BRD7, a Tumor Suppressor, Interacts with p85α and Regulates PI3K Activity  Yu-Hsin Chiu, Jennifer Y. Lee, Lewis C. Cantley  Molecular Cell  Volume 54, Issue 1, Pages 193-202 (April 2014) DOI: 10.1016/j.molcel.2014.02.016 Copyright © 2014 Elsevier Inc. Terms and Conditions

Molecular Cell 2014 54, 193-202DOI: (10.1016/j.molcel.2014.02.016) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 1 BRD7 Binds to p85α (A) HEK293T cells were cotransfected with hemagglutinin (HA)-tagged p85α and GST protein, GST-tagged wild-type BRD7 or GST-tagged NLS-deleted BRD7 as indicated, followed by GST pull-down (GST-PD) and immunoblotting analysis with antibodies against GST or HA. (B) HEK293T cells were cotransfected with Flag-tagged p85α and various GST-tagged BRD7 fragments, followed by immunoprecipitation with anti-FLAG M2-agarose and immunoblot analysis with antibodies against GST or Flag. (C) Schematic illustration of domain structures of BRD7 (upper panel). The lower panel shows the alignment of the p85-binding region of BRD7 protein sequences from human (H. sapiens), mouse (M. musculus), cow (B. taurus), chicken (G. gallus), zebrafish (D. rerio), fruit fly (D. melanogaster), and worm (C. elegans). (D) GST-tagged BRD7 and various Flag-tagged p85α fragments were transfected into HEK293T cells and then subsequently analyzed by GST pull-down and immunoblot assay with anti-GST or Flag antibodies. (E) Schematic illustration of domain structures of p85α (upper panel). Alignment of the BRD7-binding region of p85α protein sequences (lower panel). See also Figure S1. Molecular Cell 2014 54, 193-202DOI: (10.1016/j.molcel.2014.02.016) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 2 BRD7 Induces the Nuclear Translocation of p85α (A) COS7 cells were transfected with GFP-p85 and GST-tagged wild-type or mutant (ΔNLS or Δ543–604) BRD7. Antibody against GST was used to detect BRD7 in the immunofluorescence analysis. Cells were stained with DAPI and imaged with a fluorescent microscope. (B) Crude lysates from HeLa cells transfected with Flag-tagged p85α and increasing amounts of myc-tagged BRD7 (0, 1, 2, or 4 μg) were fractionated into cytosolic and nuclear fractions as described in the Experimental Procedures. Proteins in these fractions were analyzed by immunoblotting with antibodies against p85α, myc, lamin B1, and α-tubulin. (C) COS7 cells were transfected with mCherry-BRD7 and then stained with antibody against p85α and DAPI. The localization of p85α and BRD7 was analyzed by immunofluorescence. (D) HEK293T cells were transfected with GST protein alone, GST-tagged wild-type, or Δ543–604 BRD7, followed by GST pull-down and immunoblotting analysis with antibodies against p85α or GST. (E) Endogenous BRD7 was immunoprecipitated with antibodies against BRD7 from mononuclear cell lysates. Rabbit immunoglobulin G (IgG) was used as a control. Cell lysates and immunoprecipitates were analyzed by immunoblotting with antibodies against p85α and BRD7. (F) CHO-K1 cells were cotransfected with GFP-p85α and HA-tagged p110α together with GST-tagged wild-type or ΔNLS BRD7. The cells were immunostained with antibodies against GST and HA and imaged with a fluorescent microscope. (G) Cell lysates from HeLa cells transfected with GST-tagged BRD7 and HA-tagged p85α were separated by Superdex 200 using the FPLC system. Every other fraction (even-numbered fractions) was subjected to immunoblotting analysis with antibodies against GST, HA, p110α, p110β, or ARID2. The fractions containing 670 or 200 kDa proteins were indicated. (H) Fraction 14 from (G) was subjected to GST pull-down assay (GST-PD) and immunoprecipitation (IP) using rabbit IgG, p85α, p110α, or p110β antibodies. Samples were further analyzed by immunoblotting using antibodies against GST, p85α, p110α, p110β, and ARID2. See also Figures S2 and S3. Molecular Cell 2014 54, 193-202DOI: (10.1016/j.molcel.2014.02.016) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 3 BRD7 Reduces PI3K Signaling and p110 Expression (A) HeLa cells transfected with myc-tagged BRD7 or a control plasmid were under serum starvation overnight and then stimulated with 100 μM insulin (I) or 15% serum in Dulbecco’s modified Eagle’s medium (DMEM) (S). Cell lysates were separated by SDS-PAGE and analyzed with antibodies against phospho-Akt (S473), total Akt, and myc. (B) HeLa cells were transfected with increasing amounts of myc-tagged BRD7 and immunoblotted with antibodies against p110α, p110β, p85α, myc, and β-actin. (C) Stable cells expressing V5-tagged wild-type or ΔNLS BRD7 were confirmed by immunoblotting with antibody against V5. The amount of β-actin was used as a loading control (lower panel). Stable cells were stimulated with insulin or serum, followed by immunoblot analysis as mentioned in (A). (D) HeLa cells expressing wild-type or ΔNLS BRD7 were analyzed as indicated in (B). (E and F) HeLa cells were transfected with the indicated amount of BRD7 DNA. Total RNAs were extracted from those cells and analyzed with real-time PCR using primers specifically designed for detecting p110α, p110β, p85α, BRD7, and GAPDH. The mRNA levels of p110α, p110β, and p85α (E) or BRD7 (F) were normalized with the level of the housekeeping gene, GAPDH. ns, nonsignificant difference. The values are the average of triplicates ± SD. Molecular Cell 2014 54, 193-202DOI: (10.1016/j.molcel.2014.02.016) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 4 BRD7 Destabilizes p110 and Negatively Regulates PI3K Signaling (A) HeLa cells were transfected with two different pairs of siRNA oligos (a and b) against BRD7 or a control oligo. High-salt cell lysates were immunoblotted with antibodies against BRD7 (for RNAi efficiency) or β-actin (for loading control). An asterisk (∗) indicates a nonspecific band detected by antibody against BRD7. (B) Cells from (A) were serum starved overnight and subsequently treated with 100 μM insulin (I) or 15% serum in DMEM (S). Cell lysates were analyzed by immunoblotting with an antibody against phospho-Akt (S473) or total Akt. (C) BRD7 siRNA-resistant cells and the parental HeLa cells were transfected with control siRNA or siRNA against BRD7 (oligos b) and then immunoblotted with anti-V5, BRD7, or β-actin. (D) Cells from (C) were analyzed as described in (B). (E) NCI-H520 cells were transfected with control siRNA and siRNA oligos against BRD7 (a and b). Cell lysates were immunoblotted with antibodies against p110α, p110β, p85α, BRD7, or β-actin. An asterisk (∗) indicates a nonspecific band detected by antibody against BRD7. (F) Cells from (E) were examined with immunofluorescence using antibody against p85α, followed by Alexa Fluor 568 phalloidin and DAPI staining, and imaged with a fluorescent microscope. (G) HeLa cells were transfected with siRNA oligos against BRD9 or a control oligo. High-salt cell lysates from those cells were immunoblotted with antibodies against p110α, p110β, p85α, BRD9, BRD7, or β-actin. (H) Cells from (G) were serum starved overnight and then treated with 100 μM insulin (I) or 15% serum in DMEM (S). Cell lysates were analyzed by immunoblotting with an antibody against phospho-Akt (S473) or total Akt. See also Figure S4. Molecular Cell 2014 54, 193-202DOI: (10.1016/j.molcel.2014.02.016) Copyright © 2014 Elsevier Inc. Terms and Conditions