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PNAS 106 (18): 7525-7530

Copyright © 2009 by the National Academy of Sciences.

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

Miriam 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 de Madrid, 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


Figure 1
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  		Fig. 1. Interference with p110β alters S phase progression. (A) NIH 3T3 stable cell clones expressing active-p110{alpha} or β were examined in Western blotting (WB). (B) Control NIH 3T3 cells, active p110{alpha} and active p110β cells were seeded at similar densities and counted at 24 h intervals (mean ± SD, n = 6). (C) Percentage of cells with an S phase DNA cell content at different times after release from G0 arrest (mean ± SD; n = 5). (D) NIH 3T3 cells transfected with KR-p110{alpha} or β were examined in Western blot (WB) at 24 h posttransfection. (E) Cell division time for control or KR-p110{alpha}- and KR-p110β-infected NIH 3T3 cells (mean ± SD, n = 3) as in B. (F) Percentage of S phase cells (mean ± SD, n = 5) of control, KR-p110{alpha}- and β-infected cells, as in C. (G) U2OS clones expressing control, -{alpha}, or -β shRNA were induced with doxycycline for 48 or 120 h, respectively; p110{alpha} or β expression was examined in WB. Cells were subjected to thymidine block and released for different times, the profiles show cell cycle distribution. *, P < 0.05.

 

Figure 2
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  		Fig. 2. p110β is a nuclear protein and regulates DNA elongation. (A) Single-molecule analysis of DNA replication in synchronized NIH 3T3 cells treated with PIK75 or TGX221 inhibitors at 7 h postserum addition, pulse-labeled (20 min) with BrdU at 12 h postserum addition, and collected immediately for analysis. Genomic DNA fibers were stretched by DNA combing. Newly replicated DNA was detected by immunofluorescence with an anti-BrdU Ab (green); DNA fibers were counterstained with anti-DNA Ab (red). Representative fibers are shown. (Scale bar, 50 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 < 0.0001. (C) NIH 3T3 cells were cotransfected with red fluorescence protein (RFP) and control, p110{alpha}, or -β shRNA; p110 localization was examined by immunofluorescence. DAPI nuclear staining is shown in Insets. (Scale bar, 10 µm.) NIH 3T3 cells or WT-p110{alpha} or -β transfected cells were fractionated and examined in WB (Right).

 

Figure 3
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  		Fig. 3. p110β controls PCNA binding to chromatin and to DNA Pol{delta}. (A) NIH 3T3 cells were infected with KR-p110{alpha}- or -β-encoding viruses, synchronized and collected at different times. MCM2/MCM4 levels in cytosolic, nuclear and chromatin fractions were examined in WB. (B) Active p110{alpha}- and -β cells and control NIH 3T3 cells were synchronized in G0 and released for different times. PCNA in Pol{delta} immunoprecipitates and PCNA levels were examined in the chromatin fraction, total Pol{delta} and PCNA were also examined in WB. (C and D) NIH 3T3 transfected with KR-p110{alpha} or -β (C) or with control, -{alpha}, or -β shRNA (D) were collected and examined at the indicated times. Analyses were as in B (n = 3). (E) Active p110{alpha}- and β-expressing NIH 3T3 cells were synchronized in G0 and released for different times. PCNA and p21Cip levels in p21Cip immunoprecipitates and total PCNA levels in chromatin-free extracts were examined in WB. (F) NIH 3T3 cells transfected with KR-p110{alpha} or -β were examined as in E. Chr, chromatin. Percentage cells in S phase indicated below gels. The circles show the time for S phase entry. (A–F) One representative experiment of at least three with similar results.*, P < 0.05.

 

Figure 4
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  		Fig. 4. p110β controls nuclear PKB. (A) NIH 3T3 cells transfected with A145, A146, D145, or D146 p21Cip mutants were fractionated. PCNA levels were measured in p21Cip immunoprecipitates, chromatin-containing and -free fractions; p21Cip expression was also examined in chromatin-free fractions. Graphs show the percentage PCNA signal (mean ± SD) in p21Cip immunoprecipitates and that of PCNA in chromatin fractions, compared with the maximum PCNA signal in each case (n = 3). (B) NIH 3T3 transfected with control, p110{alpha}, or β shRNA were synchronized in G0 and released (9 h). WB shows pT145-p21Cip in p21Cip immunoprecipitates from chromatin extracts; graphs show the pT145-p21Cip signal (percentage ± SD) normalized to p21Cip levels and compared with the signal at 9 h in controls (100%; n = 3). WB (bottom left) shows p110 expression levels. (C) NIH 3T3 cells transfected with KR-p110 mutants were synchronized after 24 h and other cells were treated at 7 h with TGX221 (30 µM) or PIK75 (0.08 µM); cells were collected at 9 h. pPKB levels were measured in WB. (D) pPKB localization examined by immunofluorescence in cells cotransfected with KR-p110 mutants and RFP, fixed 9 h after G0 release. Graphs show fluorescence intensity in arbitrary units (AU) examined along the line in the images. Insets show expression of KR mutants. (Scale bar, 50 µm.) (E) Phosphorylation of PKB substrates was examined by WB in fractionated extracts of U2OS cells that were thymidine-arrested, then released (1 and 2 h). (F) NIH 3T3 cells expressing KR-p110β or {alpha} mutants alone or in combination with D145-p21Cip were BrdU-labeled and chased at different times. Graph shows the cell percentage remaining in S phase (mean ± SD, n = 3). *, P < 0.05.

 

Figure 5
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  		Fig. 5. p110β associates with PKB and PCNA. (A) NIH 3T3 transfected with control or p110β shRNA were selected with puromycin (2 µg/mL, 48 h), then examined. Other samples were treated with TGX221 or PKB inhibitors for 12 h before collection. Immortalized p110β–/– mouse MEF, and p110β–/– MEF reconstituted with WT- or KR-p110β were cultured in exponential growth. A fraction of the cells were pulsed-labeled with BrdU (1 h). Graphs show the percentage 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 PCNA in the chromatin fraction, as well as PCNA and p110β in the chromatin-free fraction. Graphs show the percentage of chromatin-bound PCNA normalized to total PCNA and compared with maximum signal in control NIH 3T3 or in MEF. (C) Immortalized MEF as in A were arrested by thymidine treatment, then released for different times. Cell fractions were examined in WB to test for pPKB and PKB levels; the latter was then reprobed for PTEN. The graphs show 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 fractionated. The levels of PKB, p110{alpha} and β in these fractions were examined by WB (Left). Endogenous p110{alpha} or β from cytosolic (1500 µg) and nuclear extracts (600 µg), or PKB from cytosolic (300 µg) and nuclear extracts (200 µg) were immunoprecipitated. We tested for PKB and p85 in p110 immunoprecipitates by WB. Controls 1–3, protein A plus each of the antibodies. Graph shows the percentage of p110-associated PKB signal, compared with maximal PKB signal (in PKB immunoprecipitates from an equivalent protein amount). (E) Nuclear fractions were obtained from synchronized NIH 3T3 cells (at 12 h). PCNA (800 µg) or p110 (200 µg) immunoprecipitates were tested in WB for p110. For control 1, protein A was incubated with Ab; control 2, protein A was incubated with lysate. Graphs show the percentage of p110 signal in PCNA immunoprecipitates compared with maximal p110 signal (p110 immunoprecipitated from an equivalent protein amount). *, P < 0.05.

 

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