effect. These data show that p110␤ controls PCNA binding to chromatin and to Pol␦, providing The p110␤ Is Located in the Nucleus and Controls PCNA Binding to a potential mechanism for DNA elongation impairment after Chromatin. Since DNA replication occurs in the nucleus, we exam- interference with p110␤ function. ined p110␤ localization. Subcellular fractionation (Methods) and immunofluorescence analysis showed that the majority of endog- The p110␤ Activity Regulates p21Cip Phosphorylation. PCNA loads enous p110␤, but not of ␣, concentrated in the NIH 3T3 cell nucleus Pol ␦ and ␧ to the DNA template for efficient elongation; PCNA (Fig. 2C). Both the nuclear p110␤ signal and the mainly cytosolic also binds p21Cip through the same region, p21Cip thus impairs p110␣ signal decreased with selective shRNA (Fig. 2C; Fig. S3B). PCNA association to Pol␦/␧ (22, 24). We examined PCNA-p21Cip A similar distribution was observed in MEF, COS-7, HeLa and complex formation in cells with altered p110␤ activity. Whereas U2OS cells. These results indicate that p110␤ concentrates in the interference with p110␣ did not appreciably affect PCNA-p21Cip nucleus. complexes, active-p110␤ reduced (Fig. 3E) and inactive p110␤ (or We examined the mechanisms by which p110␤ regulates repli- p110␤ inhibition) increased PCNA-p21Cip association (Fig. 3F, Fig. cation. One of the first events required to initiate replication is S4A). Phosphorylation of p21Cip on T145 and Ser-146 phosphor- MCM complex loading on origins (replication licensing, 8,13,22). ylation (by PKB and PKC) regulates its dissociation from PCNA We compared MCM loading to chromatin by cell fractionation on (25–28), nonetheless, in vivo T145 appears to be the critical residue nuclear and chromatin extracts (23). Whereas in control cells, (27, 28). We confirmed that T145 phosphorylation induced PCNA- MCM 2/4 appeared on chromatin fractions in exponential growth, p21Cip dissociation in U2OS cells and NIH 3T3 cells (Fig. 4A); but not after GF starvation or in confluence, active p110␣ or ␤ expression of the phosphomimetic D145-p21Cip mutant reduced expression induced a similar and moderate enhancement of MCM PCNA-p21Cip association increasing PCNA binding to chromatin 2/4 loading onto chromatin in starving and confluence conditions (Fig. 4A). (Fig. S3C). Accordingly, KR-p110␣ or ␤ expression induced a slight We also examined whether p110␤ regulates T145 phosphoryla- reduction in late G1 MCM loading (Fig. 3A). MCM loading onto tion. Whereas in control cells T145 was phosphorylated near S chromatin is thus modulated to some extent by p110␣ and ␤, but is phase entry, both p110␤ shRNA and KR-p110␤ expression reduced not selectively controlled by p110␤. pT145-p21Cip levels (Fig. 4B; Fig. S4 B–D). In these assays we observed that interference with p110␤ activity also resulted in The p110␤ Activity Regulates PCNA Loading onto Chromatin. After greater p21Cip expression levels. p21Cip is degraded after its release replication origin activation, Pol␣ binding to the ORC triggers from PCNA (29); the higher p21Cip levels in cells with impaired primer DNA synthesis; elongation of DNA synthesis requires p110␤ function might be due to stabilization of p21Cip in complex subsequent binding of PCNA that tethers the processive poly- with PCNA. Both KR-p110␤ and p110␤ shRNA expression in-

merases Pol␦ and ␧ to the DNA template (9, 14). In control creased p21Cip protein stability (Fig. S5A), whereas active p110␤ MEDICAL SCIENCES synchronized NIH 3T3 cells, we observed PCNA appearance in reduced p21cip stability (Fig. S5B). p110␤ activity is thus needed for chromatin extracts (22) as well as PCNA-Pol␦ association at Ϸ12 h p21Cip phosphorylation and dissociation from PCNA. after GF addition, at the onset of S phase (Fig. 3B). Active p110␣ cells behaved similarly; in contrast, active p110␤ expression accel- The p110␤ Regulates Nuclear PKB. The PI3K effector PKB phos- erated PCNA binding to chromatin and PCNA-Pol␦ association phorylates T145-p21Cip (27, 28). We confirmed that PKB phos- (Fig. 3B). Moreover, expression of KR-p110␤ (Fig. 3C), reduction phorylates T145-p21Cip in vitro (Fig. S6A) and examined whether of p110␤ levels with shRNA (Fig. 3D; Fig. S3D) and p110␤ p110␤ regulates PKB-mediated T145-p21Cip phosphorylation. We inhibition (Fig. S3 E and F) diminished PCNA loading onto found that expression of KR-p110␤ (or p110␤ inhibition) reduced

Marque´s et al. PNAS ͉ May 5, 2009 ͉ vol. 106 ͉ no. 18 ͉ 7527 Downloaded by guest on October 5, 2021 A B inhibition had a lesser effect (Fig. 4C); results were similar in U2OS Cip shRNACtr shRNAα shRNAβ cells (Fig. S6C). As an alternative approach, we examined pPKB by Ctr A145 D145 A146 D146 p21 Time, h 09 0 9 09 PCNA in pThr145 immunofluorescence. At 1 h postserum addition (G1 phase) pPKB Myc-p21Cip IP in p21Cip IP PCNA in Chr concentrated at the cell membrane and was reduced by KR-p110␣ p21Cip in IP PCNA Actin (Fig. S7A), whereas in S phase pPKB concentrated in the nucleus Myc-p21Cip 13 35 12 24 9 19 %S phase and was notably reduced by KR-p110␤ and p110␤ inhibition (Fig. rtC β α piC (*)

p ANRhs AN AN 4D; Fig. S7B). Cell fractionation confirmed TGX inhibition of S i (*) (*) rhC ni rhC 12p C Rh R 1 100 100 (*) s sh 100

2 / phase nuclear pPKB (Fig. S7C). p / / p α 50 50 p110 piC 50 ANCP A p110β 12p541Tp ␤ NCP 0 0 0 We examined other PKB substrates in S phase; GSK3 phos- 641D enoN 641D 541D 541D enoN 541A 541A 641A 641A Actin 09 09 09 ␤ Control shRNAα shRNAβ phorylation was reduced by p110 inhibition, whereas FKHRL1 phosphorylation was p110␣ activity-dependent (Fig. S7D), as is the 9 h serum

t case in G1 phase (6). WB using anti-pPKB substrate Ab showed n α β 122XGT 122XGT ec lo 011pR 011pR 57 KIP KIP 57 57

C nim s r

tnoC ␤ eiuQ that p110 inhibition reduced phosphorylation of some PKB 0 K 3 K substrates in S phase cells (such as p21Cip, Fig. S7C), while others pPKB ␣ PKB were p110 -regulated (Fig. S7D). Results were similar using S phase U2OS cells treated with PI3K inhibitors and then fraction- D ated (Fig. 4E); this assay also showed that p110␣ inhibition affected Cell 1 ␤ Control mainly cytosolic substrates and p110 nuclear substrates, suggesting Cell 3 KRp110β that p110␣ and ␤ control distinct PKB pools. p110␤ thus governs Cell 2 KRp110α nuclear S phase PKB activity. Since p110␣ is activated at the G1/S boundary (6), the early timing of phosphorylation of some PKB KR-p110α KR-p110β substrates or their cytosolic localization might determine a p110␣ )UA( ytisnetni )UA( ecnecserou Cell 1 Control Cell 2 KRp110α Cell 3 KRp110β activity requirement for phosphorylation. 150 Based on p110␤ regulation of S phase nuclear pPKB-mediated Cip 0 p21 phosphorylation, expression of the phosphomimetic D145- l 010200102001020 F Distance, µm p21Cip mutant in cells with impaired p110␤ activity could replace CYTOSOL NUCLEUS p110␤ activity in S phase. BrdU labeling of newly-synthesized DNA E ControlPIK 75 TGX221 ControlPIK 75 TGX221 ␤ Time, h in exponentially growing cells expressing KR-p110 alone or in 122 121 122 121 Cip Cip MW MW G1/S combination with D145-p21 showed that D145-p21 expression 75 75 accelerated S phase progression in KR-p110␤ cells (Fig. 4F). 50 50 D145-p21Cip expression also increased PCNA-Pol␦ association and 37 37 Cip ␤ 25 reduced PCNA-p21 complexes in KR-p110 cells (Fig. S8A). 25 Accordingly, A145-p21Cip expression corrected PCNA-Pol␦ com- WB: pPKB substrate WB: pPKB substrate plexes in active p110␤ cells (Fig. S8B). Thus, expression of phos- Cip Control Control + D145p21Cip phomimetic p21 mutants corrects the S phase defects of cells with α α Cip F KRp110 KRp110 + D145p21 100 n S 100

S ␤ ni gni ni KRp110β KRp110β + D145p21Cip altered p110 activity. i gni i

nia (*) 50 niame 50 PI3K␤ Protein Regulates PCNA Loading onto Chromatin. The recently me Ϫ/Ϫ R R % % R % % described conditional p110␤ mouse phenotype and that of 0 0 0375 9 0375 9 inactive p110␤ knock-in mice (7, 30) indicate that p110␤ kinase Chase Chase time, h time, h activity regulates mouse growth and tumor development and also that p110␤ has a kinase-independent function in embryonic devel- Fig. 4. p110␤ controls nuclear PKB. (A) NIH 3T3 cells transfected with A145, Cip opment. Kinase-independent functions often reflect the ability of a A146, D145, or D146 p21 mutants were fractionated. PCNA levels were mea- ␥ sured in p21Cip immunoprecipitates, chromatin-containing and -free fractions; protein to associate a necessary partner, as is the case for PI3K in p21Cip expression was also examined in chromatin-free fractions. Graphs show the control of cardiac stress response (31). We examined whether the percentage PCNA signal (mean Ϯ SD) in p21Cip immunoprecipitates and that p110␤ expression (independent of its kinase activity) regulates of PCNA in chromatin fractions, compared with the maximum PCNA signal in DNA elongation, studying the extent of PCNA binding to chroma- each case (n ϭ 3). (B) NIH 3T3 transfected with control, p110␣,or␤ shRNA were tin after p110␤ inhibition or p110␤ knockdown. To improve p110␤ synchronized in G0 and released (9 h). WB shows pT145-p21Cip in p21Cip immu- deletion, we transfected cells with puromycin-shRNA-encoding noprecipitates from chromatin extracts; graphs show the pT145-p21Cip signal vectors, selected them for 48 h and immediately analyzed these Ϯ Cip (percentage SD) normalized to p21 levels and compared with the signal at 9 h asynchronous cultures (synchronization requires longer culture in controls (100%; n ϭ 3). WB (bottom left) shows p110 expression levels. (C) NIH times) before reduction of cell viability. Pulse–chase BrdU analysis 3T3 cells transfected with KR-p110 mutants were synchronized after 24 h and ␤ other cells were treated at 7 h with TGX221 (30 ␮M) or PIK75 (0.08 ␮M); cells were in exponentially growing NIH 3T3 cells showed that p110 inhi- collected at 9 h. pPKB levels were measured in WB. (D) pPKB localization exam- bition reduced S phase progression, but p110␤ knockdown had a ined by immunofluorescence in cells cotransfected with KR-p110 mutants and greater effect in decelerating S phase (Fig. 5A). PCNA loading onto RFP, fixed 9 h after G0 release. Graphs show fluorescence intensity in arbitrary chromatin was also reduced by p110␤ or PKB inhibition, but was units (AU) examined along the line in the images. Insets show expression of KR drastically diminished by p110␤ knockdown (Fig. 5B). mutants. (Scale bar, 50 ␮m.) (E) Phosphorylation of PKB substrates was examined We also analyzed asynchronous cultures of p110␤Ϫ/Ϫ immortal- by WB in fractionated extracts of U2OS cells that were thymidine-arrested, then ized mouse embryonic fibroblasts (MEF) reconstituted with WT or released (1 and 2 h). (F) NIH 3T3 cells expressing KR-p110␤ or ␣ mutants alone or ␤ ␤ Cip KR-p110 (7). KR-p110 MEF progressed through S phase more in combination with D145-p21 were BrdU-labeled and chased at different ␤ ␤Ϫ/Ϫ times. Graph shows the cell percentage remaining in S phase (mean Ϯ SD, n ϭ 3). slowly than WT p110 MEF, although p110 MEF showed the slowest S phase progression (Fig. 5A). KR-p110␤ MEF had less *, P Ͻ 0.05. chromatin-bound PCNA than controls, but PCNA loading was lowest in p110␤Ϫ/Ϫ MEF (Fig. 5B). These results suggest that PCNA S phase PKB kinase activity in vitro (Fig. S6 A and B). Western blot loading onto chromatin and in turn S phase progression rate is analysis of pPKB in extracts from synchronized NIH 3T3 cells further regulated via a kinase-independent p110␤ function. expressing KR-p110␤ or treated near S phase with TGX221 pPKB was little affected by p110␤ deletion in asynchronous confirmed that p110␤ regulates S phase pPKB, whereas p110␣ cultures (7). To define whether p110␤ controls nuclear PKB in S

7528 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0812000106 Marque´s et al. Downloaded by guest on October 5, 2021 phase in these MEF, we synchronized cells at the G1/S border and 3T3 MEF

A % gniniameR ni S 100 S ni gniniameR % % gniniameR ni S 100 examined them after release. In WT p110␤-reconstituted MEF, Control WTp110β (*) TGX KRp110β pPKB was found mainly in the nuclear fraction in S phase; β (*) −/− shRNA p110β 50 50 KR-p110␤ MEF behaved similarly but had lower nuclear active (*) (*) pPKB levels (Fig. 5C). Both nuclear pPKB and PKB were unde- ␤Ϫ/Ϫ ␤ 0 0 tectable in p110 MEF (Fig. 5C), indicating that p110 expres- 0642 Chase time, h 0642 Chase time, h sion might control PKB nuclear entry. Cytosolic pPKB was more abundant in p110␤Ϫ/Ϫ MEF, but they expressed lower levels of B 3T3 MEF 3T3 MEF β .hnI BKP .hnI

β PTEN (Fig. 5C); this might represent a compensatory mechanism 011 0 −/− (*) lortnoC

rhC ot dnuob ANCP % ANCP dnuob ot rhC 1 A (*) (*) 1pRK β β (*) (*) ␤ NRhs p XGT 0 01 100 100 for p110 deletion. We also analyzed nuclear/cytoplasmic distribu- TW 1 1 1 p p tion of pPKB and PKB in NIH 3T3 cells to further examine whether PCNA 50 in Chr 50 p110␤ deletion reduces not only nuclear phospho-PKB but also PCNA nuclear PKB, as in MEF. p110␤ shRNA diminished but did not 0

0 lor .hnI BKP .hnI −/− β β A XGT β 0 011p 0 N β t completely eliminate nuclear PKB (Fig. S8C). These results do not β

p110 11p 11 no Rhs 011 p C T R

p demonstrate, but suggest that PKB nuclear entry is facilitated by K W p110␤ expression, an aspect that requires further study. In contrast, CYTOSOL NUCLEI C −/− −/− ␤ ␤ WTp110β KRp110β p110β WTp110β KRp110β p110β both p110 inhibition and p110 shRNA expression clearly re- Time, h 011 301330 011 301330 G1/S duced S phase nuclear pPKB (Fig. S8C), further confirming the pPKB function of p110␤ in control of nuclear PKB activity in S phase. PKB PKB PI3K␤ Protein Associates PKB and PCNA. To determine whether PTEN ␤ )UA( BKPp BKPp )UA( 100 100 p110 -dependent PKB nuclear activity is due to direct association, RAELCUN )UA( BKP (*) BKP )UA( (*) R

A ␤ 50 ELCUN 50 we studied PKB-p110 complex formation in cytosolic and nuclear fractions. Cells were fractionated as described (32), since the 0 0 Time, h 011303013011303013G1/S −/− −/− method used earlier (Fig. 2C) (33) destroys protein–protein inter- WTp110β KRp110β p110β WTp110β KRp110β p110β actions. NIH 3T3 cells were cotransfected with HA-gagAKT and

1 2 lortnoC 3 NUCLEI CYTOSOL ␣ ␤

WT-p110 or - , collected at 12 h post-G0 release, and examined.

D l l o o β α β α r r t t 0 0 01 Extracts 0 ␣ B B n n Although PKB and p110 associated in cytosol, this association was 11p 1 11p o o K K 1 CYTOSOL NUCLEI 1 IP P p C C p P (*) ␤ β 100 lower than that of PKB and p110 , and was not found in the BK p110α 011 PKB P 50 ␤ t

p nucleus, where only PKB-p110 complexes were observed (Fig. ot dnu ot

p110β n e

c S9A). We also analyzed association of endogenous proteins in

reP 20 PKB

PKB o synchronized NIH 3T3 cells collected at 12 h postserum addition.

b 0 β β α α 3 rt 3 1 1 2 2 BKP B 011p 011p 011p long 011p r r K Tubulin tC t ␣

P WB analysis of the fractions confirmed that p110 was mainly C exposure IP C ␤ Histones p85 NUCLEI CYTOSOL cytosolic and p110 was more abundant in the nucleus (Fig. 5D). Although immunoprecipitation concentrated the scarce nuclear 2 1 2 1 ␣ ␤ lortnoC l l

lo p110 protein, endogenous PKB associated mainly with p110 in o ort β α ANCP 100 AN 100 (*) rtnoC rtnoC 0 0 β E α n 11p 1 C oC the nuclear fraction (Fig. 5D). 11p 1p IP IP 1 P 1 p tne t 50 50 To identify other nuclear proteins that regulate DNA replication n ecr

p110α c p110β re and associate to p110␤, we performed a pull-down assay using e P0 P0 0 0 ␤ β 1 2 2 1 2 α A ANCP

mammalian GST-p110 ; we obtained a number of candidate 011p r r 01 r r NCP tC t tC IP IP tC C 1

p proteins including PCNA. Immunoprecipitates of endogenous PCNA from nuclear extracts contained associated endogenous Fig. 5. p110␤ associates with PKB and PCNA. (A) NIH 3T3 transfected with p110␤ but not p110␣ (Fig. 5E); results were similar in a reciprocal control or p110␤ shRNA were selected with puromycin (2 ␮g/mL, 48 h), then assay (Fig. S9B). To determine whether the selective association of examined. Other samples were treated with TGX221 or PKB inhibitors for 12 h PCNA with p110␤ was due to a p110␤-specific structural feature or before collection. Immortalized p110␤Ϫ/Ϫ mouse MEF, and p110␤Ϫ/Ϫ MEF ␤ to its subcellular distribution, we inserted a nuclear localization reconstituted with WT- or KR-p110 were cultured in exponential growth. A ␣ ␤ fraction of the cells were pulsed-labeled with BrdU (1 h). Graphs show the signal (NLS) in p85 and cotransfected it with myc-WT-p110 or - , percentage of cells remaining in S phase at each chase time (mean Ϯ SD, n ϭ which increased their nuclear localization. Both nuclear p110␤ and 3). (B) Lysates of cells treated as in A were analyzed in WB to determine PCNA ␣ associated with PCNA, although p110␤ association to PCNA was in the chromatin fraction, as well as PCNA and p110␤ in the chromatin-free greater than that of nuclear p110␣ (Fig. S9C). Therefore, in fraction. Graphs show the percentage of chromatin-bound PCNA normalized addition to its subcellular distribution, p110␤ has a structural to total PCNA and compared with maximum signal in control NIH 3T3 or in advantage for association to PCNA. MEF. (C) Immortalized MEF as in A were arrested by thymidine treatment, then Here, we describe a role for p110␤ in replication fork elongation released for different times. Cell fractions were examined in WB to test for in mammalian cells, providing an example of elongation control by pPKB and PKB levels; the latter was then reprobed for PTEN. The graphs show Ϯ ϭ extracellular signal-regulated molecules. The nuclear localization nuclear pPKB or PKB signal in arbitrary units (AU) (mean SD, n 3). (D) ␤ Synchronized NIH 3T3 cell cultures collected at 12 h postserum addition were and function of p110 resembles that of class IV PI3K, which are fractionated. The levels of PKB, p110␣ and ␤ in these fractions were examined recruited to DNA damage sites and mediate cell responses as DNA by WB (Left). Endogenous p110␣ or ␤ from cytosolic (1500 ␮g) and nuclear repair (34). Although some cell cycle phenotypes were moderate extracts (600 ␮g), or PKB from cytosolic (300 ␮g) and nuclear extracts (200 ␮g) (Fig. 1), complete p110␤ elimination interfered with cell survival, were immunoprecipitated. We tested for PKB and p85 in p110 immunopre- and p110␤ function was studied in partial p110␤ deletion condi- MEDICAL SCIENCES cipitates by WB. Controls 1–3, protein A plus each of the antibodies. Graph tions. p110␤ regulated DNA replication through kinase-dependent shows the percentage of p110-associated PKB signal, compared with maximal and -independent mechanisms. p110␤ associated with PKB, and PKB signal (in PKB immunoprecipitates from an equivalent protein amount). p110␤ activity regulated nuclear PKB-mediated p21Cip phosphor- (E) Nuclear fractions were obtained from synchronized NIH 3T3 cells (at 12 h). ␦ ␮ ␮ ylation, PCNA release, PCNA binding to Pol and replication PCNA (800 g) or p110 (200 g) immunoprecipitates were tested in WB for ␣ p110. For control 1, protein A was incubated with Ab; control 2, protein A was elongation. Interference with p110 activity had a slight inhibitory Cip incubated with lysate. Graphs show the percentage of p110 signal in PCNA effect on p21 phosphorylation, and might partially compensate immunoprecipitates compared with maximal p110 signal (p110 immunopre- for p110␤ activity-dependent functions. In addition, p110␤ associ- cipitated from an equivalent protein amount). *, P Ͻ 0.05. ated with PCNA and controlled PCNA loading onto chromatin in

Marque´s et al. PNAS ͉ May 5, 2009 ͉ vol. 106 ͉ no. 18 ͉ 7529 Downloaded by guest on October 5, 2021 a kinase-independent manner. Since PCNA loading onto chroma- iodide and analyzed by flow cytometry (Beckman-Coulter) using Multicycle AV tin is essential for DNA duplication, this kinase-independent func- (Phoenix Flow Systems). Cells were synchronized at G1/S by double thymidine tion explains the greater division defects in cells with reduced p110␤ block (6) or using aphidicolin (22). To determine cell division time (t1/2), cells were ␤ seeded at similar densities and counted at 24 h intervals. S phase duration was expression. The role of p110 in DNA replication could contribute ϭ ␤ calculated considering t1/2 (mean of n 6) and the proportion of cells in S phase to cause the early lethality (E2–3, ref.4) of p110 -deficient mice. in exponential growth (mean of n ϭ 12). S phase progression rates were exam- ined in exponentially growing cultures incubated with 20 ␮M bromodeoxyuri- Materials and Methods dine (BrdU; 1 h), chased at different times and stained with BrdU-FITC Ab (BD Complementary DNA and shRNA. pSG5-p110␣CAAX (active p110␣), pSG5-HA- Biosciences), then examined by three-dimensional FACS. wt-PKB and -gag-PKB were described (5, 35). pCEF2-hp110␤CAAX (active p110␤) For dynamic molecular combing, synchronized NIH 3T3 cells were treated with was a gift of Dr. Murga (Centro de Biología Molecular/CSIC, Madrid, Spain). 0.08 ␮M PIK75 or 30 ␮M TGX221 at 7 h postserum addition; 20 min before harvest PcDNA-Myc -WT and p21Cip mutants were donated by Dr. Ro¨ssig (28). pcDNA (12 h postserum addition), cells were treated with 20 ␮M BrdU. After harvest, cells Myc-S146A/T145A double mutant was generated using Quick Change Site- were embedded in LMP agarose plugs (3 ϫ 106 cells/plug) and DNA fibers were Directed mutagenesis (Stratagene). Myc-K802R-hp110␣ and myc-K805R-hp110␤ purified and stretched on silanized coverslips as described (21). BrdU tracks were mutants were subcloned into pSG5 and pRV-IRES-GFP for retroviral infection (6). detected with rat monoclonal Ab (clone BU1/75; AbCys) and an Alexa 488- We used several specific short hairpin RNA (shRNA) directed to human or murine conjugated secondary Ab (Molecular Probes). DNA fibers were counterstained p110 sequences, each assay was performed at least with two shRNA, with similar with mouse anti-ssDNA (MAB3034, Chemicon) and Alexa 546-secondary Ab results. These shRNA (6) were subcloned in pBluescript/U6 or in pTER vector; we (Molecular Probes). Signals were analyzed with MetaMorph. used control shRNA that did not reduce p110␣ or ␤ expression. We also used Statistical analyses were performed using StatView 512ϩ (Calabasas, CA). Gel Pik3cb shRNA (Origene; Fig. 5). To prepare NLS-p85, the PKKKRKV sequence was bands and fluorescence intensity were quantitated with ImageJ software. Sta- inserted 3Ј of the p85 sequence. tistical significance was calculated using Student’s t test. For DNA combing, statistical analysis was performed with GraphPad Prism 5.0 (GraphPad Software). Cell Lines, Cell Culture, and Retroviral Transduction. Active p110␣ and active For description of antibodies and reagents, cell lysis, subcellular fractionation, Western blotting, immunoprecipitation, and kinase assays, see SI Methods. p110␤ NIH 3T3 cells lines were described (6). KR-p110␣ and ␤ mutations were transduced by transient transfection or retroviral infection. We generated pTER- ␤Ϫ/Ϫ p110␣ or pTER-p110␤ U2OS clones according to manufacturer’s protocol (Invitro- ACKNOWLEDGMENTS. We thank Drs. Roberts and Zhao for sharing p110 immortal MEF, M. White for the myc-p110 plasmid, C. Murga for pCEFL -p110␤- gen); shRNA expression was induced for 2 days (p110␣) or 5 days (p110␤)in 2 CAAX, B. Vanhaesebroeck for His-p110␤, M. van de Wetering for the pTer vector, ␮ medium plus doxycycline (6 g/mL, Sigma). NIH 3T3 murine fibroblasts, U20S and Y. Shi for the pBlue/U6 plasmid, A. Klippel for anti-p110␣,J.Me´ ndez for help in COS7 cells were cultured as described (6). For retrovirus production, Phoenix cells chromatin purification, as well as E. Schwob and the DNA combing facility were transfected using JetPei-NaCl (Qbiogene). MEF were donated by Drs. Zhao (Montpellier) for silanized coverslips, and C. Mark for editorial assistance. M.M. and Roberts (7) (Dana Farber Cancer Institute, Boston, MA). has a predoctoral Formacion de Profesorado Universitario fellowship from the Spanish Ministry of Science and Innovation, and A.M.P. a postdoctoral fellowship from the Fondation Recherche Medicale. This work was supported in part by Cell Cycle, BrdU Labeling, Immunofluorescence, and Dynamic Molecular Comb- grants from the American Institute for Cancer Research Foundation, the Funda- ing. Immunofluorescence and NIH 3T3 G0 synchronization were as reported (15). cio´n Ramo´n Areces, the Asociacion Espan˜ola de la Lucha Contra el Cancer, the Briefly, cells were incubated in serum-free medium (19 h) and released by serum Centre National de la Recherche Scientifique, and the Spanish Direccio´n General addition. Cell cycle distribution was examined by DNA staining with propidium de Ciencia y Desarrollo Tecnologico Grants SAF2004-05955 and SAF2007-63624.

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7530 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0812000106 Marque´s et al. Downloaded by guest on October 5, 2021