Class I_A PI3K subunits p85β and p110β concentrate in the nucleus. (A) HeLa and SAOS-2 cells and freshly isolated MEFs were cultured in exponential growth and analyzed by IF using anti-p110β Ab. DNA was stained with DAPI (insets). The graph shows the percentage of cells with predominant p110β nuclear staining (n = 30). (B) Endogenous p110β, p110α, p85α, and p85β localization in NIH 3T3 cells was analyzed by IF using specific Ab; DNA was stained with DAPI (insets). The graph is as described in panel A. (C) NIH 3T3 cells were cotransfected with GFP plus p85α- or p85β-specific shRNA (48 h), and the cells were fixed and analyzed by IF using specific Abs. WB shows the downregulation of p85α and p85β after shRNA transfection. Insets show transfected (GFP⁺) cells. (D) HeLa cells, MEFs, and NIH 3T3 cells were fractionated into cytoplasmic nuclear and chromatin extracts, which were analyzed by WB using the indicated Abs. Tubulin and histone were used as cytoplasmic and nuclear/chromatin controls. Bar, 10 μm. Dashed lines depict cell membrane. n.s., not statistically significant; **, P < 0.001 (Student t test).

Overexpressed p110β localizes in cytoplasm. (A) PC12, U2OS, and NIH 3T3 cells were cultured alone or treated with NGF (100 ng/ml), fetal bovine serum (20%), or PDGF (50 ng/ml), respectively (30 min). Cells were fractionated, and cytoplasmic nuclear and chromatin fractions were analyzed by WB using the indicated Abs. Graphs show the p110β and p110α signal intensities in arbitrary units (AU). (B) NIH 3T3, HeLa, and SAOS-2 cells were transfected with rp110β (48 h). rp110β localization was examined by IF using anti-p110β Ab. (C) Myc-tagged rp110α, HA-rp85α, and HA-rp85β were transfected individually into NIH 3T3 cells and processed for indirect IF with appropriate tag-specific antibodies. (D) rp110β-transfected NIH 3T3, HeLa, and SAOS-2 cells were treated with cycloheximide (10 μg/ml, 5 h) before IF staining as described in panel A. Bar, 10 μm.

The p85β regulatory subunit controls p110β nuclear translocation. (A) NIH 3T3 cells cotransfected with Myc-rp110β or -rp110α in combination with HA-rp85β or -rp85α (48 h) were fixed and analyzed by IF. Catalytic subunits were stained with anti-Myc-tag Ab and p85 with anti-HA Ab; square brackets indicate channels from the same image. Graphs show the percentage of p110 nuclear signal relative to the total (100%) (n = 30). (B) WT or p85β-deficient MEFs were cotransfected with GFP plus scrambled or p85α shRNA (48 h), and the cells were fixed and analyzed by IF using specific Abs. Insets show transfected (GFP⁺) cells. The graph shows the percentage of cells with predominant p110β nuclear staining (n = 30). (C) NIH 3T3 cells were cotransfected with GFP plus p85α- or p85β-specific shRNA or both (48 h), the cells were fixed, and nuclear p110β was analyzed by IF using specific Abs. Insets show transfected (GFP⁺) cells. Dashed lines depict the cell nuclei. Graphs are as described in panel A. (D) Scheme of the rp85β-α chimera. The p85β region between amino acids 78 to 351 was replaced with amino acids 77 to 363 from p85α. NIH 3T3 cells cotransfected with the HA-rp85β-α chimera plus rp110β were stained with anti-p110β and -HA Ab. (E) Graphs are as described in panel A. Bar, 10 μm. **, P < 0.001.

p110β contains an NLS motif in the C2 domain. (A) Domain structure of p110β, potential NLS sequences, and replacement of basic with nonbasic residues in mutants 1 to 3. (B) Computational model of the p85β/p110β complex. The p85β fragment (containing the nSH2 and iSH2 domains) is indicated in blue. The following p110β domains are indicated: the p85-binding domain (brown), the Ras-binding domain (purple), the catalytic domain (yellow), the C2 domain (green), and the helical domain (cyan). NLS sequences (located in the RBD, C2 domain, and C terminus) are shown as gray spheres; intermediate sequences are indicated in red. (C) Alignment of human and mouse p110β and p110α sequences at the region surrounding the C2 domain NLS of p110β (boxed). (D) NIH 3T3 cells were transfected with rp85β and WT or rp110β mutants 1 to 3. Expression levels were examined by WB. (E) The cellular localization of rp110β mutants was analyzed by IF using anti-Myc tag Ab. (F) Percent cells with the indicated phenotypes (n = 30). (G) NIH 3T3 cells were cotransfected with rp85β with WT-rp110β or NLS-p110β-mutant1. Cytoplasmic, nuclear, and chromatin fractions were examined by WB. Tubulin and histones were used as controls. Bar, 10 μm. (H) cDNA encoding mouse rp85β and WT- or C2 mutant1-rp110β were transcribed or translated in vitro in the presence of [³⁵S]methionine. The association of rp85β with WT- or mutant1-rp110β was analyzed by HA- or Myc-tag IP. The extract composition (TT extracts) and p85/p110 complex formation were examined by SDS-PAGE and autoradiography. (I) cDNA encoding mouse rp85β and WT- or mutant1-rp110β were transcribed or translated as in panel A. rp85β/rp110β complexes were purified with anti-pan-p85 Ab and tested in an in vitro kinase assay using PtdIns(4,5)P₂ as a substrate. *, P < 0.01 (Student t test).

p85β regulates p110β nuclear export. (A) NIH 3T3 cells were transfected with HA-rp85β, HA-rp85α, Myc-rp110β, or Myc-rp110α (48 h). Transfected cells were untreated or leptomycin B treated (5 ng/ml; 2 h) before fixing. Samples were stained for IF using anti-HA or -Myc tag Ab. (B) NIH 3T3 cells were transfected with the HA-rp85β-α chimera or HA-rp65β (48 h). Cells were untreated or pretreated with leptomycin B 2 h prior to fixing, and then stained as described above. The graph shows the percentage of nuclear signal relative to total cell signal (100%) (n = 30). (C) rp85β/rp110β or p65β/p110β were expressed in NIH 3T3 cells (48 h). The cells were treated with leptomycin B as described above and examined by IF; square brackets indicate channels from the same image. The graph is as described in panel B. Bar, 10 μm. *, P < 0.01; **, P < 0.001.

The p85β subunit has a Crm1-dependent nuclear export signal. (A) Scheme of Leu-rich regions in the p85β sequence. (B) NIH 3T3 cells were transfected with cDNA encoding Δ100Np85β or NESmut-rp85β (48 h). Transfected cells were untreated or leptomycin B treated (5 ng/ml, 2 h) and processed for IF using anti-HA Ab. The graph shows the percentage of nuclear signal relative to total cell signal (100%) (n = 30). (C) NIH 3T3 cells were cotransfected with Myc-rp110β and either Δ100Np85β or NESmut-rp85β (48 h); protein localization was analyzed by IF using anti-HA or -p110β Ab. The graph is as described in panel B. Bar, 10 μm. **, P < 0.001.

Nuclear localization of p110β is necessary for cell survival. (A) NIH 3T3 cells were cotransfected with p110β-specific shRNA and rp85β plus shRNA-resistant WT-p110β or -NLS-p110β mutant1 (48 h); protein expression was examined by WB. (B) NIH 3T3 cells were transfected as described in panel A. After 24 h, irradiated (10 Gy) or unirradiated cells were cultured for a further 24 h. As a positive control, cells were treated with H₂O₂. Cell viability was determined by flow cytometry using annexin V and propidium iodide (mean ± the standard deviation [SD]; n = 3). (C) Transfected NIH 3T3 cells from panel A were lysed, and the cytoplasmic fraction was analyzed by WB. The graph shows the percentage of cytochrome c relative to the maximal signal from H₂O₂-treated cells (100%) (mean ± the SD; n = 3). n.s., not statistically significant; *, P < 0.01.